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2018-04-03T05:57:49.753Z | 2002-11-08T00:00:00.000 | 21358039 | {
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} | pes2o/s2orc | Charge attraction and beta propensity are necessary for amyloid fibril formation from tetrapeptides.
Amyloid fibrils in which specific proteins have polymerized into a cross-beta-sheet structure are found in about 20 diseases. In contrast to the close structural similarity of fibrils formed in different amyloid diseases, the structures of the corresponding native proteins differ widely. We show here that peptides as short as 4 residues with the sequences KFFE or KVVE can form amyloid fibrils that are practically identical to fibrils formed in association with disease, as judged by electron microscopy and Congo red staining. In contrast, KLLE or KAAE do not form fibrils. The fibril-forming KFFE and KVVE show partial beta-strand conformation in solution, whereas the non-fibril-forming KLLE and KAAE show random structure only, suggesting that inherent propensity for beta-strand conformation promotes fibril formation. The peptides KFFK or EFFE do not form fibrils on their own but do so in an equimolar mixture. Thus, intermolecular electrostatic interactions, either between charged dipolar peptides or between complementary charges of co-fibrillating peptides favor fibril formation.
Protein aggregates in the form of amyloid fibrils are found in about 20 diseases, including Alzheimer's disease, transmissible spongiform encephalopathies, Parkinson's disease, and type II diabetes mellitus (1). X-ray diffraction data suggest that the proteins have polymerized into a cross--sheet structure with the -strands perpendicular to the fibril axis (2). The structure of amyloid fibrils is typically very similar, although the polypeptides they are formed from can be highly dissimilar in their native states. Initially, only a few peptides and proteins were considered capable of forming fibrils, although also short fragments of amyloidogenic proteins form fibrils. More recently, it has been established that even globular all-helical proteins like myoglobin, which normally do not give rise to amyloid, can be converted to fibrils if incubated under partly denaturing conditions (3). This suggests that most proteins have the potential to form amyloid fibrils.
It is not known which features cause a few specific proteins to form amyloid fibrils in vivo. Some amyloidogenic peptides and proteins harbor specific ␣-helices that are predicted to generate -strands (4), and evidence that aggregation is initiated from particular regions of a polypeptide chain is accumu-lating (5). For example, the peptides NFGAIL from islet amyloid polypeptide, HQKLVFFAED from the amyloid -peptide (A), 1 and VQIVYK from tau form fibrils and are crucial for fibril formation of the full-length peptides (6 -8). Common to these sequences is the presence of hydrophobic amino acids of which at least one is aromatic. In this work, we aimed to explore the minimum requirements for fibril formation in short model peptides. The results show that peptides as short as 4 residues can form amyloid fibrils. Hydrophobic residues with high propensities for -strand conformation and residues with complementary charges promote fibril formation, and positively and negatively charged peptides can copolymerize.
EXPERIMENTAL PROCEDURES
Peptide Incubations-All peptides were purchased from Interactiva (Darmstadt, Germany) and purified by reversed-phase high pressure liquid chromatography using a C18 column and a linear gradient of water/acetonitrile supplemented with 0.1% trifluoroacetic acid. The identity of the peptides was verified with electrospray mass spectrometry. The peptides were incubated at 200 or 300 M for 10 days at 37°C in 50 mM phosphate buffer, pH 7.0, or in water, pH 5.0. The coincubation of KFFK and EFFE employed 100 M of each peptide.
Electron Microscopy (EM)-After incubation the peptide solutions were centrifuged at 20,000 ϫ g, and pelleted material was suspended in 40 l of water by low energy sonication for 5 s. Aliquots of 8 l were placed on EM grids covered by a carbon-stabilized Formvar film. Excess fluid was withdrawn after 30 s, and after air drying the grids were negatively stained with 1.5% uranyl acetate in water. The stained grids were examined and photographed in a Philips CM120TWIN electron microscope operated at 80 kV. Determination of fibril widths was made from high magnification micrographs (ϫ180,000 -200,000) viewed under a ϫ15 magnifier equipped with a graduated scale glass.
Congo Red Staining-Pelleted material was mixed with Congo red dissolved in Tris-buffered saline (TBS), pH 7.4, and incubated overnight. The sample was spun at 16,000 ϫ g for 30 min. The supernatant was aspirated and the pellet was resuspended in TBS and spun at 16,000 ϫ g. The pellet was resuspended in fresh TBS and transferred to a microscope slide and viewed under polarized light at ϫ200 magnification.
CD Spectroscopy-For analysis of secondary structure by CD spectroscopy peptides were dissolved at 300 M concentration in sodium phosphate buffer, pH 6.0, or water. Immediately after solubilization, CD spectra between 180 and 260 nm were recorded at 20°C with 2-s response time, 2 data points/nm, and scan speed of 20 nm/min using an AVIV model 62DS spectropolarimeter (Jacksonville, NJ). To compensate for the contributions of the phenylalanine side chains to the CD spectra, the spectrum of the dipeptide FF was subtracted from the spectra of peptides containing an FF sequence. Residual molar ellipticities () were calculated from peptide concentrations determined by amino acid analysis, and are expressed in kilodegrees ϫ cm 2 /dmol.
RESULTS
To find the minimum requirements for fibril formation, we investigated the peptides KFFE, KVVE, KLLE, KAAE, KFFK, and EFFE. Lysine and glutamate have high -sheet pair correlation, i.e. they are often found in neighboring strands in -sheets (9), and KE-rich peptides form fibrils (10). Phenylalanine and valine are frequent in -strands, whereas leucine and alanine are overrepresented in helices (4).
Fibril Formation-After incubation at 37°C for 10 days KFFE and KVVE produce sedimentable amyloid fibrils (1.2-1.6 nm in width) and fibril bundles as detected by EM (Fig. 1a). Moreover, the tetrapeptide aggregates show a classical Congo red birefringence under polarized light (Fig. 1b). The tetrapeptide fibrils are indistinguishable from disease-associated amyloid fibrils in these respects. The peptides KLLE and KAAE do not form fibrils, suggesting that hydrophobic interactions are not sufficient for fibril formation. Removal of the terminal charges of KFFE by acetylating the N terminus and amidating the C terminus significantly reduced fibril formation. Fibril formation of KFFE and KVVE was more pronounced in water than in phosphate buffer, indicating that charge attractions are important for fibril formation. The peptides KFFK and EFFE do not form fibrils when incubated individually. However, coincubation of equimolar amounts of KFFK and EFFE produce fibrils as detected by EM (Fig. 2). Typically, these fibrils were shorter and thicker (average width, 2.5-3.0 nm) than the fibrils formed from KFFE or KVVE and were also grouped into small aggregates. Fibrils virtually identical to those shown in Fig. 1a, although less abundant, were formed also from the KFFK/ EFFE mixture. Thus, heteropolymers can be formed from peptides with complementary charges.
Next, we investigated whether shorter peptides could form fibrils. The tripeptides were chosen based on optimal charge complementarity (KFE) and maximal -strand propensity (VVV and FFF). Neither of these peptides formed fibrils. Therefore, we conclude that 4 residues are necessary and sufficient for fibril formation.
Secondary Structure Analysis-CD spectra of freshly dissolved KFFE show two minima in the 216 -217 and 198 -200 nm regions (Fig. 3A), diagnostic of coexisting -strand and random structures. In contrast, non-fibrillogenic KAAE (Fig. 3B) and KLLE (data not shown) show CD spectra with one minimum at about 190 nm and a broad maximum around 215 nm, typical of an unordered structure. The comparison of the CD spectra of KVVE and KAAE (Fig. 3B) highlights the presence of -strand structure in KVVE, evidenced by the decrease in ellipticity between 210 and 220 nm, a red shift of the short wavelength minimum, and increased ellipticity around 190 nm.
The CD spectra of the non-fibrillating KFFK and EFFE (Fig. 4) show the same features as the spectrum of KFFE. The spectrum of co-incubated KFFK and EFFE was virtually identical to the spectra of the individual peptides, suggesting that a significant part of the -strand structure is present in the monomeric peptides. DISCUSSION Many proteins and peptides form fibrils. The determinants for fibril formation are not fully understood. Here we show that peptides as short as 4 residues can form amyloid fibrils and that -strand structure in solution and attractive electrostatic interactions are required for fibrillogenesis. KFFE and KVVE are the shortest fibril-forming peptides known to date and could serve as models for further studies of amyloid fibril formation and structure. The similarity in fibril formation efficiency and secondary structure of KFFE and KVVE indicates that -strand structure as such is a strong determinant of fibril formation. This suggests that the frequent occurrence of aromatic residues, in particular F, in amyloid-forming proteins is in part related to their high -strand propensities (4). However, also the contributions from interactions between phenylalanines in adjacent strands are probably important (11). The principles for fibril formation now proposed are in good agreement with previous results from studies of amyloid-forming peptides. For example, it has been found that changing the sequence KLVFF in A (positions 16 -20) to AAVFA prevents fibril formation (12), as would be predicted from the present results. Likewise, a V18A replacement in A reduces the capacity to form fibrils (13), which is in excellent agreement with the different fibrillation capacity now found for KVVE and KAAE. This agreement is noteworthy, as the peptides now studied are not derived from known fibril-forming proteins but were designed from basic structural features of amino acid residues.
The fibril formation was more pronounced at low salt concentrations, indicating that charge interactions are important. The peptides KFFK or EFFE did not form fibrils when incubated alone but did so when co-incubated. Thus, the possibility of K-E interactions seems to be essential. These results and the high pair correlation of K and E in -sheets (9) suggest that the peptides KFFE and KVVE have an antiparallel alignment in the fibrils. An energy-minimized model of KFFE in an antiparallel alignment is shown in Fig. 5.
If the present fibrils are composed of the tetrapeptides in a cross--sheet conformation, their width should be 1.4 nm (4residue -strand). This was found to be the case; the width of 10 measured fibrils was 1.2-1.6 nm. In general, the appearance of amyloid fibrils as observed by EM is similar, independent of size and structure of the native protein. The smallest widths in amyloid fibrils formed from 40-residue A and ϳ350-residue transpeptidase are 3-4 nm, and for the 100-residue C-terminal fragment of the amyloid precursor protein and the ϳ600-residue coagulation factor XIII, 4 -6-nm fibrils are found (4). The widths of the fibrils apparently do not correspond to the size of the protein of which they are composed. Possible explanations for this discrepancy include that the protein in question makes several intramolecular -strands in the direction of the fibril and that only a fraction of the polypeptide chain is incorporated The polymeric structure is stabilized by electrostatic interactions between the terminal K (blue) and E (red) side chains, van der Waals interactions between phenylalanine side chains (black), and hydrogen bonds between main chains (gray). in the fibrils. The possibility that amyloid fibrils made from different proteins and peptides can be composed of -strands as short as 4 residues may help to explain why attempts to find common motifs among these proteins have so far been largely unsuccessful. | v3-fos-license |
2020-10-06T13:33:21.978Z | 2020-09-24T00:00:00.000 | 222148998 | {
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} | pes2o/s2orc | A New Thiopeptide Antibiotic, Micrococcin P3, from a Marine-Derived Strain of the Bacterium Bacillus stratosphericus
A new thiopeptide (micrococcin P3, 1) and a known one (micrococcin P1, 2) were isolated from the culture broth of a marine-derived strain of Bacillus stratosphericus. The structures of both compounds were elucidated using spectroscopic methods, including extensive 1D and 2D NMR analysis, high resolution mass spectrometry (HRMS), and tandem mass spectrometry. Both compounds exhibited potent antibacterial activities against Gram-positive strains with minimum inhibitory concentration (MIC) values of 0.05−0.8 μg/mL and did not show cytotoxicity in the MTT assay up to a concentration of 10 μM. This study adds a new promising member, micrococcin P3, to the family of thiopeptide antibiotics, which shows potential for the development of new antibiotics targeting Gram-positive bacteria.
Introduction
Natural product-derived antibiotics have provided humankind with an effective weapon to fend off bacterial infections and diseases since their discovery in 1929. However, the popularity of antibiotics, especially broad-spectrum antibiotics, has quickly led to their overuse and misuse in people and animals, which has accelerated the emergence and development of antibiotic resistance in pathogens. Increasing resistance to antibiotics reduces the treatment options available for patients and imposes significant economic burdens on patients due to prolonged stays in hospitals. Moreover, medical procedures, such as major surgery and cancer chemotherapy, would become very risky without effective antibiotics to prevent and treat infections. According to the World Health Organization [1], antibiotic resistance is starting to complicate the combat against HIV and malaria as well. Thus, antibiotic resistance is a major global healthcare concern. In this context, it is more critical now than ever to discover new and effective antibiotics to overcome this worldwide public health crisis.
To discover new antibiotics, natural product researchers have dedicated much time and effort to explore novel bio-resources in various environments, including seas. The seas are highly complicated ecosystems with surprising microbial abundances of 10 6 per mL seawater and up to 10 9 per mL ocean-bottom sediments. As early as the 1950s, marine bacteria were found to produce antimicrobial compounds. In the 1970s, the first report on the antibiotic properties of marine bacterial metabolites Molecules 2020, 25, 4383 2 of 9 was published [2]. Thereafter, the rate of discovery of new antibiotics from marine microbes has increased significantly with the improvement of structure characterization and sampling technologies. Today, with the application of genetic information-based exploration of natural chemodiversity, marine microbes can still be regarded as excellent sources of new antibiotics.
During our ongoing program toward the discovery of new antibiotics, 32 marine-derived bacterial strains were screened by MIC (minimum inhibitory concentration) bioassays and surveyed by LC-MS (liquid chromatography-mass spectrometry) screening. Three extracts of bacterial strains displayed antibacterial activities in our MIC bioassays. In one of these antibacterially active extracts (from Bacillus stratosphericus 16L088-2), a number of new thiopeptides, albeit in very low amounts, were detected. The solvent partition of the extract followed by repeated chromatography revealed a new thiopeptide, micrococcin P3 (1), along with a known one (micrococcin P1, 2). Thiopeptides are a family of naturally occurring peptide antibiotics with a complex molecular architecture [3]. Distinctive features of the thiopeptide structure include multiple thiazole rings and a highly substituted pyridine centerpiece, or a reduced form thereof, at the junction of the characteristic macrocyclic nucleus [4]. To date, as many as over 100 members of the thiopeptide family have been obtained, but none of them have reached the clinic for humans due to their low aqueous solubility and unfavorable pharmacokinetic properties. However, the profound chemical diversity and potent activities still render thiopeptides attractive in both the chemical and biological arenas. Herein, the isolation, structure elucidation, and antibacterial activity of micrococcins P1 and P3 are described.
Structural Elucidation of the Compounds
Micrococcin P3 (1), a white amorphous powder, was determined to have a molecular formula of C 48 H 49 N 13 O 9 S 6 by high resolution electrospray ionization mass spectrometry (ESIMS) data (m/z 1144.2185, calc. for [M + H] + 1144.2179). The UV spectrum showed thiocillin-like absorptions [5] at 220, 289, and 350 nm. The UV spectral data, together with the presence of six sulfur atoms in the highly unsaturated molecule, suggested that the compound is a member of the class of thiopeptides. The 1 H NMR spectrum (Table 1) revealed deshielded singlets assignable to six thiazoles (Thia, δ 8. 13, 8.29, 8.32, 8.43, 8.46, and 8.59) and two doublets typical of a 2,3,6-trisubstituted pyridine ring (δ 8.32 and 8.45), indicating that compound 1 is a member of series d thiopeptides [3]. Other deshielded proton signals were assigned to the six N-bonded protons (δ 7.94, 7.98, 8.21, 8.69, 9.54 and 9.85). The remaining signals displayed in the 1 H NMR spectrum are attributed to seven methyls, one shielded methylene, six methines attached to heteroatoms (O or N), three O-bonded protons, and two olefinics. The 13 C and edited HSQC (heteronuclear single-quantum correlation spectroscopy) spectra showed the presence of six carbonyl carbons and 15 non-protonated aromatic carbons, 12 of which were derived from disubstituted thiazoles and the other three were assigned to 2,3,6-trisubstituted pyridine. Analysis of COSY (correlation spectroscopy) data established the spin systems of one valine (V), one 2-hydroxypropylamide (Hpa), two threonine (T), and two didehydrobutyrine (Dhb) residues. The sequence of amino acids was determined by interpreting the key HMBC (heteronuclear multiple-bond correlation spectroscopy) correlations, especially those from the nonexchangeable amide protons to their surrounding carbonyl carbons. Additional support for the sequence came from the fragmentation patterns observed in the tandem mass spectra. The sequence Thia 1 -Dhb 1 -Hpa for the C-terminal amino acid side chain was traced from the fragments at m/z 986 and 1069 ( Figure S19). a Hpa, Dhb, Thia, Pry, T, V, and OH are abbreviations for 2-hydroxypropylamide, didehydrobutyrine, thiazole, pyridine, threonine, valine, and hydroxyl group, respectively. b Proton multiplicity and coupling constants are in parenthesis. c The 13 C NMR shifts were assigned on the basis of analysis of heteronuclear single-quantum correlation spectroscopy (HSQC) and heteronuclear multiple-bond correlation spectroscopy (HMBC) data.
A literature survey on series d thiopeptides revealed a close similarity of compound 1 with micrococcin P1 (2), a representative member of the class of thiopeptides. The general features of the 1 H NMR spectrum of compound 1 are analogous to those of 2 [6], except for those assigned to the didehydrobutyrine residue (Dhb 2 ) adjacent to a thiazole (Thia 5 ). Beyond our initial exception, the atomic connections of the Dhb 2 -Thia 5 moiety are exactly the same as those corroborated by the COSY, HSQC and HMBC data. The structure was finally identified by analysis of the ROESY data of compounds 1 and 2. The NOE correlation between H-8 and H-9 showed the same intensity for both compounds, while the NOE correlation between H-44 and H-45 was not observed at all for compound 1, but was clearly observed for compound 2 ( Figure S2). These observations indicated a 42E configuration for the Dhb-Thia 5 unit of compound 1, which is different from the 42Z configuration of 2 [7].
Although the structures harbored several typical characteristics of a nonribosomal peptide, thiopeptide antibiotics have been reported to be ribosomally synthesized peptides [5,8,9]. The complex molecular architectures of the thiopeptides were concluded to arise from posttranslational modifications of a linear precursor peptide, the composition of which is confined to the 20 proteinogenic amino acids. Thus, 1 was presumed to occur in its natural configuration as shown in Figure 1. This assumption was supported by comparison of the optical rotation and NMR data of 1 with those of 2, the configuration of which was confirmed by total synthesis [6,10]. Figure 1. This assumption was supported by comparison of the optical rotation and NMR data of 1 with those of 2, the configuration of which was confirmed by total synthesis [6,10].
Antibacterial Activity
The biological activity of the compounds was evaluated against a panel of terrestrial and marine-derived bacteria using the two fold microtiter broth dilution method [11]. The terrestrial test bacteria, routinely employed in our MIC assays, included three Gram-positive strains (Staphylococcus aureus KCTC 1927, Kocuria rhizophila KCTC 1915, and Bacillus subtilis KCTC 1021) and three Gram-negative strains (Escherichia coli KCTC 2441, Klebsiella pneumoniae KCTC 2690, and Salmonella typhimurium KCTC 2515). Both 1 and 2 strongly inhibited the growth of all three Gram-positive strains with MIC values of 0.05-0.8 μg/mL (Table 2), whereas no antibacterial activity was observed against Gram-negative strains up to a concentration of 26 μg/mL. The different susceptibility of Gram-positive and Gram-negative strains to antibiotics may stem from the structure differences of their cell envelop. Gram-positive bacteria lack an outer membrane but are surrounded by layer of peptidoglycan much thicker than that of Gram-negative strains. The thick peptidoglycan layer may absorb antibiotics more easily than thin one. On the contrary, the outer membrane of Gram-negative strains may function as an evolving barrier against the penetration of antibiotics. The compounds showed no cytotoxicity in the MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assay at a concentration of 10 μM. Thus, the new bioactive compound 1 was added to the family of thiopeptide antibiotics and showed potential for the development of new antibiotics targeting Gram-positive bacteria.
Antibacterial Activity
The biological activity of the compounds was evaluated against a panel of terrestrial and marine-derived bacteria using the two fold microtiter broth dilution method [11]. The terrestrial test bacteria, routinely employed in our MIC assays, included three Gram-positive strains (Table 2), whereas no antibacterial activity was observed against Gram-negative strains up to a concentration of 26 µg/mL. The different susceptibility of Gram-positive and Gram-negative strains to antibiotics may stem from the structure differences of their cell envelop. Gram-positive bacteria lack an outer membrane but are surrounded by layer of peptidoglycan much thicker than that of Gram-negative strains. The thick peptidoglycan layer may absorb antibiotics more easily than thin one. On the contrary, the outer membrane of Gram-negative strains may function as an evolving barrier against the penetration of antibiotics. The compounds showed no cytotoxicity in the MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assay at a concentration of 10 µM. Thus, the new bioactive compound 1 was added to the family of thiopeptide antibiotics and showed potential for the development of new antibiotics targeting Gram-positive bacteria.
This is the first report on the characterization and bioactivities of secondary metabolites from the bacterium B. stratosphericus. However, B. stratosphericus is a versatile and capable bacterium. A recent example is the hydrocarbonoclastic strain FLU5 of B. stratosphericus, which produces an efficient surface-active agent, BS-FLU5, in the presence of various substrates, including residual frying oil [12]. In a previous study, strain FLU5 was also shown to degrade fluoranthene [13], a member of the U.S. Environmental Protection Agency's 16 priority pollutant polycyclic aromatic hydrocarbon (PAHs). B. stratosphericus RNCM B-11677 and B-11678 were found to produce ethanol from lignocellulosic biomass [14,15]. B. stratosphericus LT745897 showed potential for biopesticide development due to its antagonistic activity against bacterial phytopathogens [16]. Our study suggests that B. stratosphericus represents an alternative source for the discovery of new antibiotics. Furthermore, the effects of 1 and 2 on the growth of marine-derived bacteria isolated from the same area as B. stratosphericus were investigated. Six representative marine-derived bacteria, including Vibrio parahaemolyticus, Photobacterium damselae, Shewanella algae, Bacillus amyloliquefaciens ssp-plantarum, and Pseudomonas stutzeri, were employed in the MIC bioassay. Both compounds displayed strong inhibitory activities against Enterococcus faecalis, exhibiting MIC values (0.06−0.8 µg/mL) superior to those of the positive controls (vancomycin and linezolid). In contrast, the antibacterial activities of 1 and 2 against the remaining five marine-derived bacteria (0.5−8 µg/mL) were lower than those of the positive controls (Table S1).
B. stratosphericus is commonly found in high concentrations in the stratosphere, hence the name stratosphericus. However, it can also be found in various other environments, such as deep seas, estuaries, and desert soils, due to atmospheric cycling processes. B. stratosphericus has developed admirable persistence, such as halo tolerance, to adapt to adverse circumstances. In our salt tolerance study, the strain could grow well on plates and in broth with a sea salt concentration up to 15%. Compounds 1 and 2 exhibited weak inhibitory activities against marine-derived bacteria, indicating that B. stratosphericus has evolved smart chemical strategies for interspecific competition to acquire bioecological equivalence in the marine community.
Solubility Issues with Thiopeptides
Like most of the other peptides, the main issue of thiopeptides is their poor aqueous solubility, which largely hinders their application in medicine and antimicrobial treatment. This is exemplified by thiostrepton, one of the most extensively studied thiopeptide antibiotics. Although thiostrepton exerts impressive antibacterial activity via a distinct mode of action from those of current chemotherapeutics, it was approved by the US Food and Drug Administration (FDA) only as an external drug for veterinary use [17]. The lack of formulations of thiostrepton proper for human use originates from its poor water solubility and low bioavailability, which also restricts its application in animals. To overcome the obstacles faced in the applications of thiopeptides, various approaches have been tried by many research groups. For example, modification of nocathiacin I through efficient hydrolysis of the side chain generated a series of water-soluble analogues [18]. Structure-based design targeting the bacterial ribosome applied to thiostrepton led to several simpler derivatives [19]. Among the efforts on obtaining analogs with enhanced aqueous solubility, LFF571 is a noteworthy example [20]. LFF571 was developed by Novartis as a result of medicinal chemistry work on improving solubility and efficacy profile of GE2270A. For long time, LFF571 had been considered as a promising novel antibiotic for the treatment of Clostridium difficile infection (CDI). It reaches high colonic concentrations after oral administration and was proved safe and well tolerated in healthy volunteers in phase I trial. A phase II trial, started in 2015, showed higher clinical response rates than vancomycin. However, higher recurrence rates than vancomycin were also reported [21]. Thus, LFF571 was unfortunately discontinued from clinical development for CDI in 2019. Nevertheless, when considering the great endeavor carried out by researchers and the potent activities of thiopeptides, it can be believed that solubility issues with thiopeptides will be finally solved and analogs suitable for the treatment of infections will be discovered.
General Experimental Procedures
The optical rotations were measured in MeOH using a Rudolph Research Autopol III (Hackettstown, NJ, USA). UV spectra were recorded on a Hitachi JP/U-3010 UV spectrophotometer (Tokyo, Japan). All NMR spectra were recorded on a Bruker Ascend 700 spectrometer (Billerica Middlesex County, MA, USA) using DMSO-d 6 as solvent. Chemical shifts were reported with reference to the respective solvent peaks (δ H 2.50 and δ C 39.52 for DMSO-d 6 methanol-d 4 ). Electrospray ionization (ESI) source low-resolution mass data were obtained on an Agilent Technologies 6120 quadrupole mass spectrometer (Santa Clara, CA, USA) coupled with an Agilent Technologies 1260 series HPLC. High-resolution mass spectrometric data were collected on a JEOL JMS-700 double-focusing (B/E configuration) instrument (Tokyo, Japan). High-performance liquid chromatography (HPLC) was performed using a Waters HPLC (Milford Worcester County, MA, USA) equipped with a Waters 2998 photodiode array detector and Waters 1525 binary pump. HPLC-grade solvents (Daejeon, Korea) were used for HPLC. NMR solvents were purchased from Cambridge Isotope Laboratories Inc. (Andover, MA, USA).
Strain and Cultivation
The bacterial strain 16L088-2 was identified as a new member of the species B. stratosphericus on the basis of a partial 16S rRNA gene sequence using the universal primers 27f and 1492r (99.86% similarity to the sequence of B. stratosphericus strain 41KF2a; GenBank Accession No. NR042336). The bacterium was grown on SYP agar (10 g of soluble starch, 4 g of yeast extract, 2 g of peptone, and 15 g of agar dissolved in artificial seawater (sea salt 35 g/L distilled H 2 O)). The seed culture was prepared by inoculating the spores of the strain 16L088-2 into 10 mL of SYP liquid medium in a 50-mL conical tube and maintained in a shaking incubator (220 rpm) at 27 • C for three days. The seed culture (10 mL) was then inoculated into 1 L of SYP liquid medium in a 2.5-L Ultra Yield flask. Production fermentation was carried out under the same conditions as seed cultivation.
Extraction and Isolation of Compounds
In total, 40 L of SYP culture was fermented and 4.2 g of crude extract was harvested by EtOAc extraction. The EtOAc extract was then evaporated in vacuo and subjected to silica MPLC, eluting with a step gradient solvent system of 0 to 50% of MeOH-CH 2 Cl 2 , to obtain 8 fractions (1−8). Fraction 4, eluting with 5% MeOH-CH 2 Cl 2 , contained thiopeptides as detected by LC-MS. Fraction 4 was then was subjected to a semi-preparative RP-HPLC system equipped with a Phenomenex Luna C18 column (5 µm, 100 Å, 250 × 100 mm, UV = 230 nm, 2.0 mL/min) and eluted with 40% CH 3 CN in H 2 O (containing 0.05% TFA). Pure compound 1 (1.1 mg) was obtained as a minor peak following the major peak of compound 2 in the chromatogram ( Figure S1). Both compounds 1 and 2 were purified again by HPLC in the same condition and the purity (99%) was checked by evaporative light scattering detector.
Antibacterial Activity Assay
The following six microorganisms were obtained from the stock culture collection at the Korean Collection for Type Cultures: Staphylococcus aureus KCTC 1927, Kocuria rhizophila KCTC 1915, Bacillus subtilis KCTC 1021, Escherichia coli KCTC 2441, Klebsiella pneumoniae KCTC 2690, and Salmonella typhimurium KCTC 2515. The antibacterial activity was measured by the 2-fold microtiter broth dilution method. Blank sample (100 µL DMSO only) and 100-µL aliquots of the compounds and positive controls (vancomycin and ampicillin) at a concentration of 410 µg/mL in DMSO were added to different wells of column 1 of a 96-well microtiter plate. The wells of other columns were added with 50 µL of Mueller Hinton broth. Blank sample, the compounds, and positive controls were serially diluted by pipetting 50 µL of the sample in the wells of column 1 into the next wells of column 2, and the wells of column 2 into the next wells of column 3, and so on. Then, each well was inoculated with 50 µL of the test bacteria (5 × 10 5 cfu/mL). Thus, final concentrations of 205, 103, 51, 26, 13, 6.4, 3.2, 1.6, 0.8, 0.4, 0.2, 0.1, 0.05, 0.025 µg/mL of samples were made containing 50%, 25%, 13%, 6.3%, 3.1%, 1.6%, 0.8% 0.4%, 0.2%, 0.1%, 0.05%, 0.02%, 0.01%, 0.005% DMSO (v/v), respectively. The plates were incubated under static conditions at 37 • C for 24 h. MIC was measured as the concentration at which no growth was observed.
Cell Viability Assay
Cell viability was measured at 48 h post exposure to DMSO or indicated compounds by assessing the conversion of yellow MTT to purple formazan. In short, the CV-1 cells (1 × 10 4 cells/well) were cultured in 96-well plates at 37 • C for 24 h, and then each well was treated with DMSO or the compounds at the indicated concentration. After 2 days, 10 µL of MTT solution (5 mg/mL) was added to each well, and the plates were incubated at 37 • C for 3 h. The optical density values were determined at 560 nm by a standard microplate reader (Thermo Varioskan Flash, Waltham, MA, USA).
Conclusions
A new thiopeptide (1) and a known one (2) were obtained from a marine-derived strain of Bacillus stratosphericus. These compounds strongly inhibited the growth of all three tested Gram-positive strains without significant cytotoxicity up to a concentration of 10 µM, as evidenced by the MTT assay. This study adds a new promising member to the family of thiopeptide antibiotics, showing potential for the development of new antibiotics targeting Gram-positive bacteria. | v3-fos-license |
2020-11-18T14:06:57.951Z | 2020-11-01T00:00:00.000 | 226989258 | {
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} | pes2o/s2orc | Fungal Bioactive Anthraquinones and Analogues
This review, covering the literature from 1966 to the present (2020), describes naturally occurring fungal bioactive anthraquinones and analogues biosynthesized by the acetate route and concerning several different functionalized carbon skeletons. Hydrocarbons, lipids, sterols, esters, fatty acids, derivatives of amino acids, and aromatic compounds are metabolites belonging to other different classes of natural compounds and are generated by the same biosynthetic route. All of them are produced by plant, microorganisms, and marine organisms. The biological activities of anthraquinones and analogues comprise phytotoxic, antibacterial, antiviral, anticancer, antitumor, algicide, antifungal, enzyme inhibiting, immunostimulant, antiplatelet aggregation, cytotoxic, and antiplasmodium activities. The review also covers some practical industrial applications of anthraquinones.
Introduction
Anthraquinones are a group of natural compounds with a plethora of biological activities and potential practical applications. Most of them are produced by plant and micro-organisms among the living organisms [1,2]. They are acetate-derivative metabolites biosynthesized starting from a polyketide containing eight C2 units, which generates in turn with three aldol type condensations the carbon skeleton of anthraquinones except for the two carbonyl oxygens of the central ring. The latter are introduced by successive steps with an oxidation process. One example of this kind of biosynthesis is reported in Figure 1 for endocrin, a fungal anthraquinone produced by several Penicillium and Aspergillus species [3].
Among secondary metabolites anthraquinones are the most investigated natural products for their mechanism of action [4]. Plants, microorganisms, lichens, and algae are producers of metabolites possessing diverse biological activities such as phytotoxic, antibacterial, antiviral, anticancer, antitumor, algicide, antifungal, enzyme inhibiting, immunostimulant, antiplatelet aggregation, cytotoxic and antiplasmodium activities. Anthraquinones are frequently reported among the plethora of different classes of natural compounds as alkaloids, hydrocarbons, lipids, sterols, esters, fatty acids, derivatives of amino acids, terpenoids, and aromatic compounds [5][6][7]. The activity of several hydroxyland amino-anthraquinones cannot be exploited due to their weak solubility in water. Thus, some of them are converted into water-soluble analogues by biotransformation [8]. Anthraquinones have This review describes an advanced overview on anthraquinones, and related analogues grouped for the first time according to their natural sources. In particular, in addition to the isolation from fungal sources and their chemical characterization, their potential applications in different fields such as agriculture, medicine and the dyes industry are considered on the basis of their biological activities.
The first section chronologically describes the fungal anthraquinones starting from 1966 to the present day focusing on their sources, structures, and biological activities. The second section treats the industrial application of anthraquinones in a different field essentially as natural dyes. This part is focused on the comparison between natural and synthetic anthraquinone based dyes, their chemical derivatization and classification, and the advanced methods used in the treatment of the relative industrial wastewater to avoid severe negative environmental pollution. Finally, the main points described are summarized in the conclusion.
Fungal Anthraquinones and Analogues
Dothistromin (1, Figure 2, Table 1) was isolated as the main phytotoxin produced by Dothistroma pini (Hulbary), a pathogen inducing necrotic disease characterized by the formation of red bands on the infected needles of Pinus radiata and other pines [13]. The same fungus also produced six other anthraquinones: bisdeoxydothistromin; bisdeoxydehydrodothistromin; This review describes an advanced overview on anthraquinones, and related analogues grouped for the first time according to their natural sources. In particular, in addition to the isolation from fungal sources and their chemical characterization, their potential applications in different fields such as agriculture, medicine and the dyes industry are considered on the basis of their biological activities.
The first section chronologically describes the fungal anthraquinones starting from 1966 to the present day focusing on their sources, structures, and biological activities. The second section treats the industrial application of anthraquinones in a different field essentially as natural dyes. This part is focused on the comparison between natural and synthetic anthraquinone based dyes, their chemical derivatization and classification, and the advanced methods used in the treatment of the relative industrial wastewater to avoid severe negative environmental pollution. Finally, the main points described are summarized in the conclusion.
Fungal Anthraquinones and Analogues
Dothistromin (1, Figure 2, Table 1) was isolated as the main phytotoxin produced by Dothistroma pini (Hulbary), a pathogen inducing necrotic disease characterized by the formation of red bands on the infected needles of Pinus radiata and other pines [13]. The same fungus also produced six other anthraquinones: bisdeoxydothistromin; bisdeoxydehydrodothistromin; 6-deoxyversicolorin C; Macrosporin and 6-methylxanthopurpurin 3-methyl ether (8 and 9, Figure 2, Table 1) are two anthraquinones produced by Alternaria bataticola, the causal agent of a black spot of sweet potato [16]. Compound 8 was also isolated from other fungi of the same genus as A. porri, A. solani and A. cucumerina while 9 was isolated also from A. solani [16]. Then macrosporin was isolated together with another two anthraquinones, named altersolanols A and J (10 and 11, Figure 2, Table 1), as well as nectriapyrone, an α-pyrone, from the culture filtrates of Diaporthe angelicae (anamorph Phomopsis Macrosporin and 6-methylxanthopurpurin 3-methyl ether (8 and 9, Figure 2, Table 1) are two anthraquinones produced by Alternaria bataticola, the causal agent of a black spot of sweet potato [16]. Compound 8 was also isolated from other fungi of the same genus as A. porri, A. solani and A. cucumerina while 9 was isolated also from A. solani [16]. Then macrosporin was isolated together with another two anthraquinones, named altersolanols A and J (10 and 11, Figure 2, Table 1), as well as nectriapyrone, an α-pyrone, from the culture filtrates of Diaporthe angelicae (anamorph Phomopsis foeniculi), the causal agent of serious disease on fennel (Foeniculum vulgare) in Bulgaria. These four metabolites were tested on detached tomato leaves and only nectriapyrone and altersolanols A and J showed a modulate phytotoxicity while macrosporin was not toxic [17].
Stemphylin, is a phytotoxin (12, Figure 2, Table 1) produced by Stemphyfium botryosum, a fungal pathogen inducing a destructive disease in lettuce. The first structure of compound 12 was wrongly assigned by Barash et al. (1975 and1978) [18,19], and then corrected when it was isolated from the same fungus together with two other phytotoxic anthraquinones, the above cited macrosporin and dactylariol (13, Figure 2, Table 1) [20]. The latter compound (13) showed anti Adenosine TriPhosphate (ATP) catabolism in Erlich I ascite tumor cells while stemphylin showed a weak antitumor activity on the treated animal at a dose of 40 mg/kg [20]. Stemphylium botryosum, inducing leaf spot disease on beet plants, also synthesized macrosporin and dactylariol (8 and 13), together with other anthraquinones identified as stemphyrperylenol (14, Figure 2), alteroporiol, and stemphynols A and B (15-17, Figure 3, Table 1). The phytotoxicity of all the metabolites (8, 13-17) was tested on lettuce and beet evaluating the seeds elongation. Compound 13 was the most active while compound 14 exhibited a moderate inhibition while the other ones were inactive [21].
Rugulosin (18) was isolated together with emodin and skyrin from Hormonema dematioides and showed activity against survival of budworm larvae while the other two anthraquinones were inactive [24].
Aspergillus fumigatus, which is responsible for a lung disease, produced a plethora of secondary metabolites belonging to different classes of natural compounds. Among them, emodin, 2-chloro-emodin (21 and 22 Figure 3), and physcion (23, Figure 3) were also isolated. However, compound 23 appeared not to have a role in the fungal infection [25].
Physcion (23) was also isolated from the organic culture extract of the marine derived fungus Microsporum sp. and showed a cytotoxic effect on human cervical carcinoma HeLa cells and its apoptosis induction was deeply investigated. Physcion also caused the formation of reactive oxygen species (ROS) in the same cells [26].
Many Drechslera species, which are important pathogens on gramineous plants and their seeds, produced colored pigments [27]. The red pigment exudated from Drechslera teres, D. graminea, D. tritici-repentis, D. phlei, D. dictyoides, D. avenae was identified as the anthraquinone catenarin (24, Figure 3, Table 1) while that from D. avenae and Bipolaris sorokiniana were two other anthraquinones recognized as helminthosporin and cynodontin (25 and 26, Figure 3, Table 1). Catenarin (24) showed a total inhibition of Bacillus subtilis (Gram+) growth but had no effect on the Gram-bacterium Ervinia carotova but in part inhibited the mycelium growth of D. teres [28]. In particular, catanerin and emodin (24 and 21) were also found in kernels infected by Pyrenophora tritici-repentis (Died.) Drechs. (anamorph: Drechslera tritici-repentis (Died.) Shoem.), the causal agent of tan spot of wheat. Compound 24 caused the reddish discoloration with red smudge of kernels, while compound 21 indicated that P. tritici-repentis is a mycotoxigenic fungus. Compound 24 also induced non-specific leaf necrosis and appeared moderately active against some of the fungi associated with P. tritici-repentis suggesting its possible role in the life strategy of the pathogen [29]. Cryphonectria (Endothia) parasitica (Murr.) Barr, the causal agent of chestnut (Castanea sativa) canker disease and other species produces diaporthin, a phytotoxic benzopyranone pigment, together with phytotoxic anthraquinones. The hundreds of fungal strains were grouped into virulent, intermediate and hypervirulent and produced, respectively, diaporthin , rugulosin and skirin (18 and 19, Figure 3, Table 1), crysophanol (20, Figure 3, Table 1) and emodin (21, Figure 3, Table 1) [22]. Virulent and hypovirulent strains of C. parasitica also produced a main polysaccharide identified as pullulan (a polymer constituted of α-1,4-and α-1,6-glucan) and a minor fraction which Cytoskyrins A and B (27 and 28, Figure 3, Table 1) are two closely related bisanthraquinones obtained from large-scale cultures of an endophytic fungus, CR200 (Cytospora sp.), isolated from the branch of a Conocarpus erecta tree in the Guanacaste Conservation Area of Costa Rica. Previously, a substituted benzopryrone, named cytosporone, was isolated from the same fungus and showed antibiotic activity [30]. Cytoskyrin A showed strong BIA activity down to 12.5 ng in the standard assay Toxins 2020, 12, 714 6 of 30 while cytoskyrin B was inactive at the concentrations tested (<50 mg) [31]. The biochemical induction assay (BIA) measures the induction of the SOS response in bacteria and is used to identify compounds that inhibit DNA synthesis, either directly by inhibiting the DNA replication machinery or more often indirectly by modifying DNA [32][33][34]. BIA activity is highly dependent on the three-dimensional structure and not a general property of these polyphenolic compounds. In fact, close bisanthraquinones such as luteoskyrin (29, Figure 3) and rugulosin (18) are known to interact with DNA. The structural basis of these differences is not yet clear [31].
Dendryols A-D (30-33, Figure 4, Table 1), four phytotoxic anthraquinones, were produced by Dendryphiella sp. [35], a fungus isolated from an infected sample of the paddy weed Eleocharis kuroguwai (Cyperaceae) in Japan [36]. The dendryols 30-33, when tested for the phytotoxic activity by leaf-puncture assay on weeds (kuroguwai, barnyardgrass, and velvetleaf) and cultivated crops (rice, corn, and cowpea), showed toxicity only against barnyardgrass and the nercrotic area appeared to be dose-dependent. Compound 30 caused similar necrosis only on velvetleaf [35]. Rubellin A (34, Figure 4, Table 1) was isolated from the culture filtrates of Ramularia collocygni, the causal agent of leaf-spot disease of barley in central Europe [37]. From the same fungus rubellins B-F and 14-deydro rubellin D (35-38, Figure 4, Table 1) were also isolated. Biosynthetic studies carried out by the incorporation of both [1-13 C]-acetate and [2-13 C]-acetate into the rubellins Rubellin A (34, Figure 4, Table 1) was isolated from the culture filtrates of Ramularia collocygni, the causal agent of leaf-spot disease of barley in central Europe [37]. From the same fungus rubellins B-F and 14-deydro rubellin D (35-38, Figure 4, Table 1) were also isolated. Biosynthetic studies carried out by the incorporation of both [1-13 C]-acetate and [2-13 C]-acetate into the rubellins demonstrated that such anthraquinone derivatives were biosynthesized via the polyketide pathway. Rubellin A (34) increased photodynamic oxygen activation [38], while rubellins B-E exhibited antibacterial activity, as well as light-dependent, antiproliferative and cytotoxic activity in a series of human tumor cell lines [39]. Closely related anthraquinones were isolated from the same fungus and from Ramularia uredinicola and identified as uridinetubellins I and II, and caeruleoramulin (40-42, Figure 4 and Table I). Both uredinorubellins (40 and 41) showed photodynamic activity comparable to rubellin D, whereas caeruleoramularin did not display such activity [40].
1-Hydroxy-3-methyl-anthraquinone and 1,8-dihydroxy-3-methyl-anthraquinone (43 and 44, Figure 5, Table 1), were isolated together with other bioactive metabolites from Trichoderma harzianum in a study aimed to improve the production and application of novel biopesticides and biofertilizers and thus to help in the management of crop plant diseases. However, the two anthraquinones had no role in this activity [41].
Fungi belonging to the Alternaria genus are also well known as producers of a plethora of bioactive metabolites. In fact, five new hydroanthraquinone derivatives, named tetrahydroaltersolanols C-F (54-57, Figure 6, Table 1) and dihydroaltersolanol A (58, Figure 6, Table 1), and five new alterporriol-type anthranoid dimers, named alterporriols N−R (59-63, Figure 6), were isolated from the culture broth and the mycelia of Alternaria sp. ZJ-2008003.
The fungus also produced seven known analogues as tetrahydroaltersolanol B, altersolanol B, altersolanol C, altersolanol L, ampelanol, macrosporin (8, Figure 2) and alterporriol C. The fungus was isolated from Sarcophyton sp. soft coral collected from the South China Sea. All the compounds were assayed against the porcine reproductive and respiratory syndrome virus (PRRSV) and 54 and 62 showed antiviral activity with IC 50 values of 65 and 39 µM, respectively. Compound 61 exhibited cytotoxic activity against PC-3 and HCT-116 cell lines, with IC 50 values of 6.4 and 8.6 µM, respectively [47].
anthraquinones had no role in this activity [41].
Two new dimeric anthraquinones with a rare chemical skeleton, named torrubiellins A and B (65 and 66, Figure 7, Table 1), were isolated from Torrubiella sp. BCC 28517 (family Clavicipitaceae) belonging to a genus of fungus that attacks spiders, scale-insects, and hoppers. Torrubiellin B (66) exhibited a broad range of biological activities including strong antimalarial (Plasmodium falciparum), antifungal (Candida albicans), antibacterial (Bacillus cereus) activities, and cytotoxicity to cancer cell lines. Its biological activity was always higher than that of torrubiellin A (65) [49].
Two new xanthone-anthraquinone heterodimers, named acremoxanthones C and D (67 and 68, Figure 7, Table 1), were isolated from an unidentified fungus of the Hypocreales order (MSX 17022). The fungus also produced the close and already known acremonidins A and C, benzophenone, and moniliphenone. All the metabolites showed moderate cytotoxic activity in vitro. In addition, acremoxanthone D (68), and acremonidins A and C exhibited moderate 20S proteasome inhibitory activity [50]. Two new dimeric anthraquinones with a rare chemical skeleton, named torrubiellins A and B (65 and 66, Figure 7, Table 1), were isolated from Torrubiella sp. BCC 28517 (family Clavicipitaceae) belonging to a genus of fungus that attacks spiders, scale-insects, and hoppers. Torrubiellin B (66) exhibited a broad range of biological activities including strong antimalarial (Plasmodium falciparum), antifungal (Candida albicans), antibacterial (Bacillus cereus) activities, and cytotoxicity to cancer cell lines. Its biological activity was always higher than that of torrubiellin A (65) [49].
Two new xanthone-anthraquinone heterodimers, named acremoxanthones C and D (67 and 68, Figure 7, Table 1), were isolated from an unidentified fungus of the Hypocreales order (MSX 17022). The fungus also produced the close and already known acremonidins A and C, benzophenone, and moniliphenone. All the metabolites showed moderate cytotoxic activity in vitro. In addition, acremoxanthone D (68), and acremonidins A and C exhibited moderate 20S proteasome inhibitory activity [50].
,6β-triol, and 6β-methoxyergosta-7,22-diene-3β,5α-diol were isolated from the same fungus. Compound 81 exhibited antibacterial activity against Escherichia coli and S. aureus, and lethality against brine shrimp (Artemia salina) with an LC50 value of 0.5 μg/mL [56]. (81), together with three known anthraquinones 1-O-methyl averantin, averufin (9), and versicolorin C were also produced by the endophytic fungus ZSUH-36 isolated from a mangrove collected from the South China Sea. At that time, only the unambiguous structure of 79, being a new anthraquinone, was determined by advanced NMR spectra while no activity was described [54]. Previously, compounds 82 and 85, together with the two xanthones 5-methoxysterigmatocystin and sterigmatocystin (86 and 87, Figure 9, Table 1), had been isolated from the same fungus and only the unambiguous structure of compound 85, which at that time was a new anthraquinone, was determined by advanced NMR spectra [55]. Compounds 80-82 and 84 were previously isolated from the culture of Aspergillus versicolor, an endophytic fungus obtained from the marine brown alga Sargassum thunbergii.
Aspetritones A and B (108 and 109, Figure 10, Table 1) were isolated from the culture of the coral-derived fungus Aspergillus tritici SP2-8-1, together with 4-methyl-candidusin A and fifteen known metabolites belonging to different classes of natural compounds as prenylcandidusin, candidusin, and terphenyllin derivatives and anthraquinones. Bostrocyn (110, Figure 10, Table 1) and other four anthraquinones (111-114, Figure 10, Table 1) were isolated. Aspetritone A (108) showed the most significant activity against methicillin-resistant strains of S. aureus in respect to that of the positive control chloramphenicol and exhibited strong cytotoxicity against human cancer cell lines HeLa, A549, and Hep G2 [63].
The crude organic extract of the fungal culture filtrates showed cytotoxicity, using "Brine Shrimp Lethality Bioassay", antimicrobial and antioxidant activity. Among the isolated metabolites, compound 138 appeared to be the most potent anticancer and antimicrobial metabolite [77].
Industrial Application of Anthraquinones
Since 1869 with the determination of the structure of alizarin (167, Figure 13, Table 1), a yellow anthraquinone, the main industrial application of this anthraquinone was its use as a dye in textile manufacturing [85]. Compound 167 was isolated for the first time from Rubia tinctorum [84]. Thus, over a span of 20 years many analogues with different functionalities were also prepared by synthesis to obtain different dyes such as red, blue, and green mordant. Successively, the first acidic anthraquinone dye used to color wool without pretreatment with mordants was reported. At the beginning of 1900 the sulfonation and nitration of anthraquinone opened up a new era for anthraquinone based dyes.
A new phase in this development occurred with the introduction of synthetic fibers, such as polyester, polyamide, and polyacrylonitrile fibers, with the substitution of anthraquinones with other dyes. The use of acid anthraquinone dyes increased with the discovery of the first fiber-reactive dyes. At the same time, the utilization of natural substances instead of synthetic ones, increased worldwide. This satisfy the request of environmentally friendly sustainable technologies. As reported in Section 1 fungi are a significant source of pigments as several genera can produce pigments in good amounts identified as anthraquinones or analogues. The production of anthraquinones by fungal fermentation had been developed for rapid and easy growth to produce pigments useful in various industrial applications [86]. The natural anthraquinones, as well as other natural pigments, have noteworthy less toxic effects than the synthetic dyes and are easily degradable avoiding the high environmental pollution. Thus, these anthraquinone based dyes are used in medical, textile coloring, food coloring, and cosmetic industries [84,86].
On this basis also plants have been largely used as a source of natural colored anthraquinones. In fact, screening of dyeing plants was carried out for their widespread use in previous centuries. Colorimetric analysis showed that the principal color was yellow-orange shades and could be attributed to flavonoids while the red colors were due to anthraquinones. Colors from plants that contain anthocyanins varied from blue-violet through to red. The nature of the support fibers (wool or cotton) plays an important role in the perceived colors [87].
Recently water-repellent, self-cleaning and stain resistant textiles were obtained by developing anthraquinone reactive dyes which were covalently grafted onto cotton fabric surfaces obtaining bright colors with good wash-fastness properties and giving rise to breathable superhydrophobic textiles with self-cleaning properties [89].
The large number of textile dyes required a method for their classification which was based on the functional groups attached to the typical anthraquinone carbon skeleton. Thus, there are: anthraquinone, azo, phthalocyanine, sulfur, indigo, nitro, nitroso anthraquinone derivatives etc. taking into account their chemical structures. Another classification was based on the method of applying these dyes on an industrial scale, grouped as disperse, direct, acid, reactive, basic, vat dyes etc. [90].
The intensity of research focused on natural compounds has been growing over the past few decades. Anthraquinones have been most studied in China producing several publications which report different advanced extraction methods, analytical techniques, and industrial applications. These publications also describe the most used plants for anthraquinone content as Polygonaceae, Rubiaceae, and Fabaceae and report the best known anthraquinones: rhein aloe emodin, emodin, physcion, chrysophanol which are responsible for their numerous biological properties. Furthermore, the use of natural anthraquinones for industrial applications, has been described as an alternative to synthetic dyes to avoid some unwanted side effects [9].
However, the environmental contamination by wastewater containing dyes is today a severe problem to solve. The application of advanced oxidation processes (AOPs) to industrial wastewater has increased as well as an integrated approach for their biological and chemical treatment. The toxicity of the detergents and the dye have been determined in terms of effective concentration EC 50 using mixed cultures of activated sludge as well as a pure culture of luminescent bacteria Vibrio fischeri NRRLB-11177. However, the dye was not degraded without AOP pretreatment, therefore the degree of its removal (decolorization) by the AOPs is an important preliminary stage of bio-sorption on activated sludge [91].
Conclusions
The sources, structures, and the biological activities of fungal bioactive anthraquinones were reported starting from 1966 to the present day. In the introduction the previous review published on this topic was also cited which did not however treat the topic extensively. The anthraquinones were chronologically described and in some cases their isolation and biological activity was investigated in depth. Furthermore, their industrial application in different fields, essentially as natural dyes, was also reported focusing on the comparison between natural and synthetic anthraquinone based dyes, their chemical derivatization and classification, and the advanced methods used in the treatment of the relative industrial wastewaters to avoid severe negative environmental pollution.
Author Contributions: The authors equally contributed to this work. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding. | v3-fos-license |
2018-04-03T03:06:25.888Z | 2016-01-04T00:00:00.000 | 234463 | {
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} | pes2o/s2orc | A new megastigmane sulphoglycoside and polyphenolic constituents from pericarps of Garcinia mangostana
Abstract A megastigmane sulphoglycoside together with three phenolic compounds were isolated from the water-soluble fraction of the pericarps of Garcinia mangostana. The structure of the new compound was determined as 4-O-sulpho-β-d-glucopyranosyl abscisate (1) by spectroscopic data. Proanthocyanidin A2 (2) showed potent α-glucosidase inhibitory and DPPH scavenging activities with IC50 values of 3.46 and 11.6 μM, respectively.
Introduction
The plants in the genus Garcinia (Guttiferae) are well known to be rich in biologically active compounds including xanthones, benzophenones, proanthocyanidins and biphenyls (Mungmee et al. 2013;Feng et al. 2014;Fouotsa et al. 2014;Jiang et al. 2014;Anu Aravind et al. 2015). Mangosteen (Garcinia mangostana Linn.) is a plant that is widely cultivated in Vietnam and other Southeast Asian countries. The pericarp of this plant has been used as a remedy in traditional medicine for the treatment of diarrhoea, inflammation, skin infections and wounds (Vo 2012). Previous studies have shown that it contains high amounts of polyphenolic constituents, in which xanthones have been most frequently reported (Obolskiy et al. 2009;Dharmaratne et al. 2013;Morelli et al. 2015;Zhou et al. 2015). Mangosteen pericarp has exhibited a variety of interesting biological activities including antibacterial, antifungal, anti-inflammatory, antimalarial and antidiabetic (Pedraza-Chaverri et al. 2008;Obolskiy et al. 2009;Dharmaratne et al. 2013). α-Glucosidase, a membrane-bound enzyme at the epithelium of the small intestine, hydrolyses starch oligosaccharides to monosaccharides. The inhibitors of this enzyme delay carbohydrate digestion and thus cause a reduction in the rate of glucose absorption and lower the postprandial hyperglycaemia. Therefore, the inhibition of α-glucosidase plays a major role in preventing the rise of the postprandial glucose level in diabetics (Kumar et al. 2011).
Results and discussion
All isolated compounds were tested for their α-glucosidase inhibitory activity. Most of the isolated compounds are phenolic and may possess antioxidant activity, thus the compounds were evaluated for their DPPH scavenging capacity. As shown in Table 1, proanthocyanidin A2 (2) was most active with the IC 50 values of 3.46 and 11.6 μM for α-glucosidase inhibitory and DPPH scavenging activities, respectively. The known antioxidant compound (-)-epicatechin (3) and the benzofuran glycoside (4) also showed moderated α-glucosidase inhibitory effect. The new megastigmane sulphoglycoside (1) exhibited significant DPPH scavenging capacity in comparison with ascorbic acid. A previous study reported that the prenylated xanthones in the pericarps of mangosteen potently inhibited α-glucosidase (ryu et al. 2011). In the present work, proanthocyanidin A2, a polar compound isolated from the water-soluble fraction of mangosteen pericarps showed potent α-glucosidase inhibitory activity in comparison with the antidiabetic drug acarbose. This result coincided with the previous study assuming that proanthocyanidin components in the pericarps of mangosteen are potent inhibitors of α-amylase, another enzyme involving in the metabolism of carbohydrates and hyperglycaemia (Loo & Huang 2007). Although previous study showed that total proanthocyanidins from Cinnamomum cassia strongly inhibited α-glucosidase (Kang et al. 2014), the present paper is the first report for the α-glucosidase inhibitory activity of proanthocyanidin A2. A lot of studies have shown that the prenylated xanthones in mangosteen are potent inhibitors of α-glucosidase. However, these compounds are mostly soluble in organic solvents of moderate polarity. Our result showed that the water-soluble fraction of mangosteen pericarp contains attractive α-glucosidase inhibitors. Thus, these data reinforce the health benefit of mangosteen as an alternative medicine to help lower postprandial glucose absorption.
General procedures
Optical rotation values were recorded on a JASCO P-2000 digital polarimeter (JASCO, Tokyo, Japan). The uV spectra were recorded on a JASCO V-630 spectrophotometer (JASCO, Tokyo, Japan). The Ir spectrum was obtained from a Tensor 37 FT-Ir spectrometer (Bruker, ettlingen, Germany). NMr experiments were carried out on a Bruker AM500 FT-NMr spectrometer (Bruker, rheinstetten, Germany) using tetramethylsilane (TMS) as internal standard. The Hr-eSI-MS were recorded on an FT-ICr mass spectrometer (Bruker Dal-tonics, Bremen, Germany).
Plant material
The fruits of G. mangostana were purchased from markets in Hanoi in June, 2011 and identified by Prof Tran Huy Thai, Institute of ecology and Biological resources, Vietnam Academy of Science and Technology. The voucher specimens (BK-10) were deposited at School of Chemical engineering, Hanoi university of Science and Technology. The fruit pericarps were collected, air dried and powdered.
Extraction and isolation
The air-dried and powdered materials (2.0 kg) were extracted with methanol (4 L × 3 times) at room temperature for 24 h. The combined extracts were concentrated under vacuum to obtain a crude residue (152.0 g, 81% α-glucosidase inhibition at 100 μg/mL), which was then resuspended in water (1 L), and extracted by chloroform (1 L × 3 times). The organic layers were combined and concentrated to give 48.5 g of chloroform residue. The water layer (87% α-glucosidase inhibition at 100 μg/mL) was passed through a dianion HP-20 column and washed with 1.5 L water, then eluted with 50 and 100% methanol (1 L each). The eluant from 100% methanol was concentrated and chromatographed on a rP-18 column using methanol-water (1:4 v/v) as the mobile phase to afford five fractions of FA1-5. Compound 2 (60.3 mg) was purified from FA2 by using a silica gel column eluted by chloroform:methanol:water (45:10:1 v/v). Fraction FA3 was passed through a C-18 reverse-phase column using methanol:water (1:2 v/v) as the mobile phase to obtain compounds 3 (98.5 mg) and 4 (8.0 mg). FA5 was chromatographed on a silica gel column eluted with ethyl acetate:methanol:water (60:10:1 v/v) to give 1 (6.1 mg).
Acid hydrolysis of 1
Compound 1 (2 mg) was heated in 1 N HCl (1 mL) at 80 °C for 2 h, then the solution was cooled and extracted with ethyl acetate (1 mL × 3). The organic and aqueous layers were concentrated to 1 mL and their optical rotations were checked. (S)-abscisic acid was obtained from the organic layer, [ ] 25 D = +107.6 (c 0.03, MeOH). The sugar product in the aqueous layer was identified as d-glucose by silica gel TLC (rf 0.55 developed with acetone-methanol-water 100:10:1 (v/v) and was sprayed with a 10% sulphuric acid solution containing 2% vanillin), and by the positive value of optical rotation ([ ] 25 D = +3.2 (c 0.03, H 2 O) in comparison with d-glucose standard. The addition of BaCl 2 to the aqueous solution produced a white precipitate indicating the presence of SO 2− 4 anions.
Assay for α-glucosidase inhibition
The α-glucosidase (G0660-750uN, Sigma) enzyme inhibition assay was performed according to a previously described method (Luyen et al. 2013). The sample solution (2 μL dissolved in DMSO) and 0.5 u/mL α-glucosidase (40 μL) were mixed in 120 μL of 0.1 M phosphate buffer (pH 7.0). After 5 min pre-incubation, 5 mM p-nitrophenyl-α-d-glucopyranoside solution (40 μL) was added and the solution was incubated at 37 °C for 30 min. The absorbance of released 4-nitrophenol was measured at 405 nm using a microplate reader (Molecular Devices, CA). The IC 50 value was graphically measured using a plot of the per cent inhibition vs. log of the concentration of the test compound.
DPPH radical scavenging activity
The antioxidant activity of the isolated compounds was evaluated by its scavenging capacity of the DPPH radical. Briefly, the tested samples (10 μL) at various concentrations were mixed with 150 μM DPPH solution (190 μL) in 96 well plates. The plate was incubated in the dark at room temperature for 30 min. Then the absorbance of the reaction mixture was measured at 520 nm on a microplate reader. The IC 50 value is defined as the concentration of a sample required to scavenge 50% of the DPPH radical.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work is supported by a grant from the Ministry of Industry and Trade [grant number CNHD.ĐT 054/14-16]. | v3-fos-license |
2018-09-19T18:49:15.797Z | 2018-09-19T00:00:00.000 | 52293927 | {
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} | pes2o/s2orc | Partial Mechanical Unloading of the Heart Disrupts L-Type Calcium Channel and Beta-Adrenoceptor Signaling Microdomains
Introduction: We investigated the effect of partial mechanical unloading (PMU) of the heart on the physiology of calcium and beta-adrenoceptor-cAMP (βAR-cAMP) microdomains. Previous studies have investigated PMU using a model of heterotopic-heart and lung transplantation (HTHAL). These studies have demonstrated that PMU disrupts the structure of cardiomyocytes and calcium handling. We sought to understand these processes by studying L-Type Calcium Channel (LTCC) activity and sub-type-specific βAR-cAMP signaling within cardiomyocyte membrane microdomains. Method: We utilized an 8-week model of HTHAL, whereby the hearts of syngeneic Lewis rats were transplanted into the abdomens of randomly assigned cage mates. A pronounced atrophy was observed in hearts after HTHAL. Cardiomyocytes were isolated via enzymatic perfusion. We utilized Förster Resonance Energy Transfer (FRET) based cAMP-biosensors and scanning ion conductance microscopy (SICM) based methodologies to study localization of LTCC and βAR-cAMP signaling. Results: β2AR-cAMP responses measured by FRET in the cardiomyocyte cytosol were reduced by PMU (loaded 28.51 ± 7.18% vs. unloaded 10.84 ± 3.27% N,n 4/10-13 mean ± SEM ∗p < 0.05). There was no effect of PMU on β2AR-cAMP signaling in RII_Protein Kinase A domains. β1AR-cAMP was unaffected by PMU in either microdomain. Consistent with this SICM/FRET analysis demonstrated that β2AR-cAMP was specifically reduced in t-tubules (TTs) after PMU (loaded TT 0.721 ± 0.106% vs. loaded crest 0.104 ± 0.062%, unloaded TT 0.112 ± 0.072% vs. unloaded crest 0.219 ± 0.084% N,n 5/6-9 mean ± SEM ∗∗p < 0.01, ∗∗∗p < 0.001 vs. loaded TT). By comparison β1AR-cAMP responses in either TT or sarcolemmal crests were unaffected by the PMU. LTCC occurrence and open probability (Po) were reduced by PMU (loaded TT Po 0.073 ± 0.011% vs. loaded crest Po 0.027 ± 0.006% N,n 5/18-26 mean ± SEM ∗p < 0.05) (unloaded TT 0.0350 ± 0.003% vs. unloaded crest Po 0.025 N,n 5/20-30 mean ± SEM NS #p < 0.05 unloaded vs. loaded TT). We discovered that PMU had reduced the association between Caveolin-3, Junctophilin-2, and Cav1.2. Discussion: PMU suppresses’ β2AR-cAMP and LTCC activity. When activated, the signaling of β2AR-cAMP and LTCC become more far-reaching after PMU. We suggest that a situation of ‘suppression/decompartmentation’ is elicited by the loss of refined cardiomyocyte structure following PMU. As PMU is a component of modern device therapy for heart failure this study has clinical ramifications and raises important questions for regenerative medicine.
INTRODUCTION
Mechanical load is a key factor in the control of cardiomyocyte structure and subsequently controls cellular physiology (Ibrahim et al., 2013b). The cardiomyocyte is invaginated at regular intervals by transverse (t)-tubules which allow the formation of dyadic couplings between LTCC and the ryanodine receptors of the intracellular sarcoplasmic reticulum. These structures are integrally important to the control of intracellular calcium (Sanchez-Alonso et al., 2016) and secondary messengers such as cAMP (Nikolaev et al., 2010). They contribute to the faithful control of cardiomyocyte excitation contraction-coupling via the careful compartmentation of intracellular signaling processes. The PMU of healthy hearts disrupts some aspects of TT function (Ibrahim et al., 2012a). It is proposed that the transverse-axial tubules system (TATS) exists in dynamic equilibrium whereby both under and overloading scenarios can distort the system (Terracciano et al., 2009;Ibrahim et al., 2010Ibrahim et al., , 2012a. The unloading of previously overloaded failing hearts can normalize cellular structure and subsequently cellular function (Ibrahim et al., 2012b). The potentially pathological effects of PMU are clinically important due to the common deployment of LVADs as therapy for heart failure (Holmberg et al., 2017). Although, LVADs are of crucial importance as a 'bridge to transplant, ' the explant rate remains low (Drakos et al., 2012). It would therefore be useful to explore the subtleties of 'ineffective' unloading of cardiomyocytes with a view to better understanding how to tune therapies to provide the correct regenerative environment.
Previously, Ibrahim et al. (2012a) studied PMU of healthy hearts with a HTHAL model using an 8-week period of unloading. They compared this against control hearts and those of animals which had received trans-aortic constriction (hypertrophy). Isolated cardiomyocytes from healthy hearts which had received PMU were atrophied, had aberrant cytoarchitecture and calcium handling. The morphology of the calcium transient was altered (lower amplitude and longer times to peak and relaxation) in comparison to control. Importantly, like the model of overload, the cells had an increased frequency of calcium sparks and altered spark morphology. These changes were attributed to the loss of normal TATS organization. However, the localized signaling functions of molecules such as the LTCCs and β-adrenergic receptors (βARs) which are fundamental to excitation-contraction processes within the cells, were not investigated in detail in these studies.
We sought to examine the role of unloading in organizing molecular activity at the level of the cell membrane. We employed an animal model of partial myocardial mechanical unloading by performing HTHALs (Ibrahim et al., 2013a). We enzymatically isolated cells from healthy hearts and hearts after 8 weeks of unloading. The cells' structural parameters were studied with confocal and SICM. The localized signaling of LTCCs was studied with a modified SICM technique which allows the selective patching of specific membrane sub-domains such as TT openings or sarcolemmal crests (Bhargava et al., 2013). βAR function was assessed by monitoring sub-cellular cAMP responses using FRET-based biosensors (Nikolaev et al., 2004;Di Benedetto et al., 2008;Wright et al., 2018). These biosensors were delivered to either the cell cytosol or membrane regions using specific molecular tags. This allowed us to monitor the function of β 1 AR and β 2 AR sub-types within different sub-cellular microdomains. The localization of β 2 AR-cAMP activity was studied using a further modification of SICM, which combines SICM and FRET techniques (Nikolaev et al., 2010). This allows the study of cAMP activity in specific regions of the cell by permitting the acquisition of cellular membrane topography and localized application of agonist via the SICM probe. Any cAMP responses evoked by the local treatment can be measured concurrently using FRET biosensors. Using this technique, we discovered a suppression of β 2 AR-cAMP but not of β 1 AR-cAMP signaling. LTCCs signaling was suppressed by unloading, but this was dynamic and occurred without the removal of specific 'activatable' LTCC capacity from the cell.
Animals and Ethics Statement
Approval for this work was obtained from the Animal Welfare and Ethics Review Board (AWERB) of Imperial College London. This study was carried out in accordance with United Kingdom Home Office guidelines (ASPA 1986 andEU directive 2010/83) and conformed to all protocols of Imperial College London. All procedures were carried out on male, syngeneic, Lewis rats (150-250 g) obtained from Charles River Laboratories (Margate, United Kingdom). Animals were kept at 25 • C in ventilated cages and fed standard chow. Animals were randomly assigned to be either donors or recipients. Recipient animals received 'healthy' heart and lung grafts from the first group. Following 8 weeks of maintenance, and upon sacrifice, cells isolated from implanted (heterotopic) hearts are defined as Unloaded and cells from the animal's thoracic (orthotopic) heart are defined as age-matched Loaded control.
Heterotopic Heart and Lung Transplant
A more detailed description of this surgery has been published previously (Ibrahim et al., 2013a). Briefly, the recipient animal was prepared (hair removed and skin sterilized) and an incision was made in the abdomen. The mesentery and bowel were carefully removed and wrapped in damp, sterile swabbing material. The IVC and aorta were revealed and undermined. They were manipulated with 2-0 suture and clamped. A 2-3 mm incision was made in the aorta. The heart and lung block was removed from storage solution and carefully wrapped in swab, soaked in cold cardioplegia, which was refreshed periodically throughout the operation. The tissue block was maneuvered toward the abdominal cavity, an 8-0 prolene suture was passed through the aorta at the 5 o'clock position outsidein. The suture was then passed through the aortic incision at the 7 o'clock position -the anastomosis then continued clockwise-counter-clockwise, respectively, until it was sealed at the 8 o'clock position on the aorta with three surgeons' knots. Following careful removal of the swabbing protecting the heart and orientation of the tissue, the clamps were removed to allow the perfusion of the heart, as well as the restoration of circulation to the limbs distal to the anastomosis. Hemostasis was provided around the anastomosis site until bleeding ceased. The beating rate and strength of contraction of the implanted heart was assessed. Once the graft activity was deemed optimal the gut was returned and carefully orientated within the abdominal cavity. The abdominal musculature was closed with 5-0 vicryl suture in a continuous pattern. The animal's skin was closed with 4-0 vicryl sutures using a continuous subcuticular suture pattern. Carprofen was provided (1.25 mg, s.c.) for analgesia and enrofloxacin (2.5 mg, s.c.) as an antibiotic. Fluid was also given (2 ml saline s.c.). Animals were allowed to recover for about 45 min in a heated chamber, before being transferred individually to standard cages. Operated animals received carprofen and enrofloxacin s.c at 24 and 48 h postoperation and soft food. They were returned to normal chow after ∼72 h.
Unloaded Heart Harvest
After 8 weeks the recipient animals were anesthetized with isoflurane. The abdomen was incised and bowel manipulated to reveal the implanted heart. The aorta and IVC were manipulated and clamped. The heart and lung block were extracted and placed in ice cold Krebs solution. The diaphragm and thorax were incised and opened to allow the removal of the loaded heart. The heart and lung block were excised and placed in ice-cold Krebs. (C) Graph presenting mean β 1 AR-cAMP and β 2 AR-cAMP responses as a function of total AC responses for loaded and unloaded cells. cEPAC2 sensor, β 1 AR-cAMP (loaded 58.78 ± 4.82% vs. unloaded 68.12 ± 4.85% N,n 5/10-13 mean ± SEM, NS). cEPAC2, β 2 AR-cAMP (loaded 28.51 ± 7.18% vs. unloaded 10.84 ± 3.27% N,n 4/10-13 mean ± SEM * p < 0.05). (D) Illustrative traces showing β 1 AR-cAMP (ICI+ISO) and full AC-cAMP (NKH) responses measured by FRET microscopy using the RII_epac sensor in loaded and unloaded cells. (E) Illustrative traces showing β 2 AR-cAMP (CGP+ISO) and full AC-cAMP (NKH) responses measured by FRET microscopy using the RII_epac sensor in loaded and unloaded cells.
Cell Isolation
Both loaded and unloaded hearts were retrogradely perfused with Krebs-Henseleit solution (37 • C, 95% O 2 , 5% CO 2 1 mM Ca 2+ ) using a Langendorff perfusion apparatus. After beating was properly established the heart was arrested by perfusion with a low calcium buffer for 5 min. The heart was then perfused with a mixture of collagenase:hyaluronidase (1 mg/ml:0.6 mg/ml) for 10 min. After this period, the heart was cut down, the atria and right ventricle were removed. The ventricle was then disrupted and the mixture shaken mechanically in collagenase/hyaluronidase solution (35 • C). After 30 min this yielded single isolated cardiomyocytes. These mixtures were centrifuged 1,000 rpm 2 min. Cell pellets were re-suspended in buffer not containing enzymes.
Förster Resonance Energy Transfer (FRET) Microscopy
Isolated cardiomyocytes were centrifuged, re-suspended in M199 and around 5,000 plated on individual laminin coated 25 mm coverslips. After attachment they were washed with new M199 (I) Graph comparing β 2 AR-cAMP responses evoked in t-tubule (TT) or crest (C) regions by SICM/FRET in loaded and unloaded cells (loaded t-tubule 0.721 ± 0.106% vs. loaded crest 0.104 ± 0.062%, unloaded t-tubule 0.112 ± 0.072% vs. unloaded crest 0.219 ± 0.084% N,n 5/6-9 mean ± SEM * * p < 0.01, * * * p < 0.001 vs. loaded t-tubule). and cultured for 48 h in M199 in the presence of adenovirus encoding the RII_Epac or cEPAC2 cAMP sensors (Di Benedetto et al., 2008;Nikolaev et al., 2010;MacDougall et al., 2012). FRET experiments were conducted with ORCA-4.0 CCD camera's using DualView or QuadView beamsplitters. Both FRET construct consist of CFP and YFP complexed with an exchange protein activated by cAMP (epac)-derived cAMP binding domain. In the case of the RII_Epac sensor the construct also contains the regulatory II subunit of PKA, as a result this sensor aligns to the cardiomyocyte sarcomere in conjunction with A-kinase anchoring proteins. Binding of cAMP to this sensor indicates the penetration of cAMP into PKA domains, whereas cEPAC2 indicates cytosolic responses. After baseline FRET measurements in the presence of 100 nM CGP20712A (to block β 1 AR) or 50 nM ICI118, 551 (to block β 2 AR) cells were perfused with 100 nM isoprenaline to stimulate β 1 AR or β 2 AR. Following this phase NKH477 (5 µM) was perfused onto the cells to fully stimulate adenylate cyclase and allow the correction of β 2 AR-cAMP responses to maximal cAMP responses.
Scanning Ion Conductance Microscopy
Scanning ion conductance microscopy is a scanning probe microscopy technique based upon the ability to reconstruct topographical images of surfaces on the basis of changes in conductance at the tip of a nanopipette. The physical basis of this technique has been explained in exhaustive detail previously (Novak et al., 2009). Briefly, the sample in this case the cell, is rastered underneath a scanning pipette and a high-resolution image of the cell is constructed. Once this image has been acquired the tip of the pipette can be localized to very specific positions on the cell surface.
Super Resolution (Smart) Patch Clamp
This technique has been explained in detail elsewhere. Following the acquisition of a topographical image of the cell with SICM an area of interest is selected. The pipette is moved to a neutral area of the dish and clipped to give the patch-clamp resistance. It is then moved back to the original area of interest and the pipette is fused with the membrane patch and calcium channel recordings made as described in the references above. A sub-population of cells were treated with the non-specific calcium channel activator BAYK8644 (Tocris Biosciences, United States).
SICM/FRET
This technique has also been employed and described in more detail elsewhere. Following the acquisition of cell topography, as TT or sarcolemmal crest region was targeted and ISO was applied via electrophoresis. FRET response is measured as described above (Nikolaev et al., 2010;Wright et al., 2014).
Whole-Cell Patch Clamp Recordings
L-type calcium currents (ICa, L) were recorded using the whole-cell patch-clamp configuration with the external recording solution of the following composition (in mmol/L): 140 NaCl, 6 KCl, 10 glucose, 10 HEPES, 1.5 MgCl 2 , 1 CaCl 2 , pH 7.4 with 1M NaOH. An internal pipette solution contained (in mmol/L): 100 Cs-methanesulfonate, 40 CsCl, 10 HEPES, 5 EGTA, 5 Mg-ATP free acid, 0.75 MgCl 2 , pH 7.2 with CsOH. Patch pipettes had mean resistances of 3.5-5 M . Currents were recorded using an Axopatch-1D amplifier connected to a Digidata 1322A acquisition system (Axon Instruments, Foster City, CA, USA). The bath was connected to the ground via a reference electrode containing Ag-AgCl pellet. Data were low-pass filtered at 2 kHz using the Bessel filter of the amplifier and sampled at 2 kHz. All recordings were performed at room temperature (22-24 • C). ICa, L channel activity was recorded during 200 ms from a holding potential of −40 mV to test potentials ranging from −40 to +60 mV, with pulses applied every 2 s in 5 mV increments. Current amplitude at 0 mV was taken as a peak current and divided by a capacitance value for each cell (current density, pA/pF).
Measurement of Cav3, T-Cap and Junctophilin-2 Density and Regularity and Proximity Ligation Assay
Isolated cells were plated on 13 mm coverslips, fixed with methanol (−20 • C) and kept in PBS at 4 • C. Proximity ligation assay was performed with the Duolink (inSitu) kit (Sigma-Aldrich, United Kingdom) using anti-Goat, anti-Rabbit or anti-Mouse reagents as required with Duolink 'green' detection reagents (Sigma-Aldrich, United Kingdom), as per the manufacturers instructions. Primary antibodies for JPH-2 (Goat polyclonal, Santa Cruz, sc -51313), caveolin-3 (Cav3, mouse monoclonal 610421) and Cav1.2 (Alomone, Rabbit polyclonal). Cell imaging was performed with 60x oil immersion Zeiss lens, using inverted confocal laser scanning microscope (A Zeiss LSM-780), with argon laser beam at specific wavelengths for the secondary antibodies (Alexa Fluor Cav3 546 nm). Protein density and respective regularity of distribution were obtained with the same method used for TTs analysis described in more detail elsewhere (Wright et al., 2018). PLA analysis was performed by automatically thresholding cell images in ImageJ (Image J Corp, United States) and utilizing default plugins to measure area covered by particles, size of particles, number of particles and number of particles per µm.
di-8ANNEPPS Staining of Freshly Isolated Cardiomyocytes
After staining freshly isolated cells with di-8ANNEPPS image stacks were obtained with a Zeiss LSM710 confocal microscope as previously described (Schobesberger et al., 2017). A 40 × 5 µm area was selected and binarized. Density and power of regularity were analyzed as per our laboratories protocols. Density was defined as the percentage of black pixels. Power of regularity was calculated using a bespoke MATLAB code by plotting a waveform of the binarized image of the cell and the Fast-Fourier transform was used to plot a representative curve of the frequency of di8ANNEPPS stained ultrastructure. The strength of the peak at ∼0.5 micron indicated the regularity of di8ANEPPS staining and therefore TT structure.
Statistical Analysis
Where unrelated two populations were analyzed statistical difference was determined using an unpaired (two-tailed) Students' T-test. Where more than two unrelated populations were analyzed, a one-way ANOVA with a Bonferroni post-test (post hoc) was used. This was performed by using Graph-Pad 4.0 software. In the figure legends, N refers to the number of preparations, n refers to the number of measurements.
Partial Mechanical Unloading Differentially Distorts βAR-cAMP Microdomains
Loaded and unloaded cells were transfected with cEPAC2 or RII_epac sensors and cultured for 48 h. β 1 AR-cAMP and β 2 AR-cAMP were then assessed as a function of the total cAMP response. Unloading did not affect the β 1 AR-cAMP response or the overall cellular cAMP response in the cell cytosol (Figures 1A,C). Unloading reduced the β 2 AR-cAMP responses within the cell cytosol (Figures 1B,C). Withdrawal of mechanical load reduced the amount of cAMP penetrating cellular RII_PKA domains but this difference did not reach statistical significance (Figures 1D,F). No difference was observed in total RII domain cAMP responses. Specific stimulation of β 2 AR-cAMP elicited a slightly higher response in RII_PKA domains, in control cells. However, similar to experiments studying β 1 AR-cAMP this effect was also not statistically significant (Figures 1E,F). In summary, unloading only significantly reduced the cytosolic β 2 AR-cAMP response.
Partial Mechanical Unloading Selectively Reduces β 2 AR-cAMP Microdomain Responses
Scanning ion conductance microscopy scans showed similar degrees of sarcolemmal organization in loaded and unloaded cardiomyocytes (Figures 2A,B) (freshly isolated in preparation for LTCC experiments). Z-groove ratios were calculated for multiple scans and the relative degrees of cell membrane organization were analyzed. Mean Z-groove ratios were no different for the loaded and unloaded cell groups ( Figure 2C). As a result it can be reported that PMU does not produce its effect on cytosolic β 2 AR-cAMP responses by reducing the organization of the cellular sarcolemma. In control, loaded cells (cultured for 48 h) β 1 AR-cAMP responses were elicited in both tubule and crest domains after application of ISO in the presence of ICI (β 2 AR blockade). This is consistent with previous studies of this particular sensor (Figure 2D). Unloading did not appear to alter β 1 AR-cAMP responses in tubule or crest domains ( Figure 2E). Analysis revealed no statistical difference between β 1 AR-cAMP responses from the TT or crest regions of loaded or unloaded cardiomyocytes ( Figure 2F). Consistent with previous work, application of ISO in the presence of CGP (β 1 AR blockade) only elicited β 2 AR-cAMP responses on t-tubular surfaces ( Figure 2G).
Mechanical unloading significantly reduced the β 2 AR-cAMP response on cellular t-tubular structures (Figures 2H,I), which is consistent with the effect of unloading on the 'whole-cell' stimulation of β 2 AR-cAMP responses measured with the cEPAC2 sensor. Cell crests yielded very little β 2 AR-cAMP response in the control condition and the unloaded cells were no different (Figures 2G,I).
Partial Mechanical Unloading 'Silences' L-Type Calcium Channel (LTCC) Activity
TT LTCC activity was faithfully recorded from freshly isolated loaded cardiomyocytes (Figure 3A). In comparison, LTCC activity was suppressed in unloaded cardiomyocytes ( Figure 3A). The occurrence of LTCC channels, the number of channels recorded vs. successful patches, was higher in TT membranes of loaded cells than cell crests (Figure 3B and Supplementary Figure S1A). In comparison a far lower occurrence of LTCCs was observed in the TT membrane and cell crests of unloaded cells. The difference between TT and crest domains observed in loaded controls was lost after unloading. The specific LTCC channel activator BAY-K was employed to assess whether the channels had been lost or whether their activity was being suppressed. LTCC activity was recorded in TT domains in both conditions ( Figure 3A). BAY-K treated loaded cells showed the same occurrence of LTCCs as observed in untreated cells. In comparison BAY-K treatment revealed a large amount of LTCCs in both the TT and crest domains of unloaded cells (Figure 3B). The suggestion is that LTCCs are silenced by unloading and that this treatment removes the restriction of the channels to TT domains. The biophysical characteristics of LTCCs are also altered by unloading. In loaded cells the probability of a calcium channel being open is greater in the TT domain than in the crest (Figure 3C and Supplementary Figure S1B). In unloaded cells the open probability is also decreased, alongside the occurrence. To assess whether these alterations were as a result of gross changes in calcium handling, we assessed 'wholecell' calcium current. We discovered that unloaded cells had a lower capacitance in comparison to loaded cells, which is due to their smaller size ( Figure 3D). Whole-cell calcium current was no different between loaded and unloaded cells following correction for capacitance ( Figure 3E). This suggests that differences in LTCC function are directly attributable to the activity of the channels within the membrane microdomain.
Partial Mechanical Unloading Alters the Association of Structural Proteins Crucial for LTCC Function
In an attempt to understand the suppression of LTCC activity, we performed proximity ligation assays (PLA) on loaded and unloaded cells, which were isolated and fixed. PLA quantifies the association of peptides by measuring fluorescence produced by antibody constructs using confocal microscopy. The size and number of fluorescent particles were assessed. We utilized this method to explore the association of JPH-2 and Cav-3, two proteins crucial for the control of sub-cellular LTCC signaling FIGURE 6 | Schematic demonstrating the speculated state of a t-tubular microdomain in loaded or unloaded scenarios with or without isoprenaline stimulation.
(1) Loaded cell. LTCC are generally active within the tubule owning to some constitutive cAMP production and consequent PKA activity, scaffolded by Cav-3 and JPH-2. Calcium spark activity at the sarcoplasmic reticulum is nominal.
(2) Unloaded cell. LTCC are generally inactive as there is a loss of coherent confinement of cAMP and PKA activity to the t-tubular microdomain. This is due to the mechanosensitivity of the association between Cav1.2, Cav-3, and JPH-2 and the loss of t-tubules. Even though there is a smaller amount of LTCC activity spark activity increases as orphaned RyR no longer receive effective control and thus experience a gain of function.
(3) Loaded cell stimulated with ISO. After stimulation β2AR increase cAMP levels in the cytoplasm of the sarcomeric dyad and this activates a sub-population of PKA which activates almost all LTCC. Spark activity remains nominal as effective measures to control LTCC and RYR phosphorylation and the degradation of cAMP remain effective due to scaffolding by the cellular microdomain. (4) Unloaded cell stimulated with ISO. Despite a reduction in the amount of cAMP response initiated by β 2 AR in the cytosol, signals still reach RII_PKA domains, due to the loss of efficient cAMP compartmentation. This stimulates further aberrant spark activity due to the relatively uncontrolled calcium influx in the cell and EC mediator hyperphosphorylation following the loss of normal structure within the sarcomeric dyad following unloading. microdomains. We also assessed the association between Cav-3 and the Ca v 1.2 sub-unit of the LTCC itself. Unloading reduced the size of cardiomyocytes and reduced the density and regularity of Cav-3 staining (Supplementary Figures S2A-D). The staining of cells with di-8-ANNEPPS also reveals the effect of PMU on cell area and aspect ratio, as well as cellular ultrastructure. PMU causes the loss of TT regularity but not TT density (Supplementary Figures S3A-F). The JPH-2/Cav-3 PLA demonstrated that the cell size was reduced by unloading (Figures 4A,B). The number of particles per micron and total number of particles were decreased by the partial withdrawal of mechanical load (Figures 4C,D). This suggested that after unloading the degree of association between these two structural proteins was lower and less organized. Interestingly, the size of particles increased suggesting that where these associations occur there is an agglomeration of the peptides (Figure 4E). The Ca v 1.2/Cav-3 PLA demonstrated once again that cell size was reduced by unloading (Figures 5A,B). The number of particles by area and total number was also decreased following unloading (Figures 5C,D). Unlike the JPH-2/Cav-3 PLA there was no increase in particle size ( Figure 5E). This suggested that the association between Ca v 1.2/Cav-3 was simply reduced following unloading.
DISCUSSION
Partial mechanical unloading profoundly reduces cardiomyocyte size and modulates the structure and organization of TATS (Ibrahim et al., 2010(Ibrahim et al., , 2012a. It has been demonstrated to increase the frequency and persistence of calcium 'sparks.' Spark activity is speculated to be elicited by ryanodine receptors and the loss of organization of the sarcomeric dyad following the withdrawal of load. We sought to assess the effect of PMU on the physiology of LTCC's and βARs. The physiology of βAR-cAMP was altered in a sub-type and microdomain specific way. PMU did not reduce the amplitude of β 1 AR-cAMP responses elicited by whole-cell perfusion and measured in the cell cytosol or RII-PKA domains. PMU reduced the amplitude of β 2 AR-cAMP responses in the cytosol but not in the RII-PKA domains. This is an intriguing finding, as previous papers (MacDougall et al., 2012;Wright et al., 2018) which have employed these two sensors have demonstrated that β 2 AR is a compartmentalized receptor in healthy cardiomyocytes, which will produce cAMP responses in the cell cytosol but not in the RII_PKA domains. The data we present suggest that the production of β 2 AR-cAMP is load-sensitive and may involve the loss of receptor subunits. In the situation of PMU-induced atrophy this is potentially superimposed on a cell which has lost structural integrity via the loss of important components of the cAMP compartmentation machinery such as TTs. As a result we are not observing the increase of absolute β 2 AR-cAMP capacity, in fact we witness its reduction, and this is concomitant with increased persistence and subsequent enhancement of its efficacy due to the PMU-induced loss of cAMP compartmentation. We only employed a saturating level of stimulation for β 1 AR but prior reports (Agarwal et al., 2011) have demonstrated that at sub-maximal levels β 1 AR may act in a compartmentalized fashion. As a result, the effects of PMU may also affect β 1 AR-cAMP in different scenarios. We posit that the primary factor in the lack of an effect of PMU on β 1 AR activity may be a combination of its relative lack of 'compartmentation' by important subcellular structures such as TTs. Therefore their loss after PMU has less of an effect on this receptor. Equally, the supra-maximal stimulation afforded by our selected dose of isoprenaline may mean the effects of PMU on β 1 AR-cAMP have eluded us in this case.
The effects of PMU on membrane microdomains were investigated by stimulating β 1 AR or β 2 AR-cAMP responses via local application of agonist and measuring responses evoked in the cardiomyocyte cytosol. β 1 AR-cAMP responses in t-tubular or sarcolemmal crest regions were unaffected by PMU. This is concordant with our findings using whole-cell perfusion of agonist. β 2 AR-cAMP responses were of greater amplitude in t-tubular regions than on cardiomyocyte crests. This is comparable with previous reports (Nikolaev et al., 2010;Wright et al., 2014). PMU appeared to suppress the β 2 AR-cAMP responses in t-tubular domains. β 2 AR-cAMP responses in crest regions were unaltered, but were of such a low amplitude in control cells that an effect could not be seen. This is consistent with our measurements made after perfusing the whole cell with agonist. This may suggest that the effect in whole-cell measurements is purely due to the loss of t-tubular β 2 AR-cAMP responses. The effects of PMU on β 2 AR-cAMP responses occurred without an obvious loss of total cAMP production capacity within the cells. Mechanical load could therefore be an essential stimulus for the production of organized t-tubular networks which are capable of conducting normal compartmentalized β 2 AR-cAMP responses. Mechanical load may be a prerequisite to allow the cells to produce β 2 AR-cAMP responses which are faithfully produced in the TT and restricted from the RII_PKA domains. This phenomena would appear to be less significant for β 1 AR-cAMP responses. However, as suggested the relatively liberal control of this receptor may belie more significant effects at sub-maximal levels. The effects of the singular load-dependent 'suppression/decompartmentation' behavior observed for β 2 AR-cAMP responses are unclear for the β 1 AR.
Like β 2 AR-cAMP activity, LTCC seem to be confined to t-tubular microdomains. Greater numbers of LTCC were observed in TTs in normally loaded cardiomyocytes. These channels had a higher open probability, suggesting a larger more active population of LTCC in the loaded cardiomyocyte tubule in comparison to crest domains. This is consistent with our previous work (Sanchez-Alonso et al., 2016). Interestingly, following PMU, LTCC seemed to almost disappear from the TTs of cardiomyocytes. LTCC activity was also significantly reduced. This was demonstrated to be due to a 'silencing' of LTCC following PMU, as well as the loss of confinement of LTCC to the TT domains. Experiments performed in the presence of BAYK show the silencing effect of PMU. Simply adding this agent activated LTCCs in both TTs and crest domains. This once again raises the suggestion that the activation and maintenance of compartmentation pathways of molecular signaling processes within cardiomyocyte microdomains are load-dependent.
We observed that the organization and association of proteins which are usually involved in the compartmentation of calcium and cAMP in cardiomyocytes was lost following PMU (Chen et al., 2013;Wong et al., 2013). We also specifically observed the agglomeration of JPH-2 and Cav-3 following PMU, alongside the loss of normal association of Ca v 1.2 subunit of LTCC with Cav-3. JPH-2 has been reported to be essential for the normal formation of TTs (Chen et al., 2013), which in turn control both calcium handling and cAMP. Caveolae are the site of a sub-population of LTCC (Chen et al., 2013). LTCC activation is achieved by the selective phosphorylation of the channel by PKA or CaMKII, depending on the specific compartment involved (Sanchez-Alonso et al., 2016). We also observe the loss of regularity within the TAT network following PMU which is concordant with previous work in our laboratory (Ibrahim et al., 2012a). These findings suggest that mechanical load is responsible for the collection, distribution and organization of the cell membrane and structural proteins which are then able to form and arrange sub-cellular signaling microdomains. This is apparently achieved by the association and sequestration of receptors, channels and other signaling molecules. Loss of this stimulus as well as causing the atrophy of the cell, causes the loss of faithful signaling organization. This could potentially be a passive process.
Hypertrophy is stimulated by the increased demands placed upon the myocardial tissue, so-called overload, which causes two separate phases of cell behavior. The myocardium exhibits marked structural and functional plasticity, this property is largely due to the modulation of individual cardiomyocytes (Frisk et al., 2016;Watson et al., 2016). Initially, the cell grows and copes with the increased demand and 'compensates.' This is followed by an energetic crisis where the biomechanical properties of the cardiomyocytes are stretched to a point beyond which neither adequate functional capacity or maintenance can be sustained. This is referred to as decompensation and leads to myocardial failure. During these phases, the TATS is progressively altered. Atrophy most likely represents the converse of hypertrophy. It has been reported that the integrity of the TAT network is disrupted by the degree and timescales of mechanical unloading (Ibrahim et al., 2012a). As a result it can be posited that as the demand for myocardial function is withdrawn by unloading, the cardiomyocytes adapt by reducing their structural and organizational complexity, which are energetically expensive to sustain. The result is a passive rather than active loss of cardiac structure. This nonetheless results in a pathological loss of cardiac fitness and capacity. We suggest that unloading causes a situation like the one presented in our schematic in Figure 6. We suggest that the loss of TT organization removes the innate activity of molecules such as LTCC. The disorganization of TTs and structural proteins seems to result in the increased malignancy of calcium spark activity, as previously reported, (Ibrahim et al., 2012a) despite this loss of LTCC activity. We suggest this may be the effect of the loss of coherent molecular organization. It also reduces the capacity of β 2 AR to signal once stimulated with isoprenaline, the necessity of effective localization of the molecules to produce normative function suggests this effect is in someways similar to the effect on LTCC. Finally, the loss of normal activity in the cytosol but continuing stimulation of RII_PKA domains by β 2 AR after activation is further indicative of a loss of molecular control leading to a scenario in which one receives 'more bang for the buck' after excitation due to mechanosensitive decompartmentation.
The role of βAR in the pathophysiology is well characterized but somewhat poorly understood. It is known that chronic βAR stimulation is cardiotoxic and pro-arrhythmic (Engelhardt et al., 1999;Nguyen et al., 2015;Lucia et al., 2018). In settings of heart failure the sympathetic nervous system is upregulated in an effort to produce greater cardiac inotropy, the long term effects of this as described above are deleterious and this gives rise the paradoxical efficacy of 'beta-blockers' in maintaining patients with cardiac pathology (Baker et al., 2011). This aspect presents an equally complex picture as β 1 AR is desensitized and β 2 AR is uncoupled from inotropic Gs/cAMP pathways in failing hearts (Paur et al., 2012). Some beta-blockers are suggested to be effective via the Gi-coupled β 2 AR, once again paradoxically rescuing the heart by making it perform less well (Wisler et al., 2007). In our atrophic model, we see a loss of action/gain of function phenotype which is qualitatively different to hypertrophic or failing hearts. However, we also posit deleterious effects of this pathology on βAR function and a reduction of cardiac functional capacity in atrophic situations as a result of the effects of PMU on membrane structure and organization.
In this study, we employed unloaded cardiomyocytes. Unfortunately, this model system has some limitations. The removal of the cells from their normal configuration in integrated myofibers is somewhat unnatural. It removes them from the normal neurohormonal milieu which they generally experience. Perhaps, more importantly for this study it also removes them from the 'force' environment they usually experience. The cells are under cyclic stress and strain throughout the entire life-cycle of the animal and we then study them in a very quiescent state.
In some experiments we also employ culture methodology to allow the transduction of FRET reporters it is well known that culture disrupts cellular homeostasis and the TT environment in particular (Pavlović et al., 2010). However, the justification for using isolated cardiomyocytes is that many of the techniques employed within this study are not possible in isolated tissue slices or integrated ex vivo preparations. Imaging studies are made complicated by non-specific binding in integrated tissues. Equally, the measurement of cAMP and LTCC activity within cellular microdomains is also currently prohibited in tissue due to the difficulties presented by factors such as the thickness of integrated tissue. Finally, transduction of FRET reporters into isolated cells is also currently a necessity due to practicalities of infecting tissue in, for example, a slice based modality. We also lack transgenic rats expressing FRET sensors, which would be appropriate for this study. A study could be attempted in which the animals could be infected with AAV-9 vectors expressing FRET constructs of interest. This would allow FRET studies to be completed on the day of sacrifice, avoiding cells culture but the necessity for cell isolation would remain.
Mechanical support via ventricular assist devices represents an increasingly common therapy for heart failure. It has functioned as a 'bridge to transplant' for many years. Current research is geared toward repurposing mechanical support as a 'bridge to recovery.' The latter project requires mechanical unloading to provide curative relief to a heart which is theoretically capable of regenerating its normal function. Bridge to recovery is defined by the number of successful explants of LVADs following the recovery of cardiac function. The rate of explant is around 6% and is usually reliant on heart failure to be in its earliest stages, where minimal cytoarchitectural changes have occurred within the myocytes (Seidel et al., 2017). The exact interplay of the adult hearts lack of intrinsic regenerative capacity and the pathological effects of atrophy is unclear here but must be considered. Indeed, some clinicians are actively pursuing removal of LVADs and some groups are investigating 'weaning' protocols (Formica et al., 2010;Selzman et al., 2015). Currently, 'turn-down' protocols are employed to assess the heart's ability to cope on its own with or without adjuvant medical therapy prior to the decision to explant an LVAD. Hemodynamic studies are utilized during 'turn-down' to provide this assurance to physicians. The rationale of 'weaning' as part of therapy would be to allow the heart to receive enough support to provide sufficient cardiac output and adequate support to regenerate. But this 'turn-down' would also appear to offer a training program/stimulus to the damaged myocardium, providing the stimulus to prevent atrophic losses and rebuild essential structures within the cardiomyocytes. In this study, we present data showing the pathological effects of mild withdrawal of load. It would appear that myocardial βAR-cAMP and calcium handling are exquisitely load-sensitive. This should be considered during the provision of mechanical unloading therapy where functional recovery is desired.
DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.
ACKNOWLEDGMENTS
We would like to thank Prof. Manuela Zaccolo for her gift of the RII_epac FRET construct and Prof. Viacheslav Nikolaev for his gift of the cEPAC2 construct. We also would like to thank Pete O'Gara for his assistance with cell isolation. | v3-fos-license |
2017-08-15T23:18:28.713Z | 2000-10-01T00:00:00.000 | 5300972 | {
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} | pes2o/s2orc | Supercritical Fluid Extraction and Chromatographic Analysis ( HRGC-FID and HRGC-MS ) of Lupinus spp . Alkaloids
Os extratos de alcalóides de Lupinus spp., obtidos por métodos convencionais (maceração/ sonicação – extração em fase sólida; maceração/sonicação –extração líquido-líquido) e por SFE (extração com fluido supercrítico) usando CO2 e CO2 modificado (CO2/MeOH, CO2/EtOH, CO2/iPrOH e CO2/H2O) foram analisados por CGAR-DIC (cromatografia gasosa de alta resolução com detector de ionização de chama) e CGAR-EM (cromatografia gasosa de alta resolução acoplada à espectrometria de massas). As análises quantitativas por CGAR-DIC foram feitas pelo método do padrão interno, para a quantificação de lupanina, multiflorina e um alcalóide derivado da esparteína. CGAR-EM permitiu a identificação dos constituintes químicos (alcalóides e outras substâncias) destes extratos.
Introduction
Lupinus spp.has been investigated in several countries as a potential alimentary source due to its relatively high protein and oil content (35 -40% and 8 -12%, respectively), high productivity and low cost.Although lupine protein levels are similar or larger than that of the soya bean, the main problem is the quinolizidine alkaloids, which are known to provide a bitter taste and toxicity to the seeds.An usual procedure for the elimination of these alkaloids is washing the seeds with flowing water; despite simple, this method requires large volumes of water for the commercial scale Lupinus processing [1][2][3] .SFE (supercritical fluid extraction) is a valuable method both for industrial scale food processing and also for analytical scale studies, as an alternative extraction method to reduce the use of liquid solvents (mainly organic solvents).However, analytical scale SFE of polar compounds is still underexplored, mainly due to the low diffusibility and low polarity of supercritical CO 2 .Some moderately polar natural products such as alkaloids and flavonoids have been extracted by SFE 4,5 .
In this work, the extraction of Lupinus spp.alkaloids by SFE is compared with conventional methods (maceration/sonication -solid phase extraction, and maceration/sonication -liquid-liquid extraction).The extracts were analysed by HRGC-FID (high resolution gas chromatography -flame ionisation detector), for the quantification of lupanine, multiflorine and a spartein-like alkaloid.HRGC-MS (high resolution gas chromatography coupled with mass spectrometry) analysis allowed the identification of the chemical constituents (alkaloids and other compounds) from the extracts obtained by different methods from Lupinus samples obtained from markets in São Paulo, SP.
Materials
Analytical reagent grade ethanol, ethanol, isopropanol, dichloromethane and acetone were purchased from Merck, Darmstadt, Germany.The carbon dioxide and nitrogen gas were supplied by White Martins, Rio de Janeiro, Brazil.
Sample extraction: conventional method (maceration/sonication)
Extraction was performed as schematically represented in Figure 1.Each sample was extracted in triplicate, and the extracts were analysed by HRGC-FID and HRGC-MS.The yield of each extraction was determined after drying (room temperature, under nitrogen flux, followed by drying in a vacuum oven at room temperature) until constant weight.
Sample extraction: SFE
Supercritical fluid extractions were performed on an analytical scale "home-made" system, previously described 6 .Powdered Lupinus samples (0.5 g each) were extracted for approximately 20 minutes in a dynamic mode, using as extraction fluids pure CO 2 or CO 2 modified with different solvents, as indicated in Table 1.
All the extracts were collected in analytical grade CH 2 Cl 2 (Merck, Darmstadt, Germany) contained in a test tube, in an ice bath.The solvent was removed at room temperature, under nitrogen flux, followed by drying in a vacuum oven at room temperature until constant weight.Whenever possible, extractions were made in triplicate (see remarks in Tables 2 and 3).Residues were dissolved in analytical grade methanol (Merck, Darmstadt, Germany) prior to HRGC analysis.
Clean up
Clean up was done by percolating the extract (obtained by conventional extraction or SFE), solubilized in 5.0 mL analytical grade methanol, through a glass Pasteur-type pipette containing 0.5 g silica gel 60, 70 -230 mesh (Merck, Darmstadt, Germany) and 0.5 g active charcoal (Reagen, Rio de Janeiro, Brazil).
Extracts obtained by SFE using aqueous mixtures have been submitted to SPE (solid phase extraction), using Sep-Pak C-18 cartridges (Waters), preconditioned with 8.0 mL methanol followed with 8.0 mL H 2 O.The extract was Sequential mode extractions were done firstly with pure CO 2 , followed by extraction of the same Lupinus sample using CO 2 modified with 10% EtOH.Temperature and pressure conditions of each step were as reported in Table 1.percolated through the cartridge and eluted with methanol, acetone, ethyl acetate and chloroform, successively (8.0 mL each).These fractions were collected and combined for total yield determination and chromatographic analysis.Some samples were not submitted to the clean up step and were directly analyzed by HRGC; they are indicated in Table 3.
Chromatographic analysis
HRGC-MS analyses were performed using a HP 5970 mass selective detector (Hewlett -Packard, USA), (EI, 70 eV), coupled to a HP 5890 GC.The column used was a 95% methyl, 5% phenylpolysiloxane, LM-5 (50 m x 0.25 mm x 0.65 mm) supplied by L & M (São Carlos, Brazil).Samples were injected using the split mode (1:30), with injector temperature and HRGC-MS interface temperature both at 300°C.The column temperature was programmed to rise from 170 °C (3.5 min), at 6 °C min -1 , to 300 °C (held during 20 min).Helium was used as carrier gas, at the average linear velocity of 35 cm sec -1 ; MS data were processed using a CPU HP 7946 / HP 9000-300.Tentative identifications were made by comparison of the obtained spectra with literature data 7,8 .The HRGC-FID analyses were performed on a HP 5890 GC, using the same column and the same temperature program as used for the HRGC-MS analysis.Detector (FID) temperature was 320 °C, split (1:30), injector temperature was 280 °C, data were obtained on HP 3396 A integrator.Hydrogen was used as the carrier gas, at an average linear velocity of 40 cm sec -1 .All quantitative analyses were made by the internal standard method, using caffeine as an analytical standard.The Lupinus extracts were diluted to 1.0 mL in methanol, and 0.3 mL of a caffeine standard solution (1 mg mL -1 ) was added to the sample, which was analyzed by HRGC-FID.For each alkaloid quantified a corresponding calibration curve was prepared (injections in triplicate for each concentration), and linearity for internal standard quantification was checked within the range of 0.05 -0.50 mg caffeine mL -1 .
Extraction methods
The yields for each extraction procedure were determined using both Brazilian commercial and Chilean samples (Table 2).The conventional extraction gave a similar yield, both by Extrelut preconcentration and liquid-liquid extraction (Figure 1).Most of the SFE procedures gave better yields than the conventional method.Some of the SFE experiments showed a large standard deviation, due to some specific problems of some fluid mixtures.For example, water extracted polar compounds, which plugged the system due to the production of foam.Another problem was the trapping of CO 2 extracts: losses of extracts due to plugging (ice formation in the extremity of restrictor) are inherent to extract collection in solvents (the process herein adopted).The time required for SFE was 20 minutes for each extraction while for conventional methods the total time was several hours for the complete procedure.
Chemical composition of the extracts
HRGC-MS analysis of the extracts obtained from a commercial Lupinus sample allowed tentative identification of three alkaloids, by comparison with literature data 7 : lupanine (1), multiflorine (2) and a spartein derivative (3).Many other compounds were identified as alkaloids, but a more detailed tentative identification was not possible.The main feature of these unidentified alkaloids was a peak at m/z = 58, which is found in several lupane-type alkaloids 7 .Some extracts also contained other compounds, mainly fatty acids and long chain hydrocarbons, which were identified by their MS profiles 9 .The CO 2 /H 2 O mixture required slightly stronger conditions, since the critical constants of H 2 O are significantly higher than those of organic solvents 10 .SFE using CO 2 modified with 5% H 2 O was the most selective condition for alkaloid extraction; however, the critical conditions of this mixture (Pc = 89.1 atm, Tc = 69.1 o C; calculated according to the literature 11 ), require a relatively high temperature for the usual working conditions with natural products.SFE using CO 2 modified with 10% methanol showed the best yield with a lower temperature (Pc = 73.7 atm, Tc = 60.0 o C). Figure 2 (peaks key on Table 3) shows the TIC-HRGC-MS profile of these two extracts.
Quantitative analysis
Quantitative analyses were made for the main alkaloids.Lupanine was found in all of the Lupinus extracts.The regres-sion equations for the analytical curves were y= 0.0359 + 0.6647 x (r=0.999) for lupanine (1), y= -0.0236 + 0.1126 x (r= 0.999) for multiflorine (2) and y= 0.06203 + 0.02893 x (r= 0.968 ) for the spartein derivative (3).The average percentage standard error for the peak areas for replicate injections was less than 5%, showing good reproducibility.The content of each alkaloid in all Lupinus extracts is shown in Table 3.
The sum of the alkaloid content in the extract obtained by SFE using CO 2 modified with 10% ethanol and not submitted to clean-up before HRGC-FID was the greater of all extraction methods.Unfortunately EtOH usually shows problems in reproducibility as a SFE modifier, since commercial ethanol has a significant (for SFE) variation in water content.
Utilization of isopropanol as a modifier showed no significant improvement in extraction process (yield or selectivity), so this solvent should be considered only as a third option in the choice of modifiers, due to its cost and the difficulty of removing residual solvent from extracts.
Conclusions
The present results indicate that SFE can be used as an alternative to conventional methods for extraction of alkaloids from Lupinus.It is faster and had greater total yields for the extracts, and methanol was shown to be the best modifier for Lupinus extraction.Lupanin was the most abundant alkaloid in all the extracts (SFE and conventional method) from the samples studied.The utilization of water as a modifier may be a useful tool for the extraction of polar alkaloids, but only for qualitative analysis for the alkaloids herein analyzed.
Lupinus mutabilis, L. albus and L. sp.(Leguminosae-Papilonaceae) were collected in Chile by one of the authors (D. von B.).Commercial lupine samples were bought from local markets in São Paulo, SP, Brazil.The samples were dried (ca.40°C), powdered and ground (70-100 mesh).
Figure 1 .
Figure 1.Schematic drawing of the conventional extraction procedure and clean-up for Lupinus samples.
Table 1 .
Conditions of the fluid mixtures used in SFE experiments.
Table 2 .
Yield for total extracts from Lupinus samples obtained by conventional and SFE extraction methods (expressed in mg of alkaloid /g plant material).
Table 3 .
Content of alkaloids in Lupinus samples (expressed in mg of alkaloid /g plant material).
Table 4 .
Compounds and respective MS data (EI, 70 eV) found in the extracts of a commercial Lupinus sample. | v3-fos-license |
2018-09-16T08:12:25.118Z | 2018-09-12T00:00:00.000 | 52193917 | {
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} | pes2o/s2orc | A cation–π interaction in a transmembrane helix of vacuolar ATPase retains the proton-transporting arginine in a hydrophobic environment
Vacuolar ATPases are multisubunit protein complexes that are indispensable for acidification and pH homeostasis in a variety of physiological processes in all eukaryotic cells. An arginine residue (Arg735) in transmembrane helix 7 (TM7) of subunit a of the yeast ATPase is known to be essential for proton translocation. However, the specific mechanism of its involvement in proton transport remains to be determined. Arginine residues are usually assumed to “snorkel” toward the protein surface when exposed to a hydrophobic environment. Here, using solution NMR spectroscopy, molecular dynamics simulations, and in vivo yeast assays, we obtained evidence for the formation of a transient, membrane-embedded cation–π interaction in TM7 between Arg735 and two highly conserved nearby aromatic residues, Tyr733 and Trp737. We propose a mechanism by which the transient, membrane-embedded cation–π complex provides the necessary energy to keep the charged side chain of Arg735 within the hydrophobic membrane. Such cation–π interactions may define a general mechanism to retain charged amino acids in a hydrophobic membrane environment.
Vacuolar ATPases are multisubunit protein complexes that are indispensable for acidification and pH homeostasis in a variety of physiological processes in all eukaryotic cells. An arginine residue (Arg 735 ) in transmembrane helix 7 (TM7) of subunit a of the yeast ATPase is known to be essential for proton translocation. However, the specific mechanism of its involvement in proton transport remains to be determined. Arginine residues are usually assumed to "snorkel" toward the protein surface when exposed to a hydrophobic environment. Here, using solution NMR spectroscopy, molecular dynamics simulations, and in vivo yeast assays, we obtained evidence for the formation of a transient, membrane-embedded cation-interaction in TM7 between Arg 735 and two highly conserved nearby aromatic residues, Tyr 733 and Trp 737 . We propose a mechanism by which the transient, membrane-embedded cation-complex provides the necessary energy to keep the charged side chain of Arg 735 within the hydrophobic membrane. Such cation-interactions may define a general mechanism to retain charged amino acids in a hydrophobic membrane environment.
Eukaryotic V-ATPases are multiprotein complexes ( Fig. 1) that consist of a peripheral V 1 complex (subunits A-H) and an integral, membrane-embedded V 0 complex (subunits a, d, e, c, c', and c"). (14) ATP hydrolysis by V 1 drives the rotation of the central stalk (subunits D and F) and the connected ring of proteolipid subunits (c, c', and c"). (14,15) Crystal structures as well as cryo-EM reconstructions show that a glutamic acid residue is crucial for proton transport of the different subunits c (Glu 137 ), c' (Glu 145 ), and c" (Glu 108 ) (15,16). These residues might either be directly involved in transport or serve as gatekeepers for the binding pocket. (17)(18)(19)(20)(21). However, little structural information is available about the sites of entry and release of the protons into and out of the membrane, respectively. Subunit a is believed to form two water-filled hemichannels through which the protons are transported. This is supported by a recent cryo-EM structure of an assembled ATPase from Thermus thermophilus (22). Thus, protons may enter the cytoplasmic hemichannel and are transferred to a specific glutamate residue of the proteolipid c subunits. After one ring rotation, the protons are passed on to the second hemichannel and released to the lumen (4,3,6). Arg 735 , located in transmembrane helix 7 (TM7) of subunit a, is essential for proton transport (23). It is thought to interact with the glutamate residues of the proteolipid ring and thereby enables deprotonation and transfer of protons to the lumenal hemichannel (20). However, the position of charged amino acids, like arginine and glutamate, in the hydrophobic part of a membrane is energetically unfavorable. Although the negative charge of the glutamate residues in the proteolipid is neutralized by protonation, the mode of stabilization of Arg 735 within subunit a has remained elusive.
There has been great interest concerning the protonation state and localization of charged amino acids, in particular arginine, in a lipid bilayer environment. Molecular dynamics (MD) simulations have revealed that a charged arginine in a transmembrane model would experience a large free energy barrier of ϳ 17 kcal/mol to move across a lipid bilayer (24). In contrast to these calculations, a relatively low apparent free energy of ϳ2.5 kcal/mol was observed in cell biology experiments that studied the membrane partitioning of a hydrophobic protein segment containing an arginine residue (25). The situation is even more complicated by the unknown pKa value of arginine residues in a lipid environment, which usually eludes experimental determination (26). Theoretical as well as experimental work suggests that the guanidinium group of arginine either remains protonated inside the lipid membrane or resides close to the membrane-water interface (26,27).
To clarify the role of Arg 735 in V-ATPase function and to examine the environment and inter-and intramolecular interactions of this residue, we used solution NMR spectroscopy. Combined with MD simulations, this approach allows investigation of peptide structure and dynamics, providing detailed insights into Arg 735 function (28,29). Surprisingly, we found close spatial proximity between Arg 735 and the two aromatic residues Tyr 733 and Trp 737 within subunit a of the V-ATPase complex. The results suggest a transient "cationinteraction" (30 -33) between Arg 735 and these aromatic residues, which keeps the arginine residue in the hydrophobic environment and thus plays a crucial role in the catalytic mechanism. The physiological relevance of Tyr 733 and Trp 737 for V-ATPase assembly in the baker's yeast Saccharomyces cerevisiae was indeed shown by growth assays and fluorescence microscopic analyses that demonstrate the essential roles of Tyr 733 and Trp 737 for proper V-ATPase function.
Results and discussion
NMR solution structure and fluorescence spectra of TM7 reveal spatial proximity of the essential Arg 735 and the two aromatic residues Tyr 733 and Trp 737 In a previous study of TM7 of V-ATPase in membrane mimetics, we found evidence of Arg 735 being located in the hydrophobic environment (34) rather than near the polar surface, as typically found for membrane-bound peptides. To investigate the structural features of TM7 that keep the charged side chain of Arg 735 within the nonpolar core, we used a synthetic TM7 peptide with uniformly 13 C/ 15 N-labeled Arg 735 embedded in a dodecylphosphocholine (DPC) micelle. DPC is a zwitterionic detergent that has been extensively used to mimic eukaryotic membranes (35).
The solution NMR structure ( Fig. 2A, NMR statistics summarized in Table 1, PDB code 6HH0, BMRB accession number 34309) shows TM7 to adopt a transmembrane ␣-helix in the hydrophobic environment. Interestingly, Arg 735 is in close contact with two aromatic residues, Tyr 733 and Trp 737 . We observed NOEs from side chain protons Arg 735 -H⑀ and H␦ to the ring protons Tyr 733 -H␦ as well as Trp 737 -H3 and H2. The calculated structure points to a cation-interaction formed between the cationic side chain of the arginine and the polarizable electron cloud of the aromatic groups. The close spatial proximity between the arginine side chain and the two aromatic residues at positions iϪ2 and iϩ2 would not be found without a specific interaction because the side chains of iϮ2 are usually on opposite sides of an ␣-helix. This can be seen in the structure of a double-mutant transmembrane helix peptide, TM7-Y733A-W737L (Fig. S1). Very recently, two cryo-EM structures of the V 0 domain of yeast V-ATPase, reconstituted in amphipole (at 3.9-Å resolution) (15) and nanodiscs (3.5-Å resolution) (16) have been reported. In these structures, Arg 735 is embedded in a very long tilted ␣-helix (a CT7 ) and extended toward Glu 108 in the c" subunit, although the resolution of these structures does not allow to unambiguously identify side-chain interactions. The aromatic residues Tyr 733 and Trp 737 are not as close to Arg 735 as we have found them in the isolated helix (Fig. 3). However, Trp 737 is also unusually bent around a CT7 and therefore closer to Arg 735 than the typical sidechain orientation in a free helix. Helix a CT7 is kinked in the region around Arg 735 and flexible in its N-terminal part, as indicated by the missing density. In both cryo-EM structures, a snapshot of Arg 735 being close to Glu 108 of c" is captured. The increased flexibility of a CT7 could be necessary to accommodate the rotation of the nearby c-ring. As indicated in Ref. 15, as rotation of the ring continues, the glutamate residue encounters Arg 735 , possibly in a different conformation, causing deprotonation of glutamate via the
A cation-interaction facilitates proton transport
luminal half-channel. We believe that the cation-interaction observed in our structure constitutes this event, which is the preferred conformation in the absence of the nearby glutamate residue. An isolated transmembrane helix reconstituted in micelles is of course a very simple model of the corresponding helix in the whole-membrane protein. However, it shows that this interaction is strong enough to provide the energy needed to keep an arginine residue in a hydrophobic environment. It might just as well alternate with the salt bridge formed between Arg 735 and Glu 108 during c-ring rotation.
Cationinteractions were first described about 30 years ago, but their importance was firmly established only recently (30,36,37), although these noncovalent interactions can be stronger than hydrogen bonds. The formation of a cation-interaction involving a tryptophan residue in a protein can be monitored by fluorescence spectroscopy. Thereby, a cationformation in solution is characterized by a small red shift of a few nanometers, accompanied by an increase in fluorescence intensity of about 30 -40% (38,39). However, in contrast to spectra of proteins in solution, Peter et al. (40) reported even small blue shifts for a peptide embedded in different membrane mimetics. Comparing the fluorescence spectra of the WT and the R735L mutant of yeast V-ATPase, we observed the expected intensity increase of ϳ30% (max of 339.81 Ϯ 1.40 nm for the WT and 341.66 Ϯ 2.03 nm for R735L) (Fig. 2B), which corroborates the spatial proximity of the essential Arg 735 and the two aromatic residues. The lack of the red shift might be explained by the use of membrane mimetics, as mentioned above.
The Tyr 733 and Trp 737 residues influence the immersion depth of the Arg 735 side chain
We hypothesized that the loss of the cation-interaction and its charge stabilization of the arginine would directly influence the immersion depth of the Arg 735 side chain. To investigate the influence of Tyr 733 and Trp 737 on membrane immersion, we performed solvent paramagnetic relaxation enhancement (sPRE) measurements. (41-43) Gadolinium-diethylentriamine pentaacetic acid-bismethylamide (Gd(DTPA-BMA)) is an inert, water-soluble, paramagnetic agent (44). Addition to a solution renders the solvent around the micelle paramagnetic because Gd(DTPA-BMA) is unable to penetrate the micelle membrane (34). The interaction of the unpaired electron with a nuclear spin leads to enhanced relaxation (45). This sPRE is proportional to the inverse cube of the distance to the closest paramagnetic center (46,41) and is, therefore, an immersion depth indicator for nuclei in the micelle. In other words, the closer an atom is to the surface of the micelle, the stronger the decrease in its peak intensity (hence, a high sPRE) after addition of the paramagnetic molecule (47). As we could not use the guanidinium protons for sPRE measurements because of fast chemical exchange, the H␦ provides information closest to it and, hence, about its localization within the membrane. sPREs were measured by saturation-recovery 1 H-13 C HSQC experiments of the WT peptide and a double mutant lacking the aromats (Y733A/W737L). Both contained uniformly 13 C-labeled arginine for high accuracy and were embedded in DPC micelles. In the WT peptide, H␦ of Arg 735 has a rather low sPRE of 0.32 Ϯ 0.02 s Ϫ1 mM Ϫ1 . A higher sPRE value, associated with less immersion, was found for the TM7 double mutant (H␦ sPRE, 0.42 Ϯ 0.02 s Ϫ1 mM Ϫ1 ). The effect can be explained by a shallower immersion and the absence of additional shielding by the aromatic groups in the double mutant. sPREs below ϳ0.3 s Ϫ1 mM Ϫ1 are typically found for nuclei close to the core of DPC micelles, whereas values above ϳ0.5 places Figure 2. A, NMR structure of TM7 embedded in a DPC micelle (PDB code 6HH0). The essential Arg 735 (green) and the two aromatic residues (Tyr 733 in yellow and Trp 737 in orange) show the typical geometric arrangement of a cation-complex. B, fluorescence emission spectra of TM7 versus a TM7 R735L mutant show the expected intensity decrease, indicative of the loss of a cation-interaction. C, representative snapshot from MD simulations of NOE-restrained TM7 in a bilayer using the same coloring scheme for Tyr 733 , Arg 735 , and Trp 737 . The MD simulation shows the possibility that water (red/ white sticks) penetrates the bilayer, coming close to W737 (orange spheres), which might affect tryptophan fluorescence spectra. them close to the surface. A value of 0.32 s Ϫ1 mM Ϫ1 as found for H␦ of Arg 735 corresponds to an immersion depth of ϳ 12.1 Å from the surface (34), whereas 0.42 s Ϫ1 mM Ϫ1 for the mutant without aromatic groups corresponds to a localization 10.4 Å from the surface, which is just below the zwitterionic headgroup. For comparison, the sPREs of the chain CH 2 groups of DPC are 0.12 Ϯ 0.01 and 0.10 Ϯ 0.01 s Ϫ1 mM Ϫ1 for the solution containing the WT and the aromat-free TM7 peptide, respectively. This indicates that the increase in sPRE of the mutant without aromatic residues is not the result of a rearrangement in the DPC micelle.
Molecular dynamics simulations show favorable interactions of the guanidium group of Arg 735 and the aromatic systems of Tyr 733 and Trp 737
To gain further insight into the cation-interaction of TM7, especially its stability and flexibility, we used the NMR data as restraints for MD simulations. MD simulations rely on experimentally obtained physical parameters to calculate the motion of all atoms of a protein over a given time span (48). It allowed us to take a closer look at the arginine's interactions and dynamics in a bilayer.
A cation-interaction facilitates proton transport
A pronounced change in the orientation of Arg 735 relative to the aromatic groups was observed, which occurred already after a few hundred picoseconds in the simulations (Fig. 2C). More specifically, the side chains of Arg 735 and Leu 736 partly exchange positions. Upon this rearrangement, the arginine is positioned favorably to form cation-interactions with the aromatic groups. The interaction with the tryptophan occurs for 96.2% of the time and is mainly in the stacked conformation, whereas the one with tyrosine in a T-shaped conformation is observed for 95.7% of the time. The tight packing of the bilayer probably enhances the formation of cationinteractions in the MD simulation. More interestingly, a small number of water molecules were observed to penetrate the bilayer, which further stabilizes Arg 735 by microsolvation (Fig. 1B). Taken together, the NMR data and the molecular dynamics simulations show favorable interactions of the guanidium group of Arg 735 and the aromatic systems of Tyr 733 and Trp 737 , indicative of a cation-interaction.
Yeast in vivo assays confirm the biological significance of the aromatic residues for V-ATPase activity
The results from in vitro experiments and MD simulations led us to investigate the relevance of the Tyr 733 -Arg 735 -Trp 737 cation-interaction within the V-ATPase complex in a physiological context. Residues involved in cationinteractions are usually highly conserved (49). A BLAST search (50) identified a number of subunit a homologs in various organisms. A multiple sequence alignment of the homologs (ClustalX 2.1 (51)) revealed that both Tyr 733 and Trp 737 , are highly conserved in the eukaryotic kingdom, indicating their biological importance (Fig. S2). Interestingly, similar patterns of conservation can be found for the evolutionarily related Na ϩ -ATPase and F-ATP synthase, providing further proof for the functional relevance of the aromatic residues. Replacement of the tyrosine by an aromatic phenylalanine, as observed in some prokaryotes, does not affect V-ATPase activity in S. cerevisiae, ruling out a hydrogen bridge formation as a mechanism of interaction (52).
Complete loss of V-ATPase activity is lethal in all eukaryotes except in fungi, which show distinct phenotypes under defined experimental conditions (4). Thus, we chose S. cerevisiae to study the role of Tyr 733 and Trp 737 for proper V-ATPase functionality. Malfunction disables acidification of vacuoles, leading to a Vma Ϫ growth phenotype, which is characterized by sensitivity to elevated pH or calcium levels. (4,53).
We hypothesized that the removal of the aromatic residues near Arg 735 would prevent the formation of the cation-interaction and thus impair proton transport in Y733L and W737L mutants. To test this hypothesis, an S. cerevisiae V-ATPase subunit a knockout strain (lacking both subunit a isoforms encoded by VPH1 and STV1) (54) was complemented either with an empty vector (EV) or plasmids carrying genes encoding the WT VPH1 or the mutations Y733L, W737L, and Y733L/ W737L, respectively. Furthermore, the vph1 mutant variants R735K and R735L were used, as they are known to show either a mild or severe Vma Ϫ phenotype, respectively (23). To monitor the impact of the various mutations on V-ATPase activity, we performed viability tests on rich culture medium at pH 5.5 and pH 7.5, both with varying calcium concentrations (Fig. 3A).
The W737L exchange led to a more severe growth defect than the Y733L mutation, suggesting a more critical role of the tryptophan residue on V-ATPase activity. The more severe effect of the Trp 737 over the Tyr 733 mutation is consistent with cation-complex formation; its likelihood of formation and strength decreases from tryptophan to tyrosine and phenylalanine. (31) Importantly, the simultaneous exchange of both aromatic residues, Y733L and W737L, led to the same growth defects as for the EV indicating total loss of V-ATPase activity. As expected, increasing CaCl 2 concentrations, especially in combination with elevated pH levels, resulted in more pronounced growth defects for all tested constructs.
Consistent with published data (23) and the proposed formation of a cation-interaction, the strain bearing the chargeless R735L mutation led to a Vma Ϫ phenotype at a level comparable with the vph1 stv1 strain expressing the EV. Notably, replacement of the cationic arginine residue by a cationic lysine (R735K, which forms weaker cation-interactions) also led to a Vma Ϫ growth phenotype at elevated pH, especially in the presence of CaCl 2 , pointing out the importance of the highly conserved arginine residue within the Vph1 protein in vivo.
A cation-interaction facilitates proton transport
To corroborate these results in greater detail, we performed quinacrine staining to monitor V-ATPase activity in living cells (Fig. 4B). Quinacrine accumulates only in acidified vacuoles and thus indicates proper V-ATPase function in vivo (55). The vph1 stv1 strains expressing the native VPH1 and the Y733L mutant were capable to acidify vacuoles at elevated pH levels. The other mutants (W737L, Y733L/W737L, R735K, and R735L) and cells bearing the EV showed no quinacrine staining of the vacuoles, indicative of loss of vacuolar acidification and, thus, V-ATPase function (Fig. 4B).
To rule out that the loss of V-ATPase function caused by distinct mutations in VPH1 was due to disassembly of the V-ATPase complex, we used chromosomally integrated Vma6-mGFP and Vma2-mGFP fusion proteins to monitor their cel-lular localization in the vph1 stv1 double knockout strain bearing the empty vector or the distinct VPH1 mutant variants (Fig. 5). Vma6 represents subunit d of the V 0 integral membrane domain and is required for V 1 domain assembly on the vacuolar membrane, whereas Vma2 represents subunit B of the V 1 peripheral membrane domain of the V-ATPase complex. Colocalization studies of Vma6-mGFP and the vacuolar membrane dye FM4-64 (Fig. 5A) showed a high Pearson's colocalization coefficient (PCC) for the native Vph1 (0.86 Ϯ 0.01) and almost no colocalization for the empty vector control strain (0.08 Ϯ 0.05). Mutation of either Y733L or W737L did not significantly affect V-ATPase complex assembly, whereas the double mutant Y733L/W737L showed a reduced PCC (0.53 Ϯ 0.02). The R735K mutant was also slightly impaired in V-ATPase
A cation-interaction facilitates proton transport
assembly (PCC ϭ 0.64 Ϯ 0.03), and the published loss-of-function mutant R735L showed the least colocalization (PCC ϭ 0.45 Ϯ 0.02). Immunoblot analyses showed comparable protein levels for native Vph1 and the Y733L, W737L, and R735K mutants in the strain expressing Vma6-GFP, whereas decreased Vph1 protein levels were detected for the Y733L/ W737L and R735L mutants, which may be a result of lower expression or decreased protein stability (Fig. 5B). Vma1 levels were not affected by the various Vph1 mutant variants (Fig. 5B). Colocalization studies of Vma2-mGFP and the vacuolar membrane dye FM4-64 (Fig. 5C) showed similar PCC values as for Vma6-mGFP (Fig. 5A), except for the vph1 stv1 mutant strain bearing the EV. Immunoblot analyses of the WT and mutant strains expressing Vma2-mGFP (Fig. 5D) showed almost identical protein levels for the various Vph1 mutants as for the strains expressing Vma6-mGFP (Fig. 5B). It should be noted that the protein levels were analyzed from whole-cell lysates and thus only serve as a control for proper (or improper) expression of the different Vph1 variants and not for analysis of V-ATPase complex assembly. Because Vma2 is a peripheral subunit of V-ATPase, disassembly of the complex leads to cytosolic localization of Vma2 and, thus, to an unspecific overlap with vacuolar membranes. This could explain the higher PCC value for Vma2 in the vph1 stv1 strain bearing the empty vector (PCC ϭ 0.41 Ϯ 0.10) compared with Vma6, which is an integral subunit that remains localized on organellar membranes in the vph1 stv1 strain bearing the empty vector but stays strictly separated from vacuolar membranes (PCC ϭ 0.08 Ϯ 0.05).
Taken together, the in vivo experiments clearly support the physiological importance of the Tyr 733 -Arg 735 -Trp 737 cation-complex in Vph1 TM7; the two single mutant variants Y733L and W737L both showed a Vma Ϫ phenotype with a more severe impact of W737L (Fig. 4A) even though the assembly of the V 0 V 1 complex was not impaired, as monitored with live-cell fluorescence microscopy (Fig. 5C). The double mutation of Y733L/W737L in Vph1 results in total loss of V-ATPase activity (Fig. 4), as shown previously for the R735L loss-of-function mutation (23). The mislocalization of Vma6-mGFP and Vma2-mGFP in the vph1 stv1 strain expressing the Y733L/ W737L double mutant or the R735L variant indicates disassembly of the V 0 V 1 complex. It is not clear yet whether the functional impairment of the cationinteraction in Vph1 leads to V-ATPase disassembly or whether these two point mutations in Vph1 impair V-ATPase assembly, and thus its function, per se.
The in vivo experiments confirm the biological significance of the highly conserved aromatic residues for V-ATPase activity and point out the importance of the two aromatic residues for proton transport, complementing the results from MD simulations and NMR spectroscopy. Our results are also corroborated by a study using disulfide-mediated cross-linking that identified a Y733C mutation that compromises growth at pH 7.5 (56), which, according to our model, can be attributed to disruption of the cationcomplex by this mutation. Furthermore, Toei et al. (52) found a Y733F mutation to show WT growth at pH 7.5, confirming the need for an aromatic amino acid at position 733 but not the demand for a hydrogen bond donor.
Despite significant recent progress in understanding V-ATPase function, the role of Arg 735 in V-ATPase proton transport is not entirely clear. We propose a novel mechanism (Fig. 6) by which the cation-interaction helps to keep the positive charge of Arg 735 within the membrane increasing its immersion depth, as confirmed by solvent PRE measurements. This, in turn, enables interactions with the glutamic acid residues involved in proton transport. The cation-interaction might accommodate the arginine residue during the periods when no ionic interactions take place and create a counteracting force weakening the salt bridge between the arginine and the glutamate. The energy involved in the formation of a cationinteraction (57) is in the same range as the calculated free energy change (ϳ17 kcal/mol) of moving an arginine through a lipid membrane (24). Considering the high degree of conservation of aromatic residues near the arginines in ATPase-related enzymes, cationinteractions might be a general mechanism to allow the localization of charged arginine residues in membrane environments.
Conclusion
We found that the catalytically active arginine residue Arg 735 of yeast V-ATPase is positioned in the hydrophobic environment close to two aromatic residues (Tyr 733 and Trp 737 ) when embedded in membrane mimetics. These two aromatic residues in the yeast V-ATPase subunit a were identified to play a crucial role in V-ATPase activity in vivo. The findings provide strong support for a cationcomplex formed between Arg 735 and its aromatic neighbors, Tyr 733 and Trp 737 . Experimental A cation-interaction facilitates proton transport evidence indeed suggests that thermodynamic stabilization resulting from cationinteraction is sufficient to overcome the energetic cost of transferring a positive charge of the guanidinium group of an arginine residue into the hydrophobic membrane environment. Based on the highly conserved nature of the residues identified in this study, similar mechanisms for proton or sodium translocation in evolutionarily related enzymes such as ATP synthases or sodium-transporting ATPases, respectively, appear plausible.
Peptides and chemicals
The peptide TM7 (KKSHTASYLRLWALSLAHAQLSSKK) labeled with 13 C/ 15 N arginine was purchased from EZBiolab Inc. Mutant peptide TM7 R735L was synthesized by Pepnome Limited Inc. and Arg 735 13 C/ 15 N-labeled Y733A/W735L TM7 by Chinapeptides. DPC-d 38 (98%) was obtained from Cambridge Isotope Laboratories Inc. (Andover, MA). Gd(DTPA-BMA) was purified from the commercially available MRI contrast agent Omniscan as described previously (37). All other chemicals were purchased from Sigma-Aldrich in the highest purity available.
NMR spectroscopy
All NMR experiments for the solvent PRE determination, assignment, and solution structure determination were carried out at 25°C on a Bruker Avance III (Bruker BioSpin, Karlsruhe, Germany) 700-MHz spectrometer equipped with a TCI cryoprobe. Solvent PREs were determined for all peptides dissolved at a concentration of ϳ1 mM in potassium phosphate buffer (50 mM (pH 5.0) and 0.02% sodium azide) containing 100 mM perdeuterated dodecylphosphocholine (DPC-d 38 ). Proton T 1 relaxation times were determined by titrating the samples with Gd(DTPA-BMA) (60 mM) to final concentrations of 0.5, 1, 2, and 3 mM. Proton T 1 relaxation times were obtained from a series of C-HSQC spectra with a saturation recovery sequence as described previously (46). Peak intensities were then fitted to Paramagnetic relaxation enhancements were then obtained by fitting longitudinal relaxation rates as a function of gadolinium concentration as described previously (46,45). For the assignment and solution structure determination, TOCSY, NOESY C-HSQC, N-HSQC, NOESY-N-HSQC, and NOESY-C-HSQC-spectra were acquired. Data were processed with NMR-Pipe (58). Peaks were assigned manually.
Structure determination
Peak intensities in NOESY spectra were translated by the program NMRView (59) into distance restraints using the built-in median method. The median intensity was set to a distance of 2.7 Å. Additionally, ⌽ and dihedral angle restraints were obtained using the program TALOS, (60) based on H␣ proton as well as C␣ and C carbon chemical shifts. 461 NOEs and 20 dihedral angle restraints were used for the structure determination of WT TM7, whereas 254 NOEs and 10 dihedral angle restraints were used for the Y733A/W737L double mutant (Table S1). NMR solution structure calculations were performed using CNS 1.2 (61). The structure calculation was carried out using the full simulated annealing method. In the final round of structure calculations, hydrogen bonds were imposed as distance restraints exclusively for regions of regular ␣-helical structure identified in previous structure calculation runs. For each peptide, 30 structures were calculated, and the 10 lowest energy conformations, which did not show any NOE violations larger than 0.5 Å, were selected. The Ramachandran map, hydrogen bonds, secondary structure elements, and root mean square deviations were calculated and analyzed using Molmol (11) and RAMPAGE (62).
Molecular dynamics simulation
The MD simulation was performed using the GROMOS11 software package for biomolecular simulations (63) in conjunction with the 54A8 parameter set to describe interactions (64). In this force field, the partial charges for aromatic systems were fitted to reproduce the quadrupole of e.g. benzene. This means that there is a small negative charge on the aromatic carbons and a small positive charge on the aromatic hydrogens. The aromatic carbons then allow interactions with cations to potentially enable the formation of cationinteractions. The initial structure of TM7 was selected from the NMR structure bundle obtained by NOESY experiments described in this work. To mimic experimental conditions as described by Hesselink et al. (65) as closely as possible, a bilayer consisting of 51 dioleoylphosphatidylcholine and 13 dioleoylphosphatidylglycerol molecules in each leaflet was constructed and equilibrated for 30 ns in simple point charge water, and the peptide was manually inserted after removing two dioleoylphosphatidylcholine molecules in each leaflet. All four lysines and Arg 735 were fully protonated to reflect a pH of 7.0, corresponding to experimental conditions. The simulation box was solvated with approximately 5,500 simple point charge water molecules. No counterions were added in the simulation system. Nonbonded interactions were calculated using a cutoff of 14 Å, complemented with a reaction field contribution to account for a homogeneous medium outside the cutoff sphere (66). Atomic motion was integrated with the leapfrog algorithm, a time step of 2 fs, and SHAKE (67) constraints on all bond lengths. Agreement with NOE intensities was enforced through 309 timeaveraged distance restraints (68) with an attractive harmonic force constant of 2000 kJ mol Ϫ1 nm Ϫ2 . Interproton distances were averaged as Ͻr Ϫ3 Ͼ Ϫ1/3 with a memory decay time of 30 ps. Because of this averaging, the NOEs do not have to be fulfilled all the time but only a fraction of the time, so the individual snapshots of the simulations can be much more diverse than observed in a single-structure determination. The simulation was performed at a constant temperature of 300 K and a pressure of 1 atmosphere with the weak coupling scheme (69) with relaxation times of 0.1 ps and 0.5 ps, respectively. Semianisotropic pressure scaling was used with a decoupled z axis, and the isothermal compressibility was estimated at 4.575 10 Ϫ4 (kJ mol Ϫ1 nm Ϫ2 ) Ϫ1 . The simulation was performed for 100 ns. Cationinteractions were defined using the distance between the geometrical centers of the guanidinium, indole, and hydroxyphenyl groups and the angle between their respective planes. The angle was defined as the angle between the normal vectors on the planes of the sidechain rings or the guanidinium group. To qualify as a cationinteraction, the distance had to be below 5.5 Å. If the side chains encompassed an angle between 45°and 135°, the cation-interaction was defined as "T-shaped" or otherwise as "stacked" (70). The secondary structure of TM7 was classified with DISICL (71).
Tryptophan fluorescence spectroscopy
Fluorescence emission spectra of a 1 M solution of TM7 and the TM7 R735A mutant in potassium phosphate buffer (50 mM pH 5.0) containing 100 mM DPC-d 38 were recorded between 300 and 400 nm in a Cary Eclipse fluorescence spectrometer (Varian, Palo Alto, CA) at 25°C and an excitation wavelength of 295 nm. The slit widths were 5 nm and 10 nm for excitation and emission, respectively. Each spectrum is a background-corrected average of three accumulations.
Rich medium (YPD) contained (per liter) 10 g of yeast extract, 20 g of peptone (both from BD Biosciences) and 20 g of glucose. Synthetic defined selective medium lacking uracil (SDura) contained (per liter) 6.7 g of Difco yeast nitrogen base without amino acids (BD Biosciences), 20 g of glucose, 20 mg of adenine, 20 mg of arginine, 20 mg of histidine, 60 mg of leucine, 230 mg of lysine, 20 mg of methionine, 300 mg of threonine, and 20 mg of tryptophan. Media for drop tests were buffered with 100 mM MES to pH 5.5 or with 100 mM sodium succinate to pH 7.5 and solidified with 2% agar (BD Biosciences). Quinacrine dihydrochloride was purchased from Sigma and FM 4-64 from Life Technologies.
Plasmids bearing the single point mutations Y733L and W737L and the double point mutations Y733L/W737L in VPH1 were purchased from Mutagenex Inc. (Hillsborough, NJ). Plasmids bearing the single point mutations R735K and R735L in VPH1 were constructed using the QuikChange protocol from Stratagene (La Jolla, CA) and checked via sequencing of the whole VPH1 ORF. Chromosomal C-terminal tagging of VMA2 and VMA6 with monomeric GFP (mGFP) was conducted using the plasmid pFA6a-mGFP-NatMX as template, the forward primer 5Ј-CAAGAAGCTCCGGTAAGAAGAA-GGACGCCAGCCAAGAAGAATCTCTAATCgtgagcaagggcgaggagctg-3Ј, and the reverse primer 5Ј-CAAAAAAAAAACG-GACAAAATAAAAAAAGCCTTTTTCTTCAGCAACCGT-Cttaggggcagggcatgctcatg-3Ј for VMA2 tagging and the forward primer 5Ј-GTATCGCACAAAACCAAAGAGAAAGAATC-AACAATTATATTTCCGTTTATgtgagcaagggcgaggagctg-3Ј and the reverse primer 5Ј-CATATAGGTAACAATTTCTCT-TAGTGACATAAAAGAAGAAGGAACGATAACTTttaggggcagggcatgctcatg-3Ј for VMA6 tagging, respectively. The frag-ments were amplified, and chromosomal integration into BY4742 and the vph1 stv1 strains was performed using the lithium acetate method (72). Selection of mutants was performed on YPD plates containing 100 g/ml nourseothricin (clonNat; Werner BioAgents, Jena, Germany). Transformation of plasmids bearing WT VPH1 or mutated versions of VPH1 into BY4742 and vph1 stv1 strains (with or without chromosomally expressed Vma2-mGFP or Vma6-mGFP fusion protein) was conducted using the lithium acetate method, and cells were selected on SD-ura plates.
Cultivation conditions and viability test
Cells were grown at 30°C overnight in SD-ura medium to late logarithmic growth phase and then shifted to 5 ml of YPD medium with a starting optical density at 600 nm (A 600 ) of 1/ml and grown at 30°C to the logarithmic phase for an additional 3 h. Cells were harvested, washed, and resuspended in sterile water (A 600 ϭ 1/ml). 5-l aliquots of serial 1:10 dilutions starting with A 600 ϭ 1/ml were spotted on the indicated plates, and growth phenotypes were analyzed after 2 days of incubation at 30°C.
Quinacrine staining
Cells were grown as described under "Cultivation conditions and viability test." A cell aliquot at A 600 ϭ 0.5 was harvested and incubated for 10 min in 1 ml of YPD medium buffered with 100 mM sodium succinate to pH 7.5 containing 200 M quinacrine. Cells were washed with 1 ml of ice-cold glucose solution (20 g/liter) buffered with 100 mM sodium succinate to pH 7.5 and mounted on solid agar slides for fluorescence microscopy (73,74). Quinacrine fluorescence was excited at 488 nm, and fluorescence emission was detected between 500 -550 nm using a TCS SP5 confocal microscope (Leica Microsystems, Mannheim, Germany) and an HCX PL APO ϫ63 oil immersion objective (NA ϭ 1.4). Images were exported using Leica LAS Lite software (Leica Microsystems), and brightness and contrast were adjusted for improved representation.
Microscopic analysis of the V-ATPase complex
Cells were grown as described under "Cultivation conditions and viability test," with the exception that the YPD medium contained 1 g/ml FM 4-64 dye to stain the vacuolar membrane. As this dye is taken up via endocytosis, cells were grown for a total of 6 h to deplete dye from the plasma membrane and endocytic vesicles and to visualize only vacuolar membranes (75). Cells were harvested and mounted on solid agar slides for fluorescence microscopy. Fluorescence of monomeric GFP was excited at 488 nm, and fluorescence emission was detected between 500 -550 nm. FM 4-64 dye was excited at 488 nm, and fluorescence emission was detected between 590 -700 nm. Experiments were performed using a Leica TCS SP5 confocal microscope and an HCX PL APO ϫ63 oil immersion objective (NA ϭ 1.4). Images were exported using the Leica LAS Lite software (Leica Microsystems), and brightness and contrast were adjusted for improved representation. The PCC was determined using the open-source Fiji software (76) following the instructions in the manual. PCC values are presented as A cation-interaction facilitates proton transport mean Ϯ S.D. of three independent images comprising about 50 cells per image.
Immunoblot analysis of V-ATPase subunits
Cells were grown as described under "Cultivation conditions and viability test." A cell aliquot of A 600 ϭ 3 was harvested, and proteins were isolated according to Baerends et al. (77), separated on 10% SDS-polyacrylamide gels, and blotted onto nitrocellulose membranes. The protein levels of V-ATPase subunits were monitored using the mouse monoclonal antibodies 8B1-F3 against Vma1 and 10D7 against Vph1 (22), followed by a horseradish peroxidase-conjugated secondary anti-mouse antibody from goat (31430, Thermo Scientific, Waltham, MA). Yeast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal control for equal protein loading. Signals were detected by the SuperSignal WestPico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA). | v3-fos-license |
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} | pes2o/s2orc | Enhancement of the Antibacterial Activity of Natural Rubber Latex Foam by Blending It with Chitin
To enhance the antibacterial activity of natural rubber latex foam (NRLF), chitin was added during the foaming process in amounts of 1–5 phr (per hundred rubber) to prepare an environmentally friendly antibacterial NRLF composite. In this research, NRLF was synthesized by the Dunlop method. The swelling, density, hardness, tensile strength, elongation at break, compressive strength and antibacterial activity of the NRLFs were characterized. FTIR and microscopy were used to evaluate the chemical composition and microstructure of the NRLFs. The mechanical properties and antibacterial activity of the NRLF composites were tested and compared with those of pure NRLF. The antibacterial activity was observed by the inhibition zone against E. coli. NRLF composite samples were embedded in a medium before solidification. The experimental results of the inhibition zone indicated that with increasing chitin content, the antibacterial activity of the NRLF composites increased. When the chitin content reached 5 phr, the NRLF composite formed a large and clear inhibition zone in the culture dish. Moreover, the NRLF–5 phr chitin composite improved the antibacterial activity to 281.3% of that of pure NRLF against E. coli.
Introduction
Natural rubber latex (NRL), the viscous liquid from Hevea brasilensis, is the main source of commercial natural rubber. Fresh NRL comprises an aqueous colloid with 44-70 wt% water. Generally, latex is processed into high ammonia natural rubber latex (HA-NRL) for convenience of preservation and transportation [1]. The main component of natural rubber is poly(cis-1,4-isoprene). In addition to water and rubber hydrocarbons, natural rubber latex also includes proteins, lipids and other substances [2,3].
Natural rubber latex products can be divided into four categories: impregnated products, molded products, extruded products and foam products. Compared with the former three, natural rubber latex foam (NRLF) is a porous material with a low density [4,5]. NRLF has the characteristics of elastic resilience, absorbency, providing sound insulation, being shockproof and providing ventilation [6][7][8][9][10][11][12]. At present, the commercial products made from NRLF mainly include mattresses and pillows.
NRLF, widely used as bedding, is one of the economic mainstays of Southeast Asian countries [13]. However, NRLF is easily attacked by ultraviolet light due to its unsaturated carbon-carbon bonds [14]. NRLF bedding is not suitable for long-term exposure to sunlight, and slow bacterial growth might occur on the cover [15][16][17]. Therefore, it is necessary to improve the antibacterial activity of NRLF and to choose appropriate antibacterial agents to reduce the cost.
Chitin [(C 8 H 13 O 5 N)n], which widely exists in the shells of crustaceans such as shrimps and crabs, is a renewable, low-cost, antibacterial and well-sourced resource [18][19][20][21][22]. The chemical structure of chitin is shown in Figure 1. Chitin is a cationic natural polymer due to its amide groups. The positively charged polymer neutralizes the negatively charged functional groups on the surface of bacteria. Once the cell wall is damaged, the cell osmotic pressure would be destroyed and finally the bacteria would die [23,24]. Therefore, chitin could be considered an environmentally friendly antibacterial agent [25][26][27][28][29]. Most of the research on NRLF composites involves filler loading [30][31][32][33]. The focus is on exploring the mechanical properties of composite foams and emphasizing the reuse of natural fibers. Currently, the research on antibacterial NRLF mainly focuses on zinc oxide [34] and silver particles [35][36][37][38][39]. Khemara Mama [40] demonstrated that NRLF treated with silver nanoparticles of only 0.2 per hundred rubber (phr) had improved antibacterial ability by 43.8% against E. coli and 25% against S. aureus compared to NRLF that was not treated with silver nanoparticles. Few reports have been published on the utilization of natural renewable materials to prepare antibacterial NRLF composites.
Compared with zinc oxide and silver particles, chitin has the advantages of renewability, biodegradability and biocompatibility. Furthermore, the preparation of silver antimicrobials is quite complex due to the synthesis of nanoparticles [39] while blending with chitin is more simple and convenient. The poor solubility of chitin provides the NRLF composite with more durability within its service life. Therefore, chitin-NRLF composites can not only enhance the antibacterial activity of foam, but are also inexpensive and environmentally friendly [19].
In this research, 0, 1, 2, 3, 4 and 5 phr chitin-loaded NRLFs were prepared using the Dunlop method. Morphology, swelling, density, chemical composition, hardness, tensile strength, elongation at break and compressive strength are characterized to verify the antibacterial activity vs. mechanical properties of chitin-NRLF composites. This natural antibacterial composite foam has considerable prospects in commercial applications of natural rubber, which is consistent with the concept of sustainability and the goal of a green economy.
Materials
The Dunlop method was used to prepare the NRLFs. High ammonia natural rubber latex (HA-NRL) was supplied by Yunnan Natural Rubber Industry Group Jingyang Co., Ltd (Yunnan, China). The chemicals (potassium hydroxide, 2-mercaptobenzimidazole, 2-mercaptobenzothiazole, sulfur, potassium oleate, ammonium sulfate, zinc oxide, sodium fluorosilicone), Luria-Bertani solid medium (deionized water, tryptone, sodium chloride, yeast extract, agar) and chitin were supplied by Sinopharm Chemical Reagent Co., Ltd. (China). E. coli BL21 were supplied by Qincheng Biotechnology Co., Ltd. (Shanghai, China). The names, concentrations and proportions of the chemicals used in the synthesis experiment are shown in Table 1.
Sample Preparation
The Dunlop method [41,42] involves prevulcanization, foaming, gelation, vulcanization and demolding. First, potassium hydroxide, 2-mercaptobenzimidazole, 2-mercaptobenzothiazole and sulfur were added into the HA-NRL. Prevulcanization of the NRL was carried out by stirring at 30 • C for 48 h at 50 rpm. Second, potassium oleate, ammonium sulfate and chitin were blended with the prevulcanized latex (PV-NRL). The PV-NRL was stirred for 8 min at 200 rpm to form a foam, and this process continued until the volume remained the same. Third, zinc oxide was added while the whisking machine was adjusted to 100 rpm. After 10 min, the sodium silicofluonate was slowly added, and the foam was stirred to the gelling point at 70 rpm. Once the gelling point was reached, the foam was injected into the mold and solidified at 90 • C for 30 min. Finally, the NRLF was vulcanized at 100 • C for 2 h. After demolding, washing and drying, the NRLF was obtained. The NRLFs loaded with chitin at 0, 1, 2, 3, 4 and 5 phr were prepared using the above procedure. Five samples were prepared for each condition.
Morphology
The microstructure and morphology of the NRLFs were observed with optical microscopy (Leica DM500, Heidelberg, German). The magnification (objective × eyepiece) was 40 ×. The samples were sliced for light transmission and observation. From the resulting graphs, the size of the micropores and the dispersion of the chitin particles were evaluated.
Swelling
The NRLFs were made into 25 mm × 25 mm × 25 mm samples. The dried samples were weighed and then hermetically immersed in deionized water for 72 h. The surfaces of the samples were wiped with kitchen paper before the swollen samples were weighed. The testing temperature was 25 • C. The swelling is indicated as S (%). S = (mass of swollen sample−mass of initial sample)/mass of initial sample (1)
Density
The dry NRLFs were made into 50 mm × 50 mm × 25 mm (length × width × height) samples for measurement. The testing temperature was 25 • C. Five samples were tested under each loading condition, and the density is indicated as ρ (kg/m 3 ).
Chemical Composition
The chemical composition was characterized by FTIR (Nicolet iS50, Madison, WI, America). The transmittance of the samples to infrared light was measured. The chemical composition of the NRLFs with chitin loading (0, 1, 2, 3, 4 and 5 phr) was investigated in a mid-infrared region (500-4000 cm −1 ).
Hardness
The hardness was tested according to the Shore C standard. A Shore C hardness tester (LX-C, Jiangdu, China) was used to measure the hardness of the soft foam materials. The dry NRLFs were made into 10 mm × 10 mm × 6 mm (length × width × height) samples for measurement. The testing temperature was 25 • C. Five samples were tested under each loading condition.
Tensile Strength and Elongation at Break
Tensile strength and elongation at break were tested according to the Chinese National Standard GB/T 6344-2008. The NRLFs were made into 13 mm × 152 mm dumbbell-shaped samples with a gauge length of 50 mm. The speed of the universal material testing machine (XWW-20A, Beijing, China) used herein was 500 mm/min. The testing temperature was 25 • C. Five samples were tested under each loading condition.
Compressive Strength
The NRLFs were made into 50 mm × 50 mm × 25 mm (length × width × height) samples. The compression ratio was 50%. The speed of the universal material testing machine (XWW-20A, Beijing, China) was 50 mm/min. The testing temperature was 25 • C. Five samples were tested under each loading condition.
Antibacterial Activity
The antibacterial activity was characterized by the inhibition zone against E. coli. The NRLFs were made into cylindrical samples with a diameter of 8 mm and a height of 2 mm. The LB solid medium was poured into the culture dishes at 60 • C, and the samples were embedded in the medium. After the culture medium was completely solidified, bacterial liquid was added. All the dishes were incubated at 37 • C for 12 h. The deformation and fracture of the cells were detected, as shown in Figure 2e. When the loading reaches 4 phr, the rubber walls fracture due to the over-expansion of the bubbles and the obstacles provided by the chitin. In Figure 2f, the number of cell cracks and the pore size increase. The broken foam wall adheres to the chitin. The pores are not stable enough, and chitin becomes aggregated, so the rubber begins to break under an uneven force. Macroscopically, small particles can be seen on the surface of the NRLF. The pores become large and weak with high filler contents. The cell diameters of the NRLFs are shown in Figure 3. The relationship between pore size and filler loading can be seen in the diagram. First, as the loading of chitin increases, the pore size of the NRLF increases gradually. The large surface tension of the foam around the chitin results in the bursting and merging of the cells. However, the cell diameter in the NRLF-5 phr chitin case decreases. Under this loading, many bubbles become large enough to burst, and the remaining cells are weak and easily deformed. Therefore, only small cells could be measured completely. Second, with the increase in chitin loading, the standard deviation of the cell diameters increases as well. This indicates that the random dispersion of the chitin in NRLF leads to an uneven pore size. Compared with the cells in the NRLFs with a low chitin load, the difference among the cells in the NRLFs with a high chitin load is too large to be accurately estimated.
Swelling
The swelling ability could directly explain the porous structure of the NRLFs according to Equation (1). The NRLF composites were immersed in deionized water for 72 h, and the mass of the NRLF composites was measured. As shown in Figure 4, with increasing chitin content, the swelling percentage decreases. The reason might be the collapse and adhesion of the rubber foam when the loading increases. For NRLFs with a higher loading, the number of pores decreases sharply even though the cells merge and expand. Therefore, the low porosity of the NRLF composites diminishes the absorption capacity.
Density
As shown in Figure 5, the density of the NRLF increases with increasing chitin loading. The density of chitin particles is 1370 kg/m 3 , which is much higher than that of the pure NRLF. In addition, agglomerates destroy the original bubble structure in the NRLF and cause collapse of the cells.
A decrease in bubble volume leads to a decreased proportion of air, so the NRLFs with a high loading contain additional rubber and chitin. Although the density of the NRLF composite increases as the filler content increases, it is still lower than that of the other materials. This is attributed to the unique porous structure of the NRLF. The density of the NRLF composite is still less than 0.2 kg/m 3 .
Chemical Composition
The FTIR transmittance spectra of the NRLFs are shown in Figure 6. FTIR spectra are used to analyze the chemical compositions and to identify the structural transformation that occurs during blending. The peak at 2900 cm −1 is due to the stretching vibration of the C-H bonds in methyl, the peak at 1660 cm −1 is due to the stretching vibration of C = C bonds, the peak at 2850 cm −1 is due to the stretching vibration of C-H bonds in methylene, the peak at 1460 cm −1 is due to the bending vibration of C-H bonds in methylene, and the peak at 842 cm −1 is due to the out-of-plane bending of C-H bonds [43][44][45]. These peaks are the key features in the spectra for the rubber hydrocarbons. Peaks at 3280 cm −1 are probably connected to moisture in the samples.
Natural rubber latex originally contains proteins, carbohydrates, lipids and other substances, hence pure NRL possesses hydroxyl and amide peaks as well. From 1200 cm −1 to 1000 cm −1 , there are ether bond peaks [46,47]. This characteristic peak differentiates the chitin from the polyisoprene. As shown in the second local graph, the transmittance peaks of ether bonds are enhanced compared with pure NRLF. This demonstrates that chitin blends with the NRLF and does not significantly change the chemical structure of the natural rubber. It can be seen that with an increase in the chitin content, the natural rubber peaks in the transmittance spectra do not change. This indicates that there is no reaction between the chitin and natural rubber latex during blending and only physical loading occurs. In other words, the chitin has no significant impact on the spatial structure of the natural rubber.
Hardness
The effects of the chitin on the hardness are presented in Figure 7. The hardness (Shore C) mainly represents the ability of rubber to resist the pressing or intrusion of hard objects. The NRLF samples with an increased chitin loading have an increased hardness, according to the trend in Figure 7. The increase in chitin content leads to the collapse and partial adhesion of the NR, so the rubber clots provide additional resistance to the external intrusion. Chitin is a linear polymer composed of (1-4) linked 2-acetamido-2-deoxy-D-glucosamine. The hardness of chitin is 7-7.5 (Mohs) due to its regularly arranged structure. The Mohs hardness is used to describe the hardness of minerals, which is absolutely higher than that of rubber. Therefore, chitin could obviously enhance the hardness of NRLF composites.
Tensile Strength and Elongation at Break
As shown in Figure 8, the tensile strength of the chitin-NRLF composite decreases with increasing chitin content. For NRLF-5 phr chitin, the tensile strength decreases to approximately half that of the pure NRLF. This decline proves that the structure of the NRLF is destroyed during the foaming process and that additional cells appear to burst before solidification. The low compatibility between the chitin and NR leads to a large surface tension on the foam, resulting in an easy bursting of the bubbles. With increasing pore size, the cells gradually become more fragile. The increase in the filler loading could cause poor dispersion and agglomeration of the chitin. The precipitation of the chitin on the surface would form stress concentrations during tensile testing. The elongation at break of NRLFs normally depends on the crosslinking density and the foam structure. Given that the NRLF composites herein were vulcanized under the same conditions [48][49][50], the difference in elongation at break would mostly be attributed to the size and quantity of the pores. As shown in Figure 9, the elongation at break of the NRLF decreases with increasing chitin loading. Similar to the trend for the tensile strength, the elongation at break of NRLF decreases remarkably when the chitin reaches 3 phr or more. The trend of these results is analogous to other literature [30].
Stress concentrations would suddenly develop at the agglomerations of the chitin, and the thinned foam walls could accelerate the breaking. Moreover, the increased hardness of the NRLF composite might contribute to the brittleness that appears during the tensile testing, which means a decreased amount of deformation before a break occurs.
Compression Strength
As shown in Figure 10, the compressive strength of the NRLF increases substantially with increasing chitin loading. Chitin has a much higher compression strength than natural rubber, and a small amount of chitin could dramatically enhance the compressive strength of NRLF composites. In addition, when the chitin content continues to increase, the fracture of the cell might lead to adhesion of the natural rubber. For the same volume, the NRLFs with a high loading allocate an increased proportion to rubber but a decreased proportion to air. During compression, NRLFs with a high loading would have a decreased amount of inner space available to shrink. Therefore, when the samples are compressed at 50% during the compression testing, the additional rubber and chitin particles in the NRLFs with a high loading provide an increased resistance to the pressure and increased compressive strength as well.
Antibacterial Activity
As shown in Figure 11, the antibacterial activity of the NRLFs on E. coli was characterized by inhibition zone testing against E. coli. Figure 11a-f represents the NRLF composites filled with chitin from 0 to 5 phr. From Figure 11a, pure NRLF without chitin already has antibacterial activity against E. coli. This result is consistent with the previous study [40]. There are two possible explanations for this phenomenon. First, the proteins in natural rubber latex might be bacteriostatic [51]. Second, 3 phr of zinc oxide was added, as listed in Table 1, and zinc oxide is a proven antibacterial [52,53].
The increase in the inhibition zone of each of the NRLF composites can be seen in Figure 12. When the chitin loading increases, the antibacterial activity of the NRLF gradually increases. The samples were tightly embedded in the solid medium so they contacted the E. coli completely. The antibacterial activity of NRLF-3 phr chitin appears to be twice that of pure NRLF. When loaded with 5 phr, the inhibition zone is obviously enlarged and is quite clear compared with that of the former samples. Furthermore, the NRLF-5 phr chitin composite improved the antibacterial activity by 181.3% against E. coli compared to that of the pure NRLF.
Conclusions
In this research, chitin was chosen as an antibacterial agent to enhance the antibacterial activity of NRLF. NRLF composites were prepared by the Dunlop method, and chitin was blended with foam during the foaming process. In addition to the antibacterial activity, morphology, swelling, density, chemical composition, hardness, tensile strength, elongation at break and compression strength were characterized as well.
Compared with that of pure NRLF, the performance of the NRLF composite is related to the loading of chitin. When the chitin content increases, the cells expand and deform with the chitin. Overexpansion could lead to bursting of the bubbles and fracture of the rubber walls. The tensile strength, elongation at break and water swelling decrease gradually, resulting in uneven force and stress concentrations.
The chitin is inclined to aggregate due to the poor interaction between the chitin and natural rubber latex. The cracked cells and broken walls stick to the central chitin agglomerations. A decreased number of bubbles contribute to the collapse and contraction of the foam materials. With a decreased proportion of air and additional space for chitin, when the chitin loading increases, the compressive strength, density, hardness and antibacterial activity increase.
Using the natural antibacterial agent chitin as a loading filler, environmentally friendly and antibacterial NRLFs were prepared herein. The antibacterial activity of NRLF composites could be increased by up to 181.3% compared to pure NRLF. When loaded with 3 phr chitin, the microstructure and mechanical properties of the chitin-NRLF composite change moderately and the antibacterial activity reaches more than double that of pure NRLF. Therefore, chitin-NRLF composites could be widely applied to household products, such as pillows, mattresses or cushions, and possess good practical value for future applications.
Conflicts of Interest:
The authors declare no conflict of interest. | v3-fos-license |
2020-09-16T22:14:47.958Z | 2019-11-02T00:00:00.000 | 221760454 | {
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} | pes2o/s2orc | Suppressive effects of the neutrophil elastase inhibitor sivelestat sodium hydrate on interleukin-1β production in lipopolysaccharide-stimulated porcine whole blood
Objective: Sivelestat sodium hydrate (Siv) is expected to be an effective therapy for acute respiratory distress syndrome, although its mechanism of action is not understood. In this study, we investigated which myeloid cells-derived cytokines were suppressed by Siv. Methods: Continuous hemofiltration was performed by circulating fresh porcine blood through a semi-closed circuit. To ensure that leukocytes survived for 360 min, 5% glucose, heparin, and air were continuously injected. The control group received continuous administration of lipopolysaccharide (LPS) only, whereas the Siv group received LPS and Siv. Complete blood count, levels of various cytokines, and other variables were compared between the groups. Results: Interleukin (IL)-1β level was significantly suppressed in the Siv group compared with that in the control group (p<0.05). Conclusions: The results suggested that Siv suppressed the production of IL-1β and possibly other cytokines by myeloid cells. Whether this suppression of cytokine production is caused directly by Siv or mediated via suppression of granulocyte elastase should be evaluated in the future.
Introduction
Sivelestat sodium hydrate (Siv) is the first developed selective inhibitor of granulocyte elastase. Developed in Japan, it treats acute lung injury accompanied by systemic inflammatory response syndrome. In the field of intensive care, it is expected to be an effective therapy for acute respiratory distress syndrome (ARDS). 1,2 Although Siv has recently been reported to suppress the production of cytokines and other mediators, 3,4 its mechanism of action remains unclear. Cytokines are produced during inflammation for various reasons. Myeloid cells are known to be activated in ARDS. 5 It is unknown whether the suppression of cytokine production by Siv occurs because of its action on myeloid cells. We hypothesized that Siv may suppress myeloid cell-derived cytokine production.
Previously, we constructed a system for maintaining the longterm viability of blood ex vivo by circulating fresh heparinized whole blood in a circuit containing a hemofilter to create an environment with fixed pH, electrolytes, temperature, oxygen partial pressure, carbon dioxide partial pressure, glucose, and other parameters inside the flask. 6 Continuously injecting lipopolysaccharide (LPS) into this circuit and adding a column that selectively acts on activated myeloid cells (Adacolumn ® ; JIMRO Co., Gunma, Japan) 7 promotes myeloid cell-derived cytokine production. The present study aimed to use a hemofiltration system that promotes myeloid cell-derived cytokine production to investigate whether Siv, a selective inhibitor of granulocyte elastase used to treat ARDS, suppresses the production of these cytokines.
Methods
The primary objective of this study was to determine whether Siv administration suppresses myeloid cell-derived cytokine production.
Collection and processing of fresh porcine blood
Fresh blood was collected from female pigs (2-5 years old, weighing 150-250 kg) (Meat Inspection Center, Toyota, Japan). Immediately after collection, 20,000 units/L heparin and 0.5 g/L glucose were added to the fresh porcine blood, which was then placed on ice.
Apheresis circuit conditions
To ensure that leukocytes survived for 24 h, 5% glucose at 1 mL/h, heparin at 2,000 units/h, and air at 0.5 L/min were continuously injected to the reservoir. To maintain constant electrolyte and pH levels, a semi-closed circuit was built to perform continuous hemofiltration (CHF). This semi-closed circuit was connected directly to an adsorptive myeloid cell
Original Article
Open Access apheresis column (Adacolumn ® ) ( Figure 1). The FX100 ® (Fresenius Medical Care Japan, Tokyo, Japan) and Adacolumn ® instruments were primed according to the instruction in their package inserts. The amount of blood circulating in the circuit was 1,300 mL. The CHF conditions were as follows: blood flow rate, 33 mL/min; filtration flow rate, 330 mL/h; and replacement flow rate, 330 mL/h.
LPS injection
Three minutes after beginning circulation, an LPS bolus (Escherichia coli serotype 0111; Wako Pure Chemical Industries, Osaka, Japan) (5 mg/L) was injected to the circulation, followed by continuous LPS administration (0.22 mg/L/h). The amount of LPS was determined by referring to our previous research. 4
Group allotment and Siv administration
Some samples (the "Siv treatment" group) received a sivelestat (Elaspol ® ; Ono Pharmaceutical Co., Ltd., Osaka, Japan) bolus (154 mg/L) at 1 min before LPS administration, followed by continuous Siv administration (26 mg/L/h). The control group did not receive sivelestat bolus.
Examinations
Blood samples for examination were collected at the entrance to the Adacolumn ® instrument before LPS injection and at 30, 60, 90, 120, 240, 300, and 360 min after LPS injection.
Blood test
Complete blood count, biochemical tests (LSI Medience Corporation, Tokyo, Japan), and blood gas analysis were performed. Electrolyte, blood glucose, and lactic acid levels were measured.
Statistical analysis
Parametric data are expressed as mean ± standard deviation, whereas nonparametric data are expressed as median and interquartile range. Statistical analysis was performed by twoway analysis of variance (ANOVA). A probability level of <0.05 was used to indicate statistically significant differences. Stat Flex ver. 5 (Artech Corporation, Osaka, Japan) was used to conduct the statistical analyses.
Ethics
The pigs were slaughtered in accordance with Act on Welfare and Management of Animals that was established by Ministry of the Environment. Our hospital's ethical screening committee was consulted about performing the study using blood collected from pigs that were slaughtered for commercial purposes. The committee determined that there was no necessity for ethical screening.
Results
The tests were conducted using five fresh porcine blood samples each for the Siv treatment and control groups (10 samples in total).
Blood test results
No significant differences were observed between the Siv treatment and control groups in the biochemical and blood gas Figure 1 Schematic diagram of the blood preparation and treatment processes. Fresh porcine blood stimulated with LPS was maintained in a reservoir at 40°C. In addition to LPS, heparin, 5% glucose, and air were added to ensure that leukocytes survived for 24 h. The Siv treatment group received continuous administration of sivelestat sodium hydrate. The system was equipped with an Adacolumn ® (JIMRO Co. Ltd., Takasaki-shi, Gunma, Japan) and an FX100 ® instrument (Fresenius Medical Care Japan, Tokyo, Japan). Continuous hemofiltration was performed to maintain electrolyte and pH levels. Blood was collected at the entrance of the primary column. CHF: continuous hemofiltration; LPS: lipopolysaccharide; Siv: sivelestat sodium hydrate.
analysis results, nor in electrolyte, blood glucose, and lactic acid levels (data not shown).
Blood count levels
No significant differences were observed between the Siv treatment and control groups in erythrocyte, monocyte, and platelet counts (figures not shown). Regarding leukocytes, granulocyte count decreased over time in both the Siv treatment and control groups. However, the difference in granulocyte count between the Siv treatment and control groups was not significant (Figure 2).
Cytokine levels
In the control group, IL-1β level peaked at 90-120 min after LPS administration, and then remained at a high level (Figure 3). In the Siv treatment group, IL-1β level increased over time, Figure 2 Granulocyte count in the Siv treatment and control groups. Granulocyte count decreased over time in both the Siv treatment and control groups. The difference in granulocyte count between the Siv treatment and control groups was not significant. S.D.: standard deviation; Siv: sivelestat sodium hydrate. although all the values remained under 1,000 pg/mL. IL-1β level in the Siv treatment group was significantly suppressed, compared with that in the control group (p<0.05). The peak TNF-α level in the Siv treatment group was lower than that in the control group, although the difference was not significant (p=0.79; Figure 4). IL-4 and IL-6 levels in the Siv group were lower than those in the control group, but the differences were not significant (IL-4: p=0.33, IL-6: p=0.43; Figure 4). IL-8, IL-10, and HMBG1 levels increased with time in both the Siv treatment and control groups; however, these levels did not differ significantly between the Siv treatment and control groups (IL-8: p=0.27, IL-10: p=0.55, HMBG1: p=0.94; Figure 5).
Discussion
Siv is a selective inhibitor of neutrophil elastase. However, its mechanism of action in ARDS is not understood. Previously, we Figure 3 IL-1β level in the Siv treatment and control groups after LPS administration. In the control group, IL-1β level increased after LPS administration, and then remained high. In the Siv treatment group, IL-1β level remained low throughout. IQR: interquartile range; Siv: sivelestat sodium hydrate. Figure 4 TNF-α, IL-4, and IL-6 levels in the Siv treatment and control groups. TNF-α peaked at a lower level in the Siv treatment group than that in the control group, although the difference was not significant. IL-6 level tended to be lower in the Siv group than in the control group, but the difference was not significant. IL-4 level was low in both the Siv and control groups, with no significant difference. There was a large dispersion in both groups. IQR: interquartile range; Siv: sivelestat sodium hydrate.
Suppressive effects of neutrophil elastase inhibitor constructed a continuous ex-vivo hemofiltration system that circulates fresh porcine blood in a hemofiltration circuit using the FX100 instrument, which maintained homeostasis of pH, electrolytes, and other parameters for 24 h. 6 In the present study, we continuously injected LPS into this system and incorporated an Adacolumn ® , which selectively adsorbs and eliminates myeloid cells (monocytes and granulocytes), to create an experimental system that promotes myeloid cell-derived cytokine production. When activated myeloid cells pass through the Adacolumn ® , which is packed with cellulose acetate beads, they are adsorbed by the beads and eliminated. 7 However, simultaneous to this adsorption, the activated myeloid cells also release cytokines. This is an ex-vivo experimental system using blood that does not include the cytokines produced by organs in living organisms, which allowed us to focus only on hemocyte-derived cytokine production. Further, by using a column that selectively acts on myeloid cells, we believe the present study accurately reflected myeloid cell-derived cytokine production.
We compared the levels of seven types of cytokines, and the results showed that only IL-1β levels were significantly lower in the Siv group than in the control group. In addition, variation in TNFα levels was high in the control group, but low in the Siv group, thereby suggesting that increasing the sample size may reveal a significant suppressive effect. Moreover, IL-6 and IL-4 levels were low in the control group, which showed the difficulty in investigating the suppressive effects of Siv on cytokine production with this experimental system.
Although the suppressive effect of Siv on HMGB1 or IL-10 production was not observed, these cytokines are mediators secreted during the late inflammatory stage, which may be related to the results. One study examined the therapeutic effects of Siv in a rat sepsis model created by cercal ligation and puncture (CLP). The results showed that Siv administration improves survival rate and significantly reduces the serum levels of IL-1β, TNF-α, IL-6, and IL-10, although it did not reduce HMGB1 levels. However, the number of HMGB1-containing cells in lung tissues at 12 h after CLP was decreased by Siv administration. 8 In vivo studies cannot determine whether cytokines with elevated levels are organ-or hemocyte-derived. The present study examined hemocyte components ex vivo to confirm that Siv suppressed myeloid cell-derived IL-1β production. However, measurements after 6 h are difficult owing to the mechanism by which Adacolumn ® column eliminates myeloid cells. Therefore, we could capture the changes in IL-1β level in the early inflammatory stage, but not in the cytokines with suppressed inflammation peaks after 6 h.
Another consideration regarding the suppressive effect of Siv on IL-1β is that unlike the secretion of the other six cytokines, IL-1β secretion accompanies the formation of inflammasomes. Inflammasomes, which are an important mechanism in the body's inflammatory response to infection and other factors, secrete IL-1β and IL-18 via activated caspase-1. 9 Elevation of the gene expression of IL-1β and IL-18 through the inflammasome/ caspase-1 pathway has been reported in the peripheral blood of ARDS patients. 10 Furthermore, in a study using vascular endothelial cells, IL-1β secretion was significantly reduced by inhibiting neutrophil elastase, not caspase-1. 11 Taking the above findings into consideration, the results of the present study supported the hypothesis that the suppression of IL-1β production was part of the mechanism of the therapeutic effect of Siv on ARDS.
The cytokines IL-8, HMBG1, and IL-10 are produced during the late inflammatory stage. Therefore, the lack of significant differences in the results of our study may have occurred because the measurements were conducted only up to 360 min after LPS administration. Because the Adacolumn ® adsorbs and eliminates myeloid cells, they are almost completely eliminated after blood circulates in this circuit for 360 min, which is why we set 360 min as the measurement period in this study. It may be impossible to examine the effects of Siv on cytokine production after 360 min. In addition, heparin at 20,000 units/L was added to fresh porcine blood immediately after collection, which may have further Figure 5 IL-8, HMGB1, and IL-10 levels in the Siv treatment and control groups. IL-8, HMGB1, and IL-10 levels tended to increase over time in both the Siv treatment and control groups. The difference in these levels between the Siv treatment and control groups was not significant. IQR: interquartile range; Siv: sivelestat sodium hydrate. affected the results. Moreover, in the present study, we used hemocytes and an ex-vivo experimental system with no organs; therefore, it is unclear whether the same results would be obtained in vivo.
In clinical practice, Siv is administered continuously at 0.2 mg/kg/h, which is equivalent to 2.6 mg/h of Siv per liter of circulating blood volume. The amount of blood circulating in our semi-closed circuit was 1,300 mL; thus, the continuously administered dose would be 3.38 mg/h. However, in the present study, Siv was injected at 154 mg/L and then administered continuously at 26 mg/h. Because Siv is likely to be metabolized by the liver, we adopted a higher dose than that used clinically. We plan to further study the dose-dependency of Siv's effect.
A tendency toward suppressed TNF-α level was observed in the Siv treatment group, compared with that observed in the control group, but the difference was not significant. However, dispersion was large in the control group, suggesting that the lack of significant difference may have been due to the sample size. If the experiment had been performed with more than five samples in each group, we might have obtained significant differences. This was a limitation of the present study, and further studies with a larger sample size are needed to validate our results.
In conclusion, our findings suggested that Siv suppresses the production of IL-1β and possibly other cytokines by myeloid cells. Whether this suppression of cytokine production is caused directly by Siv or mediated through suppression of granulocyte elastase should be investigated in the future. | v3-fos-license |
2018-04-03T02:48:24.968Z | 2018-02-15T00:00:00.000 | 3297782 | {
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} | pes2o/s2orc | S100A4 promotes lung tumor development through β-catenin pathway-mediated autophagy inhibition
Autophagy has emerged as a critical pathway in tumor development. S100A4 plays important roles in tumor metastasis, but its role in regulating autophagy has not been well characterized. In this study, we found that S100A4 was significantly upregulated in lung adenocarcinoma tissues. Clinical investigation demonstrated that high expression level of S100A4 was associated with tumor size and advanced tumor grades of lung adenocarcinoma patients. Moreover, our results revealed that extracellular S100A4 or overexpression of S100A4 inhibited starvation-induced autophagy and promoted cell proliferation in lung cancer cells in vitro; whereas small interfering RNA (siRNA)-mediated suppression of S100A4 increased autophagy and reduced cell viability in both A549 and LLC cells. Additionally, S100A4 inhibited starvation-induced autophagy to promote tumor cell viability via the Wnt pathway. Increased expression of β-catenin consistently led to a decreased LC3-II protein abundance. Further, the inhibitory effect of S100A4 on autophagy and its promotion role in cell proliferation was abolished in A549 and LLC cells using the receptor for advanced glycation end products (RAGE)-specific inhibitor (FPS-ZM1). S100A4-deficient mice showed retarded tumor development. This effect was well correlated with increased expression of autophagy markers. Our findings demonstrate that S100A4 promotes lung tumor development through inhibiting autophagy in a β-catenin signaling and S100A4 receptor RAGE-dependent manner, which provides a novel mechanism of S100A4-associated promotion of tumor development.
Introduction
Lung cancer is a common cancer and has become the leading cause of deaths from cancer in many developed and developing countries 1 . The majority (approximately 80%) of lung cancer cases are non-small-cell lung cancer (NSCLC) cases, of which 30-50% are adenocarcinoma, the most common histological type 2 . Only 15% of patients with NSCLC adenocarcinoma survive for more than 5 years after primary diagnosis 3 . Cigarette smoking and other noxious particles and gases that favor chronic lung inflammation have been established as risk factors for lung cancer development. Aberrant molecular changes, including oncogene (HER2, BRAF, ROS1 and FGFR1) activation and tumor suppressor genes (GPRC5A, Nkx2-1) inactivation, play important roles in lung cancer development [4][5][6] . In addition, the tumor microenvironment, consisting of stromal cells, is also an indispensable participant in tumor pathogenesis 7 . Nevertheless, the precise regulatory mechanisms of lung cancer development need to be studied further. S100A4 (also known as fibroblast-specific protein 1), a member of the S100 calcium-binding protein family, was first cloned in metastatic cells and fibroblasts 8,9 . It is a marker of fibroblasts and a specific subset of inflammatory macrophages 10,11 . S100A4 is expressed in a variety of cells, such as fibroblasts, macrophages, lymphocytes and malignant cells, and plays a crucial role in mediating the interplay between the tumor and stroma 9,[12][13][14] . It is reported to function in both intracellular and extracellular forms. Intracellularly, S100A4 binds to several targets involved in the regulation of angiogenesis, cell survival, motility, invasion or metastasis [15][16][17] . S100A4 is secreted from both tumor and non-malignant cells and exerts extracellular effects in regulating angiogenesis and cell migration 18,19 . Ablation of S100A4 expression in stromal cells significantly reduces metastatic colonization by regulating the matrix protein tenascin-C and vascular endothelial growth factor-A 13 . We found that S100A4 + fibroblasts promoted skin carcinogenesis by maintaining monocyte chemotactic protein-1-mediated macrophage infiltration and chronic inflammation 12 . In addition, using a methylcholanthrene-induced fibrosarcoma model, we found that S100A4 + cells prevented carcinoma through collagen production and encapsulation of carcinogens 20 . Autophagy is a conserved self-cannibalism process in which cellular organelles and proteins are sequestered in autophagosomes and then degraded in bulk in lysosomes, after which cellular compartments are recycled to preserve cellular homeostasis 21,22 . Starvation and other stresses induce autophagy, which clears damaged proteins and organelles and provides energy and building blocks for biosynthesis, crucial for the maintenance of cellular nutrient and energy homeostasis 23 . Dysfunctions in autophagy have been associated with a variety of human diseases, including cancer. Autophagy is a double-edged sword in tumorigenesis, acting both as a tumor suppressor and a protector of cancer cell survival 24 . In epithelial cells, defective autophagy promotes tumor initiation by enhancing oxidative stress and genomic instability as well as activating the transcription factor NRF 2 25 . Defective autophagy also interferes with oncogene-induced senescence and leads to the uncontrolled proliferation of cancer progenitor cells 26 . Conversely, once the malignant phenotype has been established, autophagy serves as a survival mechanism that provides rapidly proliferating cancer cells with nutrients 27 . During autophagy, cytoplasmic LC3 (LC3-I) is enzymatically hydrolyzed and conjugated to the lipid phosphatidyl ethanolamine to form a membrane-type conjugate, LC3-II. Therefore, relative LC3-II level can be used to estimate the extent of autophagy 28 . The generation of autophagosomes can be directly observed under a transmission electron microscope (TEM) 29 . Whether S100A4 can influence tumor development by regulating autophagy is largely unknown.
In this study we showed for the first time that S100A4 plays key roles in lung cancer development by inhibiting autophagy. We found that both extracellular and endogenous S100A4 inhibited starvation-induced autophagy and promoted proliferation of NSCLC cells. Moreover, S100A4 inhibited starvation-induced autophagy and promoted tumor cell proliferation by activating the Wnt/ β-catenin pathway in a receptor for advanced glycation end products (RAGE)-dependent manner. Lung tumor growth in S100A4-deficient (S100A4 −/− ) mice was obviously delayed and that expression of autophagy markers in S100A4 −/− mice was upregulated. Thus, S100A4 may promote lung tumor development by activating β-catenin signaling and inhibiting autophagy in a RAGE-dependent manner.
Cell lines and mice
Human lung cancer cell line A549 was a kind gift from Dr. Xueqiang Zhao (Tsinghua University School of Medicine). Murine Lewis lung carcinoma cells (LLC) and human lung cancer cells (A549) were cultured in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA, USA) and RMPI-1640 (Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum and 100 U/ml penicillin-streptomycin (Invitrogen, Carlsbad, CA, USA) at 37°C in a humidified atmosphere of 5% CO 2 .
C57BL/6 mice were obtained from Vital River (Beijing, China). S100A4 knockout (S100A4 −/− ) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). All mice were bred under specific pathogen-free conditions in the animal facilities at the Institute of Biophysics, Chinese Academy of Sciences. All animal studies were performed with sex-and age-matched mice after being approved by the Institutional Laboratory Animal Care and Use Committee.
Mouse model of transplanted tumors
Exponentially growing LLC cells were harvested, washed and a suspension of 1 × 10 6 LLC cells in 100 μl of sterilized phosphate-buffered saline were injected subcutaneously into the abdomen region of the C57BL/6 (control) and S100A4 −/− mice. Two days after the injection, the tumor volumes in each mouse were measured. The mice were kept under observation for 20 days and then killed to obtain the tumors. The wet weight of the tumors was balanced for each mouse.
Tissue microarray immunohistochemistry staining
Tissue microarrays (TMAs) consisting 120 NSCLC patient cases for immunohistochemistry were purchased from Xin Chao (Shanghai, China). The company provided ethical statement to confirm that the local ethics committees approved their consent procedures and all study participants signed an approved informed consent form. The ethical statement provided by the company and the protocol of experiment had been checked carefully and approved by Ethics Committee of Beijing Jiaotong University. The tissue microarrays were stained with anti-S100A4 (Abcam, Cambridge, UK). In the 120 cases, semi-quantification was carried out for evaluating S100A4 staining according to previous study 30 . Adjacent tissues were also stained as negative control. The intensity of S100A4 expression was scored as follows: 0, negative; 1, weak; 2, moderate; 3, strong (Fig. 1a). The area of positive cancer cells in each microscopic field was categorized as follows: 1, 0 to 25%; 2, 25 to 50%; 3, 50 to 75% or 4, 75 to 100%. The final score was obtained after multiplying the Fig. 1 Upregulation of S100A4 expression in NSCLC tissues. Tissue microarray from 120 lung cancer patients was stained with anti-S100A4. a Representative S100A4 staining is shown at ×200 (upper) and ×400 magnifications (lower). Scale bar, 50 μm. b Percent of the cases expressing S100A4 in carcinoma tissues. c Percent of S100A4 negative, low and high expression in different tumor grades. d Average tumor size in NSCLC patients with different S100A4 protein levels two cores (from 6 to 80). S100A4 negative was defined as a final score from 0 to 5. S100A4 low expression was defined as a final score from 5 to 45. S100A4 high expression was defined as a final score from 45 to 80.
Reverse transcriptase-polymerase chain reaction (RT-PCR)
Total cellular RNA was extracted from cultured cells using TRIzol reagent (Invitrogen, USA). We synthesized complementary DNA using a Prime Script RT Master Mix Kit (Takara, Tokyo, Japan). Quantitative real-time PCR was performed in duplicate with a SYBR Premix Ex TaqTM Kit (Takara, Japan). The data were analyzed using the 2 −ΔΔCt method and normalized against GAPDH expression.
Analysis of cell viability
For assay of cell viability, cells were grown in a 96-well plate for 5.0 × 10 3 cells per well. The following day, cells were treated with various chemicals indicated. At 24 h after incubation, cells were measured by using Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Japan) according to the guidelines from the manufacturer. After being subjected to CCK-8, cells were incubated for 2 h, and then the OD value of cells was measured.
Transmission electron microscopy
Tissue and cells specimens were processed into approximately 1 mm cubes. Samples were fixed in 2% glutaraldehyde and 2.5% glutaraldehyde post-fixed in 1% osmium tetroxide, rinsed with sodium cacodylate trihydrate buffer, dehydrated with gradient alcohol which was then replaced with propylene oxide and, finally, embedded in Epon 812. After that, semi-thin (1 µm) sections were cut, stained with methylene blue and then viewed under a microscope. Ultra-thin sections were stained with lead citrate and uranyl acetate and examined using a JEM-1400 electron microscope (JEOL, Japan). TEM images of the sections were taken using a camera-specific imaging system for EM (830.10U3 CCD Gatan, USA).
Recombinant vector transfection and siRNA interference
The S100A4 protein was purchased from Sino Biological company (Catalog: 10185-H01H, Beijing, China). pcDNA3.1 or pcDNA3.1-β-catenin plasmids were obtained from Huiteng Gene (Guangdong, China). pEGFP and pEGFP-S100A4 plasmids were kindly provided by Dr. Yingjie Wu (College of Animal Science and Technology, China Agricultural University, China). Transfection of the vectors was performed using Lipofectamine 2000 according to the manufacturer's protocols (Invitrogen, 18292-011). S100A4 small interfering RNA (siRNA) and a nonspecific negative control were purchased from Cell Signaling Technology (USA) and transfected using Lipofectamine 2000 according to the manufacturer's protocols.
Statistics
All of the data were expressed as the mean ± SEM and analyzed using GraphPad Prism software. Significant differences between mean values were obtained using three independent experiments. Differences between two groups were compared using a two-tailed unpaired Fig. 2 Extracellular S100A4 inhibits autophagy and promotes cell proliferation in LLC and A549 cells. Cells were treated with Hank's balanced salt solution (HBSS) to induce starvation. a, b A549 cells were treated with HBSS and S100A4 as indicated. Beclin1 level and LC3-I/II accumulation was determined by western blot analysis. LLC (c) and A549 cells (f) were treated with or without 1 μg/ml S100A4 in serum-containing medium or HBSS for 4 h. Treated cells were collected and analyzed for p62, Beclin1, Bax, LC3-I/II and GAPDH expression by western blot. Real-time PCR data of the mRNA expression of Beclin1, LC3-I/II, p62 and ATG5 in LLC (d) and A549 cells (g). Cell viability of LLC (e) and A549 cells (h) treated with HBSS and S100A4 was measured using a CCK-8 kit. i Representative electron microscopy of cells treated with or without 1 μg/ml S100A4 in serum-containing medium or HBSS for 4 h. j S100A4 levels in LLC and A549 cells were determined by western blot (tumor section was used as a positive control). Scale bar, 0.25 μm; *p < 0.05, **p < 0.01 Fig. 3 In A549 and LLC cells, overexpression of S100A4 inhibited autophagy whereas siRNA-mediated suppression of S100A4 increased autophagy. a A549 cells were transfected with either a plasmid overexpressing S100A4 (pEGFP-S100A4) or a control plasmid (pEGFP). At 48 h after transfection, the cells were incubated with HBSS instead of RMPI-1640 for 4 h; mRNA expression of S100A4 was analyzed by real-time PCR. b A549 cells were exposed to S100A4 siRNA or control siRNA for 24 h, then HBSS instead of RMPI-1640 was applied in cell lines for 4 h; S100A4 mRNA expression was detected by real-time PCR. Cell lysates from A549 (c) and LLC (d) overexpressing S100A4 were subjected to western blot analysis with the indicated antibodies. A549 (e) and LLC (f) cells treated with S100A4 siRNA were collected and analyzed with the indicated antibodies. The cell viability of A549 (g) and LLC cells (h) was assessed by CCK-8 assay after exposure to pEGFP-S100A4, respectively. The effect of S100A4 siRNA on cell viability of A549 (i) and LLC cell line (j) was also assessed; *p < 0.05, **p < 0.01 Student's t-test analysis. One-way analysis of variance tests with Bonferroni correction were used for multiple comparisons. Correlations between S100A4 expression and clinicopathologic variables were obtained using Spearman's rank correlation analysis (age, sex, tumor grade and tumor size). P < 0.05 was considered statistically significant.
Results
The high expression of S100A4 in lung adenocarcinoma tissues is associated with poor prognosis To identify the relationship between S100A4 expression and lung cancer progression, the expression of S100A4 in lung adenocarcinoma tissues was performed in a TMA by immunohistochemistry. We found that only a few S100A4 + cells were detected in paracarcinoma tissues, however, various ranges of immunostaining intensities were observed in lung adenocarcinoma tissues (Fig. 1a). As shown in Fig. 1b, about 14.17% of the lung adenocarcinoma tissues were assessed to be negative expression, and 66.67% and 19.17% of the lung adenocarcinoma tissues were detected with low and high expression of S100A4, respectively. Furthermore, the high expression of S100A4 was significantly correlated with advanced tumor grades (Fig. 1c) and tumor size (Fig. 1d), while there was no significant relation between high S100A4 expression and age factors as well as sex features (Supplementary Table S1). Our results suggest that S100A4 expression may influence tumor size and advanced tumor grades in lung adenocarcinoma cases.
Extracellular S100A4 suppressed starvation-induced autophagy in both human and mouse lung cancer cells Autophagy plays important roles in tumor development 24 . We wondered whether S100A4 affects lung tumor development by regulating autophagy. S100A4 is reported to function both intracellularly and extracellularly 15,17,18 . The secretion of S100A4 by tumor and stromal cells is believed to serve as a key player in promoting the metastasis of cancer cells or affecting angiogenesis 13,31 . To determine whether extracellular S100A4 suppressed autophagy in cancer cells, A549 human lung cancer cells were starved with Hank's balanced salt solution (HBSS) to induce autophagy and then supplied with S100A4 protein. After treatment with HBSS at the indicated times or with S100A4 protein at the indicated concentrations for 4 h, Beclin1 and LC3-II levels increased most significantly after 4 h in HBSS (Fig. 2a) and decreased most significantly with S100A4 protein treatment (1 μg/ml) in the A549 cell line (Fig. 2b). Then, the expressions of LC3-II and Beclin1 were further examined in LLC and A549 cell lines after 1 μg/ml of S100A4 protein treatment for 4 h. Western blot analysis showed that after starved with HBSS, protein levels of Beclin1, Bax and LC3-II were greatly reduced, while the level of p62 was significantly elevated when S100A4 protein was supplied to LLC cells (Fig. 2c) and A549 cells (Fig. 2f). Similar results were obtained using real-time RT-PCR (Fig. 2d, g) and TEM (Fig. 2i). In addition, S100A4 led to significantly increased cell viability after cells were subjected to HBSS (Fig. 2e, h). We also found that the expression of S100A4 was relatively low in LLC cells when compared with A549 cells and tumor sections (Fig. 2j). These results strongly suggest that exogenous S100A4 is an inhibitor of autophagy and promotes cell proliferation in NSCLC cells.
Overexpression of S100A4 inhibited starvation-induced autophagy, whereas siRNA-mediated suppression of S100A4 increased autophagy in A549 cells We then investigated whether autophagy was inhibited by endogenous S100A4. As shown in Fig. 3a, the expression of S100A4 messenger RNA (mRNA) was upregulated when A549 cells were transfected with a plasmid overexpressing S100A4 (pEGFP-S100A4). The knockdown of S100A4 in A549 cells was confirmed by RT-PCR (Fig. 3b). The expression of S100A4 in purified cell fractions was also confirmed by the following western blot. Western blot data showed an enhanced inhibition of Beclin, Bax and LC3-II expression as well as elevated p62 levels in A549 cells overexpressing S100A4 (Fig. 3c), and similar results were obtained from LLC cells (Fig. 3d). Meanwhile, the effects of S100A4-siRNA on autophagy were also assessed. Silencing of S100A4 consistently led to an increased Beclin1, Bax and LC3 conversion and reduced p62 level, which was reversed by S100A4 treatment to lung cancer cells once again (Fig. 3e, f). Overexpression of S100A4 significantly increased cell viability (Fig. 3g, h) while knockdown of S100A4 reduced cellular viability in HBSS-treated cells (Fig. 3i, j). These data demonstrate that endogenous S100A4 promotes cell proliferation and has inhibitory effects towards starvation-induced autophagy.
S100A4 inhibited autophagy and promoted A549 cells proliferation via the Wnt/β-catenin pathway
We further tested the signaling pathways that S100A4 might be involved in during autophagy using the A549 cell line. A549 cells were cultured with HBSS and S100A4 to analyze the expression of pathway-related proteins. After treatment with S100A4 for 4 h, we found that both the overall protein and phosphorylation level of P65 and ERK1/2 were unaffected in A549 cells. However, supplementation of S100A4 led to a marked upregulation in phosphorylated β-catenin (Ser552) levels accompanied with the impaired Bax and LC3-II expression (Fig. 4a). The Wnt/β-catenin pathway of tumor tissues was also analyzed, and similar results were found. As shown in Fig. 4b, S100A4 deficiency resulted in a marked decrease in phosphorylated β-catenin levels in tumors from S100A4 −/− mice and increased Bax and LC3-II levels.
The effects of overexpression of β-catenin on the expression levels of phospho-β-catenin and LC3 conversion were further studied. We confirmed that β-catenin Fig. 4 S100A4 inhibited autophagy to promote tumor cell survival through β-catenin signaling. a A549 cells were treated with HBSS and S100A4 as indicated. The indicated signal pathways were analyzed by western blot in A549 cells. b Western blot analysis of β-catenin, phospho-βcatenin, Bax, LC3-I/II and GAPDH protein levels in tumors from S100A4 −/− and WT mice. A549 (c) and LLC (f) cells were transfected with plasmid overexpressing β-catenin (pcDNA3.1-β-catenin) or control plasmid (pcDNA3.1) and then treated with 1 μg/ml S100A4 in HBSS for 4 h. At 48 h after transfection, cell lysates were subjected to western blot analysis with the indicated antibodies. Cell viability of A549 cells (d) and LLC cells (g) treated with pcDNA3.1-β-catenin was measured using a CCK-8 kit. mRNA levels of target genes (CyclinD1 and c-myc) in β-catenin pathway from A549 (e) and LLC (h) cells was measured by RT-PCR; *p < 0.05, **p < 0.01 was overexpressed and found that both the cytoplasmic and nuclear expression of S100A4 were also upregulated following overexpression of β-catenin in lung cancer cells by western blot (Fig. 4c, f). Upregulation of β-catenin consistently led to a decrease in LC3 conversion as well as Bax level, differing from the control, which failed to show any effects on the steady-state levels of LC3 conversion (Fig. 4c, f). Figure 4d, g demonstrated that overexpressed β-catenin contributed to cellular proliferation in lung cancer cells. The transcriptional activity of β-catenin were further studied by monitoring the mRNA levels of CyclinD1 and c-myc, which indicated that S100A4 promoted the transcription activity of wnt/β-catenin pathway (Fig. 4e, h).
Cardamonin is a chalcone from Aplinia katsumadai Hayata that inhibits intracellular β-catenin levels 32 . To further confirm whether activated β-catenin signaling was necessary for S100A4-inhibited autophagy, A549 and LLC cells were treated with cardamonin, and the effects of S100A4 on autophagy were determined by evaluating LC3-II and Beclin1 abundance. In cells treated with cardamonin, supplementation with S100A4 protein led to abolished β-catenin levels and relatively steady Bax and LC3-II expression (Fig. 5a, d). We also found the S100A4 level was negatively influenced by cardamonin in this process. As shown in Fig. 5b, e, when A549 and LLC cells were treated with cardamonin, S100A4 failed to increase the cell viability. The decreased transcription of β-catenin pathway target genes by cardamonin was also confirmed in the two cell lines (Fig. 5c, f). Induction of autophagy was also detected by quantification of LC3 puncta in cells, where the β-catenin signaling was evaluated as inhibited cells versus neighboring cells where β-catenin signaling was uninhibited. The majority of cells with inhibited βcatenin had significantly more LC3 puncta than those without inhibited β-catenin (Fig. 5g, h).These results suggest that S100A4 inhibited autophagy to promote tumor cell survival via the Wnt/β-catenin pathway.
S100A4-induced β-catenin signaling to inhibit autophagy in A549 cells is RAGE dependent
RAGE is a well-accepted interaction partner for S100A4. We analyzed whether the S100A4-induced βcatenin signaling in A549 cells was RAGE dependent. The RAGE-specific inhibitor FPS-ZM1 was administered to A549 cells. RAGE inhibition in A549 and LLC cells both resulted in the abolishment of the inhibitory effects of S100A4 on autophagy (Fig. 6a, c). As shown in Fig. 6b, d, when starved cells were exposed to FPS-ZM1, the cell viability was not affected by S100A4. Induction of autophagy was also determined by quantification of LC3 puncta in RAGE-inhibited cells and their controls. The majority of cells with RAGE inhibition had significantly more LC3 puncta than those without RAGE inhibition (Fig. 6e, f).These results indicate that S100A4 inhibited autophagy in a RAGE-dependent manner.
S100A4 promoted lung tumor development by inhibiting autophagy
To further investigate the role of S100A4 in lung tumor development, LLC cells were implanted subcutaneously in S100A4 −/− mice and wild-type (WT) control mice. Tumor growth was monitored, and tumor volume was measured every other day. As shown in Fig. 7a, the survival rate of S100A4 −/− mice bearing LLC tumors was significantly increased as compared with WT mice. The tumors in the WT mice grew rapidly, whereas tumor development was severely impaired in the S100A4 −/− mice. On day 14, WT mice had a mean tumor volume of 957.26 mm 3 compared with 602.84 mm 3 for the S100A4 −/ − mice (Fig. 7b). At the end of the experiment, the tumor volumes were 1607.93 mm 3 for the WT group and 984.71 mm 3 for the S100A4 −/− group (Fig. 7b, c). Tumor sections were then stained with immunohistochemistry as shown in Fig. 7d. A large number of S100A4 + cells were identified in the tumors from WT mice and none were detected in the tumors from S100A4 −/− mice. Using double staining, we found that most of the S100A4 + cells were fibroblasts, i.e., 69.31% of the S100A4 + cells were α-SMA + . The S100A4 + cells rarely expressed Gr-1, CD4 or CD8, and only a small number of S100A4 + cells were positive for F4/80 + (Supplementary Figure S1). Moreover, the percentage of Ki67 + (proliferating) cells in tumors was decreased significantly in S100A4 −/− mice compared with WT mice (61.89% vs 27.64%) (Fig. 7d). In addition, the percentage of metastatic colonization in the lungs was also obviously reduced in the S100A4 −/− group compared with the WT group (5.34% vs 17.69%, Fig. 7e). Furthermore, autophagy markers were detected in tumor tissue sections from S100A4 −/− and WT mice. p62, LC3-II and Beclin1 protein expression were analyzed using western blots. As shown in Fig. 7f, autophagy levels increased significantly in tumors from S100A4 −/− mice compared with WT mice (Fig. 7f). S100A4 deficiency resulted in elevated mRNA expression of Beclin1, LC3 and decreased mRNA expression of p62, a gene which was negatively correlated with autophagy level (Fig. 7g). TEM further confirmed that the number of autophagosomes and autolysosomes obviously increased in tumors from S100A4 −/− mice (Fig. 7h). These results indicate that S100A4 promoted lung tumor development by inhibiting autophagy.
Discussion
In this study, we found that upregulation of S100A4 expression in lung adenocarcinoma tissues is closely related to advanced tumor grades. Furthermore, in vitro, S100A4 activated β-catenin signaling in lung cancer cells and increased tumor cell viability via inhibiting autophagy in a RAGE-dependent manner. S100A4 deficiency promoted autophagy and hindered lung tumor growth in vivo. Our results demonstrated that S100A4 promoted lung tumor development by upregulating the β-catenin pathway and inhibiting autophagy. S100A4 plays important roles in cancer progression and metastasis in different ways 17 . It promotes tumor growth and progression mainly by stimulating cell survival and proliferation, increasing motility and invasion of tumor cells, modifying adhesion of tumor cells, maintaining chronic inflammation and promoting angiogenesis 9,12,13,31 . 5 β-catenin inhibitor in A549 cells abolished the effects of S100A4 on autophagy. A549 and LLC cells were cultured with the β-catenin inhibitor cardamonin (20 μmol/ml) for 1 h, then cells were stimulated with or without S100A4 (1 μg/ml) in HBSS for 4 h. The treated A549 cells (a) and LLC cells (d) were collected and analyzed for β-catenin, phospho-β-catenin, Bax and LC3-I/II protein expression. Cell viability of A549 (b) and LLC (e) cells treated with cardamonin was detected by a CCK-8 kit. mRNA levels of target genes (CyclinD1 and c-myc) in β-catenin pathway from A549 (c) and LLC (f) cells was measured by RT-PCR. g A549 cells were transfected with GFP-LC3-I/II plasmid (1 ng/μl) for 6 h. h Representative images of GFP-LC3 staining and quantification of LC3 puncta numbers per cell in cells are shown; *p < 0.05, **p < 0.01 S100A4 also participates in cell apoptosis by interacting with tumor suppressors 33 . Downregulation of S100A4 enhances the sensitivity of human lung cancer cells to radiotherapy 34 . S100A4 can be one of the prognostic biomarkers in lung cancer 35 . It has been reported that elevated S100A4 expression was highly correlated with poor prognosis in lung adenocarcinoma, lung squamous cell carcinoma and other subtypes of non-small celllung carcinomas [35][36][37][38] . However, whether S100A4 can promote tumor development by regulating autophagy has not been determined. Our results demonstrated that S100A4 expression in tumor tissues from lung adenocarcinoma patients is closely linked to tumor progression patientderived tissues and this study is the first report in which S100A4 is shown to promote lung tumor growth by downregulating autophagy.
Autophagy, a cellular waste disposal process, has wellestablished tumor-suppressive properties 39 . Under metabolic stress, autophagy is a survival strategy in cancer cells. Although autophagy helps cancer cells address the increased demand for nutrients caused by cell growth and proliferation, there is more evidence that tumor suppressor genes are associated with the activation of autophagy, and the enhancement of autophagy often leads to cell death 40 . Autophagy inhibits breast cancer stem-like cells by suppressing the Wnt/β-catenin signaling pathway 41 . A defect in autophagy was sufficient to promote tumorigenesis by altering NF-κB regulation 42 . Furthermore, autophagy gene-deficient mice show high incidences of primary lung cancer 43 , which is consistent with our results that show that impaired autophagy induced rapid cell proliferation in implanted tumors. Excessive autophagy is closely linked to the low survival rate of lung cancer in lung cancer patients after surgery 44 . Here, we found that both extracellular treatment and intracellular overexpression of S100A4 suppressed autophagy and increased cell viability in lung cancer cells. Moreover, silencing S100A4 in lung cancer cells significantly increased autophagy and inhibited tumor cell proliferation. These results suggest that intracellular expressed S100A4 and extracellular S100A4 secreted by stromal cells can accelerate cell proliferation through suppressing autophagy. Our results demonstrate that S100A4 promoted tumor progression by affecting autophagy, which might be used as a therapeutic target in the development of future lung cancer therapies.
The Wnt/β-catenin pathway maintains normal tissue homeostasis and is aberrantly activated in the majority of tumors 45,46 . β-Catenin is an essential transcriptional coregulator in the canonical Wnt pathway, forming complexes with the T cell-specific transcription factor/lymphoid enhancer-binding factor 1 (TCF/LEF) family of transcription factors to control target gene expression 47 .
Mutations of Wnt pathway components prevent the destruction of β-catenin, leading to increased TCFregulated transcription of Wnt/β-catenin target genes that promote cell proliferation and facilitate tumorigenesis [48][49][50] . In this study, we found that S100A4 upregulated β-catenin expression and inhibited autophagy. The β-catenin signaling pathway regulates cell apoptosis and autophagy 51,52 . Inhibition of β-catenin significantly increased LC3-II expression and induced autophagic cell death 53 . Conversely, the upregulation of β-catenin signaling reduces Beclin1 expression and rescues endothelial cells from endostatin-induced autophagy 54 . Consistent with these reports, we showed that activated β-catenin signaling resulted in impaired autophagy which was induced by starvation and promoted proliferation in lung cancer cells, and cell autophagy was reversed by the overexpression of β-catenin, indicating the critical role of β-catenin in S100A4-induced autophagy inhibition. Furthermore, inhibition of RAGE, one of the S100A4 receptors, abolished the effect of S100A4 on autophagy and cell proliferation. However, the detailed mechanisms by which S100A4 interacts with autophagy in tumor development still need further investigation.
In conclusion, our results demonstrate that both extracellular and intracellular S100A4 promoted lung tumor development by activating the β-catenin pathway and inhibiting RAGE-dependent autophagy (Fig. 7i). The results provide direct evidence of cross talk between S100A4, autophagy and tumor growth and reveal a novel mechanism for lung tumor development. The S100A4related microenvironment may provide a promising clinical strategy for lung cancer prevention and treatment.
(see figure on previous page) Fig. 7 S100A4 promoted lung tumor development by inhibiting autophagy. Groups of C57BL/6 mice and S100A4 −/− mice (n = 9 per group) were subcutaneously injected with 1 × 10 6 LLC cells. a The survival analysis of lung tumor-bearing mice. b Tumor volumes were monitored every other day. Representative data from three independent experiments are shown. c On day 20 after tumor inoculation, mice were killed and tumors from the two groups are shown. d The tumor sections were stained for S100A4 and Ki67. Scale bar, 50 μm. HPF high power field. e Representative H&E-stained lung sections of WT and S100A4 −/− mice. The percentage of the metastatic area was quantified using Image-Pro Plus (IPP). f The levels of the autophagy-related proteins p62, Beclin1 and LC3-I/II in tumors from WT and S100A4 −/− mice were detected by western blot. g mRNA expression of Beclin1, LC3-I/II, p62 and ATG5 in tumors from S100A4 −/− and WT mice was detected by real-time PCR. h Representative electron microscopy of dissected tumors from S100A4 −/− and WT mice. Scale bar, 0.25 μm. i Schematic of the S100A4 acting model in lung cancer. S100A4 is a negative regulator of autophagy via activation of the β-catenin signaling pathway and this process is RAGE dependent. The autophagy inhibitor S100A4 contributed to lung tumor cell growth; *p < 0.05, **p < 0.01 | v3-fos-license |
2021-05-21T16:56:42.018Z | 2021-04-18T00:00:00.000 | 234835871 | {
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} | pes2o/s2orc | A rapid analysis of Bisphenol A using MISPE coupled with HPLC-FLD in tissues of food-producing animals
Bisphenol A (BPA) is a highly-produced organic compound of anthropogenic origin with a wide-range use and is ubiquitously present in both living organisms and the environment. A previously published analytical method for testing of the free (aglycone) BPA in foodstuffs was simplified and optimized for sheep muscle tissue, kidney and liver, by using only a single MISPE purification step allied with HPLC-FLD analysis. The recovery rates and RSD values over a concentration range of 1–10 µg/kg were in the range of 67‒86% and 3‒34%, respectively, while linearity in the matrix, represented by the r2, was ≥0.999. LOD values were 0.5‒1 µg/kg and LOQ values were 1 µg/kg. The analytical method used is a contribution to the field of veterinary toxicology and food testing and also proved to be applicable for other food-producing animal species, e.g. pigs, poultry and freshwater fish. The MISPE sorbent material for testing of BPA was reusable, with up to five re-use cycles without significant loss in performance characteristics.• The paper reports on a rapid determination of free (aglycone) BPA in tissues of food-producing animals by HPLC-FLD and presents a substantial improvement of analytical performance with regard to time- and cost-savings, as well as environmental protection.• MISPE is an advanced analytical technology, and the results proved that in a single and simplified SPE step it had good performance characteristics such as selectivity, recovery and precision, allied with low LOD and LOQ values.• It was proved that MISPE BPA cartridges could be used at least five times without significant loss of methodological recovery and precision.
• The paper reports on a rapid determination of free (aglycone) BPA in tissues of food-producing animals by HPLC-FLD and presents a substantial improvement of analytical performance with regard to time-and costsavings, as well as environmental protection. • MISPE is an advanced analytical technology, and the results proved that in a single and simplified SPE step it had good performance characteristics such as selectivity, recovery and precision, allied with low LOD and LOQ values. • It was proved that MISPE BPA cartridges could be used at least five times without significant loss of methodological recovery and precision. Specifications
Method details
Bisphenol A (BPA; CAS registry no. 80-05-7, synonyms 2,2-bis(4-hydroxyphenyl)propane, 4,4isopropylidenediphenol) is an organic synthetic compound, currently produced in enormous quantities throughout the world. It is primarily used as a precursor in the production of polycarbonate plastics and epoxy resins. The global market for BPA is projected to reach 7.1 million tons by the year 2027 [1] . Exposure to environmental nanomolar concentrations of BPA is ubiquitous and continuous. BPA has been shown to exert endocrine-disrupting effects and interferences with the developmental processes of humans and animal species [2] . Recent investigations refer to toxic, teratogenic, carcinogenic and particularly estrogenic mechanisms of free BPA action [3] . Low doses of BPA that are environmentally relevant might play a role in the development of chronic metabolic diseases, reproductive pathologies, hormone dependent cancers (prostate, breast, testes). High levels of BPA have recently been correlated with obesity, diabetes, cardiovascular diseases, polycystic ovarian disease and low sperm count [3 , 4] . In 2015, the European Food Safety Authority (EFSA) reduced the tolerable daily intake (TDI) for BPA from 50 to 4 μg/kg bw/day [5] . The European Chemicals Agency (ECHA) has added BPA to the Candidate List of New Substances of Very High Concern, causing adverse effects to the environment [6] .
Many analytical methods for the detection of BPA in environmental and biological materials have been developed, including chromatography and mass spectrometry-based methods, and some novel methods such as sensors. Nevertheless, sample pretreatment is a key step for the detection of BPA, but most sample preparations are time-consuming and labour-intensive [7] . Since 20 0 0, sample pretreatment methods have undergone considerable development, also due to the use of nanostructured liquids, named supramolecular solvents that combine extraction and clean-up in a single stage [8 , 9] , and due to selective sorbents, such as molecularly imprinted polymers (MIPs), which are synthetic tailor-made separation polymers having a predetermined selectivity for a given analyte, or group of structurally related compounds. MIPs are obtained by polymerizing functional and crosslinking monomers around a template molecule, leading to a highly cross-linked three-dimensional network polymer [10 , 11] . The resulting imprinted polymers are highly stable, robust and resistant against physicochemical perturbations such as pH value, solvents and temperature. Therefore, the technique of molecular imprinting provides a promising and advantageous alternative to overcome the problems associated with biomolecules such as antibodies, enzymes, and other receptor molecules [12] . MIPs have the potential to enable analyte isolation and clean-up in a single-step and provide cleaner extracts compared to, for example, "classical" SPE [13] .
Nowadays, the use of MIPs in solid phase extraction, so-called molecularly imprinted solidphase extraction (MISPE), is by far the most advanced technical application of MIPs. A review of the analytical methods for detection of BPA by Sun et al. showed that the majority of the matrices investigated were water, sediments and biofluids (urine, milk) [7] . Regarding tissue analysis, two analytical methods, by Wei et al. using a high-performance liquid chromatography (HPLC) coupled to fluorescence detection (HPLC-FLD) [14] , and by Di Marco Pisciottano et al. using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) [15] , were reported for MISPE determination of BPA in fish samples. To the best of our knowledge, only the method of Deceuninck et al. comprised using MISPE for determination of free (aglycone) BPA in meat and offal using gas chromatography coupled with tandem mass spectrometry (GC-MS/MS) [16] . This method was used for assessment of dietary exposure to BPA in the French population, by Bemrah et al., where the levels of BPA found in meat, poultry and game, and offal were in the range of 0.1-224, 0.4-49 and 0.6-395 μg/kg, respectively [17] .
The objectives of the present study were to systematically optimize the critical factors affecting the SPE step within the free BPA tissue determination in the edible tissues of sheep (i.e. muscle, kidney and liver). Within our recent research on BPA we were faced with relatively weak recovery rates for the analysis of marine mussel tissue [18] and sheep faeces [19] with a mean value of 47 and 52%, respectively. The clean-up of both analytical methods used was based on the method of Deceuninck et al. [16] , by omitting the derivatization step and using HPLC-FLD instead of GC-MS/MS. Moreover, on the basis of the reported reusability of the MIP by Gallego-Gallegos et al. [11] and a recent publication on the successful multiple use of MIP cartridges for determination of ochratoxin A in pig muscle, kidney and liver by Luci [20] , we also evaluated the possible multiple use of MISPE BPA cartridges for testing the sheep matrices investigated in the current work.
Reference standards
The certified reference standard of BPA was obtained as a powder of 99.0% analytical purity from Sigma-Aldrich (Merck, Darmstadt, Germany). The stock standard solution of 200 μg/ml was prepared in acetonitrile (MeCN) and kept frozen (at −20 °C), while the intermediate and working standard solutions ranging from 100 to 2,0 0 0 ng/ml were further prepared in a mixture of MeCN/H 2 O (35/65, v/v) and kept refrigerated (at 4-8 °C). Calibration standards of ≤50 ng/ml were prepared on a daily basis.
Reagents and consumables
The MeCN and methanol (MeOH) used, which were HPLC gradient-grade purity, were purchased from J. T. Baker (Center Valley, PA, USA). The high purity deionized water used with a resistivity of 18.2 M .cm was obtained by a PureLab Option and PureLab Classic water purification system (Elga, Woodridge, Illinois, USA). The formic acid (HCOOH), 98-100% of reagent grade purity, was supplied by Merck (Darmstadt. Germany). The solid-phase extraction (SPE) columns used were Chromabond HR-X, 6 mL, PP, with an 85 μm particle size and 200 mg of sorbent, which were supplied by Macherey-Nagel (Düren, Germany), and MIP AFFINIMIP® SPE Bisphenols, 6 ml, with 100 mg of sorbent, which were supplied by AFFINISEP (Petit Couronne, France). The centrifuge tubes (15 ml, conical, screw cap, PP) were supplied by Isolab (Wertheim, Germany), and the centrifuge tubes (15 ml, conical, glass) were supplied by Brand (Wertheim, Germany). The dark glass amber, 1.5 ml vials were obtained from La −Pha −Pack (Langerweche, Germany).
Sample extraction
An aliquot of 2.0 ± 0.005 g of a homogenized (defrosted) sample was weighed into a 15 ml plastic (PP) centrifuge tube and extracted with 8 ml of MeCN by vortexing for 2 min and ultrasonication for 13 min. After repeated vibromixing for 1 min, the sample was centrifuged at room temperature for 10 min at 2640 × g and re-extracted using 2 ml of MeCN. The combined supernatants were then evaporated under a N 2 stream at 40 °C to an aqueous residue. When performing the original method, a sample residue was dissolved using 0.3 ml of MeOH and 4.7 ml of H 2 O, while when performing a present method, it was dissolved using 1 ml of MeCN and 10 ml of H 2 O, before applying onto the SPE cartridge.
Solid-phase extraction (SPE) by the original method [16]
The workflow is presented in Table 1 . The sample extract was applied under gravity onto the SPE Chromabond HR-X cartridge, which had previously been conditioned with 10 [16] , using 6 ml of MeOH) into a 15 ml glass centrifuge tube, and the SPE eluate was evaporated under a N 2 stream at 40 °C just to dryness. Final sample extracts were re-dissolved in 0.8 ml of MeCN/H 2 O (35/65, v/v), ultrasonicated for 5 min, vibromixed, centrifuged at room temperature for 10 min at 2640 × g, and transferred into a HPLC vial.
Solid-phase extraction (SPE) by the present method
The sample extract was applied under gravity onto the MIP BPA cartridge ( Table 1 )
HPLC-FLD analysis
A 50 μl aliquot of the final sample extract was taken for the HPLC analysis. A Hypersil GOLD C18 (150 × 4.6 mm, 3 μm particle size) analytical column was used which was protected by a Hypersil GOLD 3 μ drop in guard cartridges (Thermo Scientific, Waltham, MA, USA). The chromatographic process was performed at room temperature (25-26 °C), while the mobile phase as a mixture of H 2 O (constituent A) and MeCN (constituent B) was pumped at a flow rate of 1.0 ml/min and the excitation and emission wavelengths of the fluorescence spectrometric detection were set at 230 and 315 nm, respectively [21] . For muscle tissue and kidney analysis, the mobile phase constituents were gradient pumped in the following volume ratios: time 0-2 min (35% B), time 2-12 min (gradient 35-50% B), time 12-20 min (50% B), time 20-20.5 min (gradient 50-35% B), time 20.5-21 min (35% B) [21] . For liver analysis, the last gradient ramp (50-35% B) was prolonged from 20-25 min, and thus the whole chromatographic run time for this matrix was 25.5 min. BPA sample concentrations were calculated according to the external standard calibration.
Method validation
The analytical methodology was validated to demonstrate its fitness for determination of the BPA in the edible tissues of sheep. Selectivity was evaluated by an appropriate number of representative baseline samples of sheep ( n = 11-16 per matrix) being analysed and checked for any peak interferences in the retention time region of BPA. Linearity was determined on both standard and matrix levels by the least squares method, giving the regression and correlation parameters of the calibration lines. Solvent standard concentrations ranged from 0.5-250 ng/ml with 4-8 concentration points per calibration line. Linearity in the matrix, recovery, and precision of BPA determination in the sheep muscle tissue, kidney and liver were evaluated by fortification of a baseline sample, being provided by the Infrastructure Centre for Sustainable Recultivation Vremščica of the Veterinary Faculty of the University of Ljubljana and also obtained from the city market of Ljubljana. Linearity on a matrix level was evaluated as an intra-day correlation between the mean measured and added BPA concentrations ( n = 2/fortification level over a concentration range of 0.5-100 μg/kg and 1-100 μg/kg for muscle tissue and kidney, and liver, respectively). Recovery, repeatability and intra-laboratory reproducibility were tested on three BPA concentration levels of 1, 5 and 10 μg BPA/kg. Repeatability was evaluated at the same time, while intra-laboratory reproducibility was evaluated at the two separate times. The precision of the methods was evaluated using the standard deviation (SD) and the relative standard deviation (RSD) of the determined values, and assessed in accordance with the Horwitz coefficients (RSD H ) according to the European Commission Decision 2002/657/EC [22] . Matrix effect was estimated by fortification of a baseline matrix final extract ( n = 2/concentration level) with the BPA at concentration, representing 20, 10 and 5 μg/kg in the tissue sample, and comparison of a chromatographic response with those of equivalent solvent standard. The limit of detection (LOD) value was estimated as the minimum detectable amount of BPA from matrix samples with a signal-to-noise ratio of 3:1, and was corrected for the baseline matrix response, while the limit of quantification (LOQ) value was determined as the lowest analyte content for which the analytical method proved acceptable in terms of recovery and repeatability [23] . The method's suitability for use regardless of the animal species was tested as follows: five negative samples of sheep, chicken, pig and aquaculture muscle tissue (freshwater fish) were simultaneously analysed, each at a fortification level of 10 μg BPA/kg. Additionally, five negative samples of sheep, chicken and pig liver, and sheep and pig kidney, were simultaneously analysed, each at a fortification level of 10 μg BPA/kg. The recovery and repeatability of the measurements were evaluated.
Reusability of MISPE BPA cartridges
MISPE BPA cartridges were used five times for simultaneous testing of two to five samples of sheep muscle tissue, kidney and liver, fortified at 10 μg BPA/kg. The efficiency of the MIP stationary phase with multiple use was evaluated based on the recovery achieved after individual use according to the matrix investigated.
Dealing with samples
Homogenized tissue samples were stored in PP containers at −20 °C until analysis. The importance of appropriate quality control of sample testing was considered, as blank controls were included with each batch of the fortified samples analysed to avoid and minimize possible artifacts or contamination and ensure appropriate performance characteristics of the BPA analytical method [22] . In addition, storage devices with declared absence of BPA were used, high quality glassware was used where possible, and the solvents used were mainly of HPLC grade and screened via reagent blanks.
Method development
In the present study the critical factors affecting the SPE step (nature of SPE stationary phase, nature of SPE washing solutions) within the free (aglycone) BPA tissue determination were optimized using fortified sheep tissue samples. Sheep tissues were chosen to establish the analytical conditions for testing materials obtained from a toxicokinetic and reproductive toxicity BPA study in rams (unpublished data). Moreover, on the basis of the recent discovery about the successful reusability of MISPE cartridges for determination of ochratoxin A in pig muscle, kidney and liver [20] , we also checked the possible multiple use of MISPE BPA cartridges for testing the matrices investigated.
The use of SPE for sheep muscle tissue, kidney and liver, according to the original method of Deceuninck et al. [16] , resulted in mean recoveries of BPA (fortification level 10-20 μg/kg, n = 4) of 47, 24 and 28%, respectively. Simultaneous verification of the MISPE washing with vs. without usage of 4 ml MeCN, demonstrated enhanced recovery without usage of MeCN in sheep muscle tissue from 52.4 to 95.4% and lowered relative standard deviation (RSD) from 49.1 to 1.3%, respectively (fortification level of 10 μg BPA/kg, n = 3) ( Fig. 1 ). Consequently, from this step onward, washing of MISPE BPA with MeCN was excluded from the analytical procedure to omit large and variable losses of bound BPA from the cartridge sorbent. This finding also demonstrated different MISPE BPA behaviours for clean-up of different matrix structures, e.g. for blood plasma [24] and urine [19] , where recovery rates were significantly higher ( > 76 and > 60%, respectively) after washing the cartridge with MeCN, compared to, for example, the tissue of mussels (recovery of 47%) [18] . The MISPE clean-up simplification was also systematically described for ochratoxin A determination in pig muscle tissue, kidney and liver [20] .
The second step aimed to simplify the clean-up procedure as much as possible with a hypothetical withdrawal of an SPE with HR-X stationary phase and using only the MISPE stationary phase Table 2 Optimization of SPE for BPA determination in edible tissues of sheep with regard to the cartridges' use; 3 replicates of fortified muscle tissue, kidney and liver (10 μg BPA/kg) per SPE mode were tested.
Matrix
Rec mean (%) SD (%) RSD (%) Table 3 Recovery and precision of determination of BPA in sheep muscle tissue, kidney and liver. [15] . A higher matrix background was revealed when using only a single SPE. Nevertheless, the selectivity of the chromatographic separation and FLD was sufficient, as demonstrated by Fig. 2 . Liquid chromatography-fluorescence (LC-FLD) is due to its high specificity and selectivity considered a suitable confirmatory method for organic residues or contaminants (non-prohibited) that exhibit native fluorescence (such as BPA) and to molecules that exhibit fluorescence after either transformation or derivatisation [22] . Using only the MISPE BPA step instead of a combination of both SPE HR-X and MISPE BPA significantly improved recovery for sheep muscle tissue, kidney and liver, from 71 to 89%, from 62 to 86%, and from 60 to 79%, respectively. The RSD value of determination conversely decreased markedly for muscle tissue and liver, from 10 to 1% and from 14 to 2.5%, respectively, while for kidney a slight increase was observed, from 5 to 7% ( Table 2 ).
Method validation
Very relevant analytical performance characteristics were obtained. Chromatograms obtained for baseline sheep's samples did not show any peak near the retention time of BPA as the response was < LOD for the matrices investigated. The linearity of the standard calibration curves expressed by the correlation coefficient was ≥0.9997 for the concentration range of 1-50 ng/ml, including 6 calibration points ( n = 9) and ≥0.9999 for the concentration range 1-250 ng/ml, including 8 calibration points ( n = 3). The linearity of determining the level of BPA in the matrices investigated is presented in Fig. 3 , based on the correlation curves between the found and added values of analyte. The "r-squared" values were 0.9988, 0.9996 and 0.9986 for the sheep muscle tissue, kidney and liver, respectively.
Corresponding to concentration range from 20 to 5 μg BPA/kg there was a weak matrix effect from -0.9 to -1.9% for muscle tissue, while for kidney a decrease of a chromatographic peak area from -3.6 to -19% was observed in comparison with the solvent standards. A weak matrix effect from -0.9 to -1.9% was also observed for liver at a concentration range from 20 to 10 μg BPA/kg, while at concentration of 5 μg BPA/kg of liver an increase of a chromatographic peak area for 11% was observed in comparison with the solvent standards. The recovery and precision of the method are presented in Table 3 and were determined on three levels of content for each matrix. The recovery Fig. 3. Linearity of analytical HPLC determining the level of free bisphenol A (BPA) in sheep matrices, evaluated as an intraday correlation between the mean measured and added BPA concentrations ( n = 2/fortification level); difference bars of both parallels are also presented: (A) muscle tissue, fortification range from 0.5-100 μg/kg (B) kidney, fortification range from 0.5-100 μg/kg (C) liver, fortification range from 1-100 μg/kg. values for determination of BPA in muscle tissue, kidney and liver ranged from 78 to 86%, from 67 to 85%, and from 69 to 81%, respectively. The repeatability of the measurements, represented by the daily RSD values, ranged from 6.6 to 9.3%, from 2.1 to 10.8% and from 2.1 to 8.2%, respectively. The withinlaboratory reproducibility, represented by the intr-day RSD values, ranged from 8.0 to 20.6%, from 3.0 to 34.2% and from 3.1 to 11.6%, respectively. The RSD values did not exceed the RSD H values from the Horwitz equation [22] . Moreover, with the exception for RSD value at fortification level of 1 μg/kg in kidney at the within-laboratory reproducibility conditions, of which the explanation lies in both, a very low concentration level and also a matrix effect for the kidney tissue, they were below one fourth of the corresponding RSD H values, and thus fully acceptable ( Table 3 ). The estimated LODs were 0.5 μg/kg for sheep muscle tissue and kidney and 1 μg/kg for liver, while the LOQ values determined were 1 μg/kg for all three matrices investigated in this study. This is comparable to typical reported detection limits for BPA in foods using FLD, being in the range of 0.1-2 ng/ml and 1-5 μg/kg [13] .
The method presented in this study proved to be applicable for use in various animal species, such as for the muscle tissue of sheep, chicken, pigs and freshwater fish ( Fig. 4 ), for the liver of sheep, chicken and pigs ( Fig. 5 ), and for the kidney of sheep and pigs ( Fig. 6 ). This proved its general 7. Reusability of MISPE cartridges for BPA determination in muscle tissue, kidney and liver, represented by recovery ± SD and RSD at a fortification level of 10 μg/kg ( n = 2-5 replicates/occasion) over 5 consecutive use cycles per animal matrix. applicability in edible tissues of various food-producing animals, namely ruminants, poultry, pigs and aquaculture.
Reusability of MISPE BPA cartridges
Five consecutive uses of MISPE BPA cartridges did not significantly impair the recovery rate or repeatability of BPA determination in all three matrices investigated in this study ( Fig. 7 ). This finding is in line with the reported possible multiple use of MISPE for ochratoxin A determination in muscle tissue, kidney and liver [20] , and significantly alleviates the impact of the analytical process using MIP technology from both environmental and economic aspects. However, at the fifth cycle the cartridges were blocked by the liver extracts, so a vacuum was needed at the SPE step to enable a sufficient flow rate. It is presumed, although has not been checked, that MISPE BPA cartridges could be used even more than five times for muscle tissue clean-up. | v3-fos-license |
2018-12-07T21:43:03.370Z | 2015-01-01T00:00:00.000 | 54744402 | {
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} | pes2o/s2orc | A Multicomponent Synthesis of 2-Amino-3-cyanopyridine Derivatives Catalyzed by Heterogeneous and Recyclable Copper Nanoparticles on Charcoal
An efficient and convenient method was developed for synthesis of 2-amino-3-cyanopyridine derivatives via the four-component coupling reaction between ketone, aldehyde, malononitrile, and ammonium acetate in the presence of 2 mol% copper nanoparticles on charcoal (Cu/C) catalyst. A variety of ketones and aldehydes was used to afford the corresponding products in good to excellent yields. The method is applicable to large-scale operation without any problem. The catalyst could be quantitatively recovered from the reaction mixture by simple filtration and reused at least eight times with almost consistent activity.
Introduction
2][3] Polysubstituted pyridines possess important biological and pharmacological activities and could be used as potential agrochemicals, for example as herbicides. 4In addition, the molecules containing pyridine moiety are used as non linear optical materials, 5 electrical materials, 6 and chelating agents in metal ligand chemistry. 7Among them, 2-amino-3-cyanopyridines are known as IKK-β-inhibitors. 8hey have been identified to possess multiple biological activities such as antimicrobial, 9 antiviral, 10 antibacterial, 11 antifungal, 12 antitumor, 13 anti-inflammatory, 14 as well as antihypertensive 15 properties.Besides, they are important and useful intermediates in preparing a variety of heterocyclic compounds. 12,16he interesting biological properties of these compounds have promoted a great deal of research effort toward development of new synthetic methodologies for the preparation of 2-amino-3-cyanopyridines and their derivatives.Therefore, the synthesis of 2-amino-3cyanopyridine derivatives continues to attract much interest in organic chemistry.
A survey of the literature shows that the major synthetic approaches that are used to prepare various types of 2-amino-3-cyanopyridine derivatives involve utilization of corresponding 2-chloro derivatives as substrates, 17 chalcones on treatment with ammonium acetate via the condensation reaction, 18 as well as via a one pot four components reaction 19,20 in conventional heating or under microwave irradiation, and also by some other methods. 21,22n recent efforts to develop more facile methods for the synthesis of 2-amino-3-cyanopyridines, metal-catalyzed reactions have been developed. 19n recent years, the use of heterogeneous catalysts has received considerable interest in organic synthesis.Using heterogeneous catalysts in synthetic organic routes has some advantages over their counterparts, such as great durability toward the reaction conditions, having highly active sites, recyclability and reusability of catalyst. 23][26] Based on the above mentioned reports and in continuation of our efforts to develop facile and general methods for the preparation of 2-amino-3-cyanopyridine derivatives, and as a part of our studies to utilize heterogeneous catalyst for the synthesis of organic compounds, [27][28][29][30][31] here we wish to report a heterogeneous catalyst system based on Cu/C and illustrate its application for the synthesis of 2-amino-3-cyanopyridine derivatives without any cocatalyst or activator under mild conditions.recorded on a Bruker Avance DPX-250 spectrometer ( 1 H NMR at 250 MHz and 13 C NMR at 62.5 MHz) in pure deuterated solvents with tetramethylsilane (TMS) as an internal standard.Infrared (IR) spectra were obtained using a Shimadzu FTIR 8300 spectrophotometer.Mass spectra were determined on a Shimadzu GCMS-QP 1000 EX instrument at 70 or 20 eV.Elemental analyses were performed with a Thermo Finnigan CHNS-O 1112 series analyzer.Melting points were determined in open capillary tubes in a Büchi 535 circulating oil melting point apparatus.The purity determination of the substrates and reaction monitoring were accomplished by thin-layer chromatography (TLC) on silica gel PolyGram SILG/ UV 254 plates.Column chromatography was carried out on short columns of silica gel 60 (70-230 mesh) in glass columns (2-3 cm diameter) using 15-30 g of silica gel per g crude mixture.Chemical materials were purchased from Fluka, Aldrich and Merck.The used activated carbon was also purchased from Merck (Art.No. 9631, 0.3-0.05mm).
Synthesis of copper nanoparticles on charcoal (Cu/C)
The activated carbon (1.0 g) was refluxed in nitric acid solution (5.0 mol L -1 , 30 mL) for 6 h, washed with deionized water until pH 6-7 and finally dried in an oven at 110 °C for 12 h under vacuum.For the synthesis of Cu/C, CuI (100 mg) was dissolved in absolute ethanol (30 mL), and stirred at reflux temperature for 4 h under nitrogen atmosphere in the presence of 1.0 g of purified activated carbon.The resulting solid was washed with ethanol (4 × 30 mL).Finally, the copper in charcoal was dried under vacuum in an oven overnight at 110 °C. 24neral procedure General procedure for the synthesis of 1-17 A 25 mL flask was filled with the mixture of substituted aldehyde (1.0 mmol), ketone (1.0 mmol), malononitrile (1.5 mmol), ammonium acetate (2.0 mmol), Cu/C nanocatalyst (2.0 mol%) and then stirred in acetonitrile (2.0 mL) at 80 °C under ambient atmosphere.Progress of the reactions was monitored with TLC using n-hexane/ ethyl acetate (10:1).After completion, the crude reaction mixture was filtered through a pad of Celite and washed with hot ethanol (3 × 10 mL).The recovered catalyst was dried under vacuum at 40 °C for 5 h and stored for another consecutive reaction run, and the combined filtrates were concentrated in vacuo and the resulting residue was purified by silica gel column chromatography employing n-hexane/ ethyl acetate (10:1) as eluent.
2-Amino-4,6-diphenylnicotinonitrile (1)
White solid; m.p. 176-177 °C; IR (KBr) ν max / cm -1 3465 (w), 3379 (w), 3306 (w), 3181 (w), 2207 (s), 1641 (s), 1585 (s), 1574 (s), 1550 (m), 1497 (w), 1452 (w), 1370 (w), 1259 (w), 759 (s), 699 (s); 1 We commenced our investigation with the reaction of starting materials in chloroform as solvent in the presence of Cu/C as a catalyst.Analysis of the resulting mixture revealed that the desired product 1 was formed after 6 h in only 10% yield (Table 1, entry 1).Yield of the reaction increased upon using N,N-dimethylformamide (DMF) instead of chloroform (entry 2 vs. 1).Replacement of DMF with ethanol (entry 3) did not give an improved outcome, while the formation of product 1 was markedly improved when using the same catalyst in acetone at reflux condition (entry 4).Further condition screening suggested that when the reaction was carried out in the presence of Cu/C, upon switching the solvents from acetone to ethyl acetate, tetrahydrofuran (THF) or polyethylene glycol 300 g mol -1 (PEG 300), the yield of desired product decreased (entries 5-7).However, when the reaction was carried out in toluene at reflux condition for 6 h, the expected product was achieved in 76% yield (entry 8).Interestingly, when model compounds were treated with 2 mol% of Cu/C in acetonitrile at reflux for 6 h, the expected reaction proceeded successfully to give product in 91% yield (entry 9).A further decrease in the catalyst loading resulted in 74% isolated yield (entry 10), but increase in the catalyst loading did not enhance the reaction yield any further (entry 11).Further optimization of the reaction conditions revealed that other Cu sources regardless of their oxidation states (either I or II) did not show better catalytic activity (entries 12 and 13).We speculate that the observed higher yields are probably due to the large surface area and more stability of the formed nanoparticles of Cu/C.In addition, CuI showed lower yield in comparison with Cu/C under these reaction conditions.It is possibly due to the disproportionation of this salt under optimization reaction condition.When this reaction was carried out in acetonitrile at reflux condition for 6 h, without any catalyst, no desired 2-amino-3-cyanopyridine was obtained (Table 1, entry 14).Next, experiment was carried out to distinguish the effect of temperature on the reaction.As depicted in Table 1, the desired product could not be detected at room temperature (entry 15).In addition, control experiments demonstrated that no reaction occurred in the presence of charcoal as a catalyst (entry 16).
After the optimization reaction conditions, in order to examine the scope of the reaction, we treated various aldehydes and methylketones in the presence of malonitrile and NH 4 OAc with Cu/C in refluxing acetonitrile, and desired 2-amino-3-cyanopyridine derivatives were obtained in good to high yields (Table 2).
From the results shown, aldehydes with both electronwithdrawing and -donating substituting groups afforded the 2-amino-3-cyanopyridine derivatives through reaction Vol.27, No. 4, 2016 with acetophenone, malononitrile and ammonium acetate and the isolated yields range from 86 to 94% (Table 2, entries 2-5).In an effort to apply the present reaction conditions to the synthesis of heterocycles related to 2-amino-3-cyanopyridines, the feasibility of the reaction with heteroaromatic aldehyde was examined (entry 6).It is noteworthy that the reaction of 3-thiophenecarbaldehyde as a heterocyclic aldehyde proceeded well to give the desired products in 86% yield.To expand the scope of the current method, aliphatic aldehyde was also examined as a substrate.The desired product 7 was obtained with good yields (entry 7).We further explored the potential of the present protocol from the standpoint of variety of ketones.As seen from Table 2, this strategy is effective for a great diversity of ketones such as alkylaryl (entries 8-11), cyclic (entries 12 and 13), and dialkyl ketones (entries 14-17).Acetophenone with both electron-withdrawing and -donating substituting groups fairly tolerate the reaction conditions and also afford the desired products in good yields (entries 8-10), and proves the generality of the strategy elaborated.The highly conjugated ketone derivative was also an excellent substrate for this reaction, and the corresponding compound 11 was produced (entry 11).For cyclic ketones, this transformation proceeded smoothly and afforded the products in good yields.For example, the reaction of cyclohexanone or 4-methyl cyclohexanone led to the corresponding products in 84 and 86% isolated yields, respectively (entries 12 and 13).To our delight, this method could be successfully applied to dialkyl ketones.For example, the product having t-butyl, isopropyl or methyl groups derived from the corresponding methyl ketone was formed in satisfactory yield (entries 14-16).Meanwhile, methyl acetoacetate also reacted with benzaldehyde and generated the corresponding product 17 in 85% yield (entry 17).
In addition, to evaluate the feasibility of this method on a large scale, the model reaction was performed on the 40 mmol scale and the desired product was obtained in 89% yield.
Next, we studied the reusability of the heterogeneous nano Cu/C catalyst in the model reaction (Table 3).After completion of the reaction, the catalyst was filtered from the reaction mixture, washed with hot ethanol, dried and used directly for the next round of reaction.The ease of recovery, combined with the stability of catalyst, allows the catalyst to be recycled over 8 times in reactions without any significant loss in activity.
Conclusions
In conclusion, we have reported a direct one-pot method for the construction of 2-amino-3-cyanopyridine derivatives starting from aldehydes, ketones, malononitrile, and ammonium acetate in the presence of Cu/C as a heterogeneous catalyst system, in up to 71% isolated yield.Various aldehydes and ketones were readily applied to this synthetic protocol.It is noted that in the present system the catalyst can be recycled and reused at least eight times without significant losses of activity.
Table 1 .
Optimization of reaction conditions
Table 2 .
Substrate scope for the synthesis of 2-amino-3-cyanopyridine derivatives a | v3-fos-license |
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} | pes2o/s2orc | Synthesis of New Family of Thiazoline and Thiazole Esters and Investigation of their Thermal Properties
Uma nova família de ésteres tiazolínicos e tiazóis foram sintetizados e as propriedades térmicas são apresentadas e discutidas. Os ésteres tiazolínicos foram obtidos pela reação de ciclização de benzonitrilas 4-substituídas com o amino ácido L-cisteína seguido da reação de esterificação com álcoois e fenol previamente selecionados. A oxidação dos ésteres tiazolínicos mediado pelo reagente BrCCl3/DBU permitiu a transformação dos respectivos ésteres tiazolínicos em ésteres tiazóis . Os compostos finais dos ésteres tiazolínicos e tiazóis são formados por cadeias alquílicas, (perfluoroalquil)alquílicas e p-alcoxifenilas. Alguns cristais líquidos mostraram serem mais relevantes. Um deles apresentou mesofase esmética A (SmA) monotrópica enquanto que outros apresentaram mesofase estável SmA. Como esperado, as cadeias de alcanos semifluorados induziram a formação de mesofase ortogonal via efeito de segregação.
Introduction
Liquid crystals (LC) have been long recognized as one of the most extensive and attracting fields of materials research.The first studies, dating from 150 years ago, prompted nowadays researches worldwide increasing scientific interest, focusing on obtaining and monitoring new LC compounds. 1he most common application of liquid crystal technology is in liquid crystal displays (LCDs).From the ubiquitous wrist watch and pocket calculator to an advanced computer screen, this type of display has evolved into an important and versatile interface.Since the discovery of interesting electro-optical properties in the middle sixties and the production of the first LCD panel in the beginning of the seventies, many other practical applications of these molecules have been largely studied.
As they show a variety of physical properties, these molecules find application in diverse fields of science, such as display technologies, 2 light emitting diodes 3 and photo/semiconducting materials. 4Thus, great effort has been made in developing synthetic strategies for acquisition of new and diverse mesomorphic compounds.
Heterocyclic compounds are of great importance in many fields of chemistry.It is very common the observation of interesting biological activity on compounds bearing these structures. 5Insertion of a heterocyclic unit in the skeleton of a mesogenic molecule aggregates many advantages, once one could enhance design possibilities and count up several changes in liquid crystalline properties.Also it is well-known that a minor change in molecular geometry may bring about substantial variations on mesomorphic behavior. 6Furthermore, the presence of heteroatom alters considerably the polarity and polarisability of the molecule, and the heterocycle core has the ability of impart lateral and/or longitudinal dipoles. 7In particular 1,3 thiazoles derivatives show bond angles of 153° and 133°, depending on the substitution pattern, and a dipole moment of 1.6 D. 8 In addition, the presence of the electron-rich sulfur atom attains modifications on the ionization potential that could induce smectic mesophases. 9oreover, mesophase morphologies can be further tuned by replacement of a flexible non branched alkyl chain for a perfluorinated segment. 10The increased rigidity, linearity and low surface energy of the perfluorinated chain compared to the hydrocarbon one, settles particular properties modifications, as the fluorophobic effect, which reduces the miscibility of the fluorinated moieties with other sides of the molecule. 11Thus, fluoro substituents and perfluorochains have been incorporated to LC compounds leading to a variety of modifications on these substances behaviors, as broad mesomorphic ranges, different mesophase morphologies, lower melting points and transition temperatures as well as stabilization of preexisting mesophases, due to the combination of small size, intense polarity and high strength of the C-F bond. 10 Such attributes can be successfully comprised in the structure of mesogens and are of major significance in terms of primary structure property relationships, any more than crucial to the development of novel liquid crystal materials. 12For the thiazole building block showed in Figure 1, the general structure carries always on a flexible alkyl chain from one side of the molecule and a carboxylic group at the other side to be derivatized into aliphatic or aromatic esters and amides.Among the wide range of methodologies for preparation of thiazoline derivatives, 13 the one based on cyclization of nitriles with the natural amino acid L-cysteine was elected, 14,15 once it employs readily accessfull and low cost starting materials.Also, this method delivery the target precursor bearing the desirable carboxyl group mentioned above, which enables the possibility to attach a variety of substituents by means of esterification procedures employing different alcohols phenols.
Thiazolines synthesis and their subsequent oxidation to heteroaromatic thiazole rings are an interesting way to prepare building block to be used in many branchs of the synthetic organic chemistry.As demonstrated in this work, a sequential oxidation of the thiazoline to thiazole ring leads to an enhancement of geometrical anisometry of the rigid core contributing for emergence of mesomorphic and photoluminescent properties.Thereby, we wish to report a simple and straightforward methodology for the synthesis of a new class of compounds derived from the thiazoline precursor using the chain elongation strategy as well as to investigate the liquid-crystalline behavior of the final thiazole esters.
General structure of target thiazole esters is presented in Figure 1.Primitive core is formed by aromatic and thiazole ring connected by a single bond.Variable connection means the chemical communication between additional group on the right side and the primitive core intermediated by carboxyl group presented in the target esters.Thiazolines 6a-k and thiazoles 7a-k are composed of a flexible hydrogenated alkyl chain on the left side of Figure 1, except for those from 4-bromophenyl nitrile.On the right side of Figure 1, terminal segments connected by carboxyl group are composed of flexible alkyl chains (hydrogenated chain), (perfluoralkyl) alkyl chains (semifluorinated alkane) 16 and p-alkoxyphenyl chains.Figure 1 shows the general structure of the new thiazole esters synthesized in this work.
Synthesis
The synthesis of key intermediate thiazoline carboxylic acids 4a-c is outlined in Scheme 1.The strategy applied here begins with the commercially available nitriles 1a and 1b, which were submitted to the alkylation reaction with 1-bromoctane under basic conditions, affording the alkylated nitriles 2a and 2b in almost quantitatively yields.From this point, in Scheme 1, 4-bromobenzonitrile (2c) was added to our plan of the synthesis of 4a-c.Next step nitriles 2a-c were submitted to cyclization reaction with amino acid L-cysteine (3) to generate the key intermediates thiazoline derivatives 4a-c.In general, the most common procedure for this reaction involves a system of water/ alcohol under buffer control by several days. 17In our hands the experimental condition describe above to yield the corresponding thiazoline carboxylic acids 4a-c gave very low yields of the described thiazoline derivatives.In order to achieve better results, we changed the initial protocol reaction to one that has been used by Loughlin et al.. 18 Under the new experimental conditions, we prepared the intermediate 4a-c in a good yield.Thus, exposing nitriles 2a-c to L-cysteine mediate by sodium bicarbonate, morpholine and reflux in an alcoholic solvent (ethanol or isopropanol), the cyclizated products 4a-c were afforded in excellent overall yields.This is an interesting and an alternative way to prepare thiazolines in a simple and almost inexpensive methodology.
After accomplished successfully the synthesis of 4a-c, the esters formation was initiated to complete our synthetic plan.Esterification reactions with activation of acids by carbodiimides are probably the most versatile procedures for preparation of ester compounds. 19The chemical linkage between 4a-c and 5a-d to obtain the final thiazoline esters 6a-k was firstly attempted using the dicyclohexylcarbodiimide/4-N,N-dimethylaminopyridine (DCC/DMAP) protocol. 20However, due to the troublesome to remove the by-product urea, we changed DCC to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) to promote the ester ligation.Thus, thiazoline esters 6a-k were synthesized as outlined in Scheme 2, by activation of acids 4a-c with EDCI followed by the reaction with several alcohols 5a-d in dichloromethane and DMAP as a catalyst.Table 1 summarizes the results for the thiazoline esters 6a-k.It is noteworthy to mention that EDCI/DMAP couple worked successfully for a variety of chosen alcohols, such as alkyl and (perfluoralkyl)alkyl and phenols, delivering the intermediates compounds 6a-k to be transformed into LC in good yields by oxidation process.
The 2,5-disubstituted thiazole core in the final compounds 7a-k was accomplished as described in Scheme 3. Several methods were evaluated to perform this transformation, such as reactions with 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ), 21 NiO 2 22 and MnO 2 . 23First attempts employing this later strategy resulted in poor yields of target final products, as well as a complex mixture of by-products detected by thin layer chromatography (TLC) analysis of crude reaction extract.In order to improve the obtention of these target mesogens, the oxidative dehydrogenation step was most conveniently performed using a mixture of bromotrichloromethane and 1,8-diazabicyclo[5.4.0]undec-7-ene (BrCCl 3 /DBU). 24In comparison to the ones mentioned above, this synthetic protocol is advantageous for its milder conditions and tolerating to a large variety of functional groups.Therefore, the bromination takes place first in 6a-k and then, the dehidrobromination in presence of DBU in dry DCM furnished the final products 7a-k in good to excellent yields (Table 2). 25
Liquid crystal properties
Transition temperatures of thiazoline and thiazol esters are tabulated in Table 1 and 2, respectively.Table 1 and 2 display the transition temperatures of eleven thiazoline and thiazol esters synthesized in this work.The primitive core (rigid core) contains at least a phenyl group connected to heterocyclic moiety.Esters here are composed of flexible alkyl chains (hydrogenated chain), (perfluoralkyl)alkyl chains (semifluorinated alkane) 16 and p-alkoxyphenyl chains.Thiazolines esters are not LC as we can see in Table 1, except for 6i, which displayed smectic A mesophase (SmA) upon cooling.They are mostly crystalline solid with melting point (mp) in the range between 40 °C to 140 °C, and only 6a is liquid at room temperature.Melting point was dependent on how flexible is the aliphatic chain ester.For example, 6a is a liquid, while 6b and 6c are solids with mp being bigger than 6c, which is, of course, more anisotropic but not liquid crystal.This tendency in the mp extends to the others thiazolines in Table 1.As it moves from more flexible alkyl chain to (perfluoralkyl)alkyl chains the stiffness becomes higher.Also, for 6c, 6g and 6k, the phenyl group contributes to the stiffness of the compounds.So in this sense, mp tends to be higher as the molecular rigid core becomes more anisotropic.Additionally, 6d, 6e and 6f are good examples to compare the stiffness effect caused by fluorine atom at alkyl chain.The higher mp is seen in the more rigid thiazoline 6e.Additional phenyl group has a higher contribution to molecular stiffness as expected; comparing 6d and 6g, 6d and 6h and 6g with 6k. 26 This is irrespective on the relative position of the phenyl group into the rigid rod core.The behavior was observed even in the longest and more anisotropic 6k it was not able to show some evidence of liquid-crystalline behavior.
A briefly comment is addressed to 6i, which displays a monotropic SmA mesophase.The monotropic nature of the mesophase to 6i means that mesophase appeared only upon cooling, it is thermally unstable with an unpredictable and short mesophase temperature range.The mesophase range is too narrow with DT = 2 °C.Why 6i, among all compounds listed in Table 1, displays a LC behavior?The answer of this question may be associated to perfluoroalkyl chain effect, close to the thiazoline ring.The molecular segregation is the driving force for the formation of liquid-crystalline phase of 6i.Incompatibility of totally flexible alkyl chain, rigid anisometric core and less flexible semifluorinated alkane chain are responsible for the formation of positional ordered liquid-crystalline phases 27 as observed for 6i and the thiazole series in Table 2.We have to say more about the mesomorphic properties (see thiazole discussion) but the linearity deviation and non-coplanarity of the thiazoline ring (broken anisometry) is the main force of the absence of the mesophase in this series. 28igure 2 displays a typical growth of SmA bâtonnets (bright) from the isotropic phase (black) upon cooling at 161 °C of thiazoline 6i.Bright and black colors in Figure 2 are observed due to crossed polarizers, using optical microscopy and the molecular orientation of the samples sandwiched between the glass plates.Upon cooling coalescing, bâtonnets will form full focal conic SmA fan texture.Sometimes, black areas from isotropic state remain black due to the perpendicular orientation of the molecules.Black areas of the sample have a homeotropic alignment.In this case, the long molecular axis is perpendicular to the glass plates.When the growth of bâtonnets is completed, the fan focal-conic is observed.The color is a consequence of the orientation of the director which basically lies in the plane of the substrate and the structured layers are curved across the fans. 29able 2 displays the thermal data of thiazoles 7a-k that were obtained by oxidation process from thiazolines 6a-k.As expected, some thiazoles in Table 2 display liquidcrystalline behavior as evidenced by the texture observed using polarized optical microscopy (POM).The phase transition temperatures (in °C) were obtained by POM and To the first four thiazoles 7a-d, no liquid-crystalline behavior was observed.Even for 7c, a more anisotropic one, a transition crystal to isotropic state at 128 °C was obtained.The relative compound 6c (Table 1), the melting point is DT = 21 °C below 7c, showing that in the crystal state, the packing of molecules of thiazoles is better than thiazolines molecules. 30The increase in the melting point is also observed in 7a-d in comparison to the 6a-d.The tendency noted to these compounds is also seen in the others compounds in Table 2.However, compounds 7e, 7f, 7h, 7i and 7J have also an additional mesophase intercalated between the crystal and isotropic phase.The LC thiazoles above display a structured orthogonally layer which was characterized as smectic A mesophase according to the texture analysis of POM.This smectic fluid A mesophase is characterized by positional order of molecules' centers of mass in one dimension in addition to the long range orientational order of the director phase n which is associated parallel to the long molecular axis.Locally, the SmA mesophase can be regarded as a two dimensional liquids due to the molecules' centers of mass are arranged in layers and their projection onto the smectic layer plane are isotropically distributed with no positional correlation being observed within or across the layer planes. 29igure 3 displays (left) the growth of SmA bâtonnets (bright) of fan focal-conic texture of SmA mesophase upon cooling at 176 °C of thiazole 7j; typical fan-shaped texture of a SmA phase upon cooling at 187 °C of thiazole 7i (right).Lighter and darker regions seen in Figure 3 (right), in the fans, are due to the molecular layers of sample and cross polarizers that are used in POM study.
As expected, LC properties of thiazoles are affected mainly by the flatness and coplanarity of the heterocyclic ring.Strong p-stacking in the lattice is observed in crystal phase with 3.73 Å the distance between two adjacent thiazole rings. 30Of course, to be a classical rod-shape liquid crystal the molecular shape of the structures has to be more elongated as possible to compensate bent-shape of thiazole ring. 8,31The mesophase nature is also dependent on the terminal group connected to the rigid and anisometric core.In the current study, SmA mesophase was found particularly due to the presence of two aliphatic chains with distinct chemical nature.We can see from Table 2 the predominance of orthogonal SmA mesophase for all LC compounds listed.The significance of semifluorinated alkyl chain is clearly seen when we compare compounds 7a, 7d and 7h.They have flexible hydrogenated alkyl chain in both sides of arylthiazol esters except for 7a.For the longer aryl moiety 7h, which at first would favor the appearance of stable mesophase, it is possible to observe only a monotropic SmA mesophase.Usually, molecules with longer molecular length, tend to be more elongated and consequently more anisotropic due to length-to-breadth ratio of two molecular axes.Also, it is well-known for biphenyl and others systems that, the increase of carbon atoms in the flexible alkyl chain favors the formation of smectic mesophases, usually SmA and SmC. 32The result founded in 7h definitively emphasizes the function of perfluoroalkyl chain plays on the mesomorphic properties in this study.For aryl esters 7c, 7g and 7k, and even for 7k, which has a more anisometric core, no mesophase was observed.Again, the result observed to 7c, 7g and 7k is suggesting the importance of perfluoroalkyl chain on the mesomorphic behavior.Obviously it is possible to assume that the absence of mesophase to these compounds in terms of an equilibrium of Z-and E-rotamers, that are observed in esters when they entry to the mesophase, similarly to the behavior of liquid crystal dimers where the mesophases are governed by the populations of linear and bent molecules. 33ttempt to correlate carbonyl group frequency (n C=O cm -1 ) in the IR spectrum of 6c/7c, 6g/7g and 6k/7k in the solid state (KBr pellets) with the composition of the rotamers E and Z of esters has failed.The band of carbonyl group associated to those compounds in solid state looks like strong and sharp.Thus, it is impossible to assume that the non liquidcrystalline behavior of 7c, 7g, 7k is due to the composition of the rotamers in the equilibrium between the more anisotropic Z-rotamer and the less anisotropic E-rotamer.Now we are turning our attention to the four LC: 7e, 7f, 7i and 7j.All LC compounds listed in Table 2 have one semifluorinated alkyl chain connected to the carboxyl group of thiazole ring and a flexible hydrogenated alkyl chain on the opposite side.They are LC with SmA mesophase as expected considering that the individual molecules possess specific groups which repel or attract each other strongly.This effect begins among a few molecules at molecular level on cooling and very fast amplifies to large scale, where the nanoscale segregation is the driven force.The aggregation is promoting by these groups favoring the smectic ordering.Terminal groups such as siloxanes, fluorinated, halogenated and even diol favor the smectic ordering. 34The mesophase range for 7i and 7j is bigger than 7e and 7f as a consequence of the more anisometric biphenyl group in 7i and 7j.For example, 7i displays a mesophase range of 30 °C, while for 7e is 10 °C.Transition temperatures associated to the Cr → SmA and SmA → I phase transition reveals the effect of proximity of perfluoroalkyl chain to carboxyl group.
Transition temperatures depend on how close to the carboxyl group is the perfluoralkyl chain.Comparing 7i with 7j and 7e with 7f it is possible to note that 7i and 7e display higher transition temperature from crystal to mesophase and mesophase to isotropic phase.Only 7j displays a second transition Cr → CrX and it has the biggest mesophase range (DT = 39 °C) for SmA mesophase to isotropic state.According to Höpken et al., 35 the thermal behavior of (perfluoroalkyl) alkane can be explained in terms of conformational aspect of hydrogenated and perfluorated chains.Alkyl chain tends to be more amorphous in the mesomorphic state while perfluorinated alkyl chain tends to be more organized, which of course affect the transition temperatures.In the solid state both chains self-organize in crystalline way.Of course, alkyl chain may assume predominantly an extend all-trans conformation while perfluoralkyl chain tends to be kink due to the gauche effect. 36When the solid enters to the mesophase, the hydrocarbon portion must have liquid-like conformational freedom, while perfluoralkyl chains behave as a hard molecular segment owing its polar nature.Due to stiffness, fluorinated segments are packed regularly in the solid state, which favors the high melting point and even the high clearing point.In another way, when the sample on cooling enters into the mesophase state, the conformational disordering of the hydrocarbon segments upon the transition from isotropic state to the smectic mesophase is bigger than the perfluoralkyl segments.The molecular aggregation is faster to the perfluoralkyl chains favoring the attraction between rigid segments.Soft segments in the mesophase are still mobile and conformationally disordered.And finally, when the sample starts to crystallize again, hard molecular segments are packed more efficiently than the soft molecular.It is important to mention that in the mesophase more mobility is expected to more amorphous segments.Thus, the contribution of one and two methylene carbon atoms is causing the behavior observed to that thiazoles LC ester 7e, 7f, 7i and 7j.
Conclusion
We have synthesized a new family of thiazoline and thiazole esters 6a-k and 7a-k.The final thiazoline and thiazole esters are composed by terminal flexible hydrogenated alkyl chain from one side and to the other side by terminal segments of flexible alkyl chains (hydrogenated chain), (perfluoralkyl)alkyl chains (semifluorinated alkane) p-alkoxyphenyl chains.6i was the only compound belonging to the family of thiazoline esters to display monotropic SmA mesophase.Thiazole esters family presented enantiotropic SmA mesophase for 7e, 7f, 7h, 7i and 7j.The combination of better conjugation of thiazole ring and polar effect of semifluorinated alkane chain induces the formation of structured layer mesophase by means of segregation effect.
General methods
All starting materials were purchased from commercial suppliers (Sigma Aldrich Chemical Co., Acros Organics and ABCR Chemicals) and used without further purification.All reactions were carried out under a nitrogen atmosphere in oven-dried glassware with magnetic stirring.Solvents were dried, purified and degassed under classical methods.Solvents used in extraction and purification were distilled prior to use.TLC was performed using silica gel 60 F254 aluminum sheets and the visualization of the spots has been done under UV light (254 nm) or stained with iodine vapor.Products were purified by flash chromatography on silica gel 60 M, 230-400 mesh.Melting and mesophase transition temperatures and textures of the samples points were measured using an Olympus BX43 microscope equipped with a Mettler Toledo FP82HT Hot Stage FP90. 1 H ( 13 C) NMR spectra were recorded at 300 (75) MHz on a Varian Inova and 400 (100) MHz Bruker spectrometer using CDCl 3 as solvent.The 1 H and 13 constants J [Hz] were directly taken from the spectra and are not averaged.Splitting patterns are designated as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) and br (broad).Low-resolution mass spectra were obtained with a Shimadzu GC-MS-QP5050 mass spectrometer interfaced with a Shimadzu GC-17A gas chromatograph equipped with a DB-17 MS capillary column.HRMS spectra were obtained from a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer equipped with an Infinity™ cell, a 7.0 Tesla superconducting magnet, an RF-only hexapole ion guide and an external electrospray ion source (off axis spray) and with ESI(+)-MS and tandem ESI(+)-MS/MS using a hybrid high-resolution and high accuracy MicroTOF-Q II mass spectrometer (Bruker Daltonics), or on a Micromass Q-TOFmicro instrument.
General procedure for the synthesis of thiazolines 4a-c A solution of L-cysteine hidrochloride (60 mmol; 10.8 g), NaHCO 3 (60 mmol; 5.3g), the appropriate nitrile (20 mmol) in 85 mL of ethanol was refluxed for 30 min.After, the pH was adjusted to 8.0 by adding a few drops of morpholine, and the reflux was continued for additional 12 h.The ethanol was removed, the residue dissolved in distilled water and acidified to pH 1.5 by adding concentrated HCl.The product was extracted with CH 2 Cl 2 (3 × 50 mL), the organic layers where combined, dried over MgSO 4 and the solvent removed under vacuum.The products were obtained with satisfactory purity and were used in the next step without further purification.
General procedure for esterification of thiazolines 6a-k
To a stirred solution of the corresponding thiazoline 4a-c (2.0 mmol) in dry CH 2 Cl 2 (8 mL) under N 2 atmosphere at room temperature, were added subsequently the corresponding alcohol (2.0 mmol) EDCI (2.0 mmol) and catalytic amount of DMAP.After 16 h, the organic phase was transferred to an extraction funnel, washed with saturated NaHCO 3 (2 × 10 mL), water (2 × 10 mL) and the organic layer was dried with Na 2 SO 4 .The solvent was evaporated and the remaining product was purified by chromatography (hexanes/AcOEt = 80:20).
Figure 1 .
Figure 1.General structure for the target thiazoles.
Figure 3 .
Figure 3. Growth of SmA bâtonnets (bright) of fan focal-conic texture of SmA mesophase upon cooling at 176 °C of thiazole 7J (left); typical fan-shaped texture of a SmA phase upon cooling at 187 °C of thiazole 7i (right). | v3-fos-license |
2018-12-07T08:07:07.491Z | 2013-01-31T00:00:00.000 | 55209977 | {
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} | pes2o/s2orc | Influence of Waste Cooking Oil Biodiesel on the Particulate Emissions and Particle Volatility of a DI Diesel Engine
The effect of biodiesel produced from waste cooking oil on the particulate emissions of a direct injection (DI) diesel engine was investigated experimentally and the results were compared with two diesel fuels, namely, an ultra low sulfur diesel fuel with less than 10-ppm-wt of sulfur (ULSD) and a low sulfur diesel fuel with 400-ppm-wt of sulfur (LSD). For each fuel, the number and mass based particle emissions, as well as the particle volatility, were evaluated and compared. The particulate mass emissions were measured with a tapered element oscillating balance (TEOM) and further divided into different size bins using a micro-orifice uniform deposition impactor (MOUDI). The particle number concentration and size distribution were measured with a scanning mobility particle sizer (SMPS). The size-segregated samples collected with the MOUDI were further analyzed with a thermogravimetric analyzer (TGA) to obtain the mass of volatile substances in each size bin. The SMPS was further connected in series with a thermodenuder (TD) to obtain the number concentration and size distribution of non-volatile particles, and hence the number concentration and size distribution of the volatile particles. The results indicate that the biodiesel could effectively reduce the particle mass and number concentrations, including the volatile substances, in all the measured size range, compared with LSD. Compared with ULSD, there is also a reduction in the particle mass and number concentrations, however, a higher concentration of volatile substances was found with the use of biodiesel, which should be a concern in the application of this fuel.
INTRODUCTION
Diesel particulates are composed of soot aggregates and volatile substances which include hydrocarbons and sulfate.Volatile substances have complex composition, some of which are toxic and carcinogenic.Therefore, it is important to analyze the volatility of diesel particles because it is an indication of the toxicity of these particles (Giechaskiel et al., 2009;Chuang et al., 2010;Ning and Sioutas, 2010;Wu et al., 2010;Tsai et al., 2011).There are different methods for investigating particle volatility.Maricq et al. (2002) used a scanning mobility particle sizer (SMPS) and a thermal denuder (TD) to investigate the particulate emissions of a diesel engine fueled with a diesel fuel containing 350 ppmwt sulfur.They found that most of the nuclei mode particles could be adsorbed by the TD.Rönkkö et al. (2007) compared the particle number-size distribution using a nano-SMPS and a TD and found that the nucleation mode particles have a non-volatile core with volatile species condensed on it.Kwon et al. (2003) and Sakurai et al. (2003) investigated the volatility of diesel particles using a tandem differential mobility analyzer (TDMA) and found that the volatility was size-dependent.Filter-based methods have also been used to investigate the volatile substance in diesel particles.These methods include thermal-gravimetric analysis, thermal optical carbon analysis and Soxhlet extraction (Kerminen et al., 1997;Ning et al., 2004;Collura et al., 2005;Shen et al., 2009;Mustafi et al., 2010).
Former investigations on particle volatile mainly focused on the effects of fuel sulfur content, which have been well reported (Maricq et al., 2002;Vaaraslahti et al., 2004;Ristovski et al., 2006;Rönkkö et al., 2007) and induced the tightening up of diesel sulfur content.In China, the national standard of diesel fuel follows the standard EN590.The limit of sulfur content in EN590 and its execution date in Europe and China are listed in Table 1.In China the current maximum sulfur content is limited to about 350 ppm-wt and will be further reduced, while in Europe, it has been limited to less than 10 ppm-wt.Over the past decade, using biodiesel as an alternate diesel fuel has drawn increasing interest due to its biodegradable and nontoxic properties and using biodiesel can significantly reduce particulate emissions and overall life-cycle CO 2 emission from the engine (Lapuerta et al., 2008).However, research on volatility of particles generated from the combustion of biodiesel, in particular biodiesel produced from waste cooking oil, is rare (Jung et al., 2006;Heikkilä et al., 2009;Surawski et al., 2011a, b).
In this study, we investigated the effect of a biodiesel produced from waste cooking oil on the particle number/ mass-size distributions and the volatility of the particles in the exhaust of a diesel engine.We used a SMPS and a TD to investigate the number-size distributions of the volatile substances, and used a thermal-gravimetric analyzer to investigate the mass-size distributions of the volatile substances in the particles collected with a micro-orifice uniform deposition impactor (MOUDI), and the two results were compared.Investigation on both mass-based and number-based particle volatility is rarely reported in the literature.Two diesel fuels with different sulfur contents of 10-ppm-wt sulfur and 400-ppm-wt sulfur were used for comparison.
EXPERIMENTAL METHODS
The study was performed on a naturally-aspirated, water cooled, 4-cylinder direct-injection diesel engine (ISUZU 4HF1).The specifications of the engine are shown in Table 2.The engine was connected to an eddy-current dynamometer and a control system was used for adjusting its speed and torque.The major properties of the three fuels are given in Table 3. ULSD is widely used in Europe while the properties of the LSD are similar to the diesel fuel available in China.The biodiesel used in this study was produced from waste cooking oil by Dynamic Progress Ltd. and its properties are in compliance with the standard EN14214.
The schematic of the experimental system is shown in Moreover, for minimizing cross contamination of different fuels, the engine was allowed to operate with the new fuel for thirty minutes to clean the fuel system.
Particle number concentration and size distribution was measured with a scanning mobility particle sizer (SMPS TSI Model 3934) for the size range of 10-486 nm.For each test condition, the SMPS scan was repeated 4 times.Before passing through the SMPS, the engine exhaust gas was diluted with a two-stage dilution unit (Dekati Ltd.).The transfer line from the exhaust to the diluter was heated at 170°C.The first stage was heated to keep the dilution air at 150°C to avoid condensation of volatile substances.The second diluter was directly connected to the first diluter and the diluted gas was maintained at about 25°C for direct coupling to the SMPS which counts particle concentrations on volume basis.The dilution ratio was determined from the measured CO 2 concentrations of background air, undiluted exhaust gas and diluted exhaust gas.CO 2 concentrations were measured with a non-dispersive infra-red analyzer (NDIR, CAI 300).The dilution ratio for the SMPS varied from 67.5 to 89.6, depending on the actual operating conditions.The dilution process will induce some variation of the particle size distribution in the exhaust gas, especially the nucleation mode particles which are sensitive to the dilution conditions (Shi and Harrison, 1999).
Particulate mass concentration was measured with a tapered element oscillating microbalance (TEOM, Series 1105, Rupprecht & Patashnick Co., Inc.).The engine exhaust gas passed through the TEOM with the first stage dilution and the sampling temperature was around 52°C.The dilution ratio for the TEOM was one-eighth of that for the SMPS.Classified particulate samples were also collected using a micro-orifice uniform deposition impactor (MOUDI-110R, MSP Corporation) with 10 cut-point sizes of 10, 5.6, 3.2, 1.8, 1.0, 0.56, 0.32, 0.18, 0.1 and 0.056 μm for investigating the particle mass-size distributions.The sampling conditions are the same for the MOUDI and the TEOM, both have onestage dilution.47mm quartz filters (Whatman Corporation) were used as impaction substrate for the MOUDI to collect particulate samples.The quartz filters were prebaked at 500°C for 4 hours to remove any carbon contamination.Before and after sampling, the quartz filters in the MOUDI were allowed at least 24 hours equilibration in a controlled environment with a temperature of 21-22°C and relative humidity of 40-45% and then weighed with a microbalance (Mettler-Toledo XS105).Previous studies indicated that for motor vehicles, 80-90% of the particulate mass fraction is within the fine-particle size range (Kerminen et al., 1997;Chien et al., 2009;Zhang et al., 2009).Therefore, in this study, despite particles in all size bins were collected, only those less than 1.8 μm were weighed and analyzed.Two methods were used to investigate particle volatility, namely, a mass-based method and a number-based method.The former method involves the thermogravimetry analysis (TGA) of filter samples collected with the MOUDI.TGA was conducted using the Netzch-STA 449 TGA/DSC (thermogravimetric analyzer/differential scanning calorimetry) with Al 2 O 3 crucible.The particulate samples were firstly heated in an argon environment with a heating rate of 10°C per minute to 400°C, and then held at 400°C for 10 minutes.The mass loss in the argon environment was taken as the mass of the volatile substances.The remaining part was heated at an air environment with a heating rate of 10°C per minutes to 800°C.The mass loss at the air environment was taken as the mass of the non-volatile substances.Similar approach had been used by Boehman et al. (2005) and Mustafi et al. (2010) to distinguish the volatile and non-volatile fractions of diesel particles.The numberbased method was conducted by comparing the SMPS measurements with and without the Dekati thermo-denuder (TD).The TD consists of a heated section followed by an adsorber section where the vaporized compounds are adsorbed in activated charcoal.In this study, the particle number-size distributions obtained after the TD have been corrected for diffusion losses using the measured diesel particle number concentration with the TD set at 25°C (Surawski et al., 2011b).
For the particle number and mass concentrations, the average values were presented.The standard errors were determined following the method of Kline and McClintock (1953).In order to ensure that the results are repeatable within the experimental uncertainties, for each test condition, the tests were repeated twice.In this study, the maximum standard errors are 2.5% for particle mass concentration using TEOM, 1.7% for particle number concentration, 2.3% for particle geometric mean diameter, and 4.5% for the volatile mass fraction determined with the TGA.
Particle Mass Concentrations and Size Distribution
The total particulate mass concentration for each fuel and at each engine load was measured with the TEOM.The variation of brake specific particulate matter (BSPM) with engine load is shown in Fig. 2. For each fuel, the minimum BSPM appears at some intermediate engine loads which can be attributed to the lower brake thermal efficiency at low engine load and the higher particulate emissions at high engine load.At high engine load, the increase in fuel burned in the diffusion mode leads to a rapid increase in the particulate mass concentration in the engine exhaust gas and hence a corresponding increase in the BSPM.
For the three fuels, the biodiesel and LSD generate the lowest and highest BSPM, respectively.The BSPM of biodiesel is 229.8 mg/kWh, on average of the five engine loads, which is 26.5% and 43.3% lower than BSPM of ULSD and LSD, respectively.The effectiveness of biodiesel on reducing particulate emission has been reported in the literature (Corporan et al., 2005;Tsolakis, 2006;Lapuerta et al., 2008).In general, there are three reasons leading to the lower particulate emission with biodiesel.Firstly, the advanced fuel ignition associated with biodiesel provides a longer time for soot oxidation.Secondly, the oxygen content of biodiesel enables more complete combustion and promotes the oxidation of the already formed soot.Thirdly, the absence of aromatics in biodiesel leads to a reduction in soot formation.Therefore, in this study, the engine fueled with biodiesel has lower particulate emission than fueled with the two diesel fuels.On the other hand, the lower aromatics content and the lower sulfur content are reasons for the lower particulate emission of ULSD, compared with LSD.Ullman et al. (1994) found a 3-5% particulate reduction for a 100 ppm-wt reduction in fuel sulfur on a heavy-duty diesel engine tested under a transient drive cycle.
The particulate emission at the engine load of 0.7 MPa was chosen for the investigation of particulate mass-size distribution using the MOUDI.The BSPM at different size bins are shown in Fig. 3 while the mass median diameters (MMD) and their geometric standard deviations of the diesel particulate are listed in Table 4.For each fuel, the masssize distribution of the particles exhibits a peak BSPM at the size bin of 100-180 nm.The brake specific emissions of PM 1.8 are 198.8,399.6 and 576.4 mg/kWh for the biodiesel, ULSD and LSD, respectively.Effect of the biodiesel on reducing particulate emission in comparison with the ULSD and LSD, is in line with results obtained with the TEOM.As shown in Fig. 3, biodiesel could reduce BSPM in all the size bins, while the MMD of biodiesel particles is less than those of the ULSD and LSD, which indicates that the biodiesel particles contain a larger proportion of smallsize particles and the reduction on particulate emission arises from the reduction of the large-size particles.The same reasons which cause a larger reduction of particulate emissions with biodiesel, compared with the two diesel fuels, also lead to a larger reduction in particle MMD (Mathis et al., 2005;Tsolakis, 2006).Moreover, the reduction in particulate mass concentration may also suppress particle coagulation and hence reduce the MMD.
Mass-Based Investigation of Particle Volatility
Mass-based particle volatility was investigated with the TGA to evaluate the mass fraction of volatile and nonvolatile substances in the particulate samples collected on filter papers installed inside the MOUDI.The brake specific emissions of volatile and non-volatile substances at different size bins are shown in Fig. 3 for the engine load of 0.7 MPa, and the MMD and geometric standard deviation for the non-volatile substances are listed in Table 4.For PM 1.8 , the brake specific emissions of volatility substances are 64.2,55.2 and 132.6 mg/kWh for biodiesel, ULSD and LSD, respectively, indicating that using biodiesel, the emission of volatile substances could reduce, compared with LSD, but could increase, compared with ULSD.However, in terms of mass fraction, biodiesel particles contain 32.2% volatile substances which is larger than 13.8 and 23.0% for ULSD and LSD particles, respectively.Chang et al. (1998) suggested that due to its lower volatility (higher boiling point), unburned biodiesel fuel should be more likely to condense and adsorb on the soot particles, leading to higher volatile fraction on these particles.Ballesteros et al. (2008) attributed the higher volatile fraction in biodiesel particles to their higher surface/volume ratio.The higher surface/ volume ratio implies an increment in the active surface in which the hydrocarbons can be adsorbed and causes a rise in the volatile fraction.Moreover, oxygenated fuels like biodiesel have significant effect on the reduction of soot.
On the other hand biodiesel might also lead to a reduction in hydrocarbon emissions which might reduce the volatile fraction.Further analysis shows that, with biodiesel, the non-volatile substances in PM 1.8 decrease by 69.7 and 60.9%, compared with LSD and ULSD, respectively, while for volatile substances, the corresponding reductions are 51.6% and -16.3%.It indicates more effective reduction on non-volatile substances than volatile substances.The LSD particles contain a larger percentage of volatile substances than the ULSD particles.Liu et al. (2005) suggested that higher fuel sulfur content results in higher concentration of nucleated sulfuric acid particles, which provides larger amount of sites for the condensation of volatile organic compounds.
The size resolved particle volatility is compared among the three fuels in Fig. 3.For particles with aerodynamic size less than 180 nm, the brake specific emissions of volatile substance are 40.9 and 25.7 mg/kWh, respectively, for biodiesel and ULSD, while for particles with aerodynamic size between 180-1800 nm, the corresponding values are 23.4 and 29.5 mg/kWh.It indicates that the higher brake specific emission of volatile substances from biodiesel is mainly in the smaller size bins.Fig. 3 also shows that the brake specific emission of volatile substances from LSD is larger than those from biodiesel and ULSD in all the size bins.
Fig. 4 shows the mass fraction of volatile substances at different aerodynamic size bins for the three fuels at the engine load of 0.7 MPa.For each fuel, the mass fraction of volatile substances first decreases with the particle size and then increases.The decrease of volatile fraction with particle size in the small-size range (for particles less than 100 nm in diameter) has been found by Kwon et al. (2003) who conducted an experimental investigation on particulate emissions of a medium-sized diesel truck mounted on a chassis dynamometer.It is known that nucleation mode particles with diameter less than 50 nm are mostly formed from volatile hydrocarbon or sulfuric acid in the dilution process, thus these particles contain a higher mass fraction of volatile substances.On the other hand, particles with much larger size have fractal-like structure which provides more pores and intra-particle cavities for the condensation and adsorption of volatile substances (Kerminen et al., 1997;Ristovski et al., 2006), leading to an increase of the volatile fraction in these particles.Similar result was also found by Kerminen et al. (1997) and Zhang et al. (2009) who used low-pressure impactor in their investigations.In this study, for the three fuels, biodiesel particles contain the highest mass fraction of volatile substances in the smallsize range.In the large-size range, LSD particles exhibit stronger volatility because the larger amount of large-size LSD particles promotes the adsorption and condensation of volatile substances.
Particle Number Concentration, Size Distribution and Volatility
Number-based particle concentration, size distribution and volatility were investigated using the SMPS and the TD, with the TD set to 275°C.The particles measured with the SMPS alone contain both non-volatile and volatile substances while those measured with the SMPS and the TD contain non-volatile substances only with the volatile substances being adsorbed in the TD.Typical number concentration and size distributions measured with and without the TD are shown in Fig. 5 for the engine load of 0.38 MPa, while the influence of engine load and fuel on particle geometrical mean diameter (GMD) is shown in Fig. 6.The number concentrations measured with the SMPS can be converted into brake specific particle number concentrations (BSPN) and the results are shown in Fig. 7 for different engine loads, with and without the TD.
Fig. 5 shows that the LSD and biodiesel, respectively, generate the highest and the lowest number of particles, both with and without the TD.The difference among the different fuels is mainly due to difference in particles larger than 50 nm in diameter.Number-based particle volatility was investigated using the SMPS measurements, with and without the TD.Basically, particles measured with the SMPS with the TD are non-volatile ones.As shown in Fig. 5, the loss of particles associated with the TD is mainly concentrated on particles which are less than 100 nm in size, especially for the biodiesel and ULSD.With the TD, some of the particles may be completely adsorbed if they are completely composed of volatile substances, while some of them may shrink in size to form particles with diameter less than 10 nm (Rönkkö et al., 2007).In this study, particles below 10 nm could not be measured because the SMPS measurement was set to 10-486 nm.However, particles above 100 nm in size are less affected by the TD.Alander et al. (2004) suggested that accumulation mode particles mostly exist in the form of primary particle agglomerates and the volatile substances may be collected in pores and intra-particle cavities between the primary particles.Thus the mobility size of the accumulation mode particles is not significantly influenced by the removal of the volatile substances.
Fig. 6 shows that the GMD increases with engine load for each fuel which is a consequence of the increase in mass of fuel burned in the diffusion combustion mode at high engine load (Tsolakis, 2006;Zhu et al., 2010;Srivastava1 et al., 2011).The high fuel/air ratio and local temperature associated with high engine load also promote particle formation.Moreover, at high engine load, the time available for soot oxidation after the end of the diffusion combustion period is shorter, leading to the formation of a larger number of particles (Tsolakis, 2006).With more particles being formed, they tend to coagulate to form larger particles.Furthermore, during the dilution and cooling of the exhaust gas, the volatile substances could condense on the surface of the existing particles to form larger ones (Bagley et al., 1998;Schneider et al., 2005).This effect is more significant at high engine load when the exhaust gas temperature is higher (Ning et al., 2004).Fig. 6 also shows that, with the TD, there is an increase in the GMD of the particles.On average of the different engine loads, the GMDs increased by 13.5, 12.3 and 10.4% for the biodiesel, LSD and ULSD, respectively, which could be attributed to the higher level of volatility in the small size particles.Biodiesel has a higher level of volatility in the small size particles, hence, the adsorption of the smaller particles lead to a larger increase in GMD, after passing through the TD, compared with the two diesel fuels.Fig. 7 shows that in general, with and without the TD, for each fuel, the BSPN firstly decreases with the increase of engine load and then increases, which is similar to the results of the mass-based BSPM (Fig. 2).A comparison on the results, obtained without the TD, shows that the BSPN is the highest for LSD and the lowest for biodiesel, for all the engine loads.On average of the five engine loads, with biodiesel, the BSPN is decreased by 19% and 47%, compared with ULSD and LSD, respectively.Besides a reduction in BSPN, there is also a corresponding reduction in the GMD.For the biodiesel, the explanations for its lower BSPM and MMD could also be applied to explain the lower BSPN and GMD.Fig. 7 shows both the BSPN obtained with and without the TD.If the BSPN obtained without the TD is considered as a combination of non-volatile and volatile particles, and the BSPN obtained with the TD represents the non-volatile particles, the difference between them can be used as an indication of the BSPN of volatile particles.The BSPN thus obtained is presented in Fig. 8(a) while Fig. 8(b) shows the fraction of the BSPN of volatile particles in the BSPN obtained without the TD.As shown in Fig. 8(a), for each fuel, the BSPN of volatile particles firstly decreases with the increase of engine load and then increases, which is consistent with the trend of BSPN in Fig. 7. Fig. 8(a) shows that the biodiesel particles contain a higher BSPN of volatile particles than the ULSD particles at almost all engine loads while the LSD particles contain the highest level of volatile particles, which is in line with the results obtained in the mass-based investigation on volatile particles.On average of the five engine loads, the BSPNs of volatile particles are 2.00 × 10 14 , 1.25 × 10 14 , and 3.76 × 10 14 #/kW h for biodiesel, ULSD and LSD respectively.However, the volatile particles occupy 52.3%, 40.0% and 58.7% in the total particle emissions at the engine load 0.7 MPa, for the biodiesel, ULSD and LSD, respectively, which is larger than the corresponding reduction in the mass fraction of volatile substances especial for the biodiesel and LSD.One of the reasons is that a portion of the volatile substances exists as nanoparticles which contribute significantly to number concentration reduction but much less to the mass concentration reduction.
In regard to the number-fraction of volatile particles, as shown in Fig. 8(b), the minimum percentage of volatile particles occurs at the intermediate engine load for the biodiesel and ULSD.For example, for biodiesel, there is 44.2% volatile particle in total BSPN at 0.38 MPa, while the corresponding percentages at 0.08 and 0.7 MPa are 51.9% and 52.3%, respectively.However, for the LSD, the percentage of volatile particles exhibits monotonic increase with engine load.At low engine load, due to lower incylinder gas temperature, there is a larger amount of unburned hydrocarbon and lubricating oil in the exhaust gas which could be converted to volatile particles or condense on existing soot agglomerates (Ning et al., 2004;Ristovski et al., 2006;Mustafi et al., 2010), leading to an increase in particle volatility.The increase in volatility under high engine load is uncommon but has also been observed by Meyer and Ristovski (2007) through a VH-TDMA (volatilization and humidification tandem differential mobility analyzer) investigation on emissions from a six-cylinder diesel engine fueled with commercial 500-ppm-wt sulfur diesel fuel.Meyer and Ristovski (2007) suggested that ternary nucleation involving sulfuric acid, water and ammonia might be the dominant mechanism for production of volatile substances at high engine load.Therefore, in this study, the ternary nucleation might be one of the dominant mechanisms for the formation of volatile substances when the engine is fueled with the LSD.
Particle Number Concentration and Volatility in Different Size Groups
Nanoparticles (< 50 nm) are more hazardous to human health (Peters et al., 1997;Somers et al., 2004).Thus, the particles are classified into three groups: < 50, 50-100 and > 100 nm, for further analysis.The effect of fuel type and engine load on the BSPN and fraction of the particles in each of the three size groups is shown in Fig. 9. Compared with ULSD, the BSPN of biodiesel is at similar level for particles < 50 nm but lower for the larger particles, indicating that the reduction of BSPN associated with the biodiesel is concentrated on large size particles.In comparison with LSD, biodiesel and ULSD could lead to reduction of BSPN in all size groups, and LSD has the lowest fraction of small size particles while biodiesel has the highest.The volatility of particles in different size groups is also investigated.For different size groups, the BSPN of volatile particles and their percentage in the total volatile particles are shown in Fig. 10.On average of the five engine loads, in the size group of < 50 nm, the BSPNs of volatile particles are 1.38 × 10 14 , 8.64 × 10 13 and 2.02 × 10 14 #/kW h for biodiesel, ULSD and LSD respectively.The results indicate that the biodiesel could reduce the volatile particles in the small size range, compared with LSD but leads to an increase, compared with ULSD.The number-fractions of volatile particles in the size group of < 50 nm are 57.6,35.7 and 59.5% for biodiesel, ULSD and LSD, respectively.The number fraction of volatile particles is similar between biodiesel and LSD in this size range.While for the size group of > 100 nm, the number-fractions of volatile substances are 31.3,15.2 and 42.9%, for biodiesel, ULSD and LSD, respectively.LSD particles exhibit obviously higher number-fraction of volatile substances than those from the biodiesel and ULSD.The higher volatility of the LSD particles in the large-size range is in line with the mass-based results.However, there might be different mechanisms leading to these results as a consequence of the different methods used to assess the mass-based and number-based volatile fractions.In the mass-based case, it is assessed based on the loss of adsorption and condensed volatile substances upon heating in the TGA.In the numberbased method, the volatile substances are adsorbed in the TD instead of being adsorbed or condensed on the soot particles, leading to a reduction of particle sizes and reflected in the change of BSPN, in particular for the LSD which generates a larger amount of large-size particles.Both the number-based and mass-based results show that the volatile substances in the LSD particles are distributed over a wider range of size than the biodiesel and ULSD particles.
CONCLUSIONS
In this study, the particulate emissions from a DI diesel engine fueled with a waste cooking oil biodiesel and two diesel fuels were investigated.The results indicate that the biodiesel could effectively reduce the particle mass and number concentrations, compared with the ULSD and LSD.Both the mass-size and number-size distributions indicate that the biodiesel could reduce particle number and mass emission in all the size ranges, compared with LSD, while reduction mainly concentrate in large size particles, compared with ULSD.With regard to brake specific emission of volatile substances, both the number and mass-based measurements indicate that the biodiesel could obviously reduce the emission rate of volatile substances in all the size ranges, compared with LSD.However, compared with ULSD, there is an increase in the volatile substances in the biodiesel particles and the increase in volatile substances concentrates in the small size range.In term of mass fraction of volatile substances in total particle emissions, for each fuel, the mass fraction first decreases with particle size for the small-size particles, which then increases with particle size for the large-size particles.In the small-size range, biodiesel particles contain the highest mass-fraction of volatile substances, while in the large-size range, the LSD particles contain the highest mass-fraction of volatile substances.Similar results are also found in the number-fraction of volatile particles.Moreover, number-based measurements show that the volatile fraction of biodiesel and ULSD particles first decreases with engine load and then increases, while the LSD particles exhibit increasing volatility with engine load.The different size distribution characteristics and load effect could be attributed to the different formation mechanisms of volatile substance in the use of different fuels.
Thus it can be concluded that the application of biodiesel as a replacement of LSD could effectively reduce particle emissions, both in mass and in number, including the volatile substance in all the size ranges.However, in comparison with ULSD, the usage of biodiesel could increase the emission of volatile substances, especial in the small size range, which should be a major concern in the application of biodiesel to replace ULSD, because nanosize particles are known to cause more damage to human health than the micron-size particles.
Fig. 2 .
Fig. 2. Effect of fuel type and engine load on brake specific particulate emission.
Fig. 3 .
Fig. 3. Effect of fuel type on the mass-size distribution of particulate matter (PM), volatile substances and non-volatile substances at 0.7 MPa.
Fig. 4 .
Fig. 4. Effect of fuel type on mass-fraction of volatile substances in difference size bins at 0.7 MPa).
Fig. 5 .Fig. 6 .
Fig. 5. Effect of fuel type and TD on particle number-size distribution at 0.38 MPa.D m : mobility particle diameter.
Fig. 7 .
Fig. 7. Brake specific particle number emission with and without TD.
Fig. 9 .
Fig. 9. Effect of fuel type and engine load on (a) BSPN (without TD) and (b) number-fraction distribution in different size groups
Fig. 10 .
Fig. 10.Effect of fuel type and engine load on (a) BSPN of volatile particles and (b) number-fraction distribution of volatile particles in different size groups
Table 1 .
Diesel fuel sulfur content in Euro Standards and execution date in Europe and China.
Table 2 .
Specifications of the test engine.
Table 3 .
Properties of test fuels. | v3-fos-license |
2014-10-01T00:00:00.000Z | 2012-12-17T00:00:00.000 | 263944759 | {
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} | pes2o/s2orc | Optimization of Synthesis, Characterization and Cytotoxic Activity of Seleno-Capparis spionosa L. Polysaccharide
In this study, an experiment was designed to optimize the synthesis of seleno-Capparis spionosa L. polysaccharide (Se-CSPS) by response surface methodology. Three independent variables (reaction time, reaction temperature and ratio of Na2SeO3 to CSPS) were tested. Furthermore, the thermal stability, particle size, shape and cytotoxic activity of Se-CSPS in vitro were investigated. The optimum reaction conditions were obtained shown as follows: reaction time 7.5 h, reaction temperature 71 °C, and ratio of Na2SeO3 to CSPS 0.9 g/g. Under these conditions, the Se content in Se-CSPS reached 5.547 mg/g, which was close to the predicted value (5.518 mg/g) by the model. The thermal stability, particle size and shape of Se-CSPS were significantly different from those of CSPS. Additionally, a MTT assay indicated that the Se-CSPS could inhibit the proliferation of human gastric cancer SGC-7901 cells in a dose-dependent manner.
Introduction
As an organic selenium compound, selenium polysaccharide (seleno-polysaccharide) maintains the basic configuration of the polysaccharide and physiological functions of selenium [1][2][3][4][5].Moreover, seleno-polysaccharide can improve the bio-availability of selenium and reduce the toxicity and side effects of inorganic selenium [6].Meanwhile, research shows that the biological activity of seleno-polysaccharide is higher than that of polysaccharide and selenium [7].
The natural seleno-polysaccharide is limited and scarce in plants, animals and micro-organisms [8].It is necessary to increase the yield of seleno-polysaccharide by artificial methods.Biotransformation and chemical synthesis were feasible approaches for harvesting seleno-polysaccharide [9][10][11][12].However, many defects of biotransformation can not be ignored, such as higher cost, longer time and lower yield.Therefore, chemical synthesis is the most effective way to get seleno-polysaccharide.
Capparis spionosa L., which is cultivated in China and central Asia, is a traditional medicinal plant [13].Because Capparis spionosa L. contains various active ingredients (volatile oil, sugar ligands, glucose isothiocyanates and alkaloids), it is used for treating diseases such as rheumatoid arthritis, frozen shoulder and hypertension [14].Our previous research has indicated that Capparis spionosa L. polysaccharide (CSPS) had anti-tumor activity in vivo and in vitro [15,16].We found that CSPS could prolong the survival time of H22 bearing mice in a dose-dependent manner [14].In vitro, under the laser scanning confocal microscope, we observed that the cytoplasm was leaked from intact HepG2 cells and the cells stained with AO/EB were disrupted to pieces in mid-CSPS and high-CSPS group [16].Simultaneously, we discovered that CSPS could induce HepG2 cell apoptosis by increasing intracellular Ca 2+ [17].
Up to now, little information is available on the optimal conditions for seleno-Capparis spionosa L. polysaccharide (Se-CSPS) synthesis.In addition, there are no experiments to explore the thermal stability, particle size, shape and cytotoxic activity of Se-CSPS.Therefore, this paper employed a optimized by Box-Behnken design (BBD) and response surface methodology (RSM) [18][19][20][21][22] (three factors and three levels) to optimize the effects of reaction time, reaction temperature and ratio of Na 2 SeO 3 to CSPS on the Se content in Se-CSPS.Furthermore, the thermal stability, particle size, shape and cytotoxic activity of Se-CSPS were evaluated for seeking high biological functional seleno-polysaccharide.
Single Factor Assays
The effects of single factors on Se content in Se-CSPS were shown in Figure 1.As seen from Figure 1A, the curve kept a mild slope after 7 h and the Se content decreased when the reaction time was 10 h.The reason for this result was that more and more monosaccharide and oligosaccharide were hydrolyzed from CSPS by acidic water for long time reaction.When the reacted solution was dialyzed with dialysis membrane in running water, the lower molecular weight monosaccharide and oligosaccharide were dialyzed out from solution.Se introduced into monosaccharide and oligosaccharide also was removed from dialysis membrane.This led to the decrease of Se content when the reaction time was 10 h.Therefore, 6-8 h was considered to be optimal reaction time in the designed experiment.The Se content in Se-CSPS affected by different reaction temperature (50, 60, 70, 80, 90 °C) was shown in Figure 1B.Se content in Se-CSPS reached a maximum value at 70 °C.However, with the increase of reaction temperature after 70 °C, Se content was significantly decreased.The reason was similar to that of the reaction time.Because a certain percentage of CSPS was hydrolyzed to monosaccharide and oligosaccharide at the higher temperature, Se-monosaccharide and Se-oligosaccharide were dialyzed out from reacted solution through dialysis membrane.So, the 60-80 °C was used in the present work.
As seen from Figure 1C, the result indicated that Se content in Se-CSPS significantly increased at the ratio of Na 2 SeO 3 to CSPS 0.8 g/g and then maintained a dynamic equilibrium.Therefore, the ratio of Na 2 SeO 3 to CSPS ranges of 0.6-1.0g/g were adopted for the reaction.Figure 1D showed that Se content in Se-CSPS obvious increased with the increase of water bath shaking rate, but there was no significant increase after 40 r/min.This meant that 40 r/min was sufficient for the reaction.Herein, 40 r/min was determined as the reaction water bath shaking rate in next optimization experiments.
Results of Selenylation of Optimization of the Procedure
Seventeen experimental conditions for optimizing the three individual parameters were designed in BBD (Table 1).Five replicates (Exp.No. [13][14][15][16][17] at the center of the design were used to allow for estimation of a pure error sum of squares.The response value in each trial was the average of triplicates. The Se content in Se-CSPS showed consideration variation with the reaction conditions (Table 1).As Table 1 shown, Se content in Se-CSPS ranged from 2.438 to 5.545 mg/g.
Model Fitting and Statistical Significance Analysis
By applying multiple regression analysis on the experimental data, the relationship between response variables and the test variables was obtained from the following second-order polynomial equation.
p-Value is a tool to check the significance of each coefficient, which in turn may indicate the pattern of the interactions among the variables.Table 2 showed that Se content in Se-CSPS was significantly affected by three variables (p < 0.05).It was evident that the quadratic parameters (X 1 2 , X 2 2 , X 3 2 ) and the interactive parameters(X 1 X 2 , X 2 X 3 ) were significant at the 0.05 level, whereas the interactive parameter (X 1 X 3 ) was not significant (p > 0.05).Meanwhile, the determination coefficient (R 2 = 0.9689) of the model indicated that only 3.11% of the total variations could not be explained by the calculated model.A low value 0.0577 of coefficient of the variation (C.V.) implied a high degree of precision and a good deal of reliability of the experimental values.This meant that the model could be used to analyze and predict selenylation process results.As Table 3 shown, F-value and p-value (F = 24.21,p = 0.0002) of the model, with no significant lack of fit at p > 0.05, indicated that the model used to fit response variables was extremely significant and adequate to represent the relationship between the response and the independent variables.
Optimization of Selenylation Conditions
Response surface methodology plays a key role in identifying the optimum values of the independent variables efficiently, under which dependent variables could achieve a maximum response.
Based on the regression equation obtained by Design-Expert 7.0 (State-Ease, Inc., Minneapolis, MN, USA), Se contents in Se-CSPS affected by X 1 , X 2 and X 3 were graphically presented by 3D response surface in Figure 2.
The response surface analysis predicted that the optimal reaction time, reaction temperature and ratio of Na 2 SeO 3 to CSPS were 7.53 h, 71.26 °C and 0.88 g/g, respectively.Meanwhile, the model predicted the optimized Se content in Se-CSPS was 5.518 mg/g.The interaction between reaction time (X 1 ) and reaction temperature (X 2 ) on the Se content in Se-CSPS was shown in Figure 2a (p = 0.0083 < 0.05, Table 2).At a definite ratio of Na 2 SeO 3 to CSPS, the Se content significantly enhanced with reaction time ranging from 6 h to 8 h and reaction temperature from 50 °C to 70 °C.Likewise, when the reaction time ranged from 6 h to 8 h and the ratio of Na 2 SeO 3 to CSPS varied between 0.6 g/g and 1.0 g/g, the Se content in Se-CSPS was increasing gradually (Figure 2b).However, the interactive effect between reaction time and the ratio of Na 2 SeO 3 to CSPS was not significant (p = 0.0685 > 0.05, Table 2).As shown in Figure 2c and Table 2, the interaction of reaction temperature (X 2 ) and ratio of Na 2 SeO 3 to CSPS (X 3 ) had a much higher effect on the selenylation (p = 0.0335 < 0.05).
Verification of Predictive Model
In order to validate the accuracy and reliability of the model equation for predicting the optimum response value, the validation experiments were performed in triplicate.Taking fully into account the actual operating convenience, the confirmatory experimental verification was tested under the following conditions: reaction time 7.5 h, reaction temperature 71 °C and ratio of Na 2 SeO 3 to CSPS 0.9 g/g.A mean value of (5.547 ± 0.09) mg/g (n = 3) was obtained from the confirmatory experiment.
The results showed that the actual value was very close to the predicted result.It also indicated the actual optimization parameters were reliable (Table 4).
Thermal Weight Loss Analysis
The results of TG and DTG of Se-CSPS and CSPS were illustrated in Figure 3. TG and DTG spectrum shapes of Se-CSPS and CSPS were similar.The first peaks of them were dehydration peak.In the second peak, both of them rapidly lost weight amounting to 50% in weight loss process.This indicated that Se-CSPS and CSPS took place violent decomposition reaction in the temperature range.
However, minor differences between Se-CSPS and Se could be seen from the curves.On the one hand, the temperature of dehydration peak (5% loss, around 85 °C) of Se-CSPS was significant lower than that of CSPS (5% loss, around 92 °C).On the other hand, in the decomposition peak (200-400 °C), the fastest weight loss temperatures of Se-CSPS and CSPS were 283.95 °C and 311.86 °C, respectively.This demonstrated that stability of Se-CSPS was lower than that of CSPS.
Particle Size Distribution
The pharmacological activity of polysaccharide depended on the particle size [25].Generally, particle with the smaller size could be easily absorbed and utilized by body.As seen from Figure 5, the average particle size of Se-CSPS (203 nm) was significant smaller than that of CSPS (266 nm) at the same concentration.The reason was that CSPS was hydrolyzed into some smaller pieces by acidic water in selenylation.It implied that absorption and utilization of Se-CSPS were better than that of CSPS in vivo and in vitro at the same dose.
MTT Assay
As Figure 7A shown, the inhibition rate of Se-CSPS significantly raised with the increase of dose.In addition, the inhibition rate of Se-CSPS also remarkably increased with the time prolongation.For example, the maximum inhibition rate of Se-CSPS at 24, 48, 72 h attained to 32.12%, 47.22% and 69.49%, respectively.The inhibition rate of Se-CSPS was in dose-dependent and time-dependent manner, and was higher than that of CSPS (Figure 7B).Additionally, CSPS with the low dose could temporarily induce cell proliferation at 24 h.The inhibition rate of doxorubicin (positive group) was shown in Figure 7C.Simultaneously, IC 50 of Se-CSPS (111.90 μg/mL) was obtained from the inhibition rate.The result indicated that the Se-CSPS could inhibit the proliferation of human gastric cancer SGC-7901 cells.This also suggested that seleno-polysaccharide obtained from selenylation could significantly increased cytotoxicity to tumor cells.(C) Doxorubicin
SP-752 UV-Vis Spectrophotometer was purchased from Shanghai Spectrum Instruments Co., Ltd., China.Pyris 6-thermal gravimetric analysis instrument was obtained from Perkin Elmer Co., Ltd., USA.Zetasizer Nano ZS90 nano-particle size and zeta potential analyzer was purchased from British Malvern Instruments Co., Ltd., UK.QUANTA 200-scanning electron microscope was purchased from FFI Co., Ltd., USA.WELLSCAN MK 3 Microplate Reader was obtained from Bio-Rad Co., Ltd., USA.
Extraction, Purification and Isolation of CSPS
The dried fruit of Capparis spionosa L. (2500 g) was ground in a blender to obtain a fine powder.The powder was extracted with 95% ethanol (5000 mL, ×3 cycles) at 90 °C for 3 h, and filtered through nylon cloth (pore diameter 38 μm).The residue was dried under reduced pressure and extracted by water bath in extraction temperature 90 °C, extraction time 120 min, ratio of water to sample 25 mL/g, extraction cycles 3. The extraction solution was separated from insoluble residue by centrifuge (4000× g for 5 min, at 20 °C), and then precipitated by dehydrated alcohol to a final concentration of 80% (v/v).The crude polysaccharide was collected by centrifuge (4000× g for 10 min, at 20 °C).Protein of crude polysaccharide was discarded by 0.1% (g/v) papain and Sevag.Pigment was treated with activated carbon and H 2 O 2 .CSPS collected by precipitation with dehydrated alcohol to the concentrations 80% (v/v) was washed with dehydrated alcohol, acetone and ligarine, vacuum dried.
Selenylation
Different ratios of Na 2 SeO 3 and CSPS were dissolved by HNO 3 (100 mL, 0.05%) in erlenmeyer flasks.Then, the mixed solution were reacted in the designed reaction time, reaction temperature and water bath shaking rate.After the reaction, Na 2 CO 3 was added into the reacted solution to adjust pH to 5-6.The solution was centrifuged to remove the insoluble residue (3000× g for 5 min, at 20 °C), and then dialyzed with dialysis membrane in running water.Ascorbic acid was used to detect whether free SeO 3 2− was dialyzed out from the solution.When ascorbic acid did not redden the solution, the Se-CSPS solution was collected, freeze-dried.
Design of Optimization
According to the results of mono-factor experiments for the Se-CSPS production, the proper ranges of reaction temperature, reaction time, ratio of Na 2 SeO 3 to polysaccharide were determined preliminarily.Three independent variables in a BBD (X 1 , reaction time; X 2 , reaction temperature; X 3 , ratio of Na 2 SeO 3 to CSPS) at three levels were performed.Table 5 showed that the range of independent variables and their levels.Table 5.Independent variables and their levels used in the response surface design.
Figure 1 .
Figure 1.Effect of reaction time, temperature, ratio of Na 2 SeO 3 to Capparis spionosa L. polysaccharide (CSPS) and water bath shaking rate on Se content.(A) reaction temperature 60 °C, ratio of Na 2 SeO 3 to CSPS 0.8 g/g, water bath shaking rate 40 r/min; (B) reaction time 7 h, ratio of Na 2 SeO 3 to CSPS 0.8 g/g, water bath shaking rate 40 r/min; (C) reaction time 7 h, reaction temperature 70 °C, water bath shaking rate 40 r/min; (D) reaction time 7 h, reaction temperature 70 °C, ratio of Na 2 SeO 3 to CSPS 0.8 g/g.
Figure 2 .
Figure 2. Response surface plots (3D) of reaction time, reaction temperature and ratio of Na 2 SeO 3 to CSPS (X 1 : reaction time; X 2 : reaction temperature; X 3 : ratio of Na 2 SeO 3 to CSPS).
Table 1 .
Box-Behnken design matrix of three variables and the experimental observed responses.
Table 2 .
Test result of significance for regression coefficients.
Table 3 .
Analysis of variance for fitted quadratic polynomial model.
Table 4 .
Predicted and experimental values of the responses under optimum conditions. | v3-fos-license |
2018-12-13T06:52:04.879Z | 2016-01-01T00:00:00.000 | 54729029 | {
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} | pes2o/s2orc | STUDYING PERIODIC ROLLING WAVES ON THE SURFACE OF A FALLING LIQUID FILM
In this paper the mathematical model describing the falling thin liquid film on a vertical wall is considered taking into account heat and mass transfer at the interface in the regime of periodic rolling waves. The families of exact and numerical generalized solutions, where periodic traveling waves conjugate through the strong and weak discontinuities with each other or with the residual thickness, are constructed. Evolution of exact periodic generalized solutions is studied in time. 1 Problem statement Heat transfer at condensation of immobile saturated vapor on a vertical surface was firstly considered in [1] for the case of the laminar flow of a condensate film. Later, theoretical and experimental studies of the film flows, including those with consideration of heat and mass transfer on the free surface, were carried out in many papers. It is shown [2] that the Kapitza waves are not the capillary, but the rolling ones. A possibility of traveling wave existence on the surface of a vertically falling liquid film without consideration of surface tension is shown in [3], and the equation, describing propagation of these waves, so-called kinematic equation, is also derived there (exact discontinuous solutions to this equation correspond qualitatively to the experimental results). In [4], the flow of a thin liquid film on a vertical wall was studied theoretically with consideration of condensation at the interface in the regime of the rolling waves. The families of discontinuous solutions were derived, where the traveling waves conjugate with each other or with a residual thickness through strong and weak discontinuities. In [5] the model that takes into account heat and mass transfer at the interface of a thin film of liquid flowing down a vertical wall in the regime of rolling waves for both cases – evaporation and condensation was studied. The full families of exact generalized solutions, which model the increasing and decreasing waves as well as the rolling waves, where the traveling waves are interfaced through strong or weak discontinuities with each other or with the “residual” thickness were constructed. Time evolution of these families of exact generalized solutions was studied. The maps of flow regimes of the liquid film on a vertical heat transfer surface were plotted. Based on the model developed in [3-5], the traveling waves on the surface of a falling liquid film were studied in [6], taking into account heat and mass transfer and surface tension. * Corresponding author: [email protected] DOI: 10.1051/ 01075 (2016) , 2016 72 7201075 HMTTSC MATEC Web of Conferences matecconf/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/). In this paper, we consider the model that takes into account heat and mass transfer at the interface of a thin film of liquid flowing down a vertical wall in the regime of periodic rolling waves for both cases – evaporation and condensation. We have constructed the exact generalized solutions, which model the increasing and decreasing waves as well as the periodic rolling waves, where the traveling waves are conjugated through strong or weak discontinuities with each other or with the “residual” thickness. The rolling wave equation was obtained in [5]
Problem statement
Heat transfer at condensation of immobile saturated vapor on a vertical surface was firstly considered in [1] for the case of the laminar flow of a condensate film.Later, theoretical and experimental studies of the film flows, including those with consideration of heat and mass transfer on the free surface, were carried out in many papers.It is shown [2] that the Kapitza waves are not the capillary, but the rolling ones.A possibility of traveling wave existence on the surface of a vertically falling liquid film without consideration of surface tension is shown in [3], and the equation, describing propagation of these waves, so-called kinematic equation, is also derived there (exact discontinuous solutions to this equation correspond qualitatively to the experimental results).In [4], the flow of a thin liquid film on a vertical wall was studied theoretically with consideration of condensation at the interface in the regime of the rolling waves.The families of discontinuous solutions were derived, where the traveling waves conjugate with each other or with a residual thickness through strong and weak discontinuities.In [5] the model that takes into account heat and mass transfer at the interface of a thin film of liquid flowing down a vertical wall in the regime of rolling waves for both casesevaporation and condensation was studied.The full families of exact generalized solutions, which model the increasing and decreasing waves as well as the rolling waves, where the traveling waves are interfaced through strong or weak discontinuities with each other or with the "residual" thickness were constructed.Time evolution of these families of exact generalized solutions was studied.The maps of flow regimes of the liquid film on a vertical heat transfer surface were plotted.Based on the model developed in [3][4][5], the traveling waves on the surface of a falling liquid film were studied in [6], taking into account heat and mass transfer and surface tension.
In this paper, we consider the model that takes into account heat and mass transfer at the interface of a thin film of liquid flowing down a vertical wall in the regime of periodic rolling waves for both casesevaporation and condensation.We have constructed the exact generalized solutions, which model the increasing and decreasing waves as well as the periodic rolling waves, where the traveling waves are conjugated through strong or weak discontinuities with each other or with the "residual" thickness.
The rolling wave equation was obtained in [5] 3 3 where h is the liquid film thickness, and the coefficients are determined in the following manner: . This is the rolling wave equation, which considers the capillary forces ( D > 0) and condensation at 0 4•10 6 J/kg.Calculation gives the values D =4.6•10 -5 , E 1.The discontinuous solutions to equation ( 1), limit at 0 D o , are the stable generalized solutions to hyperbolic equation 3 1 3 The similar equation was derived in [5].
Periodic solution of equation of the rolling wave at phase transition
The hyperbolic equation ( 2) admits exact solution 2 ( , , ) 2 describes a change in initial film thickness (0, ) condensation, the film thickness increases, and at 0 E , i.e., at evaporation, the film thickness decreases.We will derive the exact solutions, which model evolution of a single rolling wave at 0 E , i.e., at evaporation.These solutions are set by formula and they are presented [5] by increasing h and decreasing h waves, traveling with velocities V and V , conjugated with depths ( , , ) , ) through weak discontinuities on lines ( ) x a t and ( ) x c t , where ( , , ), 0 ( , ), ( , , ) 0, ( , ).
Here, 2 ( , ) / (2| |) T K E K E is time, required for complete evaporation of the residual layer with initial depth K .It follows from formulas (3)-( 4) that complete evaporation of the residual layer from the left of the rolling wave, i.e., at ( ) x a t d , occurs at ( , ) l T K E , and from the right of this wave, i.e., at ( ) We will study time evolution of infinite succession of waves of type (3) rolling in the residual layer ( , , ) H t K E , where l r K K K , in the regime of evaporation at 0 E .We assume that the initial amplitude of these waves is relatively small.To set the initial position of rolling wave succession, we consider section * * , and expand solution (3), limited in this section, to periodic solution ( , ) In the case of condensation, i.e., at 0 E ! , time evolution of infinite succession of waves of type (3) rolling in the residual layer ( , , )
Results of the calculations
The solution to Cauchy problem is presented in the figure for eight successive time points for equation (2) with periodic initial conditions (5).This solution is continuous (lines 1-3 in fig. 1
Conclusion
Here, we examine the model problem concerning the flow of a thin liquid layer on a vertical wall with consideration of heat and mass transfer at the interface in the regime of periodic rolling waves.The families of generalized periodic solutions to the rolling wave equation are presented, the evolution of exact solutions is studied. | v3-fos-license |
2020-06-25T09:09:02.220Z | 2020-06-01T00:00:00.000 | 220048223 | {
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} | pes2o/s2orc | Changes in Physical Meat Traits, Protein Solubility, and the Microstructure of Different Beef Muscles during Post-Mortem Aging
This study was performed to compare the differences in pH, myofibril fragmentation index (MFI), total protein solubility (TPS), sarcoplasmic protein solubility (SPS), myofibrillar protein solubility (MPS), and the microstructure of seven beef muscles during aging. From the six beef carcasses of Xinjiang brown cattle, a total of 252 samples from semitendinosus (ST), longissimus thoracis (LT), rhomboideus (RH), gastrocnemius (GN), infraspinatus (IN), psoas major (PM), and biceps femoris (BF) muscles were collected, portioned, and assigned to six aging periods (1, 3, 7, 9, 11, and 14 day/s) and 42 samples were used per storage period. IN muscle showed the highest pH (p < 0.05) from 1 to 14 days and the lowest TPS (p < 0.01) from 9 to 14 days with respect to the other muscles. Moreover, the changes in IN were further supported by transmission electron microscopy due to the destruction of the myofibril structure. The highest value of MFI was tested in ST muscle from 7 to 14 days. The total protein solubility in PM, RH, and GN muscles were not affected (p > 0.05) as the aging period increased. The lowest TPS was found in the RH muscle on day 1, 3, and 7 and in the IN muscle on day 9, 11, and 14. The pH showed negative correlations with the MFI, TPS, and MPS (p < 0.01). The results suggest that changes in protein solubility and muscle fiber structure are related to muscle location in the carcass during aging. These results provide new insights to optimize the processing and storage of different beef muscles and enhance our understanding of the biological characteristics of Xinjiang brown cattle muscles.
Introduction
Meat and meat products are important nutrient-intensive foods that are widely consumed worldwide [1]. For consumers, tenderness is a critical indicator that drives their willingness to repurchase and acceptability [2,3]. There are still great variations in the tenderness between different parts of beef muscles [4,5]. In the beef industry, aging is often considered to be one of the important factors determining the ultimate tenderness of the meat [6,7]. Due to the differences between the biochemical characteristics of these muscles, they may cause different degrees of response to aging [4]. Therefore, the inconsistency of the tenderness of different muscles has become the main problem that currently exists in the meat processing industry during the aging process.
Reducing the intake of fat in meat is essential for maintaining consumer health and reducing the risk of illness [8]. In fact, the type of muscle fiber is one of the factors that affects the lean meat rate [9]. The muscle fiber type differs greatly among various body parts and is generally divided into slow-oxidized fiber (type I), fast oxido-glycolytic fiber (type IIA), and fast glycolytic fibers (type IIB and type IIX) [10,11]. Studies indicated that muscle fiber type was closely associated with muscle quality and more type IIB fiber might lead to poorer muscle quality and lower lean meat productivity [12,13]. Our previous unpublished study has shown that semitendinosus (ST) and longissimus thoracis (LT) muscles of Xinjiang brown cattle have large percentages of type IIB fibers (approximately 47.89% and 40.85%, respectively), rhomboideus (RH) and gastrocnemius (GN) muscles have high percentages of type IIA fibers (approximately 61.43% and 42.10%, respectively), and infraspinatus (IN), psoas major (PM), and biceps femoris (BF) muscles have large percentages of type I fibers (79.69%, 43.06%, and 39.75%, respectively). In the current study, we focused on these seven muscles, representing the characteristics of the three muscle fiber types.
Protein in skeletal muscle is a key factor in determining the type of muscle fiber. Establishing the relationship between muscle protein structure and functional properties will contribute to the understanding of the modification mechanism of meat products during processing and storage [14]. According to the solubility and location of protein, it can be divided into myofibrillar protein, sarcoplasmic protein, and matrix protein, which maintain the structural integrity of myofibrillar fiber [15,16]. These proteins are susceptible to oxidative degradation during aging, which may explain the meat tenderization after muscle fiber breakage [17]. Oxidation affects the chemical properties of proteins and changes in protein solubility can reflect the degree of protein denaturation [18,19]. Sarcoplasmic protein solubility (SPS) is sometimes used as a measure of muscle quality [20]. It has been well documented that precipitated or denatured sarcoplasmic proteins may bind to myofibrils, leading to a decrease in water holding capacity [21][22][23]. It is vital to the production of Xinjiang brown beef, for the lives of Xinjiang people, and for local meat production. However, there are still limited studies examining the changes in protein solubility between various parts of muscles of Xinjiang brown cattle during post-mortem aging.
Therefore, the aim of this research was to evaluate the changes in pH, myofibril fragmentation index (MFI), total protein solubility (TPS), SPS and myofibrillar protein solubility (MPS), and the microstructure of seven beef muscles (ST, LT, RH, GN, IN, PM, BF) during aging from 1 to 14 days.
Animals and Muscle Samples Preparation
Six Xinjiang brown bull calves of approximately 30 months of age were slaughtered at a local slaughterhouse (Yining, Xinjiang, China) according to the commercial procedures. The mean weight at slaughter was 566 ± 32 kg. All corn-fed calves were raised on the same farm to ensure background consistency and then slaughtered using electrical stunning on the same day. The carcasses were overhung in a cold room (3 ± 1 • C) for 24 h. All procedures were undertaken following the guidelines given by the Animal Care and Ethics Committee for animal experiments, Institute of Animal Science, Chinese Academy of Agricultural Sciences.
The semitendinosus (ST), longissimus thoracis (LT), rhomboideus (RH), gastrocnemius (GN), infraspinatus (IN), psoas major (PM), and biceps femoris (BF) muscles were collected from each carcass. The samples were kept on ice and transported to the laboratory after 50 min and all visible intermuscular and subcutaneous fat was removed. Prior to packaging, each sample was stamped with a date mark. Each muscle was further separated into three equal-length sections, resulting in six muscle Sections (5 cm × 3 cm × 2 cm, 50 ± 0.05 g) per carcass, with 42 slices per aging period (six replications per muscle and per aging period). Afterwards, the samples were individually vacuum-packaged in a polyolefin bag and randomly assigned to aging at 3 ± 1 • C (relative humidity: 70-80%) for 1, 3, 7, 9, 11, and 14 days (within muscles, samples from the same carcass were not aged for the same time). Upon completion of each postmortem aging period, samples were taken to determine pH and transmission electron microscopy (TEM). The remaining samples were stored at −30 • C for further analysis within 2 weeks.
pH Measurement
The pH value of the beef muscles was measured at 1, 3, 7, 9, 11, and 14 days postmortem following the method described by Kim et al. [24], with minor modifications. Chopped meat (10 g) was mixed with 100 mL of distilled water for 15 s and then homogenized using an Ultra-Turrax T25 homogenizer (IKA-Werke, Gmbh & Co., Staufen in Breisgau, Germany) at 2800 g. The pH of the homogenate was measured using a pH meter equipped with an electrode (PB-10, the precision was 0.01, made by Sartorius Group, Goettingen, Germany). Each sample was determined three times.
Myofibril Fragmentation Index (MFI)
MFI was determined by modification of the method by Hou et al. [25]. Two gram samples were homogenized with a homogenizer (FJ200-S, Shanghai Specimen Model Co., Shanghai, China) at 10,000 g for 60 s (3 × 20 s with a 60 s break between bursts) at 4 ± 2 • C in 20 mL ice-cold buffer (100 mM KCl, 20 mM K 2 HPO 4 , 1 mM EGTA, 1 mM MgCl 2 , and 1 mM NaN 3 , pH 7.0). The homogenates were centrifuged using a LG10-24A model centrifuge (Peking Medical centrifuge Factory, Beijing, China) at 3000 g for 15 min at 4 • C and the supernatant was discarded. The pellets were homogenized in 20 mL of homogenizing buffer and centrifuged and the supernatant was discarded again. The resulting pellets were then resuspended in 5 mL of homogenizing buffer and filtered through a polyethylene strainer (200-mesh) to remove the fat and connective tissue. Then, 5 mL buffer was used to promote the passage of myofibrils through the strainer. The protein concentration of suspension was determined by the biuret method [26]. The protein concentration was diluted to 0.5 mg/mL and measured spectrophotometrically at 540 nm (UV 6100, Metash, Shanghai, China). MFI was calculated by multiplying A540 by 200.
Protein Solubility
The total protein solubility (TPS) and sarcoplasmic protein solubility (SPS) were measured using the procedure of Li et al. [27]. A 1 g sample of each muscle (six replications per muscle and per aging period) was homogenized with 10 mL cold 0.025 M potassium phosphate buffer (pH 7.2), which was then shaken at 4 • C for 12 h and centrifuged at 1500 g for 20 min. The total protein concentrations of the supernatants were assessed using the biuret method [26]. The step of SPS detection was that the muscle sample (1 g) was homogenized in ice-cold 20 mL 0.025 M potassium phosphate buffer (pH 7.2) and then carried out using the same shaking, homogenization, and centrifugation procedures mentioned above. Myofibrillar protein solubility (MPS) was calculated by TPS minus SPS. The value was expressed as micrograms of soluble protein per gram of meat.
Microstructure Analysis
At each storage time point, strips (approximately 5 × 1 × 1 mm) were cut off from three muscles (ST, RH, and IN) and were selected using transmission electron microscopy (TEM). Muscle samples were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) and 2% osmium tetroxide. Then, the gradually increasing concentrations of ethanol (50-100%, v/v) were used for dehydration. The solvent was then switched to acetone and the samples were dehydrated with acetone gradient, followed by replacing with propylene oxide and being embedded with resin. The samples were trimmed, sectioned (50-70 nm), and stained with uranyl acetate and lead citrate. The samples were viewed under TEM (Hitachi, H-7500, Japan) at required magnification as per the standard procedures.
Statistical Analysis
The experimental design was a randomized complete block design, where each carcass (n = 6) served as a block. Each measurement was performed in triplicate. To determine the significant effect by two factors (muscles and aging period), data analysis was performed using the SAS 9.2 program (SAS Institute, Cary, NC, USA) with muscles and aging period as the main effects, using two-way analysis of variance (ANOVA). Analysis of variance was performed on all the variables using the General Linear Model (GLM) procedure. Duncan's multiple range test (p < 0.05) was used to determine the significance of the differences in the mean values for different samples. Pearson's correlation coefficients were calculated for variables.
Changes in pH Value
The changes in pH values of beef muscles during aging are shown in Table 1. There was a considerable variation in pH between muscles. Compared to the IN muscle, the pH values in ST, LT, PM, and BF were lower at 1 day (p < 0.05). The difference in pH may be due to the rate of glycolysis between muscles [28]. Similar results were obtained in some studies of beef and lamb [29,30], indicating that the rate of decrease in the pH of PM accelerated. Aging had no significant impact on pH in PM muscles (p > 0.05) from 1 to 14 days. Except for IN and PM, no significant changes were observed (p > 0.05) in the pH of the remaining six muscles from 3 to 14 days of aging. In terms of value, the pH of IN showed the highest value (p < 0.05) of all the muscles in the whole aging period. IN had more type I muscle fibers and its main characteristics were higher oxidative metabolism and lower glycogen content [31]. Therefore, in this study, we can speculate that lower glycolysis capacity would result in a higher pH of IN muscle, which might influence proteolysis.
Changes in Myofibril Fragmentation Index (MFI)
Changes in MFI of different beef muscles during aging are displayed in Table 2. The highest MFI was observed in the RH muscle at 1 day (p < 0.05). The ST muscle had the highest MFI from day 7 onwards (p < 0.05). Compared with the other muscles, the MFI values of IN and LT were found to be lower from 3 to 11 days (p < 0.05). MFI is used as an important indicator of I band rupture and interstitial fibril connection breakage [32]. These results seem to indicate that the degree of rupture of muscle fibers in the I band is greater in ST muscles during aging, resulting in changes in the integrity and solubility of myofibrillar proteins [33,34]. The MFI of each individual muscle increased significantly with the aging period (p < 0.05) and this is in agreement with Li et al. [35]. However, the difference in MFI change was very small in the later days of the aging period. This might be due to the fact that as the aging period increases, the degree of variation in myofibril breaks becomes smaller [36].
Changes in Protein Solubility
The results of the changes in TPS, SPS, and MPS during aging are shown in Table 3. No significant changes (p > 0.05) were evidenced in TPS of RH, GN, and PM muscles and in MPS of IN and BF muscles during aging. In regard to SPS, there were no significant differences in SPS between different muscles at day 11 and 14 (p > 0.05). IN muscle exhibited a lower TPS at day 9, 11, and 14 than that at day 7 (p < 0.05). LT muscles had the highest TPS and MPS among all muscles at day 14 (p < 0.05). The SPS of RH was significantly lower than that of IN and GN at day 7 and 9 (p < 0.05). Protein solubility was an important indicator of protein properties, which was attributed to protein denaturation [37]. In this study, the results indicated that IN muscle had a high extent of protein denaturation after 9 days [19]. The solubility of myofibrillar protein increased with the aging period up to day 7 and then did not change significantly. This is related to the characteristics of myofibrillar proteins [38,39]. During aging, myofibrillar protein bonds were weakened and more protein hydrolysis was released, which resulted in higher MPS [40]. However, myofibrillar proteins are gradually unfolded as the aging period increases, exposing hydrophobic groups and resulting in almost no change in MPS [41].
Pearson Correlations
Correlation coefficients among pH, MFI, TPS, SPS, and MPS of all muscles during aging are presented in Table 4. MFI, TPS, and MPS are negatively correlated with pH (p < 0.01). MFI is positively correlated with MPS (p < 0.05), whereas no correlations with TPS and SPS are observed (p > 0.05). The correlation coefficients of TPS with MPS are higher than those of SPS, indicating that TPS was affected to a larger extent by the denaturation of MPS than that of SPS. These results suggest that pH is a more important determinant for protein solubility than MFI in this study. Moreover, the correlation can further explain the results of protein solubility and confirm that the variation in protein solubility of different muscles may be due to pH changes.
Changes in Muscle Microstructure
The changes in the microstructure of muscle (ST, RH, and IN) by TEM are shown in Figure 1. In ST, RH, and IN muscles, the sarcoplasmic reticulum around the sarcomeres can be clearly distinguished and the myofibrils are tightly combined with the visible I-band and A-band and the Z-disk and M-line can be differentiated on day 1 and day 3 (Figure 1(a1,a2,b1,b2,c1,c2)). After day 7, the overall integrity of the myofibrils diminishes (Figure 1(a3,b3,c3)). The M-line located on the centre of the A-band looks vague and the Z-disk and I-band junctions are weakened after day 9. The Z-disk is distorted but undamaged, although some longitudinal splits are evident in RH muscles (Figure 1(b4,b5)). The worst myofibrillar structure is observed in IN muscle, in which the overlapping structure of thick and thin filaments is destroyed, the skeletal muscle structure is severely broken, and the Z-disk is distorted and weakened (Figure 1(c4,c5)). At day 14, IN muscle shows fractures in the Z-disk, as well as fragmentation at the junction of the I-band and Z-disk (Figure 1(c6)). Pan and Yeh [42] found that cracking of muscle fibers and shortening of sarcomere could largely reduce the tenderness of the meat. Aside from mechanical damage, muscle fiber structure was also affected by endogenous proteases during aging [43,44]. Moczkowska et al. [45] had reported that BF muscles were more sensitive to oxidation than LL muscles and the extent of this phenomenon depends on the type of muscle examined. In this study, TEM results showed that IN muscle had a longer sarcomere length and a greater degree of rupture in the muscle fiber structure, as expected because this muscle has a higher proportion of type I fiber (approximately 79.69%), which predicted that the aging rate would be faster. These results are in accordance with the MFI described above. On the other hand, previous research shows that the concentration of coenzyme Q10, carnosine, and taurine in IN muscle was higher than that in LT muscle [46], implying a higher concentration of functional bioactive compounds in oxidized muscle. These results suggest that oxidized muscle accelerates the improvement of meat tenderness.
Conclusions
In this study, it can be concluded that the meat characteristics of different beef muscles responded differently to the storage time and were significantly affected by carcass position. The meat quality parameters of IN muscle were improved, which was due to its higher pH value, lower MFI and TPS, and greater degree of myofibril rupture, followed by ST and RH. However, there is
Conclusions
In this study, it can be concluded that the meat characteristics of different beef muscles responded differently to the storage time and were significantly affected by carcass position. The meat quality parameters of IN muscle were improved, which was due to its higher pH value, lower MFI and TPS, and greater degree of myofibril rupture, followed by ST and RH. However, there is little difference in meat quality parameters among other muscles. Therefore, for IN, it can be suggested that the characteristics are particularly suitable for producing high-quality beef products. Additionally, a significant interaction between pH and protein solubility was observed. Further research should be conducted to explore the mechanism of how pH affects protein solubility (for example, to assess its effect on cathepsin activity). | v3-fos-license |
2016-05-12T22:15:10.714Z | 2012-03-31T00:00:00.000 | 208941814 | {
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} | pes2o/s2orc | 2-Chlorobenzohydrazide
The asymmetric unit of the the title compound, C7H7ClN2O, contains two molecules in which the chlorophenyl and the formic hydrazide units are almost planar (r.m.s. deviations of 0.0081 and 0.0100 Å, respectively, in one molecule and 0.0069 and 0.0150 Å in the other) and are oriented with respect to each other at dihedral angles of 56.8 (2) and 56.9 (2)°. In the crystal, the molecules are consolidated in the form of polymeric chains extending along [010]. R 3 3(10) ring motifs exist due to N—H⋯O and N—H⋯N hydrogen bonds.
The asymmetric unit of the the title compound, C 7 H 7 ClN 2 O, contains two molecules in which the chlorophenyl and the formic hydrazide units are almost planar (r.m.s. deviations of 0.0081 and 0.0100 Å , respectively, in one molecule and 0.0069 and 0.0150 Å in the other) and are oriented with respect to each other at dihedral angles of 56.8 (2) and 56.9 (2) . In the crystal, the molecules are consolidated in the form of polymeric chains extending along [010]. R 3 3 (10) ring motifs exist due to N-HÁ Á ÁO and N-HÁ Á ÁN hydrogen bonds.
In (I), two molecules are present in the asymmetric unit, which differ slightly from each other geometrically. In one molecule, the chlorophenyl group A (C1-C6/Cl1) and the formic hydrazide moiety B (O1/C7/N1/N2) are planar with r. m. s. deviations of 0.0081Å and 0.0100 Å, respectively. The dihedral angle between A/B is 56.8 (2)°. In second molecule, the chlorophenyl group C (C8-C13/Cl2) and the formic hydrazide moiety D (O2/C14/N3/N4) are also planar with r. m. s. deviation of 0.0069Å and 0.0150 Å, respectively and the dihedral angle between C/D is 56.89 (20)°. Each molecule is connected to symmetry related neighbors through classical intermolecular H-bonding of the N-H···O or N-H···N type (Table 1, Fig. 2) with R 3 3 (10) ring motifs (Bernstein et al., 1995) to generate one-dimensional polymeric chains along the Experimental 2-Chlorobenzoic acid (3.44 g, 22 mmol) was converted to methyl 2-chlorobenzoate by refluxing in methanol (20 ml) in the presence of a catalytic amount of sulfuric acid. This ester was converted into the title compound, 2-chlorobenzoylhydrazide, by refluxing with hydrazine hydrate (80 %, 10 ml) in dry methanol using the literature procedure (Moise et al., 2009). M.p. 379-380K.
Refinement
The coordinates of the H-atoms of the NH 2 groups were refined. The remaining H atoms were positioned geometrically with (N-H = 0.86 and C-H = 0.93 Å) and refined as riding with U iso (H) = xU eq (C, N), where x = 1.2 for all H-atoms.
Figure 2
Partial packing diagram (PLATON; Spek, 2009) which shows that the molecules form polymeric chains extending along the [0 1 0] direction forming R 3 3 (10) ring motifs. Special details Geometry. Bond distances, angles etc. have been calculated using the rounded fractional coordinates. All su's are estimated from the variances of the (full) variance-covariance matrix. The cell e.s.d.'s are taken into account in the estimation of distances, angles and torsion angles Refinement. Refinement of F 2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F 2 , conventional R-factors R are based on F, with F set to zero for negative F 2 . The threshold expression of F 2 > σ(F 2 ) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F 2 are statistically about twice as large as those based on F, and R-factors based on ALL data will be even larger.
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å 2 )
x y z U iso */U eq | v3-fos-license |
2017-09-15T21:36:04.402Z | 2010-01-01T00:00:00.000 | 98253702 | {
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} | pes2o/s2orc | Chiral Auxiliary-Mediated Enantioenrichment of ( ± )-Ibuprofen , under Steglich conditions , with Secondary Alcohols derived from ( R )-Carvone
A síntese de uma série de álcoois secundários derivados da (R)-carvona, assim como o curso estereoquímico da esterificação destes derivados com (±)-ibuprofeno é apresentada. O composto comercial racêmico foi transformado nos respectivos ésteres diastereoisoméricos através do acoplamento mediado por DCC/DMAP, fornecendo o par de diastereoisômeros derivados de (S)ou (R)-ibuprofeno em relação até 5.7:1, dependendo do tipo de auxiliar quiral empregado.
Introduction
During the past two decades, the preparation of optically active drugs has received considerable attention both, from academics and industry.The reasons behind this interest are threefold, including the medical benefit from using a single chemical entity, the changing pharmaceutical regulations, which now require the development of optically active drugs as single stereoisomers, and also advances in strategies for the synthesis of optically pure compounds, which facilitated the task. 1 Ibuprofen (1, Figure 1) is a non-steroidal antiinflammatory agent of the class of the a-arylpropionic acids, which also includes flurbiprofen (2, Figure 1), fenoprofen (3, Figure 1), ketoprofen (4, Figure 1) and naproxen (5, Figure 1), amongst its most prominent members.The drug is marketed mostly as the racemate despite that it has been shown that the (S)-enantiomer is the main responsible for the therapeutic effect.The pure enantiomer is also available, being prescribed only for certain specific conditions. 2t has been demonstrated that (R)-ibuprofen as well as its congeners can be stereoselectively interconverted in vivo into their active enantiomers through a thioester-mediated epimerization process which involves the Co-enzyme A (CoA). 3 However, these acyl-CoA thioester derivatives are chemically reactive and may become involved in a transacylation reactions with endogenous nucleophiles, leading to covalent binding of the drug to proteins, the clinical consequences of which relate to the toxicity of this pharmaceutically active ingredient. 4It is also suspected that the acyl-CoA intermediate derived from (R)-ibuprofen may be responsible for the formation of mixed glyceryl esters, which once deposited in fatty tissue could cause unknown long-term effects, transforming (R)-ibuprofen into a pharmacological uncertainty. 5herefore, the selective preparation of (S)-ibuprofen has been the subject of intense research and numerous stereoselective chemical approaches have been explored for that purpose. 6In addition, resolutions of the racemate by means of crystallization of diastereomeric salts, 7 as well as chemoenzimatic transformations including esterificacion, trans-esterification, 8 enantioselective hydrolysis of esters, 9 amides 10 and nitriles 11 and other enzyme-mediated strategies, 12 and a yeast-mediated selective degradation of the unwanted enantiomer, 13 have also been reported.
Carvone is a naturally-occurring cyclohexylic terpenoid which is isolated from Carum carvi, Anethum graveolans and Mentha spicata, being available in both of its enantiomeric forms.It has been extensively employed as building block in organic synthesis. 14Interestingly, however, the use of carvone derivatives as chiral auxiliaries has only few and scattered precedents. 15he chiral auxiliary-mediated dynamic kinetic resolution of a-substituted carboxylic acids has been recently recognized as an approach towards the preparation of enantioenriched a-aminoacids, as well as their a-mercapto-, a-haloand a-hydroxycongeners.This is an effective alternative, provided configurational lability can be induced at the stereogenic a-carbonyl center. 16ecently, Amoroso and coworkers 17,18 reported the preparation of different a-arylpropionic acids in their enantioenriched forms, by using lactamide-type chiral auxiliaries.The diastereomeric mixtures of the corresponding lactamic esters were obtained in good yields and reasonable diastereoselectivities depending upon the solvent and chiral auxiliary used.Therefore, here we wish to report the synthesis of chiral secondary alcohols derived from (R)-carvone (6, Figure 1) and their reaction with racemic ibuprofen under Steglich conditions, 19 as a stereodivergent entry to enantioenriched mixtures of esters derived from either (S)-or (R)-ibuprofen, depending on the type of chiral auxiliary employed.
Results and Discussion
Three series of secondary alcohols were prepared.In the first one, (R)-carvone was submitted to a conjugate addition reaction with thiophenol under thermodynamic conditions, 20 furnishing thioethers (7a-c, Scheme 1) in 4, 34 and 16% yield, respectively.On the other hand, reaction with PhSH and 2-naphthalenethiol under kinetic 21 conditions afforded sulfides (7a and 8, Scheme 1) in 60 and 92% yield, respectively.
The stereochemistry of these products was deduced by comparison with literature data, 22 analysis of the enhancement of signals in nuclear Overhauser effect (nOe) experiments and examination of their 1 H NMR spectra, which revealed that H-3 was considerably more deshielded in 7a (d 3.89 ppm) than in 7b (d 2.91 ppm) and 7c (d 3.29 ppm), pointing out to a pseudo-axial orientation of the heteroatomic substituent.The preferential generation of 7a and 8 under kinetic conditions is consistent with an axial Michael addition of the thiols from the a-face of carvone, driven by stereoelectronic effects, and subsequent protonation of the resulting enolate from the opposite and less hindered b-face.Compounds 7b and 7c may result from the base-mediated equilibration of 7a under the reaction conditions. 23iastereoselective reduction of ketones 7a, 7b and 8 with K-Selectride in anhydrous THF, 24 from the less encumbered b-face of their corresponding carbonyl moiety, 25 provided alcohols 9a, 9b and 10 (Scheme 1) in 58, 66 and 56%, respectively.
For the second series, where the aromatic substituent is directly attached to a cyclohexenic six-membered ring, (R)-carvone was subjected to the direct addition of three different Grignard reagents.The procedure stereoselectively furnished alcohols 15a-c (Scheme 2), as result of an antiperiplanar attack of the Grignard reagent with respect to the isopropenyl group. 27In turn, these tertiary allylic alcohols were treated with PCC/Al 2 O 3 , producing an oxidative allylic transposition 28 to the corresponding 3-phenyl-(S)-carvone derivatives 16a-c (Scheme 2) in yields ranging from 71 to 86%. 29 Figure 1.Chemical structures of (±)-ibuprofen (1), other relevant a-arylpropionic acid type non-steroidal anti-inflammatory agents (2-5) and R-carvone (6).Vol.21, No. 6, 2010 These ketones were stereoselectively reduced with sodium borohydride in a MeOH-THF mixture, furnishing the secondary allylic alcohols 17a-c (Scheme 2) in 83-94% yield.Their configuration was determined as 1S,5S based on literature precedents 30 and the results of a nOe experiment, where signal enhancement of H-5 was observed upon irradiation of H-1.
Me
The third group of chiral alcohols was obtained after subjecting (R)-carvone to conjugate addition reactions with aryl and 1-naphthyl Grignard reagents in the presence of copper(I) iodide 31 to which TMSCl was added in order to ensure efficient transformations. 32ese gave the corresponding silyl enol ether intermediates 18a and 18b (Scheme 3) in high yields, as single diastereomers to which the (3S,5R)-configuration was assigned on the basis of literature precedents, 33 an exhaustive NMR analysis of 18b and knowledge of the role of the isopropenyl group in determining the stereochemistry of the conjugate addition product.The stereochemical result of this reaction is consistent with the outcome of the reductions of ketones 16a-c, resulting from a nucleophilic attack anti-periplanar to the bulky isopropenyl group.
In turn, these were desilylated by careful treatment with TBAF in Et 2 O in order to avoid the production of diastereomeric mixtures at the stereocenter adjacent to the carbonyl, due to the base-catalyzed epimerization of the initial product. 34In this process, protonation of the trapped intermediate enolate from the less hindered face anti-periplanar to the C-5 substituent, furnished ketones 19a and 19b (Scheme 3) in 96 and 90% yields, respectively.Finally, ketones 19a,b were stereoselectively reduced with K-selectride to the corresponding secondary alcohols 20a and 20b (Scheme 3) in 80% yield.Interestingly, the stereochemical outcome of the reduction was different from that previously observed with the sulfur-containing alcohols and could be attributed to a different preferred conformation of the starting ketones, where the bulky aryl substituents are located equatorially, flipping the isopropenyl moiety to a pseudo-axial position, thus hindering the approach of the reducing agent from the side of the isopropenyl group.
This speculation was confirmed by nOe experiments on 19a and 19b, which revealed that irradiation of the methylene carbon of the isopropenyl moiety excerted signal enhancement of H-3 (Figure 2).On the other hand, detection of H-3 signal enhancement in 20a upon irradiation of the C-2 methyl group and also of H-9b confirmed that they were located on the same side of the molecule.Furthermore, the axial nature of the hydroxyl group was established from the values of the coupling constants between H-1 and its neighbours in its 1 H NMR spectrum (4.5 and 5.9 Hz) and its lack of nOe signal enhancement of H-1 upon irradiation of H-9b.
The performance of the chiral auxiliaries was evaluated through their submission to esterification reactions with (±)-ibuprofen, under the conditions described by Steglich, 19 which employ the DCC-DMAP reagent system as condensing agent.
As shown in Table 1, alcohol 9a provided up to a 28:72 mixture of diastereomeric esters in 89% combined yield, when the reaction was carried out in CHCl 3 at -50 ºC (entry 1).Furthermore, when the bulkier 2-naphthyl thioether 10 was used as chiral auxiliary, a slight improvement in the diastereomeric ratio (26:74) was observed, at the expense of a slight decrease in yield of product (entry 10).On the other hand alcohol 9b, a diastereomer of 9a, furnished essentially equimolecular mixtures of diastereomeric esters (entries 4-9), according to NMR and HPLC peak area integration, regardless the solvent and temperature conditions employed.
In an attempt to improve the product diastereomeric ratio, the reactions with the related sulfoxide 13 and sulfone 14 were explored.However, essentially no chiral induction was observed among the resulting esters (entries 11-14).
Chiral auxiliaries 17a-c gave diastereomeric ratios nearing 2:1 (Table 2), always favouring the same ester which as discussed below, was demonstrated to be that derived from (S)-ibuprofen.The best results in terms of chemical and optical yields were obtained with alcohol 17a, when subjected to esterification in toluene at -20 ºC (entry 3).
When chiral alcohols 20a and 20b were subjected to esterification with (±)-ibuprofen under Steglich conditions (Table 3), it was observed that the best results were obtained when chloroform was employed as solvent (entries 1, 2 and 13) and that the product was enantioenriched in the ester derived from (R)-ibuprofen.Comparison with esters prepared with pure (S)-ibuprofen indicated that the major diastereomer is that derived from (R)-ibuprofen.Interestingly, slightly improved diastereomeric ratios were obtained when a five-fold excess of ibuprofen was employed, while changing the order of addition of the reagents, preincubation of a mixture of the acid and the alcohol with activated 4Å molecular sieves and the addition of triethylamine (entries 3-5) did not improve the diastereomeric ratios of ester products.
In addition, a trend was observed, indicating that the bulkier 1-naphthyl derivative 20b slightly outperformed its congener 20a, probably by leading to a chiral auxiliary with a conformationally more rigid cyclohexyl ring.Under the typical conditions, a ratio of products up to 82:18 was observed (entry 13).These levels of enantioenrichment are similar to those previously informed by the group of Amoroso. 17or the sake of comparison, the effect of placing a bulkier group near the secondary alcohol was examined with (-)menthol (31, Scheme 4), the esterification of which with (±)-ibuprofen in CHCl 3 at 0ºC gave 55% of a nearly 1:1 mixture of diasteromeric esters 32 (Scheme 4).
The group of Amoroso has demonstrated that ibuprofen and other a-substituted carboxylic acids undergo a dynamic kinetic resolution process when subjected to the Steglich esterification. 19As shown in Scheme 5, when an a-substituted carboxylic acid (I) reacts with DCC and DMAP, enantiomeric acyl-DMAP derivatives II and III are formed as intermediates.Under these conditions, it has been proposed that the stereogenic center becomes labile and the enantiomeric acyl-DMAP derivatives are thus capable of being interconverted.When a chiral alcohol (R*OH) is added to the reaction medium, both acyl-DMAP intermediates can react to furnish diastereomeric ester products (IV and V).Two main conditions are required for a successful dynamic kinetic resolution.One of them is that interconversion between the acyl-DMAP derivatives II and III should proceed at a faster rate than formation of the ester products and the other is that the acyl-DMAP intermediates should react at very different rates with the chiral auxiliary.
In order to gain further understanding on the studied esterification, a series of esterifications were carried out with pure (S)-ibuprofen.After examination of the products, it was observed that when the reaction was carried out with alcohol 20a the resulting major diastereomers where those carrying the (R)-configuration on the ibuprofen a-carbonyl stereocenter.On the contrary, when the esterifications where carried out with alcohols 17a-c, the prevailing diastereomers were those derived from (S)-ibuprofen (Tables 1-3).However, composition of the mixtures was different from those arising from the racemic acid, pointing out to poor compliance of the studied transformations with the requirements for a successful dynamic kinetic resolution.Particularly, the experiments revealed that interconversion of the enantiomeric acyl-DMAP intermediates is not fast enough, compared with their reaction with the chiral auxiliary, to grant a more optically efficient transformation.
In conclusion, it was demonstrated that secondary alcohols derived from naturally-occurring (R)-carvone are able to provide good yields of enantioenriched mixtures of ibuprofen esters, that their performance is similar to that of other chiral alcohols and that depending on the chosen chiral auxiliary, production of (R)-or (S)-ibuprofen derived esters may be favoured.
Experimental
Melting points were taken on an Ernst Leitz Wetzlar model 350 hot-stage microscope and are reported uncorrected.Specific rotation data were obtained with a Jasco DIP 1000 photopolarimeter, fitted with 1 dm cells.FT-IR spectra were determined employing a Shimadzu Prestige 21 spectrophotometer as solid dispersions in KBr disks, or as thin films held between NaCl cells.The 1 H and 13 C NMR spectra were acquired in CDCl 3 in a Bruker Avance spectrometer (300.13 and 75.48 MHz for 1 H and 13 C, respectively), with tetramethylsilane (TMS) as internal standard.The chemical shifts are reported in ppm downfield from TMS and coupling constants (J) are given in Hertz.DEPT 135 and DEPT 90 experiments aided the interpretation and assignment of the fully decoupled 13 C NMR spectra.In special cases, 2D-NMR (COSY, HMBC, HMQC and J-resolved spectra) and selective nOe experiments were also employed.Pairs of signals marked with "#", " ‡" or with an asterisk, "*", as superscripts indicate that their assignments may be exchanged.
In the conventional work-up procedure, the reaction was diluted with brine (5-10 mL) and the products were extracted with EtOAc (4-5 × 20 mL).The combined organic extracts were then washed once with brine (5 mL), dried over Na 2 SO 4 and concentrated under reduced pressure.The residue was submitted to flash column chromatography with silica gel 60 H. Elution was carried out with hexane-EtOAc mixtures, under positive pressure and employing gradient of solvent polarity techniques.
All new compounds gave single spots on TLC plates run in different hexane-EtOAc and CH 2 Cl 2 -toluene solvent systems.Chromatographic spots were detected by exposure to UV light (254 nm), followed by spraying with ethanolic ninhydrin (amines) or with ethanolic p-anisaldehyde/ sulfuric acid reagent and careful heating of the plates for improving selectivity.
The diastereomeric esters of ibuprofen were best separated on a Varian ProStar liquid chromatograph fitted with a 250 × 4.6 mm C-18 Luna column (Phenomenex, 5 mm particle size), using a 80:20 mixture of MeOH:50 mmol L -1 phosphate buffer, pH 5.5 as mobile phase, pumped at 1 mL min -1 .Detection wavelength was 254 nm.
S , 3 R , 5 R ) -5 -I s o p r o p e n y l --m e t h y l -3phenylcyclohexanone 19a:
CuI (500 mg, 2.64 mmol) was added to a stirred 1.18M THF solution of PhMgBr (8 mL, 5.28 mmol), cooled to 0 ºC.After stirring 15 min, a solution of (R)-carvone (209 mg, 1.39 mmol) and TMSCl (772 mg, 7.1 mmol) in THF (2 mL) was introduced dropwise and the reaction was further stirred 45 min at 0 ºC.Then, the mixture was treated with saturated NH 4 Cl and the reaction products were extracted with EtOAc (4 × 50 mL).The combined organic extracts were washed with brine (10 mL), dried (Na 2 SO 4 ) and concentrated under reduced pressure.The resulting residue was filtered through a short plug of silica gel, furnishing 18a (380 mg, 96%), as a yellowish oil.Without further purification, a stirred solution of the silyl ether 18a (100 mg, 0.35 mmol) in Et 2 O (10 mL) was cooled to 0 ºC and treated with 1M TBAF in THF (0.42 mL, 0.42 mmol).After 2 h at this temperature, the volatiles were removed under reduced pressure and the residue was chromatographed affording ketone 19a (72 mg, 95%), as a pale yellow oil.[a] D 25 -55.
Scheme 5 .
Scheme 5. Proposed mechanism of the DCC-DMAP mediated dynamic kinetic resolution of a-substituted carboxylic acids.
Table 2 .
Esterification a Deduced by comparison with esters prepared with pure (S)-ibuprofen.
1 H enone 16c: 18% PCC on Al 2 O 3 (1.09g, 0.91 mmol) was added portionwise to a solution of alcohol 15c (98 mg, 0.38 mmol) in anhydrous CH 2 Cl 2 (15 mL), and the resulting suspension was stirred 1 day at room temperature.The solids were separated by filtration through Celite and the filter was washed with CH 2 Cl 2 (3 × 10 mL). | v3-fos-license |
2020-08-06T09:09:18.561Z | 2020-06-30T00:00:00.000 | 225649150 | {
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} | pes2o/s2orc | Cytotoxicity Assay of 2,4-Dihydroxide-4’-Methoxychalcone Against Cervical (HeLa) Cancer Cell by MTT Assay
Chalcone is one of the phenolic group secondary metabolic with numerous biological activities. Many studies have shown that chalcone derivatives compound has anti-cancer, anti-inflammatory, antimalarial, and antibacterial activities. The purpose of this research was to study the prediction potency unsaturated carbonyl system of chalcone derivative against HeLa cell by MTT assay. Those activities assumed can inhibit the mechanism action of NF-kB that caused cervical cancer. The 2,4-dihydroxide-4’methoxychalcone has done synthesis as a target compound by a sonochemical for 7 hours. The results showed that chalcone derivative most active against the HeLa cell.
Introduction
Cancer is a disease caused by abnormal and uncontrolled cell growth (Anwar et al., 2018). In its development, cancer cells can spread to other parts of the body and end in death. In general, the cause of cancer cannot be ascertained. Various factors both genetic and environmental factors (Shanmugam et al., 2016) have a certain contribution to increasing the risk of cancer. Various types of cancer, including breast cancer and cervical cancer, are contributors to sufficient mortality, tragically, cervical cancer is one of the most cancer that diagnosed among woman (Bak et al., 2016), however, new drug and new technologies or all-combined have been allowed as solve a problem (Bedell et al., 2020).
Treatment efforts in terms of dealing with cancer certainly continue. Given the predicted increase in the surge in cancer patients by 300 percent worldwide in 2030 with a quantity of 70 percent in developing countries including Indonesia. Based on the Ministry of Health data, the prevalence of cancer can reach 4.3 to 1,000 people. For 2020, about 13,800 new cases of cervical cancer diagnosed in women between 35 until 44 ages year old and 4,290 women die caused cervical cancer based on The American Cancer Society's estimates. Chalcone as flavonoids classes of plant-derived compounds containing than 4000 secondary metabolites (Syed et al., 2016) which several biological effects, in recent years, have biochemical and pharmacological as well as anti-cancer (Alper et al., 2019). Flavonoids as a natural product such as oxorylin A, wogoni, and vitexin were reported for the treatment of cancer diseases and undergoing phase I/II clinical trials in China (Liu et al., 2020). The conjugation, hydroxylation, and substitutions in the chemical structure of chalcone derivates may give significant biological activities and flavonoids classes were bearing hydroxyl group and double bond of ring recently designed synthesized as biochemical properties as against cancer (Hoang et al., 2015).
Various treatments for many types of cancer, chemotherapy surgery, radiotherapy, or combinations, however, multidrug resistance happens in some cases. The development of another new drug for design and discovery needs to solve the problem (Abbas et al., 2019). Chalcone is an important anti-cancer agent that discovery in the synthesized drug-using mechanism of cytotoxic activity of chalcone included by apoptosis, cell cycle disruption, inhibition of tubulin polymerization, blockade of nuclear factor-kappa B and inhibition of kinases for cervical cancer cell (Abbas et al., 2019). Nuclear factor-kappa B (NF-kB) one of the transcription factors as a molecular target that critical factors and important studies intervention biological, pharmacological and development new drug since they involved in the control of immune responses that causes cancer diseases (Kaneko et al., 2019).
The aim of the study was to predicted and investigate of the compound that obtained by hydroxyl and unsaturated carbonyl system in chemical structure in term of anti-cancer by blockade nuclear factor-kappa B (NF-kB) that the first classical pathway (Escárcega et al., 2007) induced genes. This general structure of synthesized is shown in Figure 1. and cytotoxic properties shown by the MTT assay data as a cytotoxic assay that used directly (Abel & Baird, 2018) to determine cell viability.
In Vitro anti-cancer, HeLa cells (Luong et al., 2017) (human cervical cancer) in this research were culture in Roswell Park Memorial Institute (RPMI) from Parasitology Laboratory, Universitas Gadjah Mada. The procedure was started in RPMI, 100 mL of cell suspension containing 106 cells was added into microplate 96 each well and incubated for 24 h at 37 0 C and 5% CO2. A test chalcone compound solution was inserted into a cell suspension that had been incubated previously. Added 100 mL of RPMI and cancer cells in wells as media and cell control. After the incubation, the culture media was removed and washed by PBS, then MTT solution of 100 mL (9.5 mL of RPMI and 1 mL of MTT solution) was inserted into wells and incubated again for 4 h with the same condition. 100 mL of 10% SDS in 0,1 N HCl as stopper reaction added into each of wells.
Next, the wells left at room temperature overnight were wrapped by paper tightly. Then, absorbance reading by ELISA reader at 595 nm (wavelength optimum) and Percentage of survival cells were determined and calculated by eq. 1. The compound of 2,4-dihydroxide-4'-methoxychalcone has been synthesized by (Suryani et al., 2019) with yield product 98 %, respectively. Sonochemical method using to achieve optimal yield and this method an alternative to a short time reaction of synthesis. Methanol as a solvent (Figure 1) for synthesis growth a yield as good as possible and evaporated easily. The result showed that the sonochemical method was efficient (Monsef et al., 2020) since the conventional method needs a long time reaction for 24-72 hours. Figure 1. General one-pot synthesis method with reagents; 1) acetophenone, 2) benzaldehyde and product; 3) 2,4-dihydroxide-4'-methoxychalcone Chalcone compound was identified by spectral data IR, EI-MS, 1 H, and 13 C-NMR (Suryani et al., 2019). By 1 H-NMR, H with 7,44 ppm and H 7,86 ppm with doublet peak (J = 15 Hz) described the unsaturated carbonyl of chalcone were performed. That peak with chemical shift was specific for unsaturated carbonyl of chalcone. Furthermore the chemical shift of H presenced downfield since resonance by electronwithdrawing side with H .
Hence we assumed that activity in vitro of chalcone against a panel of human cervical cell lines (HeLa) by applying 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) assay as the most popular assessment of viability and cytotoxicity (Pascua-Maestro et al., 2018). Chalcone has an active compound as inhibition of cell growth with IC50 value less than 20 g/mL, moderate active with IC50 value 20-100 g /mL and not active in the inhibition of cell growth if IC50 value than 100 g /mL (Anwar et al., 2018). Based on IC50 references, chalcone synthesized has moderate activity with IC50 (percentage at which 50% of HeLa cells are dead) value shown in Table 1, but that activity of the compound is not selective because of that activity against normal cell (Vero) by 42,29 g /mL with the low selectivity index 0,57.
Discussion
Chalcone compound, 2,4-dihydroxide-4'-methoxychalcone was prepared by the Claisen-Schmidt according to Scheme 1. To obtain chalcone in good yield, the residues were treated with 10 % in methanol. The synthesis compound was evaluated for its activity as anti-cancer by showed IC50 value may be influenced to such factors like the nature of substitutions in the ring of chalcone, the genetic and biochemical specific compound (Solomon & Lee, 2012).
Based on Table 1, it showed that the IC50 value chalcone derivative is 74,24 g/mL with 0,57g/mL of selectivity index. The IC50 value calculated, represents that the concentration of the result in a 50 % decrease in cell growth since the chalcone compound is the most widely known as anti-cancer agents. The range as moderate activity, the chalcone compound showed as generally better against HeLa cell lines cancer. The addition of the hydroxyl group at positions 2 and 4 in ring A may affect the activity cancel line inhibitor, also the methoxy group presence increases inhibitory activity too.
As a whole, chalcone structure consists of two aromatic rings (A and B) (Han et al., 2019) with disubstituted of a hydroxyl group in ring A and monosubstituted of methoxy in ring B (Mahapatra et al., 2015) have activities moderate level since that substituted important as active site (Venkateswararao et al., 2012). Moreover, it was also has a double bond opening ring of benzene. Accordingly, the chalcone compound should undergo mechanism investigation to understand the active sites as potentially more drug agents. Nuclear factor-kB (NF-kB) one of a key role in cancer disease (Folmer et al., 2008), then as a subunit of the transcription factor in cancer cells, NF-kB, controlled immune responses, proliferation, apoptosis and tumorigenesis (Wu et al., 2019).
Investigated of chalcone, particularly that hydroxyl group in the aromatic ring did not give a significant activity (Zhang et al., 2018), but unsaturated carbonyl of chalcone predicted inhibiting the activity of transcription activity in an uncontrolled cell (Bazzaro et al., 2011) From Scheme 2, unsaturated carbonyl of chalcone attacked by Ik-kB, that it was IKK protein-mediated of the NF-kB activation pathway (Yadav et al., 2011). Based on the mechanism reaction of Scheme 2, bonding between protein Ik-kB and unsaturated carbonyl of chalcone may stop NF-kB pathway as caused proliferation of cancer cells in humans. As a candidate drug, a mechanism the activity chalcone assumed by blocked the NF-kB pathway was identified since the in-vitro MTT assay test.
In other particularly research, the hydroxyl group of chalcone as flavonoid metabolic secondary, interaction too with Ik-kB of human and it useful to synthesis a novel drug (Moulishankar & Lakshmanan, 2020). When we treat cancer cases, activation of NF-kB should be blocked and the Ik-kB activation pathway did not bind with NF-kB when unsaturated carbonyl bind with Ik-kB than NF-kB transcription factor will be inhibited (Yoon & Rui, 2007). Despite chalcone effects, it potency (Meng et al., 2014), the active site for inhibited NF-kB was an important role in determining chalcone to docking molecular as an anti-cancer drug in future (Ren et al., 2017). To rapidly, when the compound of drug identify inhibiting the NF-kB signaling pathway currently these drugs provide have development.
Conclusion
Briefly, Chalcone derivative was synthesized successfully by the sonochemical method, and presence yields 98 % with IC50 value 74,24 g/mL. The biological activity was studied, from data can be seen that the IC50 value of chalcone has a moderate activity towards cervical (HeLa) cancer cell lines. According to the IC50 and selectivity index value, it can be indicated that chalcone derivative against a cervical cancer cell line may affect since unsaturated carbonyl of chalcone and that assumed as the active sites for inhibiting growth cancer line by binding with Ik-kB protein then blocked NF-kB pathway activation as caused cancer growth. This suggests that chalcone derivative is a potential for studies as a new anti-cancer candidate drug | v3-fos-license |
2020-10-06T13:35:29.940Z | 2020-09-29T00:00:00.000 | 222165162 | {
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} | pes2o/s2orc | A Functional K+ Channel from Tetraselmis Virus 1, a Member of the Mimiviridae
Potassium ion (K+) channels have been observed in diverse viruses that infect eukaryotic marine and freshwater algae. However, experimental evidence for functional K+ channels among these alga-infecting viruses has thus far been restricted to members of the family Phycodnaviridae, which are large, double-stranded DNA viruses within the phylum Nucleocytoviricota. Recent sequencing projects revealed that alga-infecting members of Mimiviridae, another family within this phylum, may also contain genes encoding K+ channels. Here we examine the structural features and the functional properties of putative K+ channels from four cultivated members of Mimiviridae. While all four proteins contain variations of the conserved selectivity filter sequence of K+ channels, structural prediction algorithms suggest that only two of them have the required number and position of two transmembrane domains that are present in all K+ channels. After in vitro translation and reconstitution of the four proteins in planar lipid bilayers, we confirmed that one of them, a 79 amino acid protein from the virus Tetraselmis virus 1 (TetV-1), forms a functional ion channel with a distinct selectivity for K+ over Na+ and a sensitivity to Ba2+. Thus, virus-encoded K+ channels are not limited to Phycodnaviridae but also occur in the members of Mimiviridae. The large sequence diversity among the viral K+ channels implies multiple events of lateral gene transfer.
Introduction
After the discovery that the M2 protein of influenza virus A exhibits ion channel function [1], many more viruses were discovered to contain genes encoding channel-forming proteins [2][3][4][5][6]. Structure and function studies have shown that these proteins have little in common. In terms of function, the known viral channels exhibit a large diversity ranging from some with high ion selectivity [1,7] to The results of this study confirm that functional K + channels are present not only in the members of Phycodnaviridae, but they also occur in viruses in the Mimiviridae family. The phylogenetic similarity between K + channel genes from the different viruses advocates a common evolutionary origin.
3D Modeling
The 3D model structure for the TetV-1 protein was created by using the web portal for protein modeling Phyre2 [30] on the basis of the X-ray template structure of the K + channel of Streptomyces lividans (KcsA; Protein Data Bank (PDB) ID: 1K4C). The 3D model was further refined [31] by using the web server 3Drefine [32].
Planar Lipid Bilayer Experiments
These experiments were done either on a vertical bilayer set up (IonoVation, Osnabrück, Germany) as described previously [6,22] or in horizontal bilayers using an eNPR amplifier, equipped with a BLM_chip flowcell (Elements srl, Cesena, Italy).
Channel Protein Synthesis
All channels were synthesized cell-free with the MembraneMax TM HN Protein Expression Kit (Invitrogen, Carlsbad, CA, USA) as reported previously [22]. In vivo synthesis occurred on a shaker with 1000 rpm at 37 • C for 1.5 h in the presence of nanodiscs (NDs) with 1,2-dimyristoyl -sn-glycero-3-phosphocholine (DMPC) lipids (Cube Biotech GmbH, Monheim, Germany). The scaffold proteins of the NDs were His-tagged, which allowed purification of channel/ND complexes via metal chelate affinity chromatography. The concentration of His-tagged NDs in the reaction mixture was adjusted to 30 µM. For purification of the channel/ND complexes, the crude reaction mixture was adjusted to 400 µL with equilibration buffer (10 mM imidazole, 300 mM KCl, 20 mM NaH 2 PO 4 , pH 7.4 with KOH) and then loaded on an equilibrated 0.2 mL HisPur nickel-nitrilotriacetic acid agarose (Ni-NTA) spin column (Thermo Scientific). For binding of His-tagged NDs to the Ni-NTA resin, the columns were incubated for 45 min at RT and 200 rpm on an orbital shaker. In the subsequent step, the buffer was removed by centrifugation. To eliminate unspecific binders, the column was washed three times with 400 µL of a 20 mM imidazole solution. Finally, the His-tagged NDs were eluted in three fractions with 200 µL of a 250 mM imidazole solution. All centrifugation steps were performed at 700× g for 2 min. After purification, the preparations were stored at 4 • C. For the reconstitution of channel proteins into the lipid bilayer, a small amount (~2 µL) of the purified channel/ND conjugates (1:1000 dilutions) was added directly below the bilayer in the trans compartment.
Ion Channel Recordings, Data Analysis, and Statistics
After the insertion of an active channel into the bilayer, the membrane was routinely clamped for periods between 10 s and 15 min to a range of positive and negative voltages (usually from +160 mV to −160 mV in 20 mV steps). Data analysis was performed with KielPatch (version 3.20 ZBM/2011) and Patchmaster (HEKA Electronik, Lambrecht, Germany). Experimental data are presented as mean ± standard deviation (sd) of n independent experiments. Statistical significances were evaluated by one-way ANOVA and Student T-tests.
Putative K + Channels
The amino acid sequences of the putative K + channels from TetV-1, CsV-KB1, FloV-SA1, and RhiV-SA1 viruses exhibit a low degree of identity/homology with the exception of a small cluster of amino acids around the conserved motif (TXXTXGYG) characteristic of the selectivity filter of K + channels ( Figure 1). All of them have the conserved GYG sequence, but some have unusual substitutions or features at other positions. For example, the third variable position of the selectivity filter motif is often a threonine but may be substituted by serine, leucine, or valine [33]. In CsV-KB1, this position is replaced with a cysteine. The second conserved threonine (position 4 of the motif) is occasionally a serine or cysteine [33], but in CsV-KB1, it is substituted with a leucine, which is unusual for K + channels. A BLAST search shows that a leucine is not present at this position in the consensus sequence of human K + channels. In all but one of the sequences (RhiV-SA1), the amino acid immediately following the GYG is aspartate, which is found in the same position in many K + channels [7,33], with the exception of those in which the tyrosine of GYG is substituted with a phenylalanine (GFG).
In addition to having the consensus motif itself, all of the sequences have a cluster of aromatic amino acids upstream of the motif (Figure 1). This is consistent with structural requirements of a K + channel pore in which aromatic side chains in this position keep the pore at the appropriate diameter for K + transport [34]. Hence, aside from a few unusual residues in or immediately after the GYG motif in two of the sequences, it would appear at first glance that any of these sequences could form functional K + channels. To better understand the relationship of these four putative K + channels with similar proteins from other viruses, we constructed a multisequence alignment and a phylogenetic tree ( Figure 2). The tree includes sequences from all known viral K + channel prototypes ( [8]; Supplementary Table S1). Because of the small size of the proteins, most nodes only have bootstrap values. However, the tree still suggests that sequences from phage proteins are separated from the proteins from eukaryote-infecting viruses. While TetV-1, CsV-KB1, and FloV-SA1 cluster with proteins from other mimivirids (AaV1, OLPV2, and YLPV2), RhiV-SA1 is closer to the chlorovirus channels. The residues in blue indicate functionally important aromatic amino acids in K + channels upstream of the consensus motif. In the alignment, identical amino acids are indicated by "*" and conserved (amino acids with similar characteristics) and semi-conserved (amino acids having similar shape) amino acids are indicated by ":" and ".", respectively.
Figure 2.
Phylogenetic tree of viral K + channels including new sequences of putative K + channels (underlined) from Figure 1. A possible evolutionary history was inferred by using the maximum-likelihood method and JTT matrix-based model [35]. The tree with the highest log likelihood (−7636.59) is shown. Initial trees for the heuristic search were obtained automatically by applying neighbor-join and BioNJ algorithms to a matrix of pairwise distances estimated using the JTT model, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The proportion of sites where at least 1 unambiguous base is present in at least 1 sequence for each descendent clade is shown next to each internal node in the tree. This analysis involved 25 amino acid sequences. The final dataset contained a total of 366 positions. Evolutionary analyses were conducted in MEGA X [29]. Percentages are data coverage for each node. Color coding of the proteins is as follows: green = phycodnaviruses/chloroviruses, dark green = other phycodnaviruses, blue = phages, red = mimivirids.
We then searched among viral and non-viral sequences in the protein databank for candidates that are most similar to the putative channels from the four Mimiviridae isolates using BLAST ( Table 1). The best hits within the viruses confirm the results of the phylogenetic tree, in that, the putative channels show a distinct relationship to channel proteins from freshwater and marine phycodnaviruses as well as to a putative channel from other members of the Mimiviridae (Aureococcus anophagefferens virus; AaV). The high degree of diversity among the mimivirid sequences already suggests multiple independent events of lateral gene transfer. The BLAST results also show that the putative channel proteins share up to 40% sequence identity with bacterial proteins. Most interesting in this context is the similarity between the primary sequences (low E values, Table 1) of some of the viral channel candidates to proteins with unknown function in bacteria in the phylum Actinobacteria. These organisms are thought to be very ancient, dating back to a pre-oxygen atmosphere on earth~2.7 billion years ago [36,37]. It is possible that the recurrent similarity between viral and Actinobacteria proteins and the fact that both Actinobacteria and NCLDVs have ancient ancestors [36][37][38][39] could provide clues on the evolutionary origin of the viral K + channels.
In addition to the consensus motif, which is part of the selectivity filter, a functional K + channel requires additional structural elements. This includes at least two transmembrane domains that are able to span the bilayer and a short α-helix upstream of the selectivity filter motif [33,34]. The latter forms the pore helix and is essential for the correct positioning of the filter motif in the ion conducting pore [34]. A visualization of the bacterial channel KcsA ( Figure 3A) serves as a reference for the pore structure. KcsA was the first crystalized K + channel and serves as a model structure for K + channel pores with all the aforementioned essential structural elements [34]. The same building elements can be recognized in the primary sequence of viral K + channels by structural prediction algorithms. In the case of the functional viral channel Kcv PBCV1 [7] these algorithms predict with high propensity two transmembrane domains and an α-helix upstream of the selectivity filter ( Figure 3B). To examine whether the new sequences fulfill these requirements, we applied the same structural prediction algorithms. The results show that TetV-1 has the predicted transmembrane domains ( Figure 3B) and required structural elements in the exact positions that are expected for a functional K + channel ( Figure 3C). The combination of the overall structural architecture of a K + channel pore and the presence of a consensus sequence suggest that this protein could indeed be a functional K + channel. The same analysis conducted with all four mimivirid sequences shows a more diverse picture ( Figure 4A). Based on TMHMM and the consensus results from four additional prediction programs, both TetV-1 and RhiV-SA1 are robustly predicted to have both transmembrane domain (TMD)1 and TMD2 in the expected positions. In FloV-SA1, all programs predict an upstream TMD1-like domain, but there is no consensus prediction of a TMD2. CsV-KB1 also has a predicted upstream TMD, but the predicted second TMD is in the wrong position with the selectivity filter in the center of the transmembrane helix. We then examined whether a structural prediction algorithm provides evidence for an α-helix region prior to the canonical filter motive of K + channels (Figures 1 and 4B). In a previous study on other viral K + channels, the prediction of α-helices in this position was very robust [17]. The data show α-helices in the correct position in 3 of the 4 viral protein sequences; only CsV-KB1 lacked this structure.
Collectively, the data indicate that at least three of the four protein sequences have the major structural elements consistent with a K + channel function. TetV-1 and RhiV-SA1 show the most convincing evidence. FloV-SA1 is somewhat less convincing, because of disagreements in the prediction of TMD2, but it has the other expected features. The CsV-KB1 protein seems unlikely to be a functional K + channel; it shows some deviation from the consensus sequence of K + channels and the bioinformatic scrutiny of the amino acid sequence reveals no strong evidence for some essential structural elements.
The TetV-1 Protein Has K + Channel Function
To test all four proteins for channel function, they were synthesized in vitro into nanodiscs and, after purification, reconstituted into planar lipid bilayers. Typical channel fluctuations were routinely obtained with the 79 amino acid TetV-1 protein ( Figure 5A). None of the other proteins generated any perceivable channel activity in multiple repetitions ( Table 2). These results establish that TetV-1 is a protein with typical K + channel functions and that the other proteins do not work under conditions in which most viral K + channels function [6,12,22,40]. Since it is known that the activity of some ion channels depends on factors such as the presence of anionic lipids [41], we cannot completely exclude the possibility that these other three proteins might function as K + channels under different conditions. Table 2. Ratio of measurements with channel activity (n c ) divided by the number of attempts of reconstituting (n a ) a putative channel protein in a bilayer of either DPhPC or DPhPG.
Source of Putative Channel Protein n c /n a in DPhPC n c /n a in DPhPG
RhiV-SA1 0/3 not tested FloV-SA1 0/3 0/1 CsV-KB1 0/3 not tested TetV-1 39/39 not tested We then conducted a basic characterization of the TetV-1 K + channel protein, named Ktv1 for K + channel from Tetraselmis virus 1. The channel generated in a solution with 100 mM K + on both sides of the membrane an asymmetric current/voltage (I/V) in which the conductance at positive voltages (57 ± 7 pS) was about 2 times larger than that a negative voltages (27 ± 5 pS) ( Figure 5B). This value of unitary conductance is similar to that of other viral K + channels [12]. The Ktv1 channel also exhibited an appreciable voltage dependency; it was nearly always open at negative voltages and exhibited increasingly long closed times at positive voltages ( Figure 5A). The open probability/voltage (P 0 /V) plot shows that this results in a progressive decrease in channel activity with increasing positive voltages. If we assume that, like the other viral K + channels, the Ktv1 channel inserts preferentially with the n-and c-terminus into the membrane [22,42], the low open probability at positive voltages would imply that the channel is an inward rectifier.
To test the selectivity of Ktv1, the K + on the cis side of the bilayer was replaced by Na + (Figure 6A). As a result, the channel activity was only visible at negative voltages. This means that the channel conducts K + inward, but there is no Na + outward current. The assumption that the channel has a high preference for K + over Na + was confirmed by experiments in which the K + on the trans side was replaced by Na + . In this situation, only outward K + current but no Na + inward current was visible. The mean I/V relations obtained from experiments with K + /Na + on different sides of the membrane ( Figure 6B) confirm that the channel transports exclusively K + . The I/V curves do not intersect with the voltage axis indicating perfect selectivity of the channel for K + over Na + . Hence in agreement with the structure of its selectivity filter, Ktv1 is a highly selective K + channel.
K + channels are typically blocked by Ba 2+ in a voltage dependent manner [43,44]. To test whether Ktv1 also exhibited this Ba 2+ sensitivity, channel activity was recorded in 100 mM KCl and BaCl 2 added at 5 mM first to the trans and then also to the cis chamber. The results indicate that the presence of the divalent cation on the trans side had no effect on the open channel amplitude ( Figure 6C) but greatly reduced the open probability of the channel. The effect of the blocker is seen in Figure 6C: In the absence of the blocker the channel typically exhibits only short closures, while in its presence only short openings are seen. This mode of block and the voltage dependency of this effect, which increased with negative voltages ( Figure 6D), is typical for the Ba 2+ block of K + channels [44]. Further addition of Ba 2+ to the cis chamber resulted in a complete block of channel activity at positive voltages ( Figure 6D). This effect is also typical for K + channels in which Ba 2+ blocks the channel with a higher affinity from the cytosolic than from the external side of the protein [45].
In summary, the combination of structural and functional data confirms that the Ktv1 79 amino acid protein from virus TetV-1, a member of the family Mimiviridae, functions as an ion channel with the typical hallmarks of a K + channel. Modeling indicates that Ktv1 has the same architecture as the KcsA channel with all the structural elements of a K + channel pore. However, like all other functional viral K + channels, the transmembrane helixes are significantly shorter than those of the reference channel. This is most obvious for the inner transmembrane domains, which form the so-called bundle crossing gate in KcsA [34]. In the case of Ktv1, they are too short to form this gate ( Figure 3C).
Conclusions
We provide the first direct evidence of a functional K + channel encoded by a member of Nucleocytoviricota outside of the family Phycodnaviridae. All four members of the Mimiviridae family that were examined here code for proteins in which the structural hallmarks of functional K + channels were either completely or partially conserved. Hence, like other giant viruses, the two families not only share a set of core genes that are typical for Nucleocytoviricota but also share subsets of other genes [46]. Scrutiny of the proteins of interest revealed high conservation of the selectivity filter with the typical K + channel consensus sequence plus the upstream aromatic amino acids and the common aspartic acid after the GYG motif. However, this central domain was the only region common to all four proteins. Such structural diversity among the proteins from Mimiviridae and their distinct similarities to K + channels from Phycodnaviridae tentatively suggests independent events of lateral gene transfer. A long evolutionary history of these proteins is possible since these viruses and their hosts are ancient. However, these evolutionary details cannot be resolved given the small datasets available so far.
We demonstrated that channel activity having the typical functional features of highly evolved K + channels from mammals was present in the virus-encoded channel named Ktv1. Ktv1 had a remarkable high selectivity for K + over Na + , channel gating, and a distinct voltage dependent sensitivity to Ba 2+ block. These results indicate that the protein is not generating an unspecific leak conductance in the membrane but rather has all the structural and functional features of a bona fide selective K + channel. That the other three putative channels did not generate detectable K + channel activity does not rule out a channel function of these proteins. From an experimental point of view, there are several reasons for why these proteins were not generating measurable channel activity in our recording system. Based on experience with other channels, it is possible, for example, that these proteins require a distinct composition of the lipid bilayer [41] or the correct thickness of the bilayer in relation to the length of the transmembrane domains [47] for function. It is also possible that they are not properly folded and/or inserted into the membrane in the in vitro translation system with the result that they are not functional in the host bilayer. Another reasonable explanation for the negative results is that the channels may have a very small unitary conductance and/or very high flicker type open probability, which is not resolved with the standard measuring equipment [48,49].
We find it intriguing that all viral genes encoding K + channels in Nucleocytoviricota are present in viruses that infect algae even though they are not necessarily obligatory for these viruses [50]. The hosts for the viruses with K + channels vary from unicellular to multicellular [18] and from freshwater to marine algae [17,20,23]. It is possible that the K + channel provides a function in these viruses that is of particular benefit when infecting algal hosts. However, the apparent exclusivity may simply reflect the very limited phylogenetic diversity of non-algal protists that have been used to isolate viruses in the phylum Nucleocytoviricota thus far.
For the PBCV1/Chlorella system, it is well established that the activity of the viral K + channel is crucial early during infection when the viral membrane fuses with the plasma membrane of the host [51]. This triggers a depolarization and a loss of osmolytes and water from the host, which in turn lowers the high internal turgor pressure of host and promotes ejection of the viral DNA into the host [51,52]. Since marine algae do not have the same high internal pressure of freshwater algae [53], it is unlikely that the same mechanism is employed by all viruses that code for K + channels. This implies these proteins may display alternative functions in the infection/replication cycle. An attractive hypothesis is that the activity of viral channel could indirectly alter crucial cellular parameters such as pH or Ca 2+ in order to favor the activity of viral proteins [54]. This could be a successful and parsimonious way for individual virus to take command over their large hosts in the early steps of infection. If generally useful, K + channels may yet turn up in nucleocytoplasmic viruses infecting non-photosynthetic protists with additional targeted isolation and screening. | v3-fos-license |
2018-04-03T02:42:55.142Z | 2007-01-01T00:00:00.000 | 19804310 | {
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} | pes2o/s2orc | Multivariable Difference Gel Electrophoresis and Mass Spectrometry
Multivariable DIGE/MS was used to investigate proteins altered in expression and/or post-translational modification in response to activation of transforming growth factor (TGF)-β receptors in MCF10A mammary epithelial cells overexpressing the HER2/Neu (ErbB2) oncogene. Proteome changes were monitored in response to exogenous TGF-β over time (0, 8, 24, and 40 h), and proteins were resolved using medium range (pH 4–7) and narrow range (pH 5.3–6.5) isoelectric focusing combined with up to 2 mg of protein to allow inspection of lower abundance proteins. Triplicate samples were prepared independently and analyzed together across multiple DIGE gels using a pooled sample internal standard to quantify expression changes with statistical confidence. Unsupervised principle component analysis and hierarchical clustering of the individual DIGE proteome expression maps provided independent confirmation of distinct expression patterns from the individual experiments and demonstrated high reproducibility between replicate samples. Fifty-nine proteins (including some isoforms) that exhibited significant kinetic expression changes were identified using mass spectrometry and database interrogation and were mapped to existing biological networks involved in TGF-β signaling. Several proteins with a potential role in breast cancer, such as maspin and cathepsin D, were identified as novel molecules associated with TGF-β signaling.
2D 1 gel-based approaches are often used to survey the proteome on a global scale, typically resolving thousands of intact proteins based on charge (using isoelectric focusing) and apparent molecular mass (using SDS-PAGE). Despite its popularity for differential display proteomics, 2D gel-based strategies have until recently lacked the ability to directly quantify abundance changes in the same fashion as in stable isotope strategies using liquid chromatography coupled with tandem mass spectrometry (1)(2)(3), i.e. by multiplexing samples into a single run to remove analytical (gel-to-gel or columnto-column) variation. Multiplexing samples labeled with stable isotopes have been used in gel-based proteomics (4), but in this case abundance changes are monitored during the mass spectrometry stage on each individual protein prior to the knowledge of which proteins are changing. DIGE technology (first described by Unlu et al. (5)) adds an essential quantitative component to 2D gel-based strategies and allows for the detection of subtle changes in protein abundance with statistical confidence (6 -8). DIGE uses three spectrally resolvable fluorescent dyes (Cy2, Cy3, and Cy5) to label up to three samples to be run together on the same 2D gel. A pooled mixture containing an equal aliquot of all samples is made and labeled in bulk with Cy2 and used as an internal standard to coordinate between multiple DIGE gels with each gel containing two samples from the experiment that have been individually labeled with Cy3 or Cy5. This use of a pooled sample internal standard provides every resolved protein form under survey with a unique internal standard across a coordinated set of DIGE gels (Refs. 9 and 10; for a review, see Ref. 11) and allows for the quantitative comparison of proteomic changes with statistical confidence afforded by analyzing replicate samples relative to the same internal standard.
As with any complex system, pre-and postfractionation allows for access to lower abundance proteins by increasing the total amount of protein analyzed without overloading the analytical system. Prefractionation is useful for subproteomes but can often introduce additional non-biological variation into the samples that must be controlled. Postfractionation of complex samples is perhaps most popular in complementary peptide-based proteomics strategies using multidimensional HPLC separations prior to mass spectrometry (e.g. multidimensional protein identification technology (MudPIT) (12)). In a similar fashion, resolution and sensitivity can be improved in a 2D gel experiment by postfractionating complex proteomes with medium range (e.g. pH 4 -7) and narrow range (e.g. pH 5.3-6.5) isoelectric focusing gradients with commensurate increases in protein load (13). By resolving intact proteins, 2D gels can resolve multiply charged isoforms (that may result from phosphorylation or other charged post-translational modifications) and biologically significant proteolytic products. Subsequent mass spectrometry can verify that a set of isoforms is in fact related without necessarily identifying the modified peptide(s), whereas such changes may be completely overlooked in the more sensitive peptide-based approaches without mass spectral information on the modified peptide(s).
The main objective of this study was to demonstrate the technical advantage of DIGE/MS to facilely quantify changes in protein abundance and/or post-translational modification on a global scale from multiple experimental variables, each with independent biological repetition. To this end, we used as a model system transforming growth factor- (TGF-) stimulation of human mammary epithelial cells transfected with a HER2 expression vector. This model system is of particular biological interest because overexpression of the tyrosine kinase receptor HER2/Neu (ErbB2) is detected in ϳ25% of breast cancers (14). TGF- is a cytokine that suppresses early tumor formation but promotes tumor progression and metastasis at later stages (15) and has been shown to synergize with ErbB receptor tyrosine kinases. For example, overexpression of active TGF-1 (or active receptor mutants) in transgenic mice also expressing murine mammary tumor virus/Neu (ErbB2) accelerates metastases from Neuinduced mammary cancers (16 -18). A genetic modifier screen in non-tumorigenic mammary epithelial cells identified TGF-1 and TGF-3 as molecules that cooperate with HER2 in inducing cell motility and invasion (19), and inhibition of HER2 with the antibody trastuzumab blocked the promigratory effect of TGF- on HER2-overexpressing mammary epithelial cells (20). Despite these and other studies, little is known regarding the molecular mechanisms or downstream effectors of cross-talk between TGF- and ErbB receptor signaling, making the findings herein of potential value for further investigation.
Changes due to TGF- stimulation were assessed over time (0, 8, 24, and 40 h) in triplicate experiments that were analyzed coordinately by DIGE. High resolution information on over 1500 protein forms was surveyed in the pH 4 -7 range (0.5 mg of total protein per gel), and in some cases increased sensitivity and resolution were afforded by using narrow range isoelectric focusing (pH 5.3-6.5) in conjunction with up to 2 mg of total protein per gel. Principle component analysis (PCA) and unsupervised hierarchical clustering (HC) of the individual Cy3-and Cy5-labeled DIGE expression maps provided independent confirmation of distinct expression patterns from each group and demonstrated high reproducibility between the replicate samples. Each experimental condition was measured using independent replicates for statistical confidence and to rule out false-positive results due to nonbiological variation. Proteins of interest were identified using mass spectrometry and database interrogation, and identified proteins were mapped to existing biological networks and pathways to reveal additional information regarding the relationship of the proteins identified in these studies.
Extraction of Total RNA and RT-PCR-Total RNA was extracted using the RNeasy minikit (Qiagen). RT-PCR was carried out using the Titanium one-step RT-PCR kit (BD Biosciences). For each RT-PCR, 100 ng of RNA were added to a 50-l reaction system according to the manufacturer's protocol (50°C for 1 h followed by 30 cycles of PCR amplification) using primers 5Ј-gcaatggatgccctgcaactagcaaattc and 5Ј-cacttaaggagaacagaatttgccaaag specific for maspin cDNA. The PCR products were analyzed in 1.2% agarose gels.
DIGE Experimental Design-The mixed internal standard methodology of Friedman et al. (10) and Gerbasi et al. (22) was used with the following modifications. Each experiment contained four conditions repeated in triplicate, generating 12 individual samples that were co-resolved across six DIGE gels all coordinated by the same pooled sample internal standard. Experiments utilizing 24-cm pH 4 -7 IEF gradients contained 0.5 mg of total protein equally divided between any two samples and an aliquot of the internal standard as follows. For each individual sample, 0.25 mg of protein was separately precipitated with methanol and chloroform (23) and resuspended in 30 l of labeling buffer (7 M urea, 2 M thiourea, 4% CHAPS, 30 mM Tris, 5 mM magnesium acetate). One-third of each sample (10 l, 83.3 g) was removed and combined into a single tube to comprise the pooled sample internal standard. Thus for each six-gel experiment comprising 12 individual samples, the pooled standard contained 1000 g. The remaining two-thirds of each individual sample (20 l, 166.7 g) was labeled with 200 pmol of either Cy3 or Cy5, whereas the pooled sample was labeled en masse with 1200 pmol of Cy2. A dye-swapping scheme was used such that the three samples from any condition were never labeled all with Cy3 or Cy5 to control for any dyespecific labeling artifacts that might occur. Experiments utilizing 24-cm pH 5.3-6.5 IEF gradients contained 2 mg of total protein treated essentially as above, only starting with 1 mg of extract from each sample, labeling individual samples with 400 pmol of Cy3/Cy5, and labeling the pooled sample internal standard with 2400 pmol of Cy2.
The N-hydroxysuccinimidyl ester forms of Cy2, Cy3, and Cy5 were used following standard methods for minimal labeling. Briefly labeling was performed for 30 min on ice in the dark after which the reactions were quenched with the addition of 10 mM lysine (2 l for each 200 pmol of dye) for 10 min on ice in the dark. The quenched Cy3-and Cy5-labeled samples (each containing 166.7 g of protein) were then combined and mixed with a 166.7-g aliquot of the Cy2-labeled pooled standard after which an equal volume of 2ϫ rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 4 mg/ml DTT) was added. The mixtures were brought up to final volume of 450 l with 1ϫ rehydration buffer (same as above except for 2 mg/ml DTT) after which 0.5% IPG buffer 4 -7 or 5.3-6.5 was added and mixed thoroughly. Although the dye:sample ratios are skewed from the manufacturer's recommendation, we have validated that these ratios provide sensitive labeling comparable to SYPRO Ruby total protein staining while allowing for optimal protein amounts to facilitate subsequent mass spectrometry (10, 22, 24 -26).
2D Gel Electrophoresis and Imaging-For each six-gel DIGE experiment, tripartite-labeled samples for each gel (450-l final volume) were passively rehydrated into 24-cm pH 4 -7 and pH 5.3-6.5 IPG strips (Amersham Biosciences/GE Healthcare) for 24 h followed by simultaneous isoelectric focusing using a manifold-equipped IPGphor IEF unit (Amersham Biosciences/GE Healthcare) according to the manufacturer's instructions for a total of 60 kV-h. The cysteine sulfhydryls were reduced and carbamidomethylated while the proteins were equilibrated into the second dimension loading buffer by incubating the focused strips in equilibration buffer (30% glycerol, 2% SDS, 6 M urea, 50 mM Tris, pH 8.8, trace bromphenol blue) supplemented with 1% DTT for 20 min at room temperature followed by 2.5% iodoacetamide in fresh equilibration buffer for an additional 20-min room temperature incubation. Second dimensional SDS-PAGE was performed on hand-cast 12% SDS-PAGE gels using low fluorescence glass plates with one plate presilanized to preferentially affix the gel, thereby ensuring the accuracy of subsequent robotic protein excision. Electrophoresis was carried out at 0.2 watts/gel for 3 h followed by 20 watts/gel until completion using a DALT-12 unit (Amersham Biosciences/GE Healthcare).
The differentially labeled co-resolved proteome maps within each DIGE gel were imaged at 100-m resolution separately by dyespecific excitation and emission wavelengths using a Typhoon 9400 variable mode imager (Amersham Biosciences/GE Healthcare). 16-bit tagged image file format images were cropped and exported for analysis using the DeCyder version 6.5 suite of software tools. After imaging for CyDye components, the non-silanized glass plate was removed, and the gels were fixed in 50% methanol, 7% acetic acid for 2 h and then incubated in SYPRO Ruby (Invitrogen) in the dark overnight. This poststain also visualizes approximately 97% of unlabeled protein and ensures accurate protein excision as the molecular weight and hydrophobicity of the CyDyes influence the apparent molecular mass of proteins during SDS-PAGE. SYPRO Ruby images were acquired on the same imager as well as reimaged postexcision to ensure accurate protein excision.
DIGE Analysis-The DeCyder version 6.5 suite of software tools (Amersham Biosciences/GE Healthcare) was used for DIGE analysis. The Differential In-gel Analysis module was used to quantitatively compare the normalized volume ratio of each individual protein spot feature from a Cy3-or Cy5-labeled sample on a given gel relative to the Cy2 signal from the pooled sample internal standard corresponding to the same spot feature. Within each gel, the co-resolved fluorescent signals from each protein (two individual samples and one internal standard) are co-detected by the software, and abundance measurements are made directly to the internal standard without interference from gel-to-gel variation, obviating the need to run analytical replicates for each sample.
The Differential In-gel Analysis datasets for each individual gel were then collectively analyzed using the Biological Variation Analysis module, which allowed for the facile matching of protein migration patterns and normalization of Cy3:Cy2 and Cy5:Cy2 quantitative abundance ratios for each protein between gels of a coordinated set, again using the unique signal of each protein from the pooled internal standard. In this way, multiple variables each with independent experimental repetition were coordinately quantified with statistical confidence and without the requirement that every pairwise comparison be made within a single 2D DIGE gel. Statistical significance was associated with each change in abundance or charge-altering posttranslational modification using Student's t test and analysis of variance (ANOVA) analyses that compare the variation of expression within a group to the magnitude of change between groups. Many statistically significant changes were observed within the 99.9th percentile confidence interval (representing less than one false positive for approximately 1500 proteins resolved in a gel), but changes within the 95th percentile were also considered.
Unsupervised PCA and HC was performed using the DeCyder Extended Data Analysis module. These multivariate analyses clustered the individual Cy3-and Cy5-labeled samples based on the collective comparison of expression patterns from the proteins identified in Table I. These groups of protein expression characteristics are represented by each data point in the PCA plots and by each column in the HC expression matrixes (heat maps). PCA reduces the complexity of a multidimensional analysis into two principle components, PC1 and PC2, which orthogonally divide the samples based on the two largest sources of variation in the dataset. Values within the circles of the PCA plots are within the 95th percentile confidence interval. HC performs a similar unsupervised clustering of the samples based on similarities of expression patterns in the selected proteins, which are visually presented as horizontal lines in an expression matrix "heat map" using a standardized log abundance scale ranging from Ϫ0.5 (green) to ϩ0.5 (red). HC expression matrixes were calculated using Euclidean correlation and average linkage. Mapping of proteins identified by mass spectrometry and database interrogation (see below) onto existing networks and pathways was accomplished using Ingenuity Pathway Analysis software (Ingenuity Systems, Inc.).
In-gel Digestion, Mass Spectrometry, and Database Interrogation-Proteins of interest were robotically excised and digested into peptides in-gel with modified porcine trypsin protease (Trypsin Gold, Promega), and peptides were applied to a stainless steel target using an integrated Spot Handling Workstation (Amersham Biosciences/GE Healthcare) according the manufacturer's recommendations. Peptide samples (0.3 l) were robotically mixed wet on the target with an equal volume of ␣-cyano-4-hydroxycinnamic acid (5 mg/ml in 60% acetonitrile, 0.1% trifluoroacetic acid supplemented with 1 mg/ml ammonium citrate).
MALDI-TOF MS and data-dependent TOF/TOF tandem MS/MS was performed on a Voyager 4700 mass spectrometer (Applied Biosystems, Framingham, MA). MALDI-TOF mass spectra were acquired in reflectron positive ion mode, averaging 1500 laser shots per spectrum. Peptide ion masses (M ϩ H) were accurate to within 20 ppm after internal calibration using the trypsin autolytic peptides at m/z 842.51 and 2211.10. TOF/TOF tandem MS fragmentation spectra were acquired in a data-dependent fashion based on the MALDI-TOF peptide mass map for each protein, averaging 2000 laser shots per fragmentation spectrum on each of the 20 most abundant ions present in each sample (excluding trypsin autolytic peptides and other known background ions).
The resulting peptide mass maps and the associated fragmentation spectra were collectively used to interrogate sequences present in the Swiss-Prot and National Center for Biotechnology Information non-redundant (NCBInr) databases to generate statistically significant candidate identifications using GPS Explorer software (Applied Biosystems) running the MASCOT search algorithm (Matrix Science). Searches were performed without constraining protein molecular weight or isoelectric point, with complete carbamidomethylation of cysteine, with partial oxidation of methionine residues, and with one missed cleavage also allowed in the search parameters. Evidence of proteins derived from mycoplasma was found in the course of these studies but did not have an effect on the statistical significance of the proteins presented in this study. Significant Molecular Weight Search (MOWSE) scores (p Ͻ 0.05), number of matched ions, number of matching ions with independent MS/MS matches, percent protein sequence coverage, and correlation of gel region with predicted molecular weight and pI were collectively considered for each protein identification (all data are presented in Table I and Supplemental Table 1).
Time Course of TGF--induced Proteome Changes
Measured by DIGE/MS-MCF10A/HER2 cells were treated with 2 ng/ml TGF-1 for 0, 8, 24, and 40 h, and efficacy of ligand treatment was confirmed by monitoring phosphorylation of Smad2, which occurs directly by the TGF- type I receptor upon ligand binding (Fig. 1A). To assess proteomic changes in these samples, we performed DIGE/MS analysis using a pooled sample internal standard present on every gel (see "Experimental Procedures"). With this experimental design, every DIGE gel in a six-gel set contains two of the 12 samples (labeled with either Cy3 or Cy5) along with an equal aliquot of the Cy2-labeled internal standard (Fig. 1B, 12-mix). Although an 8-and 24-h sample may be co-resolved on the same gel, the quantitative comparison for a resolved protein is between the Cy3 or Cy5 signals and the Cy2 internal standard signal for this protein, not between the Cy3 and Cy5 signals directly. The intragel ratios for each resolved protein (Cy3:Cy2 and Cy5:Cy2) are then normalized to the cognate ratios from the other gels. In this way, for a given protein form, the normalized abundance ratios from all 12 samples can be intercompared with statistical confidence in a concerted six-gel experiment all coordinated by the same mixed sample internal standard. A schematic of the loading matrix, indicating how the individual samples were labeled and loaded into the six DIGE gels along with the internal standard, is shown in Fig. 1C. Within this loading matrix, we have depicted a theoretical protein that is up-regulated at the 40-h time point and indicated (with dotted lines), how the relative abundance is quantified for this protein within each gel relative to the cognate signal from the protein in the Cy2-labeled internal standard (without gel-togel variation), and how the Cy3:Cy2 and Cy5:Cy2 ratios are then normalized between the gels using the Cy2 signal for that protein. The graphical readout for this theoretical change is shown in Fig. 1D where the normalized abundance ratios (i.e. relative to the internal standard that is normalized across all six gels) are all graphed together to easily visualize the relative magnitude and reproducibility of the expression data.
DIGE analysis flagged 26 protein forms that exhibited statistically significant (ANOVA, n ϭ 3) changes in abundance or charge-altering post-translational modification that were greater than 1.2-fold displaying early, late, or biphasic kinetics ( Fig. 2A and Table I, lines 1-26). Many of the changes fell within the 99.9th percentile confidence interval where only one false positive is expected for over 1000 features, but changes within the 95th percentile were also considered (Table I, lines 1-26).
Proteins were excised from a subset of the six gels and subjected to protein identification using mass spectrometry and database interrogation as described under "Experimental Procedures," the results of which are summarized in Table I (lines 1-26) and detailed in Supplemental Table 1. The 26 identified features specified 23 unique proteins including redundancies due to post-translational modification or proteolysis. Many of these 23 proteins were also found as additional charge-related isoforms (migrating with closely related isoelectric points but indistinguishable apparent molecular masses, data not shown) consistent with a charge-altering post-translational modification. However, the expression patterns of these isoforms were overall similar to those listed in Table I, and in most cases these were omitted from Table I for brevity.
The above analysis used medium range pH 4 -7 gradients for the first dimension isoelectric focusing because this condition offers increased resolution and sensitivity (loading 0.5 mg of total protein into each gel) compared with broader range gradients (e.g. pH 3-11) (13). To increase further the pI resolution and to gain access to lower abundance proteins in these samples, we reanalyzed the same samples using narrow range isoelectric focusing (pH 5.3-6.5) in a second six-gel set with 4-fold more (2 mg) total protein per gel ( Fig. 2B and Table I, lines 27-59). We identified 33 additional features (specifying 31 proteins) in the narrow range DIGE analysis exhibiting kinetics similar to those observed above that were subsequently identified by mass spectrometry and database interrogation (Table I and Supplemental Table 1 (Table I, similarly confirmed by positional matching and inspection of the DIGE profiles but were not further confirmed by mass spectrometry (data not shown). The remaining 20 features listed in Table I for the narrow range experiment displayed only marginal changes (in magnitude and/or statistical significance) that may not have been selected in the first pH 4 -7 experiment due to the presence of stronger candidates in the dataset.
Principle Component Analysis, Hierarchical Clustering, and Pathway Analysis-We sought to further validate the experimental samples for relevancy of ligand binding and to establish biological significance of the resulting protein changes (as opposed to stochastic changes) by performing multivariate statistical tests and network mapping of the proteins identified by DIGE/MS. PCA reduces the dimensionality of a multidimensional analysis to display the two principle components that distinguish between the two largest sources of variation within the dataset.
In both pH range analyses, PCA indicated distinct expression patterns from the four groups and demonstrated high reproducibility between the replicate samples (Fig. 3, A and B). Each data point in the PCA plots describes the collective expression profiles for the subset of proteins listed in Table I for each pH range (Fig. 3, A and B). For the 26 features identified in the pH 4 -7 analysis, the first principle component distinguished 65.5% of the variance with 19.4% additional variation distinguished by the second principle component. For the pH 5.3-6.5 analysis, these values were 81.1% of the variance and 8.3% additional variance for PC1 and PC2, respectively. In addition, the PCAs demonstrate that the greatest amount of variation in the experiment is what distinguishes the 40-h time point from the others. Although quite similar, the different relative orientations of the groups between the pH 4 -7 and 5.3-6.5 analyses are most likely due to a different subset of proteins used for the analysis in each pH range.
These grouping assignments were reiterated in an unsupervised HC analysis of the protein expression patterns within each sample (Fig. 3, C and D). HC compares groups based on similarity of the collective expression patterns of the selected proteins with similarity being proportional to the lateral distance depicted in the branched dendrograms above each expression matrix (heat map). Each column in the HC expression matrix is effectively the same as each data point in the PCA plots. For clarity, expression and identification information for each of the individual proteins in the HC analysis (grouped into horizontal bars in the expression matrix and related via a similar dendrogram on the left) is directly tied to the numerical line entry in Table I, which summarizes the mass spectrometry search results and details the DIGE results. Similar PCA and HC results were found for both pH ranges when expanding the dataset to over 100 features by relaxing significance thresholds (data not shown). The PCA and HC results validate the biological significance of the protein ex-pression changes detailed in Table I as we would not expect these individual samples to cluster in this way if these individual changes arose stochastically.
In a similar fashion, we sought to validate the significance of the proteins listed in Table I by mapping them to pre-existing mammalian networks and pathways ( Fig. 4 and Supplemental Table 2). Of the 51 unique proteins specified by the 59 protein forms identified in the pH 4 -7 and pH 5.3-6.5 TGF- time course experiments, 46 mapped to a network of pathways involving TGF-1 as a major hub (Fig. 4). Associated with this network were intercalating pathways involving MYC, p53, and the peroxisome proliferative-activated receptor-␣ (PPARA) that in turn affected many proteins that were independently identified in the DIGE experiments (Fig. 4, shaded proteins). Although additional validation is necessary to establish biological significance, the mapping of these proteins to established networks also provides new insight into potential TGF- effectors and pathways that might otherwise have gone unnoticed based solely on the list of proteins presented in Table I.
Maspin and Cathepsin D-Several proteins identified by DIGE/MS as potential new TGF- effectors had demonstrated roles in breast cancer. Maspin, a tumor-suppressing serpin (serine protease inhibitor; Table I, lines 13 and 14, highlighted in Fig. 5, A-D), is expressed in non-tumor mammary epithelial cells but not in most human breast cancer cell lines or primary breast tumors (27). Restoration of maspin expression or treatment with recombinant maspin protein in MDA-MB-435 and MDA-MB-231 cancer cells reduces Rac1 activity and cell invasiveness as well as metastases in nude mice (27,28). We found two isoforms of maspin that were increasing 1.65-1.67fold (65-67% increase, p ϭ 0.003 and 0.0069, respectively) after 40 h of treatment with TGF-.
These findings were validated by Western and RT-PCR analyses using a new time course of TGF--induced maspin expression with or without concomitant overexpression of HER2 (Fig. 6). Maspin protein levels were up-regulated by exogenous TGF- in both HER2-overexpressing cells and controls (Fig. 6A), implying that this effect does not depend on high levels of HER2. TGF- treatment induced similar increases in maspin mRNA levels, indicating that the level of regulation was transcriptional (Fig. 6B).
In another example, two charge-related isoforms of cathepsin D, a lysosomal aspartyl endoproteinase, were altered by treatment with TGF- (features 21, 22, and 53; highlighted in Fig. 5, E-H). In this case, both the magnitude and direction of the change differed between the two isoforms of cathepsin D, indicating a change in a charge-altering modification. At 40 h after treatment, the acidic isoform (feature 21) decreased by 34% (1.51-fold decrease, p ϭ 0.021), whereas the basic isoform increased by as much as 195%, or 2.95-fold (p ϭ 0.0000017; Table I, lines 22 and 53). MS and database interrogation unambiguously identified both isoforms as cathepsin D despite low protein expression Table I (26 proteins for pH 4 -7 and 33 proteins for pH 5.3-6.5). A, principle component analysis discretely clustered the 12 individual Cy3-and Cy5-labeled DIGE expression maps into the four time treatment groups differentiated by two principle components: PC1, which distinguishes 65.5% of the total variance in the analysis, and levels and associated weak signals in the mass spectrometer (Fig. 5, F-H, Table I, and Supplemental Table 1, lines 21, 22, and 53). Although there was no indication in the mass spectral data as to the nature or location of the charged modification, without mass spectral data on a modified peptide, this significant change in protein expression may well have been overlooked in standard peptide-based proteomics analyses (e.g. LC/MS/MS shotgun analysis with spectral counting or stable isotope labeling strategies).
These results were validated by Western analysis using an independent time course of TGF- treatment (Fig. 6C). The antibody used cannot distinguish between isoforms as we did with DIGE/MS. However, an overall increase in cathepsin D levels is predicted if both isoforms are considered collectively, and this increase is evident in the Western blot results. Treatment with TGF- increased the mature 31-kDa isoform in MCF10A/HER2 cells but not in control MCF10A/vector cells, indicating that the observed changes in cathepsin D were dependent on high levels of HER2 signaling.
DISCUSSION
In this study we have shown the utility of the DIGE/MS approach for the quantitative analysis of over 1500 resolved proteins (including modified isoforms) across multiple conditions using TGF- and HER2 signaling as a model system. Analyzing replicate samples enabled statistical confidence to be assigned for each measurement of altered protein expression over multiple variables. Multiplexing samples into the same gel separations along with a pooled sample internal standard allowed for these quantitative measurements to be made with statistical confidence across all samples without interference from gel-to-gel variation while significantly reducing the number of gels necessary compared with conventional 2D gel-based proteomics.
The expression patterns of the proteins identified in this study most likely work in concert to promote phenotypic PC2, which distinguishes an additional 19.4% of the variance. Related samples are encircled for illustrative proposes only. B, similar PCA plot shown for the pH 5.3-6.5 range analysis with PC1 and PC2 distinguishing 81.1% of the variance and 8.3% additional variance, respectively. C, unsupervised hierarchical clustering of the 12 independent samples based on the global expression patterns of 26 proteins in the pH 4 -7 range that are detailed in Table I. Hierarchical clustering of individual samples is shown on top, and clustering of individual proteins is shown on the left with relative expression values displayed as an expression matrix (heat map) using a standardized log abundance scale ranging from Ϫ0.5 (green) to ϩ0.5 (red). The gel number (1-6) and Cy3/5 dye labeling for each sample is listed below, and the protein identifications with corresponding line entries in Table I (lines 1- 26) are listed along the right-hand side. D, similar expression matrix from an unsupervised hierarchical clustering analysis of33 proteins from the pH 5.3-6.5 range that are also detailed in Table I ( Table I were occurring stochastically. In addition, mapping the proteins identified by DIGE/MS to previously characterized networks and pathways revealed new insight into the inter-relationships of these proteins as well as identified additional potential effectors that are members of these pathways but not identified in the DIGE/MS analysis due to a variety of reasons (e.g. low abundance, low molec-ular weight, or basic pI). MYC and PPARA are nuclear transcriptional regulators affecting the expression of target proteins governing cell proliferation and differentiation, adhesion, apoptosis, cell cycle progression, and inflammation responses. It was recently found that p53 promotes the activation of multiple TGF- target genes during Xenopus embryonic development (29). A model has been proposed describing cooperation between p53 and Smads in TGF-mediated gene transcription where Smad2 enters into the nuclei and associates with Smad4 and specific cofactors to bind target sequences (30). Thus it is not surprising that many FIG. 4. Networks and pathways associated with proteins identified by DIGE/MS from the TGF- time course study (both pH ranges combined, Table I). Ingenuity Pathway Analysis software (Ingenuity Systems, Inc.) was used to map identified proteins onto existing mammalian pathways and networks that associate proteins based on known protein-protein interactions, mRNA expression studies, and other biochemical interactions established in the literature. Shaded features depict proteins identified in the present study, whereas unshaded features depict additional members of these networks and pathways that were not detected by DIGE/MS. Full names and annotations for the proteins represented in this network are listed in Supplemental Table 2. of the proteins identified by DIGE/MS were secondary effectors of the TGF- pathway.
Several proteins identified in these studies are known targets or downstream effectors of TGF- signaling. For example, proliferating cell nuclear antigen (PCNA), a cell proliferation marker, was down-regulated by TGF- by 1.72-fold (42%, p ϭ 0.003) at 40 h (Table I, line 18). This is consistent with the known proliferation suppressive function of TGF- that is retained in MCF10A/HER2 cells (20). In another example, Hsp27, which was up-regulated in response to TGF- by as much as 4.43-fold (343%, p ϭ 0.000075) at 40 h (Table I, lines 24 and 57), mediates TGF--induced cell motility as demonstrated using small interfering RNA to block TGF-mediated cell invasion in human prostate cancer (31). TGF- has also been shown to induce Hsp27 phosphorylation in osteoblast-like MC3T3-E1 cells (32). Determining whether the observed increases found in the current study result from specific phosphorylation or overall protein abundance will require further investigation.
Many novel TGF- or HER2 effectors were also identified in these studies; several of these have demonstrated roles in breast cancer. One example is the tumor suppressor maspin (27), the expression of which has been shown to be regulated by the p53 tumor suppressor family (33,34). Furthermore mutant p53 and aberrant cytosine methylation cooperate to silence maspin expression in cancer cells (35). This relationship between maspin (SERPINB5) and p53 is also reflected in the pathway/network map for TGF- treatment (Fig. 4) where p53 is also shown to modulate the expression of lamin A/C (LMNA), PCNA, Hsp105 (HSPH1), and stress-induced phosphoprotein Hsp70 (STIP1), all of which were independently identified in the DIGE analysis (Table I, lines 3, 18, 31, and 32). We demonstrated that TGF--induced maspin expression was at the level of transcription (Fig. 6B), and subsequent experimentation indicates a direct role for p53 in maspin expression. 2 In another example, cathepsin D, which is implicated in a number of cancers, including breast (Ref. 36, for a review, see Ref. 37), was identified as two isoforms that were differentially expressed, indicating a change in post-translational modification as well as an overall increase in expression (Fig. 5, E-H, and Fig. 6C). The observed apparent molecular mass and isoelectric point of the basic isoform (features 22 and 53) was consistent with the mature 31-kDa heavy chain that is produced after several rounds of post-translational processing (Ref. 38, predicted pI ϭ 5.5). The substantial shift in pI for the acidic isoform is consistent with the addition of a single negative charge or with the removal of approximately 10 amino acids from the C terminus as proposed to occur in the lysosome (38,39). One interpretation of these results is that not only are cathepsin D expression levels increasing at the later time points but also that the protein is undergoing a differential modification/processing. An alternate interpretation is that although more cathepsin D is being expressed at the later time points no more is being modified/processed into the acidic isoform.
That this type of quantitative change in isoforms may well be overlooked in peptide-based strategies exemplifies the complementary nature of these two proteomics technologies. Any single proteomics strategy provides access only to a subset of the proteome under study, and each has unique strengths and weaknesses. Although DIGE provides statistically significant quantitative information from replicate samples on over 1000 intact proteins and modified isoforms in a single run, very hydrophobic proteins remain difficult to resolve, and proteins outside of the pH and molecular weight ranges will not be surveyed. Using complementary technologies can also provide corroborative evidence. For example, a recent study of TGF- treatment of human lung cancer cells using a global quantitative peptide-based profiling strategy (iTRAQ (isobaric tags for relative and absolute quantitation)) identified several of the proteins described here by DIGE/MS (tropomyosin, Hsp27, EF-Tu, and cofilin) and mapped proteins to similar networks that also spoke to a cellular program of adhesion/invasion known to be up-regulated by TGF- in transformed cells (40).
Although complementary, an overriding limitation that is universal to all proteomics platforms for global scale experiments is the constraint imposed by abundant proteins that limits the total amount of sample extract that can be loaded into the analytical system without compromising resolution. This problem is most often addressed by postfractionation of the sample with commensurate higher protein loads. For DIGE/MS, this is accomplished using medium range (e.g. pH 4 -7, 7-11) and narrow range gradients (e.g. pH 5.3-6.5) that offer greater resolving power and sensitivity (as demonstrated here). In this regard, pH 3-11 gradients offer the lowest resolution and are most biased toward the high abundance proteins despite being inclusive to a wide range of protein pI values.
Newer technologies that couple in-solution isoelectric focusing with molecular weight separations hold great promise for resolving even higher protein loads to access lower abundance proteins while retaining the protein content of all pH fractions for subsequent analysis. Calcitonin and tumor necrosis factor ␣, estimated at levels below 0.1 ng/ml, were recently reported from human serum using this approach after major protein depletion (41). Utilization of the maleimide Cy-Dyes for saturation labeling of cysteine sulfhydryls offers about a 10-fold increased sensitivity, although there are currently only two CyDyes with this chemistry, and generating usable signals in the mass spectrometer usually requires increased material. Combining these technology platforms to maximize their complementary nature while minimizing the Table I to illustrate the normalized relative abundance change and consistency across the independent replicates for each time point. Expression data are reported as the average percent change of normalized volume ratios for the indicated condition relative to 0 h or control with associated Student's t test p value (calculated using DeCyder version 6.5 software). Similar values were found for the basic isoform (Table I, line 14). B, MALDI-TOF mass spectrum of the trypsin-digested peptides derived from the protein excised from gel 4, feature 13 (arrow in A). The spectrum was internally calibrated to Ͻ20 ppm mass accuracy using the trypsin autolytic peptide ions at m/z 842.51 and 2211.10. The other labeled peptide ions (M ϩ H) were used to generate statistically significant matches to maspin as indicated in Table I and Supplemental Table 1 (line 13). C, TOF/TOF tandem mass spectrum of the fragment ions of the peptide ion at m/z 1870.93. Fragment ions with the charge retained on the N terminus (b-ions, numbered left to right) and with charge retained on the C terminus (y-ions, numbered right to left) are indicated using the displayed amino acid sequence of the predicted maspin peptide. D, the peptide ion at m/z 1653.81 was not matched to a predicted peptide from the database entry for human maspin, but the TOF/TOF fragmentation pattern was consistent with a predicted maspin peptide containing a missense mutation of Asp to Thr or Ile to Val, neither of which was reported in the existing database entry. E, SYPRO Ruby gel image of gel 4 indicating proteins excised for the identification of cathepsin D isoforms (acidic feature 21 and basic feature 22) that were changing in opposite directions relative to the 0-h samples as indicated. F, MALDI-TOF peptide mass map resulting from the in-gel digestion of the basic isoform feature 53 excised from gel 7, internally calibrated as above. Peptide signals used for the statistically significant identification of cathepsin D (CATD) are labeled and listed in Table I and Supplemental Table 1 (lines 21, 22, and 53). The ion at m/z 1045.57 is derived from trypsin autolysis, and the ions labeled Bg were background ions present in every spectrum. G and H mark two ions for which the TOF/TOF tandem MS/MS spectra are also shown. G, tandem TOF/TOF mass spectrum of the peptide ion at m/z 1462.66 with b-and y-ions annotated for the predicted amino acid sequence as described above. H, similarly annotated tandem TOF/TOF mass spectrum of the peptide ion at m/z 1601.83. Similar MS and MS/MS spectra were acquired for protein features 21 and 22 that were comparable both with respect to m/z values and relative ion intensity albeit with lower scores (Table I and Supplemental Table 1).
FIG. 5-continued
individual weaknesses will undoubtedly be necessary to increase the scope and depth of quantitative differential display proteomics. | v3-fos-license |
2018-12-15T05:35:08.251Z | 2013-01-01T00:00:00.000 | 58914429 | {
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} | pes2o/s2orc | Elimination of interferences from surface active substances in voltammetric determination of trace amounts of titanium in environmental water samples
A cathodic stripping voltammetric method for determination of Ti(IV) in water samples containing high concentrations of surfactants is described. The linear calibration plot for Ti(IV) was achieved in the simultaneous presence of 5 mg L anionic, 1 mg L cationic and 2 mg L nonionic surfactants for an accumulation time of 30 s in the range 2.5 × 10 to 5 × 10 mol L, the detection limit for accumulation time of 30 s was about 8.4 × 10 mol L. The developed method was successfully applied to Ti(IV) determination in environmental waters, such as river water (Bystrzyca, Czerniejówka), stagnant water (Lake Zemborzyce) and rain water (collected from eastern areas of Poland) with satisfactory results.
Introduction
The excellent properties possessed by titanium make it a highly useful material.Titanium alloys have been increasingly widely used in aviation, aerospace, shipbuilding and other industrial departments whereas the main use of TiO 2 is as a white powder pigment used in products such as paints, coatings, plastics, paper, inks, fibers, food and cosmetics [Akram et al., 2011;Chunxiang et. Al., 2011].Because application of titanium has been growing steadily, the possibility of rise of its concentration in environmental water samples increases.Therefore the need for simple, cheap and sensitive procedure of titanium determination in environmental water samples grows.
The electrochemical behavior of titanium makes its classical voltammetric determination very difficult, because at the common potentials it cannot be reduced to the metallic state and deposited onto an electrode.The voltammetric procedures for titanium determination are based on adsorption of the Ti(IV) complexes and next its reduction to Ti(III).For this purpose various complexing agents are exploited, e.g.mordant red, cupferron, beryllon III, pyrocatechol, calcein, xylenol orange, methylthymol blie, chromotripic acid, pyridylazoresorcinol.
The analysis of titanium at the trace level in various environmental samples has been achieved by adsorptive stripping voltammetric method which is based on the formation and accumulation of complexes with chloranilic acid.
However, it should be noted that although voltammetric methods are simple and sensitive, they are vulnerable to numerous interferences arising mainly from the environmental sample matrix.In voltammetric procedures even low concentrations of organic matter, inevitably present in environmental samples, limit the applicability, e.g.surfactants can foul and passive the electrode causing a decrease or total decay of the analytical signal [Hoyer and Jansen, 2003].Because production and use of surfactants has been growing steadily, their concentration in environmental water samples increases [Chebotarev et al., 2004].Therefore the need for voltammetric stripping procedures making possible the titanium determination in the presence of surfactants grows.
This work provides a simple and fast procedure for determination of Ti(IV), which allows for the analysis of environmental water samples with a complicated matrix containing high concentration of nonionic, anionic and cationic surfactants.For this purpose Amberlite XAD-7 resin was used for surface active substance removing by means of their adsorption on the resin while Ti(IV) remains in the sample and it is possible to directly determine it by adsorptive stripping voltammetry.
E3S Web of Conferences
The measurements were made using an EA-9 electrochemical analyzer and a controlled growth static mercury drop electrode in the HMDE mode, both made by MTM-ANKO Cracow, Poland.A three electrode system consisting of an Hg drop working electrode (drop surface area 1.4 mm 2 ), a Pt auxiliary electrode and an Ag/AgCl reference electrode were used.Measurements were performed using differential pulse adsorptive stripping voltammetry according to the following procedure.A natural water sample or synthetic sample for the testing procedure (containing Ti(IV) and surface active substances), 2 mL of 1 mol L -1 acetate buffer pH = 3 and an adequate volume of triply distilled water, so that the final volume of the solution was 20 mL were added to a glass vial of volume 25 mL and finally 0.5 g of XAD-7 resin was inserted.Then, a magnetic stirring bar was put into the vial, and the solution was mixed for 5 min.Next, after sedimentation of resin 9.8 mL of the solution was pipetted into the electrochemical cell and then 200 µL of 1 × 10 -2 mol L -1 chloranilic acid was added and deaeration for 5 min.was performed.A mercury drop was formed, and the accumulation of the Ti(IV)-chloranilic acid complex was carried out at -0.25 V for 60 s from the stirred solution.After the equilibration time of 5 s, the differential pulse voltammogram was recorded, while the potential was scanned from -0.25 V to -0.85 V, with the intensity of the obtained peak directly proportional to the concentration of Ti(IV) in the sample.The scan rate and pulse height were 20 mV s -1 and -50 mV, respectively.
The elimination of influence of nonionic surface active compounds
Triton X-100 is a high-purity, water-soluble, liquid nonionic surfactant that has come to be recognized as a performance standard among similar products.Triton X-100 is widely used in numerous commercial and industrial products as a substrate for detergents, in textile and fiber manufacture because of its excellent detergency, excellent wetting ability and excellent grease and oil removal from hard surfaces [Mei-Hui, 2008].Considering such a common utilization, Triton X-100 is released into the environment in relatively great amounts.Taking this into account Triton X-100 was chosen for the examination of the influence of nonionic surface active compounds on the voltammetric titanium signal and next elimination of these interferences using XAD-7 resin.
As can be observed, the voltammetric signal of titanium is very sensitive to the presence of even small amounts of the nonionic surfactant, 0.5 mg L -1 of Triton X-100 causes almost total decay of the Ti(IV) peak.The addition of resin eliminates the unwanted negative influence of the nonionic surfactant.In the presence of Amberlite XAD-7 resin inherency of even 2 mg L -1 of Triton X-100 does not affect the titanium signal at all and 5 mg L -1 of Triton X-100 causes a decrease of the titanium signal to 60 % of its original value.
The elimination of influence of cationic surface active compounds Cationic surfactants are widely used in household products such as fabric softeners, hair conditioners, soaps, shampoo and other hair products.Other applications of cationic surfactants ensue from the fact that they are effective anti-bacteria agents, therefore, they are generally employed as disinfectants and antiseptic agents and are used in germicide and sanitizer products.Because of their positive charge, cationic surfactants adsorb strongly to the negatively charged surfaces of sludge, soil and sediments.The widespread use and sorption behavior of cationic surfactants implies that these substances are expected to be present in many environmental compartments [Agrawal et al., 2004].One of them is a well-established commercial synthetic cationic surfactant cetyltrimethylammonium bromide (CTAB) and it was chosen for this work.
As can be observed, similarly as in the case of Triton X-100, the voltammetric signal of titanium is very sensitive to the presence of even small amounts of the cationic surfactant, 0.1 mg L -1 causes a decrease of the Ti(IV) peak to 60 % of its original value and 0.5 mg L -1 causes total decay of the titanium signal.
The addition of Amberlite XAD-7 resin minimalize this negative influence.In the presence of resin inherence of even 2 mg L -1 of CTAB does not affect the titanium signal at all.
The elimination of influence of anionic surface active compounds
Anionic synthetic surfactant sodium dodecylsulfate (SDS) was selected for this work due to its high solubility in water and first of all because it is a well-established commercial anionic synthetic surfactant.SDS is commonly used as an ingredient in household and personal care products as well as in specialized applications [Mei-Hiu L, 2008].
SDS to a lesser extent influences the voltammetric signal of titanium, 5 mg L -1 causes a decrease of the peak to 60 % of its original value.In the presence of Amberlite XAD-7 resin even 40 mg L -1 of SDS does not affect the titanium signal.
Environmental samples analysis
Four fresh environmental water samples, such as river water (Bystrzyca, Czerniejowka), stagnant water (Lake Zemborzyce) and rain water were collected from eastern areas of Poland and Ti(IV) determination by the proposed procedure was performed.No Ti(IV) was detected in the samples in concentrations above the detection limit.To confirm the accuracy of the proposed procedure the analysed samples were spiked with Ti(IV) at different concentration levels and the titanium content was determined by the standard addition method.Three replicate determinations using the standard addition method gave average recovery values between 97.3 and 102.8 % with relative standard deviation between 4.8 and 2.6 %, what proves a satisfactory accuracy and precision of the proposed method for the determination of titanium 09006-p.2ICHMET 2012 in environmental water samples.
Conclusion
Analysis of titanium in natural water samples is of great relevance from the environmental point of view.In the literature data there are many voltammetric methods devoted to this case but the problems arise from the matrix of natural samples.The performed experiments demonstrate that the Amberlite XAD-7 resin can be effectively applied to eliminate interferences connected with the presence of surface active substances.The proposed procedure can be carried out directly in environmental water samples containing nonionic, anionic and cationic surfactants, without matrix removal.Additionally the method is simple, quick and requires no expensive equipment, which makes it available even for small laboratories.
Web of Conferences DOI: 10.1051/ C Owned by the authors, published by EDP Sciences, | v3-fos-license |
2018-04-03T03:25:37.092Z | 2017-07-24T00:00:00.000 | 20090765 | {
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} | pes2o/s2orc | Novel Molecule Exhibiting Selective Affinity for GABAA Receptor Subtypes
Aminoquinoline derivatives were evaluated against a panel of receptors/channels/transporters in radioligand binding experiments. One of these derivatives (DCUK-OEt) displayed micromolar affinity for brain γ-aminobutyric acid type A (GABAA) receptors. DCUK-OEt was shown to be a positive allosteric modulator (PAM) of GABA currents with α1β2γ2, α1β3γ2, α5β3γ2 and α1β3δ GABAA receptors, while having no significant PAM effect on αβ receptors or α1β1γ2, α1β2γ1, α4β3γ2 or α4β3δ receptors. DCUK-OEt modulation of α1β2γ2 GABAA receptors was not blocked by flumazenil. The subunit requirements for DCUK-OEt actions distinguished DCUK-OEt from other currently known modulators of GABA function (e.g., anesthetics, neurosteroids or ethanol). Simulated docking of DCUK-OEt at the GABAA receptor suggested that its binding site may be at the α + β- subunit interface. In slices of the central amygdala, DCUK-OEt acted primarily on extrasynaptic GABAA receptors containing the α1 subunit and generated increases in extrasynaptic “tonic” current with no significant effect on phasic responses to GABA. DCUK-OEt is a novel chemical structure acting as a PAM at particular GABAA receptors. Given that neurons in the central amygdala responding to DCUK-OEt were recently identified as relevant for alcohol dependence, DCUK-OEt should be further evaluated for the treatment of alcoholism.
). The β1 subunit residue 265 seems to play an important role in determining the effect of certain GABA A receptor modulators: when S265 in β1 is mutated to N (homologous residue in β2 and β3) on the GABA A receptor complex, the modulators' potentiation is increased, and vice versa, when N265 in β2 or β3 is mutated to S, the potentiation is reduced [16][17][18] . When we tested DCUK-OEt on α1β2(N265S)γ2 compared to α1β2γ2, the effect of DCUK-OEt as a PAM was significantly reduced (Table 3), but not to the extent seen with drugs such as etomidate (no GABA potentiating effect of etomidate at concentrations up to 1 mM was evident with the α1β2(N265S)γ2 receptor combination) 16 .
To further investigate potential binding sites for DCUK-OEt on the GABA A receptor, we performed computationally-based small molecule docking studies to compare the potential interactions of DCUK-OEt with those of DCUKA, flunitrazepam, and etomidate, with either the classical benzodiazepine binding site (located at the α + γinterface of the pentameric receptor) or an alternative binding site (at the α + βinterface) (Fig. 3a). The corresponding binding energies are shown in Table 4. These studies indicated that DCUK-OEt exhibited the highest predicted affinity for an alternative binding site, while, as expected, flunitrazepam exhibited the highest predicted affinity for the benzodiazepine site.
The modeling studies predicted both the carboxylate of DCUKA and the ethyl ester moiety of DCUK-OEt to be oriented towards the α subunit in the region of α:Tyr160 in the alternative site ( Fig. 3b and c). The ethyl ester was predicted to participate in additional hydrophobic interactions with the residues of this region, and there exists a potential π-σ interaction with α:Tyr160. These additional interactions of the ethyl ester also appeared to 50 and Ki values were obtained by non-linear regression analysis of radioligand binding isotherms. Ki values are reported as estimates from the non-linear regressions and their associated standard errors (n = 10 points in the binding isotherms). optimize the positioning of the head group and amide linker within the binding pocket to allow for additional potential H-bond and π-π interactions with β:Asp43 and Tyr62, respectively, leading to the higher affinity for DCUK-OEt compared to DCUKA for the GABA A receptor. The predicted binding and interactions of flunitrazepam in the benzodiazepine site ( Supplementary Fig. S4) were consistent with previous studies [19][20][21] , and flunitrazepam made a number of favorable contacts, including H-bond interactions with α:Tyr160 and γ:Thr142, and π-π interactions with α:Tyr160 and Tyr210. DCUKA shared a number of these predicted contacts, while the ethyl ester of DCUK-OEt appeared to impair the optimal positioning of the head group in the benzodiazepine binding pocket (Fig. 3d and e). Flunitrazepam bound somewhat more deeply into the pocket, compared to the other tested compounds, with the fluorbenzene ring predicted to be locked in place by a three-way π-stacking interaction with α:His102 and γ:Tyr58, Phe77. An additional π-σ interaction with α:Phe100 and π-π stacking with γ:Phe77 not only distinguish the predicted binding of flunitrazepam from DCUKA, but also represent the crucial aspects of interaction of flunitrazepam with the receptor Table 3. Comparison of DCUK-OEt induced changes in EC 10 GABA responses between receptors differing in a single subunit. These comparisons were executed with the linear mixed model using linear contrasts. Correction for multiple pairwise comparisons was by a Bonferroni adjustment.
that lead to its pharmacological function. The presence of the phenyl (C ring) substitution at the 5 position of the benzodiazepine ring structure is necessary for the PAM actions of the benzodiazepine derivatives 22,23 . Therefore, even though DCUKA and DCUK-OEt may bind to the benzodiazepine binding site on the GABA A receptor (with lower affinity), the lack of the fluorbenzene ring on the DCUKA and DCUK-OEt structures would predict the lack of functional effect of DCUKA and DCUK-OEt via the benzodiazepine site. It is important to note that, due to the method by which binding energies are calculated, comparisons of relative binding affinity can only be reliably assessed between different molecules within the same binding site. The studies showing that potentiation of GABA responses by DCUK-OEt cannot be blocked by flumazenil do not preclude the possibility, suggested by the docking experiments, that DCUK-OEt could bind to the benzodiazepine site as an antagonist, while producing potentiation via binding to a different site (the alternative, extracellular site or a transmembrane one). We tested this hypothesis by co-applying DCUK-OEt (1 µM) and Dashed lines indicate predicted non-bond interactions (green = H-bonds, orange = electrostatic or π-cation/anion, magenta = π-π, purple = π-σ, pink = hydrophobic).
SCientifiC REPoRtS | 7: 6230 | DOI:10.1038/s41598-017-05966-x flunitrazepam (0.1 µM). The combined effect was larger than the sum of their individual effects ( Supplementary Fig. S5), suggesting that the functional effects of the two drugs may be mediated by actions at two different sites.
The significant effects of DCUK-OEt on particular subunit combinations of the GABA A receptor led us to test the effects of this compound on neurons in the rat central amygdala (CeA). The CeA is primarily composed of GABAergic neurons and changes in CeA GABAergic neurotransmission have been implicated in the development and maintenance of alcohol dependence 24 . Focal application of DCUK-OEt (0.5 µM) significantly increased the holding current in medial CeA neurons ( Fig. 4a and b), while producing no significant effect on spontaneous inhibitory postsynaptic current (sIPSC) frequency, amplitude, rise or decay times (Fig. 4c).
To confirm that the changes in holding current that we observed were due to increases in tonic signaling at the GABA A receptor, the GABA A receptor antagonist gabazine (GBZ, 100 μM, Sigma Chemical Co., St. Louis, MO) was focally applied following DCUK-OEt application. GBZ produced a significant reduction in holding current when applied after DCUK-OEt, suggesting that the changes in holding current that were observed with DCUK-OEt, were due to DCUK-OEt-induced increases in tonic conductance via GABA A receptors on medial CeA neurons. In addition, we found that the increase in holding current with DCUK-OEt was positively correlated with the reduction in holding current seen with GBZ application (Pearson correlation coefficient = 0.838; p = 0.0094; n = 8; Fig. 4d).
Discussion
DCUK-OEt acts as a subunit-selective PAM at the GABA A receptor, and our ligand binding studies produced no evidence of interaction of DCUK-OEt (<10 μM) with 32 other receptors/transporters/channel proteins. DCUK-OEt exhibited its most robust effects on submaximal GABA-induced currents when applied to the α1β2γ2 GABA A receptor, the subunit combination most highly expressed in mammalian brain 2 . Similar PAM activity of DCUK-OEt was exhibited with GABA A receptors composed of α1β3δ subunits. On the other hand, DCUKA, which lacks the ester moiety at the 2 position of the carboxyquinoline, and is the major metabolite of DCUK-OEt, was 10 times less potent than DCUK-OEt in acting as a PAM at the α1β2γ2-containing GABA A receptors.
The most studied PAMs at the GABA A receptor are benzodiazepine derivatives and other compounds (e.g., zolpidem) which act at the interface of extracellular domains of the α and γ subunits 4 . Our data produced no evidence for DCUK-OEt action at this site. DCUK-OEt did not displace flunitrazepam in ligand displacement experiments, and the electrophysiological effects of DCUK-OEt (and DCUKA) on GABA A receptors expressed in oocytes were not modified by the selective benzodiazepine antagonist, flumazenil. Additionally, the substitution of a δ subunit for a γ subunit in the GABA A receptor complex greatly diminishes the effects of benzodiazepines 25 but the effects of DCUK-OEt were similar in receptors containing either γ2 or δ subunits (compare α1β3γ2 and α1β3δ in Tables 2 and 3). Finally, when DCUK-OEt and flunitrazepam were applied together, the PAM effect was supra-additive.
Assessment of the possibility that DCUK-OEt acted at the "neurosteroid" site on the GABA A receptor produced somewhat equivocal results. 17PA, which has been reported 15,26 , and in our hands also shown, to be a weak partial antagonist at the "neurosteroid" site on the GABA A receptor, produced a statistically significant but modest (35%) inhibition of the PAM actions of DCUK-OEt, while inhibiting the effects of allopregnanolone by 56%. This difference in potency of 17PA could be due to differences in the affinity/efficacy of DCUK-OEt compared to allopregnanolone at the "neurosteroid" site(s). However, neurosteroid agonists acting at a "neurosteroid"
Compound
Binding energy H-bonds/Electrostatic π-π π-Anion/Cation π-σ Hydrophobic π-Amide Table 4. Docking binding energies and interactions at GABA A receptor sites. Summary of the binding energies and non-bond interactions of the top scoring predicted binding orientations for each compound docked into the homology model of the benzodiazepine binding site at the α + γsubunit interface or the "Alternative" binding site at the α + βsubunit interface of the human GABA A receptor shown in Fig. 2 and in Supplementary Fig. S4. Binding orientations were predicted using the Discovery Studio flexible docking protocol and energies were calculated using the distance-dependent dielectric model, as outlined in the methods.
site 26 are particularly effective as PAMs, and are also direct agonists at GABA A receptors composed of the α4βxδ subunits, while DCUK-OEt had no significant effect on this subunit combination. Furthermore, the modulatory action of neurosteroids at low concentrations does not differ among β subunits 27 . On the other hand, both β2 and β3 subunits in combination with α1 and γ2 subunits responded to the addition of DCUK-OEt with a significant increase in the current induced by submaximal GABA, but the substitution of the β1 subunit for either β2 or β3 resulted in a notable decrease of the PAM activity of DCUK-OEt ( Table 2). The negative effect of the β1 subunit is reminiscent of the selectivity for β subunits shown by modulators such as loreclezole 18 and etomidate 28, 29 , among others. Three amino acids in the transmembrane domains of the β subunit, distinguish the sequence of β1 from β2/β3 30 , and mutation of the asparagine at position 265 in the β2 sequence, located at the interface of α/β transmembrane domains, has been demonstrated to interfere with the potentiating action of etomidate and other anesthetics at GABA A receptors 16,17,30,31 . The introduction of a mutated β2 (N265S) into a complex containing α1 and γ2 subunits significantly reduced (Table 3) the PAM activity of DCUK-OEt. However, this mutation has been shown to eliminate etomidate's PAM action 28,32 . Mutation of β2N265 also decreases alcohol PAM activity on GABA A receptors 33,34 . However, ethanol potentiates GABA effects at receptors composed of dimeric αβ GABA A receptors, and does not discriminate between β1 versus β2 subunits 35 . Reports on the concentrations of ethanol necessary to potentiate the effects of GABA on α4β3δ GABA A receptors expressed in Xenopus oocytes have been contradictory [36][37][38] , but the ethanol effect on the α4β3δ subunit combination is always potentiation of the GABA actions, in contrast to the lack of any significant effect of DCUK-OEt. At the EC 10 concentration of GABA, DCUK-OEt exhibited PAM effects on α1β3δ GABA A receptors similar to effects seen with α1β2γ2. However, DCUK-OEt also enhanced the current produced by saturating concentrations of GABA with the α1β2/3δ subunit combination, but not with the α1β2/3γ2 combination (Fig. 2c). GABA has been shown to be a partial agonist at δ subunit-containing receptors 39 , and DCUK-OEt, and some other PAMs 40 , may allow for further activation of the GABA A receptor at concentrations seemingly maximal in the absence of PAMs. It also should be stressed that we detected no effect of DCUK-OEt at any concentration on any of the subunit combinations we tested in our paradigm, without the addition of GABA.
Overall, as noted above, there seems to be some overlap in the characteristics of DCUK-OEt with properties exhibited by allopregnanolone, CGS 9895, LAU-177 41,42 , loreclezole, etomidate and ethanol, but other characteristics regarding subunit selectivity of DCUK-OEt mitigate against assuming that DCUK-OEt binding/activity occurs specifically through the currently described site(s) for binding of these agents. Additionally, DCUK-OEt characteristics do not conform to what would be expected if DCUK-OEt were utilizing the canonical barbiturate, or intravenous or inhalation anesthetic sites to affect GABA action at the GABA A receptor 31,[43][44][45] .
Our models to ascertain the docking of DCUK-OEt to interfaces between the various subunits of the GABA A receptor (composed of α1β2γ2 subunits), indicated that a binding site for DCUK-OEt may exist between the α + βinterface in the pentameric receptor. The free energy (−ΔG) of binding at this site was highest for DCUK-OEt and lowest for etomidate and flunitrazepam. When examining the docking at the benzodiazepine site located between the α + γinterface, the order was reversed, with flunitrazepam showing the highest binding energy and DCUK-OEt and etomidate showing the lowest −ΔG. If the function of DCUK-OEt was dependent on binding at a single site at the α + βinterface, one would expect that GABA A receptors composed of only α and β subunits would respond as well as the receptors which also contain the γ or δ subunit. This was not the case, and the presence of the γ or δ subunit was necessary to exhibit the PAM action of DCUK-OEt. In fact, the type of γ subunit expressed with the α and β subunits was important, with the γ1 subunit being significantly less effective than the γ2 subunit. Because of the absence of the phenyl ring substituent (C ring) that generates functional (PAM) benzodiazepine derivatives, DCUK-OEt would not be expected to be an agonist at the benzodiazepine site, and our electrophysiologic experiments in the presence of flumazenil support this contention. It was, however, interesting that the combined effects of flunitrazepam and DCUK-OEt produced significantly more than an additive effect, possibly indicating an allosteric interaction between the benzodiazepine site and the site on the α + βinterface which binds DCUK-OEt with higher affinity.
The radioligand binding studies that led us to the electrophysiological examination of DCUK-OEt on the GABA A receptor, also produced some insight into the possible mechanism by which DCUK-OEt may generate its effects. DCUK-OEt produced a decrease in the affinity for muscimol at the GABA A receptor. Such action may be expected if DCUK-OEt is shifting the GABA A receptor into a state more likely to be in an open channel configuration. The GABA A receptor has been shown to display two affinity states for agonists such as muscimol 46,47 and the high affinity state of the GABA A receptor has been proposed to represent stabilization of the desensitized form of the receptor 48 . One can speculate that DCUK-OEt is increasing the proportion of receptors in a low affinity state at any particular concentration of agonist (muscimol). This speculation will require more investigation, but it is interesting that ethanol 49 and the anxiolytic/anticonvulsant etifoxine 50 , which both can act as PAMs at lower concentrations, reduce muscimol affinity at GABA A receptor in rat brain membrane preparations.
The α1β2γ2 combination of subunits is the primary combination of synaptically localized GABA A receptors in brain that mediate phasic inhibition, while α1/4/6βxδ receptors have been considered to be the primary type of extrasynaptic GABA A receptors that mediate tonic inhibition 51 . Given our results with GABA A receptors containing α4 and α1 subunits together with the γ2 or δ subunit, one could assume that DCUK-OEt would well affect the function of synaptically localized GABA A receptors as well as certain extrasynaptic GABA A receptors. We noted two characteristics of DCUK-OEt that suggest that its primary effect may be at extrasynaptic receptors containing either a γ2 or δ subunit together with an α1 and β3 subunit. These combinations of subunits (α1β3γ2 and α1β3δ) display a low EC 50 for GABA (see Supplementary Fig. S6) and DCUK-OEt can produce highly significant potentiation of α1β3γ2 and α1β3δ-mediated currents at the EC 10 concentration of GABA in our assays, and probably at concentrations of GABA consistent with those encountered in locations outside of the GABA synapse. This observation would be quite compatible with significant potentiation at extrasynaptic sites where concentrations of GABA have been considered to be in the high nM range, as opposed to the high concentrations (mM) of GABA that are present in the synapse 52 . We saw no measurable effect of DCUK-OEt on α1β2γ2 receptors at high concentrations of GABA (EC 60 and above), and non-significant effects on α1β1γ2 and α1β2γ1 GABA A receptors at low GABA concentrations (EC 10 ). Since αβγ is responsible for the major portion of the phasic actions of GABA, and relatively high amounts of β1 and γ1 were reported at synaptic sites in CeA [10][11][12]14 , it is plausible that phasic effects of GABA through these subunit combinations would not be modulated by DCUK-OEt. In fact, when we applied DCUK-OEt focally to CeA neurons, we found no change in sIPSC frequency, amplitude, rise or decay time, indicating no effects on phasic transmission (Fig. 4c).
There is strong evidence for the existence of α1βxδ receptors located extrasynaptically in particular areas of brain (i.e., the interneurons of the hippocampus and particularly those of the dentate gyrus) [53][54][55] . Tonic inhibition mediated by GABA A receptors containing the α1 subunit has also been noted in the CeA 56 . Our prior studies using slices of the CeA demonstrated that CRF1 receptor-positive (CRF1+) neurons express the α1 GABA A receptor subunit, and this subunit is integral for the GABA-mediated tonic conductance in these neurons as well as being involved in the phasic synaptic response to GABA 56 . When we measured tonic conductance in CeA neurons, focal application of DCUK-OEt produced an enhancement of the recorded tonic current, suggesting local effects of DCUK-OEt at extrasynaptic GABA A receptors. To further ascertain whether the effects of DCUK-OEt were mediated particularly by extrasynaptic GABA A receptors, we performed a comparison of the change (increase) in current produced by DCUK-OEt and the decrease generated by the subsequent co-application of 100 μM gabazine 57 . The strong correlation indicated that DCUK-OEt was indeed stimulating a tonic conductance in these neurons by actions at extrasynaptic GABA A receptors. Recently, de Guglielmo et al. 58 reported that inactivation of an ensemble of neurons in the CeA resulted in abrogation of excessive alcohol consumption by alcohol-dependent rats. The anatomical area of the CeA from which we obtained our electrophysiologic data coincides with the area containing the ensemble described by de Gugielmo et al. 58 . An increase in the tonic conductance through extrasynaptic GABA A receptors, mediated by DCUK-OEt, may engender reduced activity of the neurons identified by de Guglielmo et al. 58 and be an effective mode for reducing alcohol intake by dependent animals.
In all, our characterization of DCUK-OEt indicates that this molecule has characteristics that resemble those of etomidate, other anesthetics, ethanol and neurosteroids, but the full profile of DCUK-OEt actions speaks to an interaction with a site or sites on the GABA A receptor that distinguish DCUK-OEt from currently known PAMs and direct agonists acting at GABA receptors.
Radioligand binding. [ 3 H]Flunitrazepam Binding and Displacement by DCUK-OEt. Membrane
Preparation. These experiments were performed at the University of Colorado Health Sciences Center, Denver, CO. Experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Colorado, Denver, and were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Male Sprague-Dawley rats (200-250 g) were maintained in an AAALAC-accredited facility and sacrificed by CO 2 exposure and decapitation. Brains were removed, and membranes were prepared from the forebrain as described previously 6 .
Ligand binding assay. The binding of [ 3 H]flunitrazepam was assayed in triplicate, using final incubation volumes of 0.55 ml consisting of protein (approx 200-300 mg/ml), [ 3 H]flunitrazepam (New England Nuclear) at a concentration of 1 nM, 10 µM GABA and DCUKA or DCUK-OEt at 0, 5, 10, 20, 50, 100 and 200 µM in DMSO solution (final DMSO concentration 0.2%). Nonspecific binding was measured in the presence of 10 µM diazepam. Binding was initiated by addition of protein, followed by incubation at 4 °C for 30 min. Bound and free ligand were separated by rapid filtration under vacuum over Whatman GF/B filters presoaked in buffer in a 24 port Brandel Cell Harvester. Filters were washed with 2 × 5 ml of ice-cold HEPES buffer and dried prior to measurement of bound radioactivity by scintillation counting (Beckman LS3800 scintillation counter) using Ultima Gold XR scintillation cocktail.
Displacement of [ 3 H]muscimol binding by DCUK-OEt or DCUKA.
The assays of [ 3 H]muscimol binding were performed by the Psychoactive Drug Screening Program/NIMH (PDSP). Rat brain membranes were prepared as described in the Protocol Manual on the PDSP website (https://pdspdb.unc.edu/pdspWeb/). DCUK-OEt or DCUKA were dissolved in 1.0% v/v DMSO and assayed at 11 concentrations ranging from 0.05 nM to 10 μM (final DMSO concentration, 0.2%). The final concentration of [ 3 H]muscimol in the assay mixture was 5 nM. Displacement of [ 3 H] muscimol by GABA at concentrations ranging from 10 nM to 10 μM was measured to provide a positive control. Table 1), were also performed by PDSP and in our laboratories (batrachotoxin binding) 6 . The experimental details for all of the PDSP binding studies can be obtained by connecting to the PDSP website (https://pdspdb.unc.edu/pdspWeb/) and clicking on "Assays" (binding or functional) on the menu bar. PDSP initially performed ligand displacement studies at a default concentration of 10 μM DCUK-OEt. For any receptor/ transporter at which the compound generated a 50% or greater displacement of the receptor/transporter-selective ligand, a secondary binding assay was performed to calculate K i values (see below).
Screening for binding of DCUK-OEt to other receptors/transporters/enzymes. Additional ligand binding studies (Supplementary
Analysis Oocyte electrophysiology. Xenopus laevis frogs were obtained from Nasco (Fort Atkinson, WI, USA). All surgery was performed in accordance with a protocol approved by the University of Texas, Austin IACUC and the NIH Guide for the Care and Use of Laboratory Animals. The complementary DNAs encoding the GABA A subunits rat α1, β1, β3, γ2 s, δ, and human β2 were provided by Drs Myles H. Akabas, Paul J. Whiting and Richard W. Olsen. Human γ1 cDNA was synthesized de novo, optimized for Xenopus laevis oocyte expression and subcloned in pGEMHE by GenScript (Piscataway, NJ). The in vitro transcription of GABA A subunits was performed using mMessage mMachine (Life Technologies, Grand Island, NY). After isolation of Xenopus laevis oocytes, they were injected with capped complementary RNAs encoding wild-type or mutant subunits in different ratios, depending on the subunits: α1β2γ2 s, 2:2:20 ng; α1β2γ1, 2:2:6 ng; α1β2, 3:3 ng; α1β1γ2, 0.5:0.5:5 ng; α1β3γ2, 0.1:0.1:1 ng; α1β3, 0.5:0.5 ng; α1β3δ, α4β3γ2 and α4β3δ, 0.4:0.4:4 ng.
Electrophysiology. The injected oocytes were incubated at 15°C in sterilized Modified Barth's solution for 1-7 days before recording, and the responses of GABA A receptors expressed in oocytes were studied using two-electrode voltage clamp as described earlier 33,60 . Oocytes were discarded if the maximal current was over 20 µA or if the baseline was unstable or drifted to positive values.
Modulator application. DCUK-OEt and DCUKA stock solutions were prepared in DMSO weekly, then sonicated for 15 min, and stored at 4 °C, protected from light. On the day of the experiment, dilutions were prepared, sonicated for 5 min, and used immediately. The final DMSO concentration in the buffer bathing the oocyte was ≤0.1%. In order to test the effects of DCUKs, the agents were first pre-applied for 1 min and then co-applied with GABA. To verify the presence of a third subunit in expressed subunit combinations, the responses to GABA in the presence of Zn++ (1, 10 or 100 µM) were evaluated (Supplementary Table S2). The application sequence in each instance was as follows: Maximal GABA (20 s application, 15 min washout), EC 10 GABA (30 s application, 5 min washout), EC 10 GABA, pre-application of the drug followed by a co-application with EC 10 GABA, EC 10 GABA, pre-application of Zn++ immediately followed by a co-application with EC 10 GABA, EC 10 GABA. In most cases, we limited to one DCUK application per oocyte. Flumazenil and 17PA were pre-applied before their co-application with GABA. When co-applying with DCUK, the antagonist and DCUK were pre-applied together before their co-application with GABA. Flunitrazepam was not pre-applied before co-application with GABA.
Statistical Analysis. Responses to DCUK-OEt were quantified as the percent change in current between the response to the EC 10 concentration of GABA and the response to the EC 10 concentration of GABA in the presence of 0.3 μM concentration of DCUK-OEt. To control for batch effects a linear mixed model was implemented in SAS (version 9.4) to calculate the normalized percent change in current for each receptor subunit combination (each receptor combination was examined in two to nineteen separate experiments). Because each receptor was examined across several experiments, a random effect of batch was included in the model. For each receptor, the estimated percent change in the GABA EC 10 -induced current produced by addition of DCUK-OEt was compared to zero by ascertaining whether zero percent change was outside the confidence interval of the measured values. This was accomplished by using a single sample t-test in the MIXED procedure of SAS, and a Bonferroni adjustment to control for multiple comparisons across receptors. Comparisons between receptors with a single subunit difference were executed within the linear mixed model using linear contrasts. A Bonferroni adjustment was used to control for multiple pairwise comparisons. Significant effects are those with a Bonferroni adjusted p-value < 0.05 and marginal effects are those with a Bonferroni adjusted p-value < 0.2.
Other statistical tests (t-test and ANOVA) were applied as indicated in the corresponding table or figure legend.
The GABA concentration response curves (CRCs) were fitted to the following equation: Brain slice electrophysiology. Brain slice preparation. All procedures were approved by the Scripps Research IACUC and were consistent with the NIH Guide for the Care and Use of Laboratory Animals. Slices were prepared from brains of 5 adult male Wistar rats (250-350 g) as described by Herman et al. 56 . A single slice was transferred to a recording chamber mounted on the stage of an upright microscope (Olympus BX50WI).
Brain slice electrophysiological recording. Neurons were visualized and whole cell patch clamp recordings were made as previously described 56 . Series resistance was typically <15 MΩ and was continuously monitored with a hyperpolarizing 10 mV pulse. Electrophysiological properties of cells were determined by pClamp 10 Clampex software online during voltage-clamp recording using a 5 mV pulse delivered after breaking into the cell. The resting membrane potential was determined online after breaking into the cell using the zero current (I = 0) recording configuration and the liquid junction potential was included in the determination.
DCUKA and DCUK-OEt were prepared as described for the experiments on oocyte electrophysiology. Other drugs were dissolved in aCSF, and all drugs were applied by Y-tubing application for local perfusion primarily on the neuron of interest. To isolate the inhibitory currents mediated by GABA A receptors, all recordings were performed in the presence of glutamate and GABA B receptor blockers 56 . All voltage clamp recordings were performed in a gap-free acquisition mode with a sampling rate per signal of 10 kHz or a total data throughput equal to 20 kHz (2.29 MB/min) as defined by pClamp 10 Clampex software.
Data Analysis. Frequency, amplitude and decay of spontaneous inhibitory postsynaptic currents (sIPSCs) were analyzed and visually confirmed using a semi-automated threshold based mini detection software (Mini Analysis, Synaptosoft Inc.). Averages of sIPSC characteristics were determined from baseline and experimental drug conditions containing a minimum of 60 events (time period of analysis varied as a product of individual event frequency) and decay kinetics were determined using exponential curve fittings and reported as decay time (ms). All detected events were used for event frequency analysis, but superimposed events were eliminated for amplitude and decay kinetic analysis. In voltage clamp recordings, tonic currents were determined using Clampfit 10.2 (Molecular Devices) and a previously-described method 61 . Responses were quantified as the difference in holding current between baseline and experimental conditions. Events were analyzed for independent significance using a one-sample t-test and compared using a two-tailed t-test for independent samples, a paired two-tailed t-test for comparisons made within the same recording, and a one-way ANOVA with a Bonferroni post hoc analysis for comparisons made between 3 or more groups. All statistical analysis was performed using Prism 5.02 (GraphPad, San Diego, CA). Data are presented as mean ± SEM. In all cases, p < 0.05 was the criterion for statistical significance.
Molecular modeling.
All molecular modeling studies were conducted using Biovia Discovery Studio 2016 (Biovia Inc., San Diego, CA) and all crystal structure coordinates were downloaded from the protein data bank (www.pdb.org). The homology model of the human GABA A receptor pentamer was generated with the MODELLER protocol 62 utilizing the crystal structures of the human GABA A receptor β3 homopentamer as a template (PDB ID: 4COF 63 , Uniprot accession: P28742). Homology models of the human α1 (Uniprot accession: P14867) and γ2 (Uniprot accession: P18507) subunits were superimposed over the template, with the crystal structure of two β3 subunits, so that the final pentameric model consisted of two α1, two β3, and one γ2 subunits, arranged in an γβαβα pattern (counterclockwise, as seen from above). The resulting final structures were subjected to energy minimization utilizing the conjugate gradient minimization protocol with a CHARMm forcefield and the Generalized Born implicit solvent model with simple switching (GBSW) 64 . The minimization calculations converged to an RMS gradient of <0.01 kcal/mol. The Flexible Docking protocol 65 , which allows flexibility in both the protein and the ligand during the docking calculations, was used to predict the binding orientations of both known and candidate GABA A PAMs in the binding site located at either the classical α-γ benzodiazepine site (α + γinterface) or the alternative α-β site (α + βinterface). Predicted binding poses were energy-minimized in situ using the CDOCKER protocol 66 prior to calculation of binding energies using the distance-dependent dielectric model. All numeration refers to the corresponding mature protein. | v3-fos-license |
2019-06-26T14:12:55.486Z | 2018-12-18T00:00:00.000 | 195401952 | {
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} | pes2o/s2orc | Gingival Necrosis Caused by the use of Paraformaldehyde-Containing Paste: Case Series
Many of the medicaments used historically in root canal treatment have been shown to be cytotoxic [1]. Toxic devitalising agents such as arsenic trioxide (As2O3), a water-soluble compound, forming arsenious acid (H3AsO4) and paraformaldehyde were commonly used in the past to devitalise inflamed pulps when effective anaesthesia could not be obtained [2]. Among these substances an important role was played by paraformaldehyde pastes. In the oral cavity paraformaldehyde agents are used both as disinfectants and to devitalise inflamed pulps when local anaesthesia is ineffective. Despite the clinical benefits paraformaldehyde is not confined to the pulp, but penetrates through dentine and is gradually released as formaldehyde. Formaldehyde released through dentine has a destructive effect on periodontal and bone tissues [3-8].
Introduction
Many of the medicaments used historically in root canal treatment have been shown to be cytotoxic [1]. Toxic devitalising agents such as arsenic trioxide (As2O3), a water-soluble compound, forming arsenious acid (H3AsO4) and paraformaldehyde were commonly used in the past to devitalise inflamed pulps when effective anaesthesia could not be obtained [2]. Among these substances an important role was played by paraformaldehyde pastes. In the oral cavity paraformaldehyde agents are used both as disinfectants and to devitalise inflamed pulps when local anaesthesia is ineffective. Despite the clinical benefits paraformaldehyde is not confined to the pulp, but penetrates through dentine and is gradually released as formaldehyde. Formaldehyde released through dentine has a destructive effect on periodontal and bone tissues [3][4][5][6][7][8].
Formocresol is advocated as an intracanal medicament because of its antibacterial and tissue fixative properties [9,10]. Liquid formaldehyde compounds, such as formocresol may be expressed through patent apical, lateral and accessory canals and cause soft tissue and bony injury within the periodontium [10][11][12][13]. Since the advent of effective methods of securing anaesthesia, the use of paraformaldehyde or other toxic preparats for pulp devitalization has declined [14]. However, contrary to the trends within modern endodonties, it has yet to disappear from clinical practice, being advocated in vital primary endodonties and sporadically associated with severe tissue breakdown in adults [15][16][17][18]. This article describes the effects on the periodontal and bone tissues of the paraformaldehyde used as a devitalising or disinfectant agent.
Advances in Dentistry & Oral Health
A 20-year-old female presented to her dentist complaining of pain in the maxillary right quadrant. The practitioner elected to devitalize tooth 16, which had shown signs of pulpitis, with a paraformaldehyde-containing paste since local anaesthesia had been ineffective. Shortly after the placement of the paste, the patient experienced increasing pain and noticed that there was blackening of the gingiva (Figure 1). She was referred to Ataturk University, Department of Periodontology for advice regarding gingival necrosis associated with the distal interproximal area of tooth 16. A decision was made to attempt to preserve the tooth by root canal treatment. Oral hygiene procedures were instituted, and the necrotic tissues were removed from the area. The operative area was irrigated with saline. Root canal treatment was completed, and the patient discharged for 1 week. One week later, there was no distal gingival papilla at tooth 16 and interproximal bone was exposed to a height of approximately 2-3mm (Figure 3). At this stage a coronally repositioned flap was planned in order to remove of the necrotic tissues and to cover the root surface. A sulcular incision was performed initially and then a vertical incision was made between distal aspect of teeth 17 and 15. Exposed necrotic bone was removed with a curette and water-cooled bur. To protect the exposed bone and expedite healing, the affected area was covered with a coronally repositioned flap. The patient was reviewed to check healing. After 1 week the sutures were removed and the operative area irrigated with saline. At 1-year recall, the patient reported that the tooth was functional without any problem. Clinical examination revealed nothing abnormal -no symptoms, no detectable mobility, no periodontal pocketing. The tooth was in functional occlusion.
Case 2
A 35-year-old female with a history of severe pain in the left mandibular molar region was referred for assessment. The patient reported symptoms of irreversible pulpitis around from tooth 36 ( Figure 4A & 4B). His general dental practitioner was unable to obtain adequate anaesthesia to extirpate the pulp from this tooth and applied paraformaldehyde paste to effect devitalisation. Subsequently the patient stated that she experienced severe pain, the lower left molar area. She was referred to Ataturk University, Department of Periodontology for advice regarding gingival necrosis associated with the distal interproximal area of tooth 36-37. Clinical examination revealed hard, inflamed gingiva with probing depths not exceeding 3mm on the distal aspect of tooth 36. There was no measurable tooth mobility. Radiographic examination demonstrated expansion of the periodontal ligament and loss of lamina dura distal to the tooth 36. Despite all the persuasive efforts, the patient decided to extract the tooth and the tooth was extracted.
Discussion
The primary cause of pulpal pathosis is bacterial infection. Without the presence of bacteria in the root canal system, periradicular inflammation will not develop or persist [19]. Consequently, the purpose of root canal treatment is the elimination of bacteria and their substrates from the pulp canal system. During this treatment the pulp is removed and the root canal is cleaned and shaped. Various chemicals used in medicating root canals can create adverse tissue reactions [10]. This fact, combined with the knowledge that the pulp and periodontal ligament are interconnected via accessory canals, dentinal tubules and iatrogenic communications suggests that overzealous use of intracanal medicaments can lead to deleterious effects to the host tissue with resultant postoperative discomfort [20,21]. In the past, effective anaesthesia was either unavailable or rudimentary [2]. Today there are methods of anaesthesia and reliable anaesthetic agents able to control most pulpal pain for treatment. Therefore, it is unnecessary to use chemicals for devitalising pulps. However, they are still in use and dental complications continue to be reported, even in developed countries [7,13,18]. Paraformaldehyde has well-recognised toxic effects, which predictably occur on direct contact with gingiva and bone. The recovery of paraformaldehyde from a sequestrum is an indication of the local distribution of this toxic material in the tissues [14]. In the present study toxic effects of paraformaldehyde was seen on periodontal tissues and the tooth preserved by a combination of periodontal, endodontic and maintenance therapy.
Conclusion
As there is no longer either any indication or need to utilize devitalising preparations in dental practice, its continued and unjustified use must be condemned and should be prohibited. However, in a mobile world population, dentists should be aware of such materials and their adverse consequences and be prepared to recognise and manage the tissue injuries that may result from their use. | v3-fos-license |
2019-05-26T13:55:13.845Z | 2019-03-28T00:00:00.000 | 164474492 | {
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} | pes2o/s2orc | ANALYTICAL METHODS FOR THE QUANTITATION OF AMLODIPINE BESYLATE AND ATORVASTATIN CALCIUM IN PHARMACEUTICAL DOSAGE FORM AND BIOLOGICAL FLUIDS
Amlodipine is the best-prescribed medication for cardiovascular disease major risk factor for hypertension and atorvastatin well known for diabetic. First discussed low cost ultraviolet-visible technique for the determination and quantitation of drugs in pharmaceuticals and biological fluids. Chromatographic techniques have an application with respect to trace analysis. Different types of chromatography such as high-performance liquid chromatography, high performance thin layer chromatography have most frequent applications in the field of pharmaceutical as well as biomedical analyses. Chromatography combined with mass spectrophotometry has the ability to collect molecular ion, followed to prepare a spectrum to assess molecular weight as well as structure. High-performance liquid chromatography coupled with mass spectrophotometry is a reliable and dynamic technique for the analysis of small and large drugs molecule. The advantages and disadvantages of all techniques are compared with each other with respect to sensitivity, reproducibility and other important parameters. The investigation also focused for the quantitation on both drugs in pharmaceutical preparations and plasma samples with the help of all available analytical techniques.
INTRODUCTION
Amlodipine besylate ( fig. 1a) is scientifically known as (RS)-3-ethyl-5-methyl-2-(2-aminoethoxymethyl)-4-(2-chlorophenyl)-1,4-dihydro -6-methyl-3,5-pyridinedicarboxylate benzene sulfonate. It was first introduced and prescribed for coronary artery disease. It was also helpful for angina and peripheral artery disease [1]. Now days mostly targeted for the patient having hypertension. The European Society of Cardiology was conducted survey until the year 2000 and their report based on statistical analysis showed nine hundred seventy-two million people were in this category. The number will increase with time and expected by the year 2025, approximately 1.56 billion [2]. This drug is under the umbrella of calcium channel blocker. The mechanism of such blockers to control the transportation of calcium to coronary (mainly smooth muscle) and arteries, that reflects on muscles became relaxes, reduces peripheral resistance and ultimately lowering the blood pressure [3]. Atorvastatin ( fig. 1b) is chemically known as (βR, δR)-2-(4-fluorophenyl)-β,δ-dihydroxy-5-(1methylethyl)-3-phenyl-4-[(phenylamino)carbonyl]-lH-pyrrole-1heptanoic acid. The synthetic drug, atorvastatin is generally under the class known statins. These reductase inhibitors are familiar as 3-hydroxyl-3methylglutaryl-coenzyme A (HMG-CoA). All drugs are prescribed for cardiovascular disease and reduction of heart attack, as well as for clinical demand [4][5][6][7][8][9][10]. Active pharmaceutical ingredients (APIs) such as amlodipine are commonly manufactured as their acid addition salts to promote solubility and improve both stability and bioavailability. The objective of this review article was to research more about ultraviolet-visible and chromatographic technique's applications, specifically for the quantification of amlodipine and atorvastatin in bulk, pharmaceutical formulations, and biological fluids. Compared the result between all the developed method for amlodipine and atorvastatin about the limit of detection and quantitation value. Discussed briefly the importance of all analytical techniques with respect to cost, time of analysis, sensitivity and limitations.
UV-visible spectrophotometer
Spectroscopic methods are mostly used for the determination of drugs in bulk as well as with pharmaceutical formulations. Ultraviolet (UV) and visible spectrophotometry are important and common techniques for the quantitative analysis of drugs. Because these are low-cost techniques, simple and no requirement of pretreatment as well as any elaborate preparatory step prior to assay. The visible spectrophotometer is depending on the redox and complex formation reaction. However, some deficiencies are also with the techniques with respect to the 6 presence of two or more drugs have similar UV characteristic. The detailed literature survey studies elaborate on the advantages and disadvantages among all developed method for both drugs using UVvisible spectrophotometer are presented in table 1.
High performance liquid chromatography
High-performance liquid chromatography (HPLC) technique is more accurate and based on the properties of the analyte with the existing mobile phase and stationary phase. Depending on the stationary and mobile phase, different types of chromatography techniques are developed. Recently high-performance liquid chromatography has achieved lots of attention in the field of pharmaceutical analysis in dosage forms and biological fluids because of its simplicity, sensitivity and high specificity. The conventional rule for the analysis of polar compounds by utilizing a non-polar stationary phase with the polar mobile phase and vice versa for the non-polar compounds. Stationary phase binding the analyte is directly proportional with the surface area of the nonpolar segment of the analyte associate the ligand as well as with aqueous eluent. One of the main parameters found as evidence about the quality and efficacy of drug products and formulations is stability testing. The products nature is changing with humidity, temperature, light, retesting time, storage conditions, shelf life, so it is necessary to control the environmental factors. To develop method different types of the column (ODS C 18 , BDS C8, ODS C8, BDS C18 and Discovery HS C18 The ultra-performance liquid chromatography (UPLC) is a new and modern technique for liquid chromatography. Worldwide HPLC was a predominant technique for the last 30 to 40 y for the drug analysis. Speed, sensitivity, and resolution are the main keywords for the drugs analysis with UPLC compare to HPLC. The particle size can play a significant role in this type of chromatography that governed by the well-known Van Deemter equation. For UPLC, the preferred size diameter is less than 2μm to get more sensitivity, short analysis time and improved resolution. The quality of the product analyzed by UPLC will give better with less time. However, the main disadvantage is related to the column life. The analysis requiring high pressure (100 M Pa) that damage the column efficiency. The novel and selective methods were developed for the determination of both drugs with a marketed formulation as single or combined dosage form [84][85][86][87][88][89][90][91][92][93][94][95][96][97][98] (table 3).
Thin layer chromatography
) with a combination of different mobile phases containing buffers, organic modifier (acetonitrile, methanol) can be used. Amlodipine and atorvastatin were quantified using HPLC with ultraviolet in bulk and pharmaceutical dosage form (table 2).
Ultra pressure liquid chromatography
Thin-layer chromatography (TLC) is a simple separation technique typically for the mixture of nonvolatile compounds. The adsorbent material, specifically cellulose, silica gel, aluminum oxide is covered on plastic or glass sheet, known as the stationary phase. Aleppo bentonite with modified phenyl support can also be applied as the stationary phase, has the ability to separate amlodipine and atorvastatin with a mobile phase consisting of sodium phosphate buffer and acetonitrile (50:45, v/v) in pharmaceutical dosage form [99]. Methanol, toluene triethylamine combination and silica gel adsorbent distinct atorvastatin in pharmaceutical formulation with high resolution [100]. However, triethylamine can be replaced with chloroform and acetic acid in tablet dosage form on the aluminum plate [101]. However, propanol and water system (70:30, v/v) has the capability to separate amlodipine with a detection limit of 0.4 µg [102].
248
Bulk drug and tablets [83] High-performance thin layer chromatography High-performance thin layer chromatography (HPTLC) is an accelerated separation technique and flexible enough to determine the drug sample. The advantages associated with HPTLC are a short analysis time for the complex sample without pretreatment, independent construction of chromatogram with multiple samples, easy to transfer samples that will increase the confidence and reliability of the technique. It can be employed for qualitative and quantitative purposes. Several combinations of mobile phases with different size of plates have been successfully studied by HPTLC in pharmaceutical preparations [103][104][105][106][107][108][109][110][111][112][113][114][115] (table 4).
Capillary electrophoresis
Capillary electrophoresis (CE) is based on charge and size under electric field separate molecules. The capillary tube is made of glass loaded with an electrolyte solution. Electrophoretic mobility is an important parameter for the separation, chemical constituents as well as solvent viscosity. However, the limitation with gel electrophoresis with regards to applied voltage due to ohmic heating damage the gels and restrict the separation. Need to require large voltage often 10-20 thousand for experiments with CE. Different types of capillary electrophoresis are capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), micellar electrokinetic capillary chromatography (MEKC), capillary electro chromatography (CEC), capillary isoelectric focusing (CIEF) and capillary isotachophoresis (CITP).
Phosphate buffer (pH 6.5) and methanol mixture combined (80:20, v/v) used as background electrolyte with capillary fused silica column under 15 KV voltage at room temperature for the determination amlodipine and atorvastatin [116]. In tablet formulation no interference from present common excipients [117]. Phosphate buffer can separate both drugs in 5 min with high precision but in the presence of acidic products need to involve borate buffer [118][119]. It is likewise possible to quantify within 3 min in combined dosage form with high efficiency and resolution [120]. CZE method validated for solid dosage form in the presence of electrolyte as a methanol borate buffer [121]. MEKC succeeded by controlling surfactant, sodium dodecyl sulfate (SDS) concentration and acquired within 2 min [122] compared to CZE [117] 13 min migration time having comparable resolution. High and ultra-pressure liquid chromatography combined with mass spectrophotometry High pressure and ultra-performance liquid chromatography with the mass spectrometry (LC-MS, UPLC-MS) are analytical techniques that combine liquid chromatography with mass spectrophotometer. The aim to develop a fast and reliable analytical method for the determination of atorvastatin and amlodipine together with the presence of metabolites using the above techniques. The MS combined with LC has high selectivity and sensitivity. The techniques are commonly applied for bioavailability and pharmacokinetics investigation. Mostly quantification of parent drug, active and inactive metabolites are of interest for various studies with biological fluids. It is also required to verify the interaction between drug to drug and side effects as well as the toxicity of different metabolites after metabolism in the human body. The LCMS [123][124][125][126][127][128][129][130][131][132][133][134][135][136] (table 5) and UPLC-MS [137][138][139][140][141][142] (table 6) were used for the determination of amlodipine, atorvastatin and their metabolites in pharmaceutical dosage forms and biological fluids.
DISCUSSION
The UV-visible spectrophotometer does not require any pH adjustment and very close to the pharmacopeia method for both drugs. Recently HPTLC can be applied as an alternative option for traditional TLC method. It is easy to handle with software, which is not possible with TLC. HPTLC enhanced the capability to determine impurity with the help of the hydrophilic phase combined silica gel, an important parameter in all pharmacopoeias. One single run is enough to achieve two parameters such quantity and its impurity by HPLC, main pharmacopeias method to quantify the assay percentage. The setting of parameters for the reaction is not easy as well as for characterization with CE that is why maybe not recommended in official method. UPLC is a high-cost instrument compare with other chromatographic methods, ability to study pharmacokinetics such as adsorption, metabolism. The limit of detection (LOD) and limit of quantitation (LOQ) value is very less with chromatographic method combined with mass spectro-photometry (table 7). Amlodipine and atorvastatin concentration in pharmaceutical formulations and biological fluids are based on different parameters and the calibration curve of the analyte (table 8). During method development and validation, accurately quantified the analyte as well provide brief information related to impurity profiling, a key equipment for the formulation process of the drug. Simple operation procedure, fast, cost effective, accurate in readings and used widely
CONCLUSION
The main objective of this review is to provide sufficient information for the researcher, academic, scientist about the analysis of important calcium channel and statins receptor. After going through with the proposed review, people can understand the common method for the analysis of drugs. These medications are very significant due to its growing demands in our daily life that is why it was one of the fastest growing products in the pharmaceutical industry. The ultraviolet and visible spectrophotometer are low-cost instrument as well as easy to handle and available everywhere. It is more suitable for pure pharmaceutical formulations. The biological fluids and trace analysis will go for highly sensitive instrument combined with the mass spectrophotometer. Therefore, the present review will give an idea for determination and quantification of both drugs using UV-visible, HPLC, HPTLC, UPLC, LCMS and UPLC-MS in bulk, pharmaceutical formulations, and biological fluids.
AUTHORS CONTRIBUTIONS
All the author have contributed equally
CONFLICT OF INTERESTS
The authors report no conflicts of interest | v3-fos-license |
2017-08-02T18:41:40.319Z | 2016-11-14T00:00:00.000 | 9395382 | {
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} | pes2o/s2orc | Matchout deuterium labelling of proteins for small-angle neutron scattering studies using prokaryotic and eukaryotic expression systems and high cell-density cultures
Small-angle neutron scattering (SANS) is a powerful technique for the characterisation of macromolecular structures and interactions. Its main advantage over other solution state approaches is the ability to use D2O/H2O solvent contrast variation to selectively match out specific parts of a multi-component system. While proteins, nucleic acids, and lipids are readily distinguished in this way, it is not possible to locate different parts of a protein–protein system without the introduction of additional contrast by selective deuteration. Here, we describe new methods by which ‘matchout labelled’ proteins can be produced using Escherichia coli and Pichia pastoris expression systems in high cell-density cultures. The method is designed to produce protein that has a scattering length density that is very close to that of 100% D2O, providing clear contrast when used with hydrogenated partner proteins in a complex. This allows the production of a single sample system for which SANS measurements at different solvent contrasts can be used to distinguish and model the hydrogenated component, the deuterated component, and the whole complex. The approach, which has significant cost advantages, has been extensively tested for both types of expression system.
Introduction
Small angle neutron scattering (SANS) and small-angle X-ray scattering (SAXS) provide important low resolution structural information on biological macromolecules in solution (Jacrot 1976;Glatter and Kratky 1982;Serdyuk et al. 2007). SANS approaches have the unique advantage of being able to exploit solvent contrast variation through the use of buffers containing specific D 2 O/H 2 O ratios (Stuhrmann 1974;Svergun et al. 2013). This capability arises from the different neutron scattering properties of hydrogen ( 1 H, neutron coherent scattering length b c = −3.7423 fm) and its heavy isotope deuterium ( 2 H or D, neutron coherent scattering length b c = 6.675 fm) (Shull 1962). This results in the very different scattering length densities (SLDs) of −0.562 × 10 10 cm −2 and 6.404 × 10 10 cm −2 for H 2 O and D 2 O, respectively. When specific H 2 O/D 2 O solvent mixtures are made, it is therefore possible to make solutions having any SLD in this range (Fig. 1, black line). In the normal (hydrogenated) context, the major classes of biomolecules (protein, lipid, nucleic acid) exhibit naturally occurring differences in SLD. By changing the SLD of the solvent (buffer) to match specific parts of the biomolecule being studied, each part of the complex can be individually 'matched out' and rendered invisible to SANS data collection (Jacrot 1976). Figure 1 shows the variation of SLD as a function of D 2 O/H 2 O composition by volume for the different classes of biomolecules, including deuterated proteins. Hydrogenated protein shows a match point at approximately 40% D 2 O, while nucleic acids show a match point of ~62%. This means, for example, that SANS data Abstract Small-angle neutron scattering (SANS) is a powerful technique for the characterisation of macromolecular structures and interactions. Its main advantage over other solution state approaches is the ability to use D 2 O/H 2 O solvent contrast variation to selectively match out specific parts of a multi-component system. While proteins, nucleic acids, and lipids are readily distinguished in this way, it is not possible to locate different parts of a protein-protein system without the introduction of additional contrast by selective deuteration. Here, we describe new methods by which 'matchout labelled' proteins can be produced using Escherichia coli and Pichia pastoris expression systems in high cell-density cultures. The method is designed to produce protein that has a scattering length density that is very close to that of 100% D 2 O, providing clear contrast when used with hydrogenated partner proteins in a complex. This allows the production of a single sample system for which SANS measurements at different solvent contrasts can be used to distinguish and model the hydrogenated component, the deuterated component, and the whole complex. The approach, which has significant cost advantages, has been extensively tested for both types of expression system. recorded for a protein-DNA complex in 40% D 2 O buffer will reveal only the DNA structure. Likewise, SANS data measured using ~62% D 2 O when the DNA is matched out will reveal the protein component alone (Fig. 1). This powerful approach enables different parts of the same complex to be modelled both separately and together (Callow et al. 2007;Niemann et al. 2008;Obarska-Kosinska et al. 2008;Rochel et al. 2011;Vijayakrishnan et al. 2011;Taylor et al. 2012;Cuypers et al. 2013;Compton et al. 2014;Appolaire et al. 2014).
To distinguish between biomolecules having the same SLD, such as protein-protein complexes, deuterium labelling is necessary. Perdeuterated protein, where all the hydrogen atoms are replaced by deuterium, has an SLD higher than that of D 2 O (Fig. 1, magenta line) and cannot be fully solvent matched in SANS experiments. However, protein that is part-deuterium labelled such that its SLD is the same as that of 100% D 2 O can be readily exploited in SANS studies. We refer to such sample material as being matchout labelled. This type of labelling is particularly advantageous in studies of protein-protein complexes in which one protein partner is hydrogenated and the other is matchout labelled. Here, as illustrated schematically in Fig. 2, SANS data would typically be recorded in three different buffers: (a) where the full complex is observable (e.g., 0% D 2 O), (b) where only the matchout labelled component is visible (~40% D 2 O), (c) where only the hydrogenated component is visible (100% D 2 O). In (a), there should be good correspondence between SANS and SAXS analyses of the complex, although a lower radius of gyration may be expected for the SANS analysis of the matchout-labelled protein; this will occur because it will be measured in conditions where a high fraction of the surface solvent layer around the complex will be H 2 O which has a SLD close to zero (Svergun et al. 1998;Perkins 2001).
Methods to prepare appropriately deuterated proteins for SANS are required. The Escherichia coli and Pichia pastoris expression systems are both well characterised with numerous cell lines and expression plasmids available. The effect of D 2 O in E. coli growth media on the deuteration of RNA polymerase and ribosomal proteins has been studied in detail (Lederer et al. 1986;Leiting et al. 1998). A method Fig. 1 The scattering length densities (SLDs) of the four major biomolecules are depicted as a function of the volume percentage of D 2 O, assuming all labile hydrogen atoms are exchanged. The black line represents the variation of the solvent SLD. The match point of each biomolecule corresponds to the intersection of the solvent SLD with that for each biomolecule. Perdeuterated protein, in which all the hydrogen atoms are replaced by deuterium, has an SLD that is higher than that of D 2 O and cannot be solvent matched. to estimate deuteration levels in whole E. coli cells and cellular proteins by NMR has been described (Perkins 1981). For P. pastoris (yeast), fewer studies have been reported, although it is clear that this yeast can grow in deuterated media (Haon et al. 1993). Here, following development work in the Deuteration Laboratory (D-Lab) of the Life Sciences group at the Institut Laue-Langevin (Haertlein et al. 2016), we describe new deuteration techniques by which matchout labelled proteins can be routinely prepared using E. coli and P. pastoris. Both expression procedures utilise cell growth in minimal media based on 85% D 2 O and a hydrogenated carbon source. The matchout labelled proteins were validated by mass spectrometry and SANS. In the case of the E. coli system, maltose binding protein (MBP) was used as a representative model system. MBP is a well-studied model protein that plays an important role in the metabolism of E. coli (Sharff et al. 1992) and is essential for the energy-dependent translocation of maltose and maltodextrins through the cytoplasmic membrane. For P. pastoris, a model system based on the C-terminal domain pair of human complement Factor H (CFH) was used. CFH is a key regulator of the complement system of innate immunity, in which its two C-terminal short complement regulator domains (SCR-19/20) are crucial for protecting host cells against undesired immune destruction (Rodriguez et al. 2014).
Protein production from E. coli in matchout conditions
MBP was expressed using the E. coli BL21(DE3) cell line (Laux et al. 2008). High cell-density cultures were achieved using fermenters for cell growth. Minimal medium was prepared with the composition 6.86 g L −1 (NH 4 ) 2 SO 4 , 1.56 g L −1 KH 2 PO 4 , 6.48 g L −1 Na 2 HPO 4 ·2H 2 O, 0.49 g L −1 diammonium hydrogen cit- and 5 g L −1 glycerol. The medium was supplemented with 40 mg L −1 kanamycin to select for the recombinant plasmid. The BL21(DE3) cells containing the DNA construct were firstly adapted to growth in minimal media using a stepwise process in which cells were inoculated into minimal media, grown for 36 h and then transferred into fresh minimal media. This was repeated until a sufficient growth rate was obtained. For the preparation of deuterated minimal media, the mineral salts were dried out in a rotary evaporator (Heidolph) at 60 °C, then dissolved in a mixture containing 85% D 2 O/15% H 2 O. Cells were then adapted to growth in deuterated media. When a sufficient growth rate was achieved, large scale expression was carried out. Then 1.5 L of deuterated medium was inoculated with 100 mL pre-culture of adapted cells in a 3 L fermenter (Labfors, Infors). During the batch and fed-batch phases, the pH was adjusted to 6.9 by addition of NaOD, and the temperature was adjusted to 30 °C. The gas-flow rate of sterile filtered air was 0.5 L min −1 . Stirring was adjusted to ensure a dissolved oxygen tension of 30%. The fed-batch phase was initiated when the optical density at 600 nm reached 5.1. Glycerol was added to the culture to keep the growth rate stable during fermentation. When the OD 600 reached 14.7, over-expression was induced by the addition of 1 mM IPTG and incubation continued for 24 h. Cells were then harvested and stored at −80 °C.
Protein production from P. pastoris in matchout conditions
SCR-19/20 was expressed in yeast P. pastoris using the X-33 cell line. Cloning and expression of hydrogenated SCR-19/20 were described in detail elsewhere (Cheng et al. 2005). Cells, which had previously been transformed with the plasmid construct, were adapted to growth in a minimal media. Minimal media consisted of 13.4% yeast nitrogen base, 0.02% biotin, 1% glycerol and 100 mM potassium phosphate, pH 6.0. Adaptation was carried out by a similar protocol to that of the E. coli system, with the cells grown for 48 h before transferral into fresh minimal media. When high-density cell growth was achieved, cells were then adapted to growth in 85% deuterated minimal media by the same process. Deuterated minimal media was prepared as for the hydrogenated media, but was dissolved in 85% D 2 O. Large scale expression was then carried out by sustaining cell growth for 72 h in deuterated minimal media containing glycerol. Cells were then harvested by centrifugation and re-suspended in deuterated minimal media with 0.5% methanol in place of glycerol to induce protein expression. Expression was sustained for 96 h by feeding with 0.5% methanol every 24 h. All cell growth and expression was carried out using baffled flasks with shaking at 220 rpm at 29 °C. Final cell cultures were centrifuged to remove the cells from the supernatant containing the secreted protein.
Protein purification
MBP from E. coli was expressed in a soluble form. Cells were broken by sonication and the insoluble fraction removed by centrifugation. For both proteins, purification was carried out using hydrogenated buffers according to the same protocol used for the hydrogenated protein. In the case of the MBP purification immobilized metal ion affinity chromatography (IMAC) on TALON (Clontech) was 1 3 used. The supernatant was loaded on a column filled with 10 mL of TALON beads. This column was washed with 20 column volumes of lysis buffer containing 5 mM imidazole in 10 mM Tris-HCl, 100 mM NaCl, pH 7.5. MBP was then eluted with 100 mM imidazole. Fractions were analysed by polyacrylamide gel electrophoresis (PAGE), pooled and dialysed against 10 mM Tris-HCl, 100 mM NaCl, pH 7.5. 300 mg of MBP was obtained from 1 L of media. For the SCR-19/20 purification, cation exchange chromatography was used on an SP FF column (GE Healthcare). The supernatant was dialysed against 50 mM Tris, 25 mM NaCl, 1 mM EDTA, pH 7.4 and loaded onto the column. The column was washed with five column volumes of the same buffer. Elution was achieved by applying a NaCl salt gradient from 25 mM NaCl to 1 M NaCl. Fractions were analysed by PAGE and were pooled and dialysed against 10 mM Hepes, 137 mM NaCl, pH 7.4. Approximately 7 mg of SCR-19/20 was obtained from 1 L of start media. Further details for the MBP and SCR-19/20 purifications are given in Laux et al. (2008) and Dunne (2015) respectively.
Mass spectrometry
Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectroscopy was carried out on the deuterated MBP and SCR-19/20 proteins. The matrix consisted of sinapinic acid in acetonitrile/water-0.1% TFA (50:50). For both proteins, measurements were carried out at a concentration of 0.5 mg/mL in hydrogenated buffers. This meant that the calculation of the deuteration levels for the two matchout labelled proteins made the assumption that all of the labile deuterium atoms were replaced by hydrogen.
SANS data collection
Data were collected on the SANS instruments D22 (MBP) and D33 (SCR-19/20) at the Institut Laue-Langevin, Grenoble, France (Dewhurst et al. 2016). To calculate the match point, samples were prepared in a range of D 2 O concentration in the appropriate buffer. SANS data from MBP were collected in 0, 20, 40, 60, 80% D 2 O buffers; SANS data for SCR-19/20 were collected in 0, 25, 40, 75 and 100% D 2 O buffers. Data reduction, buffer subtraction, and transmission calculations were carried out using the program GRASP. The curves were fitted using Guinier plots (Guinier and Fournet 1955) yielding the radius of gyration R G and the scattering intensity at zero angle I(0). From this the normalised scattering amplitude of the proteins at each D 2 O concentration was calculated using the following expression: where T is the sample transmission, l is the cuvette path length (cm) and c is the concentration (mg/mL). This scattering amplitude was plotted as a function of D 2 O percentage and a linear fit carried out. The contrast match point was taken as the intersection of this plot on the abscissa.
Results
Escherichia coli and Pichia pastoris cultures were successfully adapted to growth in deuterated minimal media. For both organisms, five changes of media were sufficient for adaptation. High cell densities were achieved using 3 L fermenters in which the conditions for growth were tightly controlled. For the production of E. coli cells in high celldensity cultures, the growth conditions were controlled and monitored using IRIS software (http://www.infors-ht.com). The induction of expression commenced at an OD of about 15. Both proteins were expressed in their soluble forms and successfully purified using a similar protocol to that used for the same hydrogenated counterparts.
Maltose binding protein (MBP)
SDS-PAGE results before and after purification showed that MBP of high purity was obtained after a single IMAC purification step (Fig. 3A). The mass of normal hydrogenated MBP was measured to be 42,360 Da by mass spectrometry, and is identical to the value predicted from the sequence taking account for methionine aminopeptidase processing (Laux et al. 2008). Mass spectrometry measurements of the partially deuterated analogue (Fig. 4a) showed an increase (Gasteiger et al. 2005); the deuteration level of the MBP was estimated to be 64.1% of the non-exchangeable hydrogen atoms. The smaller peak to the right of this in Fig. 4a is attributed to incomplete aminopeptidase processing of the matchout labelled MBP. The SANS data collected from MBP showed no evidence of aggregation at any contrast, and Guinier analyses were used to determine the I(0) and R G values for each contrast (Fig. 5A). The R G value of MBP was 2.6 nm. The variation of scattering amplitude √ I(0) as a function of solvent D 2 O composition for the partially deuterated MBP gave the contrast match point (Fig. 5B). That for the partially deuterated MBP expressed in a high cell-density culture with 85% D 2 O as the only source of deuterium was determined to be 99.5% D 2 O.
Complement factor H SCR-19/20
Deuterated SCR-19/20 of high purity was obtained by cation exchange chromatography followed by size exclusion chromatography. This was confirmed by the PAGE results after purification (Fig. 3B). The yield of deuterated SCR-19/20 was calculated from the absorbance (A280 nm) of the pooled size exclusion chromatography fractions corresponding to the protein peak. From 1 L of glycerol growth media, an average of 7.2 mg protein was obtained. This yield was similar to the average of 7 mg obtained for hydrogenated SCR-19/20 in hydrogenated nutrient rich media. The mass of hydrogenated SCR-19/20 was 14,734 Da. Mass spectrometry showed that the mass increased to 15,281 Da following deuteration (Fig. 4b).
Because perdeuterated SCR-19/20 has an expected mass of 15,749 Da, the deuteration level was deduced to be 71.4% of the non-exchangeable hydrogen atoms. An additional peak was observed at 15,490 Da, corresponding to two additional amino acids, and was attributed to erroneous Kex protease processing of the signal peptide.
The SANS data for SCR-19/20 also showed no evidence of protein aggregation, and resulted in the I(0) and R G values for each contrast in Guinier plots (Fig. 5A). The R G value was 2.2 nm for SCR-19/20. The scattering amplitude plot of √ I(0) for the partially deuterated SCR-19/20 gave a contrast match point of 97% D 2 O and confirms that our protocol was optimal for the production of matchout labelled protein (Fig. 5B).
Discussion and conclusion
The novel deuteration methods described here allow the efficient production of matchout labelled proteins that are optimised for SANS structural studies in solution. For both proteins studied, the yields were similar to those observed for hydrogenated nutrient rich media. The difference in the deuteration levels (for the non-exchangeable hydrogen atoms) of the two proteins, as observed by mass spectrometry, are attributed to their different amino acid compositions and to differences in the growth media used. In both cases a ~100 Da broadening of the main peak was observed by mass spectrometry, compared to the hydrogenated or perdeuterated proteins, and this reflects the nature of the random fractional deuteration regime used. For a typical SANS experiment, this labelling heterogeneity is not normally a problem. Proteins that are matchout labelled in this way are particularly useful in structural analyses of protein-protein complexes for which one protein of the pair would be labelled.
The advantages of this method of labelling are: 1. Matchout labelled proteins are very effective in SANS studies of complexes. Only a single preparation of complex containing a matchout labelled component is required to allow measurements for a complete SANS structural analysis. Measurements at 100%, ~40%, and 0% D 2 O solvent contrasts will yield structural information on the unlabelled component, the labelled component, and the full complex respectively, i.e., on conformational changes in either protein after complex formation, as well as the relative orientation of the two proteins in the complex. Such a study requires that the For SCR-19/20, the central peak is associated with the properly processed SCR-19/20 protein (15,281 Da) complex is stable (i.e., a low dissociation constant) and that there is no propensity for aggregation in all the required solvents. The approach has significant advantages over SANS experiments in which two separately labelled complexes are produced in which one of the two components is typically perdeuterated, and has already been successfully applied to numerous protein complexes. 2. The adaptation of host cells to growth conditions containing a hydrogenated carbon source and 85% D 2 O is much more efficient than the culture conditions needed for perdeuteration. 3. Significant cost advantages are accrued, because no deuterated carbon source is needed in the culture medium. Since the solvent used is ~85% D 2 O, this permits the effective use of recycled D 2 O. Previouslydescribed methods used more expensive deuterated glycerol and deuterated methanol to obtain similar yields of protein (Massou et al. 1999;Pickford and O'Leary 2004).
The ability to label proteins using either E. coli or P. pastoris expression systems offers versatility in the labelling of recombinant proteins. For those systems where there are difficulties in expressing folded protein in E. coli, the use of P. pastoris offers a robust alternative in which issues associated with co-translational and post-translational modification are addressed. Future work of this type will focus on the development of similar matchout labelling approaches for insect cells and mammalian cells. GR/R47950/01 that allowed associated diffractometer developments at the Institut Laue-Langevin. We also thank the Institut Laue-Langevin for beamtime on instruments D22 and D33. We thank University College London and the Institut Laue-Langevin for joint funding of studentship support to OD. The European Union is thanked for its support of methodological developments that occurred under contracts RII3- CT-2003505925 and NMP4-CT-2006-033256. This work used the platforms of the Grenoble Instruct centre (ISBG; UMS 3518 CNRS-CEA-UJF-EMBL) with support from FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). We thank Luca Signor for assistance and access to the Mass Spectrometry facility. We also acknowledge Joe Zaccai for advice and for numerous helpful discussions.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of interest.
Open Access This article is distributed under the terms of the Creative Commons 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. | v3-fos-license |
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} | pes2o/s2orc | Influence of Catalytic Formulation and Operative Conditions on Coke Deposition over CeO 2-SiO 2 Based Catalysts for Ethanol Reforming
In this work, a series of CeO2-SiO2 (30 wt % of ceria)-based catalysts was prepared by the wetness impregnation method and tested for ESR (ethanol steam reforming) at 450–500 ◦C, atmospheric pressure and a water/ethanol ratio increasing from 4 to 6 (the ethanol concentration being fixed to 10 vol %); after every test, coke gasification measurements were performed at the same water partial pressure, and the temperature of the test and the gasified carbon was measured from the areas under the CO and CO2 profiles. Finally, oxidation measurements under a 5% O2/N2 stream made it possible to calculate the total carbon deposited. In an attempt to improve the coke resistance of a Pt-Ni/CeO2-SiO2 catalyst, the effect of support basification by alkali addition (K and Cs), as well as Pt substitution by Rh was investigated. The novel catalysts, especially those containing Rh, displayed a lowering in the carbon formation rate; however, a faster reduction of ethanol conversion with time-on-stream and lessened hydrogen selectivities were recorded. In addition, no significant gain in terms of coke gasification rates was observed. The most active catalyst (Pt-Ni/CeO2-SiO2) was also tested under different operative conditions, in order to study the effect of temperature and water/ethanol ratio on carbon formation and gasification. The increase in the water content resulted in an enhanced reactor-plugging time due to reduced carbonaceous deposits formation; however, no effect of steam concentration on the carbon gasification rate were recorded. On the other hand, the increase in temperature from 450–500 ◦C lowered the coke selectivity by almost one order of magnitude improving, at the same time, the contribution of the gasification reactions.
Introduction
In order to encourage an economy based on clean energy, the demand for hydrogen fuel cells is expected to grow rapidly in the near future [1].Hydrogen is a valid alternative to conventional fuels due to its ability to burn without emitting environmental pollutants, as well as its high energy content per unit of weight (142 KJ/g [2], 2.75 times greater than that of hydrocarbon fuels).Nowadays, methane steam reforming is the most conventional and economical process for hydrogen production [3,4] but other technologies, including biofuels conversion, have been intensively investigated, also due to the bio-feedstock's ability to act as an emission sink within a carbon-balanced life-cycle [5,6].Moreover, among the various renewable sources, biomass is highly promising due to its abundance and low cost [7,8].Biofuels can be produced both from food crops (first generation), as well as the residuals of agricultural and forestry wastes (second generation).Bioethanol presents several advantages, such as a high octane number, high heat of vaporization and, most importantly, reduction of greenhouse gas emissions.In fact, the CO 2 generated during bio-fuels conversion can be recycled for growing plants through photosynthesis, resulting in a carbon neutral process.In addition, bioethanol provides an important route for gasoline replacing, due to its easy handling and hydrogen storage ability [9].Currently, starch and sugar-rich materials are mainly employed for large-scale bioethanol production which, however, are not desirable due to their value as food [10].On the other hand, lignocellulosic biomass is cheap and widely available and can be converted to bioethanol by a pre-treatment-hydrolysis-fermentation cycle [11].Ethanol can be efficiently converted into H 2 by means of its catalytic reaction with steam, according to Equation (1): Despite ethanol steam reforming (ESR) theoretically producing only H 2 and CO 2 , other reactions, such as methanation, water gas shift, dehydration, dehydrogenation, and cracking, can led to the formation of several side products, including methane, carbon monoxide, acetaldehyde, ethylene, acetone, and coke [12,13].For example, unsaturated hydrocarbon (C 2 H 4 ) can be polymerized to coke while the decomposition of saturated hydrocarbons, such as CH 4 , promote carbidic species formation that act as intermediates in the filamentous carbon growth [14].Hence, major challenges of catalyst design for ethanol steam reforming is to maximize hydrogen productivity, inhibiting, at the same time, coke formation.In addition, operating at low-temperatures are highly desired from the point of view of the hydrogen economy and energy consumption [15].
Several metals have been effectively used for the steam reforming of ethanol, due to their capability to break C 2 H 5 OH molecule.In particular, less-oxophilic metals (Pd and Pt) are known to activate α-C-H bonds, while more-oxophilic metals (Co, Ni, Rh, Ru) promote molecule activation via O-H [16].Despite noble metals catalysts being more active and stable than base metal catalysts in ESR reaction, the high costs hinder their practical applications.Conversely, nickel and cobalt-based catalysts are widely used and recognized as appropriate catalysts for ethanol reforming due to their low price and high activity towards C-C bond cleavage [17,18].However, to overcome the considerable coke selectivity observed over these catalysts and to increase the hydrogen yield, the synergistic combination of transition metals with very low amounts of noble metals has been reported as a viable route [19][20][21].It was also shown that adding promoters to Ni-based catalysts is an effective method to prevent carbon deposition.In fact, alkali metals (K, Cs, Na, Li), being electron donators, display excess mobile electrons, which enhances hydrogen spillover on the catalyst surface, thus reducing the amount of coke deposits [22,23].Moreover, due to the neutralization of catalyst acid sites, alkali metals are able to suppress hydrocarbon (CH 4 and ethylene) decomposition and Boudouard reaction.Rass-Hansen et al. [24] found an optimum potassium doping (0.5%) over Ni/MgAl 2 O 4 for maximizing steam adsorption onto the catalyst surface and reduce the carbon formation while Akiyama et al. reported that the cesium-doped Ni/ZrO 2 catalysts improve H 2 yield with respect to Li-and K-based samples and were more effective in decreasing carbon deposition [25].
The nature of the support is also of great importance for the final catalyst performances.It was shown that acidic supports, such as Al 2 O 3 , preferentially promote dehydration while basic support (MgO) favor dehydrogenation reactions.Conversely, reducible supports, including rare earth oxides, display low by-products selectivity and improved hydrogen yield.The superior activity of CeO 2 -based support can be linked to the good metals particles dispersion and the prevention of sintering phenomena; moreover, their capability to add and remove oxygen in a reversible manner facilitates carbon gasification [26].Mobile oxygen of ceria can also activate water, with regard to the formation of hydroxyl groups, resulting in higher ESR efficiency [27].In particular, ceria was shown to accelerate the reaction of steam and absorbed species at the metal-support interface, giving place to -O and -HO species, which are transferred to the surface carbon, thus promoting coke conversion to gaseous products (CO, H 2 , and CO 2 ) [28,29].However, the low intrinsic surface area of reducible supports has encouraged the deposition of thin films of these oxides on porous supports.To that end, silica is a very interesting material due to its high surface area and chemically-inert nature [30].Moreover, the high number of oxygen vacancies in CeO 2 -SiO 2 mixed oxides may increase the lattice oxygen mobility and improve reducibility, activity, and stability of the catalyst [31].
In our previous works [32,33], we demonstrated the efficiency of a bimetallic Pt-Ni catalyst supported on CeO 2 -SiO 2 for hydrogen production via ethanol reforming: the combination of two metals, as well as the choice of a mixed oxide as a support having enhanced redox properties with respect to ceria alone resulted in interesting activity and stability.
In this study, a series of CeO 2 -SiO 2 based catalysts were prepared and characterized.The effect of alkali metals (K and Cs) modification, as well as the addition of Rh (or Pt substitution by Rh) on catalyst activity and stability for ethanol steam reforming was investigated.Moreover, once selected the most interesting sample, coke formation and gasification rates were evaluated and compared at different operative conditions (temperature and water/ethanol ratio).
Catalyst Preparation and Characterization
Five CeO 2 -SiO 2 based catalysts (Pt-Ni, Rh-Pt-Ni, Rh-Ni, Pt-Ni-K, Pt-Ni-Cs) were prepared by sequential wetness impregnation.More details about the preparation method are reported in [32].CeO 2 content in the catalyst was previously optimized and fixed to 30 wt %; Ni and noble metals loading (in the bimetallic catalysts) were equal to 10 wt % and 3 wt %, respectively, calculated on the basis of ceria mass.K, Cs, and Rh content in trimetallic catalyst was 0.5 wt % on ceria and potassium hydroxide, caesium chloride (both from Sigma-Aldrich, Saint Louis, MO, USA), and rhodium chloride (Strem Chemicals, Newburyport, MA, USA) were selected as metals salt precursors.K and Cs were directly impregnated on the support, followed by Ni and then Pt while the trimetallic catalysts were prepared by depositing Rh on the Pt-Ni/CeO 2 /SiO 2 sample.
The CeO 2 , as well as the metal loading (wt %), were determined by X-ray fluorescence on an ARL (Air Resources Laboratory) QUANT'X ED-XRF (energy-dispersive X-ray diffraction) spectrometer (Thermo Fisher Scientific, Waltham, MA, USA).N 2 adsorption-desorption isotherms at −196 • C were obtained for the determination of the specific surface area (SSA) of the catalytic samples, according to Branauer-Emmet-Teller (BET) method by using a ThermoScientific Surfer (Thermo Fisher Scientific, Waltham, MA, USA).The calcined catalysts, as well as the bare support, were characterized by powder XRD (X-ray diffraction, model D8 Advance Bruker (Bruker, Billerica, MA, USA) using nickel-filtered CuKα radiation over the 2θ range 20-80 • .The Scherrer equation, (d = 0.9•λ•β −1 •cosθ −1 ), where λ is the wavelength of incident radiation (nm), β is the half-height width of the most intense peak for the species (radians) and θ is the Bragg angle ( • ) of that peak, was employed to determine the particle size of the various phases based on their most intense diffraction peaks.The hydrogen temperature programmed reduction (H 2 -TPR) experiments were performed in the laboratory apparatus described in the Section 2.2.The reduction was investigated in the temperature range of 25-500 • C using a constant flow (500 N•cm 3 •min −1 ) of 5% H 2 -N 2 gas mixture.The maximum temperature was held for 1 h in order to assure complete reduction of metals oxides.The H 2 consumption was obtained by integrating the area under the TPR profile.
Catalyst Testing
An amount of the calcined sample (4.5 g) was pressed, sieved into a 45-80 mesh, and loaded in a stainless steel tubular reactor (Officina Elettromeccanica Mormile, Casavatore, Italy) having an annular configuration (internal diameter = 15 mm, external diameter = 17 mm).The sample was treated in an H 2 -N 2 mixture as described above and the catalytic activity was evaluated at atmospheric pressure, 450 • C and water/ethanol molar ratio f.r.(feeding ratio) of 3 (10 vol % of ethanol).Further tests on the Pt-Ni/CeO 2 /SiO 2 sample were carried out at 500 • C and f.r. of 4 and 6.The reaction temperature was measured by means of a thermocouple that was inserted into the tube reactor, in contact with the catalyst bed.A differential pressure sensor (Nagano Sensortechnik, Ottendorf-Okrilla, Germany) allowed monitoring the pressure drop through the catalytic bed.The test was performed until measuring a pressure drop of 500 mbar; then, a N 2 -steam mixture having the same water concentration of the feeding substituted the reacting stream and gasification measurements were carried out.Water/ethanol mixture, stored in a tank under N 2 pressure, was fed into a boiler (T = 200 • C, Officina Elettromeccanica Mormile, Casavatore, Italy) and then into the reactor by means of a mass flow controller for liquids (Bronkhorst High-Tech, Bronkhorst, Ruurlo, The Netherlands) with a flow-rate of 13.4 g•h −1 .N 2 (300 N•cm 3 •min −1 ) was supplied into the boiler as a carrier gas; the resulting weight hourly space velocity (WHSV), defined as the ratio of the ethanol mass flow-rate and catalytic mass, was equal to 4.1 h −1 .The inlet and the outlet lines of the reactor were heated to 160 • C for complete vaporization of the reactant species.The composition of the outlet gas (i.e., CO, CO 2 , CH 4 , H 2 O, and C 2 H 5 OH concentration) was analysed with an on-line FT-IR (Fourier transform-infrared spectroscopy) Spectrophotometer (Antaris IGS Analyser, Thermo Fisher Scientific, Waltham, MA, USA).Unreacted water and ethanol as well as condensable reaction products eventually present were trapped in a condensate collector while the hydrogen concentration in the dry outlet gas was measured with an advanced optima gas analyser from ABB (Caldos 27, Alamo, TX, USA).Ethanol conversion (X), hydrogen yield (Y), carbon formation (CFR), and carbon gasification (CGR) rates were calculated following Equations ( 2)-( 5), respectively, where mass coke,oxidised (in grams) is determined through thermo-gravimetric analysis carried out on the spent catalyst after the reaction + gasification step; mass catalyst (in grams) stands for the catalytic mass; mass carbon,fed (m c,fed in grams) refers to the total mass of carbon fed as ethanol during the test; and time reaction is the time-on-stream in hours.Concerning CGR, the gasification time was set to 60 min (time gasification ) and the mass of carbon oxidized (mass coke,oxidized ) was evaluated by integrating the area under CO and CO 2 signals.
Textural/Structural Properties and H 2 -TPR Measurements
The textural and structural properties of the bare CeO 2 -SiO 2 (CeSi) and of all the catalysts are summarized in Table 1.The ceria content in all the samples was close to the nominal value.A fairly good agreement was also observed for Ni and noble metals loading (the expected values of 10 wt % and 3 wt %, respectively, refers to ceria mass).Due to the very low content of Rh in the trimetallic catalyst and of the alkali metals, their value was not verified by means of XRF (X-ray fluorescence).BET surface area decreases because of active species composition, except for the Pt-Ni/CeO 2 /SiO 2 catalyst, which displayed a negligible area variation with respect to the bare support.Upon Rh deposition on Ni/CeO 2 -SiO 2 sample, a decrease in BET area was observed, probably due to partial pore blockage and to a worst dispersion of rhodium with respect to platinum.The trimetallic catalyst containing Rh, as well as the alkali-doped samples, displayed lower surface area than the bimetallic sample and this difference is ascribable to the further calcination step.However, the lowest area was recorded over the K-containing sample while a similar area was determined for the Pt-Ni-Cs and Rh-Pt-Ni catalysts.The crystalline structure of the calcined support and of all the catalysts is reported in Figure 1.The broad peaks originated from about 2θ = 15 • to 35 • are the reflections ascribable to amorphous silica framework [34].In addition, the XRD data of all the samples display the presence of characteristic lines of ceria cubic phase [35] (at 2θ = 28.5 • , 33.0 • , 47.4 • , and 56.3 • ).No other peaks were detected over the samples after active species deposition: NiO and PtO x phase are not visible and this may be due to the low metals amount (the experimental composition of the samples is reported in Table 1) or well-dispersed oxides [36].The crystallite sizes of ceria particles (d) were not affected by active species deposition and by the number of calcination steps occurred during catalyst preparation; as a result, all the samples displayed ceria particles between 7 and 8 nm.The crystalline structure of the calcined support and of all the catalysts is reported in Figure 1.The broad peaks originated from about 2ϴ = 15° to 35° are the reflections ascribable to amorphous silica framework [34].In addition, the XRD data of all the samples display the presence of characteristic lines of ceria cubic phase [35] (at 2ϴ = 28.5°,33.0°, 47.4°, and 56.3°).No other peaks were detected over the samples after active species deposition: NiO and PtOx phase are not visible and this may be due to the low metals amount (the experimental composition of the samples is reported in Table 1) or well-dispersed oxides [36].The crystallite sizes of ceria particles (d) were not affected by active species deposition and by the number of calcination steps occurred during catalyst preparation; as a result, all the samples displayed ceria particles between 7 and 8 nm.TPR measurements were carried out in order to investigate the reducibility of the calcined support and to examine the interaction strength of metal species with the surface of the support.H2-uptake profiles of CeO2-SiO2 based catalysts in comparison with the TPR curve of the bare support are shown in Figure 2. The Gaussian-type deconvolution of the profile was also carried out, thus allowing the evaluation of hydrogen consumption.All the catalytic samples had similar reduction behaviour with two broad reduction zones between 50-250 °C and above 250 °C, respectively ascribable to noble [37] metals (rhodium oxides are commonly reduced at lower temperatures compared to the Pt ones [38]) and non-noble metal [39] reduction.An additional shoulder always accompanies the major peak observed at lower temperature.In fact, the well-dispersed noble metals nanoparticles were easily reduced below 130 °C while the Pt and/or Rh nanoparticles strongly interacting with Ni and/or with CeO2-SiO2 support were reduced in the temperature range of 150-300 °C [36,40].On the other hand, for all the prepared catalysts, the reduction band observed at high temperature can be deconvoluted into two hydrogen consumption peaks, which indicates that TPR measurements were carried out in order to investigate the reducibility of the calcined support and to examine the interaction strength of metal species with the surface of the support.H 2 -uptake profiles of CeO 2 -SiO 2 based catalysts in comparison with the TPR curve of the bare support are shown in Figure 2. The Gaussian-type deconvolution of the profile was also carried out, thus allowing the evaluation of hydrogen consumption.All the catalytic samples had similar reduction behaviour with two broad reduction zones between 50-250 • C and above 250 • C, respectively ascribable to noble [37] metals (rhodium oxides are commonly reduced at lower temperatures compared to the Pt ones [38]) and non-noble metal [39] reduction.An additional shoulder always accompanies the major peak observed at lower temperature.In fact, the well-dispersed noble metals nanoparticles were easily reduced below 130 • C while the Pt and/or Rh nanoparticles strongly interacting with Ni and/or with CeO 2 -SiO 2 support were reduced in the temperature range of 150-300 • C [36,40].On the other hand, for all the prepared catalysts, the reduction band observed at high temperature can be deconvoluted into two hydrogen consumption peaks, which indicates that NiO particles differently interacted with the support: free NiO particles can be reduced earlier than NiO strongly bonded on CeO 2 -SiO 2 support [41].
NiO particles differently interacted with the support: free NiO particles can be reduced earlier than NiO strongly bonded on CeO2-SiO2 support [41].Rh-containing samples displayed the lowest temperature for consumption peaks, due to the improved catalyst reducibility upon Rh addition [42].Conversely, the support doping by alkali addition (especially in the case of potassium) was accompanied by a leftward shift peaks, which attested that catalyst basification may increase the metals-support interactions and retard oxide reduction [43].
The results shown in Table 2 demonstrate that the quantity of consumed H2 during the TPR analysis surpassed the value needed for complete reduction of deposited NiO, PtOx, and/or RhOx to metals, according to the theoretical chemical composition of the tested materials.Consequently, partial reduction of the support must have occurred simultaneously to metal oxide reduction because of hydrogen spillover.The latter effect [44], in fact, promotes the migration of hydrogen atoms from the reduced metal particles (especially Rh and Pt) to the nearby ceria surface, thus assuring the reduction of lattice oxygen in the ceria surface, as well as bulk, along with the metal oxides [45].In fact, the reduction of the Ce 4+ -O ions is shifted towards lower temperature range.Moreover, in the case of CeO2-SiO2 supported catalysts, the high oxygen storage capacity (OSC) of ceria, as well as oxygen mobility improved by silica, further increased catalyst reducibility [46].The H2 consumption at low temperature recorded over Pt-Ni catalyst increased by almost 200 µmol/gcat upon Rh deposition; rhodium addition to the catalyst also improved the reducibility of NiO particles and hightemperature uptake grew from 1636 to 2089 µmol/gcat.The highest hydrogen consumption was measured over the bimetallic catalyst containing rhodium (3209 µmol/gcat), again demonstrating the better reducibility properties of Rh over Pt.Conversely, alkali addition partially inhibited Pt-Ni/CeO2/SiO2 reducibility, lowering the H2 uptake of almost 10% in the case of potassium-based sample.The TPR analysis of the bare support (Figure 2 and Table 2) also confirmed the marked effect of the spillover phenomena: for the CeO2-SiO2 sample, a very low H2 consumption was calculated (298 µmol/gcat), with it being interesting to highlight that reduction peaks are observed at higher temperatures compared with the metal-containing sample.This result highlights the key role of the active component in promoting support reduction.Rh-containing samples displayed the lowest temperature for consumption peaks, due to the improved catalyst reducibility upon Rh addition [42].Conversely, the support doping by alkali addition (especially in the case of potassium) was accompanied by a leftward shift peaks, which attested that catalyst basification may increase the metals-support interactions and retard oxide reduction [43].
The results shown in Table 2 demonstrate that the quantity of consumed H 2 during the TPR analysis surpassed the value needed for complete reduction of deposited NiO, PtO x , and/or RhO x to metals, according to the theoretical chemical composition of the tested materials.Consequently, partial reduction of the support must have occurred simultaneously to metal oxide reduction because of hydrogen spillover.The latter effect [44], in fact, promotes the migration of hydrogen atoms from the reduced metal particles (especially Rh and Pt) to the nearby ceria surface, thus assuring the reduction of lattice oxygen in the ceria surface, as well as bulk, along with the metal oxides [45].In fact, the reduction of the Ce 4+ -O ions is shifted towards lower temperature range.Moreover, in the case of CeO 2 -SiO 2 supported catalysts, the high oxygen storage capacity (OSC) of ceria, as well as oxygen mobility improved by silica, further increased catalyst reducibility [46].The H 2 consumption at low temperature recorded over Pt-Ni catalyst increased by almost 200 µmol/g cat upon Rh deposition; rhodium addition to the catalyst also improved the reducibility of NiO particles and high-temperature uptake grew from 1636 to 2089 µmol/g cat .The highest hydrogen consumption was measured over the bimetallic catalyst containing rhodium (3209 µmol/g cat ), again demonstrating the better reducibility properties of Rh over Pt.Conversely, alkali addition partially inhibited Pt-Ni/CeO 2 /SiO 2 reducibility, lowering the H 2 uptake of almost 10% in the case of potassium-based sample.The TPR analysis of the bare support (Figure 2 and Table 2) also confirmed the marked effect of the spillover phenomena: for the CeO 2 -SiO 2 sample, a very low H 2 consumption was calculated (298 µmol/g cat ), with it being interesting to highlight that reduction peaks are observed at higher temperatures compared with the metal-containing sample.This result highlights the key role of the active component in promoting support reduction.
Ethanol Steam Reforming Tests: Effect of Catalytic Formulation
The stability tests carried out at 450 • C and f.r.= 3 were performed for different times, selected in order to reach 500 mbar of pressure drops through the bed and guarantee almost the same mass of carbon deposited.The results shown in Table 3 revealed a very fast reactor plugging for the K-based catalyst, which also displayed a CFR of 0.017 g coke,oxidized •g cat −1 •g c,fed −1 •h −1 .A quite high coke selectivity was also observed over the Pt-Ni sample: pressure drops reached the critical value of 500 mbar within 310 min.However, it is interesting to observe that Rh deposition on Pt-Ni/CeO 2 -SiO 2 catalyst reduced carbon formation rate from 0.0030 to 0.00084 g coke,oxidized On the other hand, over the sample basified by Cs deposition, CFR was halved with respect to the Rh-Pt-Ni sample.The lowest activity towards carbon formation reactions was measured over the bimetallic catalyst containing Rh: also other authors found that Rh is less selective than Pt towards coke precursors formation during reforming [47].However, looking at the coke gasification measurements, differently form the result previously reported about the clear role of alkali metals [48] as well as Rh in carbon gasification reactions [49], CGR was unaffected by catalyst formulation and very similar values were recorded over the five catalysts (Table 3).Figure 3 displays the trend of ethanol conversion as a function of time on stream: TOS (time-on-stream) for all the samples was equal to critical plugging time, except for the Pt-Ni catalyst, which was tested for 5000 min, in order to evaluate in more detail the dependence of X vs TOS.It was found that Pt-Ni and Rh-Pt-Ni catalysts maintained initial catalytic performances for at least 240 min without apparent deactivation.Conversely, similar C 2 H 5 OH profiles were recorded over the Rh-Pt-Ni and Pt-Ni-Cs samples, with an activity reduction from 100% to 97% in the time range of 0-1900 min.The K-containing catalyst displayed the worst performances among the prepared catalysts with a very fast conversion decrease to 93%.The Pt-Ni conversion profile was higher than that recorded over the other samples, with an X value of 95% after 5000 min.It is also interesting to note that the bimetallic catalyst containing Pt also assures an initial hydrogen yield better than the other samples (26.5%) and very close to the value predicted by thermodynamic equilibrium.
The unsatisfactory behaviour of the Pt-Ni-K catalyst can be ascribed to its low surface area (Table 1) and a subsequent worse active species dispersion, which reduced ethanol conversion in reforming reactions [50].be ascribed to its low surface area (Table 1) and a subsequent worse active species dispersion, which reduced ethanol conversion in reforming reactions [50].On the other hand, Rh addition had no positive effect on the stability of Pt-Ni catalyst while the bimetallic catalyst containing Rh showed slightly improved activity with respect to the Cs-containing sample.The described results suggest that, despite Rh and alkali metals are able to reduce coke formation rate and, consequently, enhance reactor-plugging time, no improvements can be drawn under the point of view of gasification reactions, which spurs the investigation of other parameters suspected to affect coke removal by steam (water partial pressure and reaction temperature).Moreover, a negative effect of support basification, Rh addition as well as Pt substitution by Rh on the long-term stability and H2 yield of Pt-Ni/CeO2-SiO2 catalyst was observed.Based on the above discussion, the Pt-Ni catalyst was selected as the most interesting sample in terms of catalytic performances and employed for further tests.
Ethanol Steam Reforming Tests: Effect of Operative Conditions
The impact of different amount of steam and the influence of reaction temperatures on the Pt-Ni/CeO2-SiO2 catalyst performances during ethanol steam reforming at WHSV = 4.1 h −1 was also investigated.C2H5OH conversion was not affected by water partial pressure change and 100% of conversion (as predicted by thermodynamic analysis) was measured during all the tests, carried out for a TOS equal to the critical plugging time.A stable product gas distribution was also recorded, with a mean hydrogen yield increasing from 26.5% at f.r.= 3 to 30% and 36%, at f.r.= 4 and 6, respectively; this trend is reasonable since the added steam could favour reforming reactions [51].In addition, a strong variation in the pressure drop profile with the growth of the amount of steam was observed (Figure 4).The plugging time measured during the test at f.r.= 6 was almost eight times On the other hand, Rh addition effect on the stability catalyst while bimetallic containing Rh showed slightly improved activity with respect to the Cs-containing sample.The described results suggest that, despite Rh and alkali metals are able to reduce coke formation rate and, consequently, enhance reactor-plugging time, no improvements can be drawn under the point of view of gasification of other parameters suspected to affect coke removal by steam (water partial pressure and reaction temperature).Moreover, a negative effect of support basification, Rh addition as well as Pt substitution by Rh on the long-term stability and H 2 yield of Pt-Ni/CeO 2 -SiO 2 catalyst was observed.Based on the above discussion, the Pt-Ni catalyst was selected as the most interesting sample in terms of catalytic performances and employed for further tests.
Ethanol Steam Reforming Tests: Effect of Operative Conditions
The impact of different amount of steam and the influence of reaction temperatures on the Pt-Ni/CeO 2 -SiO 2 catalyst performances during ethanol steam reforming at WHSV = 4.1 h −1 was also investigated.C 2 H 5 OH conversion was not affected by water partial pressure change and 100% of conversion (as predicted by thermodynamic analysis) was measured during all the tests, carried out for a TOS equal to the critical plugging time.A stable product gas distribution was also recorded, with a mean hydrogen yield increasing from 26.5% at f.r.= 3 to 30% and 36%, at f.r.= 4 and 6, respectively; this trend is reasonable since the added steam could favour reforming reactions [51].In addition, a strong variation in the pressure drop profile with the growth of the amount of steam was observed (Figure 4).The plugging time measured during the test at f.r.= 6 was almost eight times higher than the value recorded at stoichiometric feeding conditions (Table 4).Moreover, for a water concentration growth in the range 40-60 vol % in the reacting mixture, CFR was reduced of almost one order of magnitude (from 0.0013 to 0.00011 g coke,oxidized •g cat −1 •g c,fed −1 •h −1 ).However, the carbon gasification rate, as it is possible to observe also from the slope of pressure profile during gasification measurements (Figure 4) and from the data shown in Table 4, was not affected by water partial pressure.This phenomenon can be explained considering that, even when the steam to ethanol molar ratio is adjusted to favour the gasification reaction, the kinetics of the reaction can be very slow, resulting in a negligible effect of water partial pressure [52].Based on the above results, the effect of temperature was also studied and the results are shown in Figure 5 and Table 4.
Energies 2017, 10, 1030 9 of 13 higher than the value recorded at stoichiometric feeding conditions (Table 4).Moreover, for a water concentration growth in the range 40-60 vol % in the reacting mixture, CFR was reduced of almost one order magnitude (from 0.0013 to 0.00011 gcoke,oxidized•gcat −1 •gc,fed −1 •h −1 ).However, the carbon gasification rate, as it is possible to observe also from the slope of pressure profile during gasification measurements (Figure 4) and from the data shown in Table 4, was not affected by water partial pressure.This phenomenon can be explained considering that, even when the steam to ethanol molar ratio is adjusted to favour the gasification reaction, the kinetics of the reaction can be very slow, resulting in a negligible effect of water partial pressure [52].Based on the above results, the effect of temperature was also studied and the results are shown in Figure 5 and Table 4. higher than the value recorded at stoichiometric feeding conditions (Table 4).Moreover, for a water concentration the range 40-60 vol the reacting CFR one 0.0013 gcoke,oxidized•gcat −1 •h −1 ). is possible to also (Figure the data shown Table 4, was not affected pressure.phenomenon can to gasification reaction, the kinetics of the reaction can be very slow, resulting in a negligible effect of water partial pressure [52].Based on the above results, the effect of temperature was also studied and the results are shown in Figure 5 and Table 4.The trend of pressure drops during stability tests and the carbon formation rate variation with temperature in the range 450-500 • C suggest that the most favourable conditions for carbon deposition are always at the lowest catalyst temperature, where the reactions leading to coke formation are much higher than the rates of carbon gasification reactions [53].The temperature growth from 450 to 500 • C reduced CFR, due to the lower activity towards coke deposits formation and/or the increased contribution of gasification reaction during the test.Moreover, the results of gasification measurements revealed a strong kinetics improvement, which allowed the increase of CGR from 0.035 to 0.25 g •gcat −1 •h −1 .
Conclusions
CeO 2 -SiO 2 -based catalysts prepared by wetness impregnation have been tested in an ethanol steam reforming reaction.The physiochemical properties of the prepared catalysts (Pt-Ni, Rh-Pt-Ni, Rh-Ni, Pt-Ni-K, and Pt-Ni-Cs) and the effect of catalytic formulation, as well as test conditions on the performances for ESR, were investigated.Among the tested samples, the catalysts containing Rh and Cs displayed the lowest carbon formation rate.However, lessened ethanol conversions and hydrogen yields were measured over the above samples (the worse performances being observed over the K-based sample) while the Pt-Ni/CeO 2 -SiO 2 catalyst displayed the best results in terms of activity and stability; the total conversion was recorded for more than 10 h and the initial hydrogen yield was very close to the thermodynamic predictions.The latter sample, which displayed relevant resistance towards deactivation, was further investigated under different operative conditions (H 2 O/C 2 H 5 OH ratio and T).The increase of water amount in the feed promoted hydrogen production and reduced carbon formation rate over the Pt-Ni catalyst, without, however, bringing any relevant improvement in terms of carbon gasification.Conversely, the temperature rise enhanced both the plugging time and coke gasification rates.
Figure 1 .
Figure 1.X-ray diffraction patterns of the CeO2-SiO2 support and the catalysts.
Figure 1 .
Figure 1.X-ray diffraction patterns of the CeO 2 -SiO 2 support and the catalysts.
Table 1 .
Chemical composition, CeO 2 crystallite sizes (d), and specific surface area of the calcined samples.
Table 1 .
Chemical composition, CeO2 crystallite sizes (d), and specific surface area of the calcined samples.
Table 2 .
Comparison between experimental and theoretical amounts of hydrogen consumption during TPR. | v3-fos-license |
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} | pes2o/s2orc | Modulation of Re-initiation of Measles Virus Transcription at Intergenic Regions by PXD to NTAIL Binding Strength
Measles virus (MeV) and all Paramyxoviridae members rely on a complex polymerase machinery to ensure viral transcription and replication. Their polymerase associates the phosphoprotein (P) and the L protein that is endowed with all necessary enzymatic activities. To be processive, the polymerase uses as template a nucleocapsid made of genomic RNA entirely wrapped into a continuous oligomer of the nucleoprotein (N). The polymerase enters the nucleocapsid at the 3’end of the genome where are located the promoters for transcription and replication. Transcription of the six genes occurs sequentially. This implies ending and re-initiating mRNA synthesis at each intergenic region (IGR). We explored here to which extent the binding of the X domain of P (XD) to the C-terminal region of the N protein (NTAIL) is involved in maintaining the P/L complex anchored to the nucleocapsid template during the sequential transcription. Amino acid substitutions introduced in the XD-binding site on NTAIL resulted in a wide range of binding affinities as determined by combining protein complementation assays in E. coli and human cells and isothermal titration calorimetry. Molecular dynamics simulations revealed that XD binding to NTAIL involves a complex network of hydrogen bonds, the disruption of which by two individual amino acid substitutions markedly reduced the binding affinity. Using a newly designed, highly sensitive dual-luciferase reporter minigenome assay, the efficiency of re-initiation through the five measles virus IGRs was found to correlate with NTAIL/XD KD. Correlatively, P transcript accumulation rate and F/N transcript ratios from recombinant viruses expressing N variants were also found to correlate with the NTAIL to XD binding strength. Altogether, our data support a key role for XD binding to NTAIL in maintaining proper anchor of the P/L complex thereby ensuring transcription re-initiation at each intergenic region.
Introduction
Measles virus (MeV), a member of the Morbillivirus genus, belongs to the Paramyxoviridae family of the Mononegavirales order [1]. These viruses possess a non-segmented RNA genome of negative polarity that is encapsidated by the nucleoprotein (N) to form a helical nucleocapsid. Not only does N protect viral RNA from degradation and/or formation of viral dsRNA, but it also renders the latter competent for transcription and replication. Indeed, the viral polymerase cannot processively transcribe nor replicate RNA unless the viral genome is encapsidated by the N protein within a helical nucleocapsid [2,3]. Transcription and replication are ensured by the RNA-dependent RNA polymerase complex made of the large protein (L) and the phosphoprotein (P), with P serving as an essential tethering factor between L and the nucleocapsid. The complex made of RNA and of the N, P and L proteins constitutes the replication machinery. In order to perform messenger RNA synthesis, the polymerase has not only to bind to the 3' transcription promoter, but also to re-initiate the transcription of downstream genes upon crossing each intergenic region (IGR). Following polyadenylation, which serves as gene end (GE) signal, the polymerase proceeds over three nucleotides (3'-GAA-5' or 3'-GCA-5') without transcribing them and then restarts transcription upon recognition of a downstream gene start (GS) signal.
Within infected cells, N is found in a soluble, monomeric form (referred to as N 0 ) and in a nucleocapsid assembled form [4]. Following synthesis, the N protein requires chaperoning by the P protein so as to be maintained in a soluble and monomeric form. The P N-terminal region (P NT ) binds to the neosynthesized N protein thereby simultaneously preventing its illegitimate self-assembly and yielding a soluble N 0 P complex the structure of which have been characterised for MeV [5] as well as for four other members of the Mononegavirales order [6,7,8,9]. N 0 P is used as the substrate for the encapsidation of the nascent genomic RNA chain during replication [10], (see also [4,11,12,13] for reviews on transcription and replication). In its assembled homopolymeric form or nucleocapsid, N also makes complexes with either isolated P or P bound to L, with all these interactions being essential for RNA synthesis by the viral polymerase [14,15,16]. Throughout the Mononegavirales order, P and P+L binding to the nucleocapsid is mediated by interaction of the C-terminal region of P with either the C-terminal tail of N (Paramyxoviridae members), or to the N-terminal globular moiety (or core) of N (see [11,17] for review).
The MeV N protein consists of a structured N-terminal moiety (N CORE , aa 1-400), and a Cterminal domain (N TAIL , aa 401-525) [18,19] that is intrinsically disordered, i.e. it lacks highly populated secondary and tertiary structure under physiological conditions of pH and salinity in the absence of a partner (for a recent review on intrinsically disordered proteins see [20]). While N CORE contains all the regions necessary for self-assembly and RNA-binding [10,21,22] and a binding site for an α-MoRE located at the N terminus of the P protein, N TAIL is responsible for interaction with the C-terminal X domain (XD, aa 459-507) of P [11,18,21,23,24,25,26,27] (Fig 1A). N TAIL binding to XD triggers α-helical folding within a molecular recognition element [28,29] of α-helical nature (α-MoRE, aa 486-502) located within one (Box2, aa 489-506) out of three conserved N TAIL regions [18,24,27,30,31,32,33,34,35,36,37]. XD-induced α-helical folding of N TAIL is not a feature unique to MeV, being also conserved within the Paramyxoviridae family [38,39,40,41,42,43]. XD consists of a triple α-helical bundle [27,34,44], and binding to the α-MoRE leads to a pseudo-four-helix arrangement that mainly relies on hydrophobic contacts [18,27,44]. The α-MoRE of N TAIL is partly preconfigured as an α-helix prior to binding to XD [31,32,35,37,45] and adopts an equilibrium between a fully unfolded form and four partly helical conformers [37]. In spite of this partial pre-configuration, N TAIL folds according to a "folding after binding mechanism" [45,46]. Previous mutational studies showed that Box2 is poorly evolvable in terms of its binding abilities towards XD, in that amino acid substitutions therein introduced lead to a dramatic drop in the binding strength, as judged from a protein complementation assay (PCA) based on split-GFP reassembly (gfp-PCA) [47]. In particular, substitutions within the N-terminal region of Box2 (aa 489-493) and at position 497 were found to lead to the most dramatic drops in the interaction strength [47].
In the context of the viral nucleocapsid, N TAIL points towards the interior of the latter and then extrafiltrates through the interstitial space between N CORE moieties, with the first 50 residues (aa 401-450) being conformationally restricted due to their location between successive turns of the nucleocapsid [37]. The N TAIL region spanning residues 451-525 and encompassing the α-MoRE is, by contrast, exposed at the surface of the viral nucleocapsid and thus accessible to the viral polymerase. Binding of XD to N TAIL has been proposed to ensure and/or contribute to the recruitment of the viral P/L polymerase complex onto the nucleocapsid template. However, its precise function has remained enigmatic so far with reports of apparent conflicting observations. From the analysis of four N TAIL variants it was concluded that the accumulation rate of primary transcripts is rather insensitive to a drop in the apparent XD to N TAIL affinity [26], while an XD variant showing a 1.7 times stronger interaction with N TAIL was associated with a 1.7-fold reduction in the accumulation rate of viral transcripts [48]. Furthermore, deletions studies of N TAIL have indicated that the interaction between XD and N TAIL may be dispensable for transcription and replication [49].
In the present work, we further investigate the molecular mechanisms by which substitutions in critical positions of N TAIL previously identified by a random approach [47] affect the viral polymerase activity. We did so by combining biochemical studies and molecular dynamics (MD) simulations on one hand with functional studies that made use of minigenomes and recombinant viruses on the other hand. Results identify positions 491 and 497 as the most critical in terms of both binding affinities and functional impact. In addition, thanks to the availability of a newly conceived minigenome made of two luciferase reporter genes, with the second one being conditionally expressed via RNA edition of its transcript by the viral polymerase, we could quantify the efficiency of transcription re-initiation after polymerase scanning through each of the five IGRs of MeV genome and on an elongated un-transcribed IGR (UTIGR). A low N TAIL -XD affinity was found to be associated to a reduced ability of N to support expression of luciferase from the second gene. Furthermore, in infected cells, the accumulation rate of primary transcripts and transcript ratios were found to correlate with the equilibrium dissociation constant (K D ) of the N TAIL /XD pair. Altogether obtained data argue for a key role of the N TAIL /XD interaction in transcription re-initiation at each intergenic region.
Binding strengths of N TAIL variants from two protein complementation assays
In a previous random mutagenesis study that made use of a PCA based on split-GFP re-assembly (gfp-PCA) [47], we identified N TAIL variants that either decrease or increase the interaction strength towards XD [47]. Variant MX208, which bears the D437V, P485L and L524R substitutions that are all located outside the α-MoRE, is an example of the latter group. We previously reported the generation and assessment of binding properties by gfp-PCA of six singlesite variants (R489Q, R490S, S491L, A492T, D493G and R497G) bearing each a unique substitution within the α-MoRE [47]. Here, we additionally designed and generated the MXSF variant, which bears D437V, R439S, P456S and P485L substitutions that are all found in variants displaying an increased fluorescence [47]. Gfp-PCA in E. coli showed that the binding strength of these variants towards XD is scattered over a wide range, with the S491L and R497G variants showing the lowest interaction and with variant MXSF displaying interaction strength only moderately higher than wt N TAIL (Fig 1B). Incidentally, this latter finding indicates that the effects of the substitutions are not cumulative.
We then sought at assessing to which extent results afforded by the split-GFP assay in E. coli cells reflect N TAIL /XD binding occurring in the natural host cells of MeV. To this end, the interaction between XD and N TAIL variants was measured using the split-luciferase reassembly assay [50]. This technique is based on the same principle as the split-GFP reassembly assay. The reporter (i.e. Gaussia princeps luciferase) and the measured parameter (luminescence) are however different, and the assay is performed in human cells. Moreover, contrary to the split-GFP reassembly assay where reporter reassembly is irreversible, in the split-luciferase assay (glu-PCA), association of the two luciferase fragments is reversible. As such, while the measured parameter in the former assay is dominated by the k on , the measured parameter in the latter assay does reflect the equilibrium between a k on and a k off and hence a true K D . A significant correlation was obtained between the two PCA methods (Fig 1C), a finding that provides additional support for the significance of the observed differences in binding strength among variants. Furthermore, a significant correlation was also observed when comparing binding strengths as obtained using monomeric constructs (i.e. N TAIL /XD) and binding strengths obtained using their natural multimeric counterparts, i.e. P multimerization domain (PMD)-XD (P303-507) and full-length mutated N protein constructs (Fig 1D). The rationale for using P multimeric constructs devoid of the N-terminal region (P NT ) was to eliminate the binding site to N CORE located within P NT and involved in P chaperoning of N protein to form N 0 P complexes [5] Generation, purification and characterization of the single-site Box2 N TAIL variants In order to characterize Box2 variants (Fig 2A) at the biochemical level, we expressed and purified six α-MoRE variants of N TAIL as N-terminally hexahistidine tagged proteins. All N TAIL variants were purified to homogeneity from the soluble fraction of the bacterial lysate through immobilized metal affinity chromatography (IMAC) followed by size exclusion chromatography (SEC) (Fig 2B). The identity of all purified proteins were checked and confirmed by mass spectrometry. Even if their molecular mass is~16 kDa, they all migrate on a denaturing gel with an apparent molecular mass of approximately 20 kDa (Fig 2B, inset). This aberrant electrophoretic migration has been systematically observed for all N TAIL variants reported so far [26,30,31,32,46] including wt N TAIL [19]. This anomalous migration is frequently observed in IDPs and is due to a high content in acidic residues [51] and/or a large extension in solution [43].
All N TAIL variants, including wt N TAIL, have the same SEC elution profile (Fig 2B). In particular, they are all eluted with an apparent molecular mass higher than expected and typical of a premolten globule (PMG) state [52], as already observed in the case of wt N TAIL [19]. Thus, the amino acid substitution(s) causes little (if any) effect on the hydrodynamic volume sampled by the protein.
Analysis of the secondary structure content of the N TAIL variants by far-UV circular dichroism (CD) shows they are all disordered, as judged from their markedly negative ellipticity at 200 nm (Fig 2C). In addition, they are all similarly able to gain α-helicity in the presence of 20% 2,2,2 trifluoroethanol (TFE) (Fig 2D), as already observed for wt N TAIL [19]. All variants have an estimated α-helical content similar (within the error bar) to that of wt N TAIL , with the only exception of variant R489Q that exhibits a lower α-helicity both in the absence and in the presence of TFE (Fig 2E). Thus, most of the amino acid substitutions cause little (if any) effect on the overall secondary structure content and folding abilities of N TAIL .
Assessment of binding affinities of single-site Box2 N TAIL variants towards XD by ITC
The binding abilities of the N TAIL variants, including wt N TAIL , were assessed using isothermal titration calorimetry (ITC). To this end, the purified N TAIL proteins were loaded into the sample cell of an ITC200 microcalorimeter and titrated with wt XD. For each variant, two independent experiments were carried out. Fig 3 shows, for each variant, one representative ITC curve along with the relevant binding parameters. The XD/N TAIL molar ratios achieved at the end of the titration were 2.0 (wt, R489Q, A492T, D493G), 2.5 (R490S) or 3.0 (R497G) (Fig 3). The data, following integration and correction for the heats of dilution, were fitted with a standard model allowing for a set of independent and equivalent binding sites.
Consistent with the unfavorable entropic contribution associated to the disorder-to-order transition that takes place upon N TAIL binding to XD, whenever binding parameters could be determined, they revealed a decrease in entropy, with a ΔS ranging from -13 to -29.5 cal mol -1 deg -1 (Fig 3). Binding reactions were all found to be enthalpy-driven, with ΔH values in the same order of magnitude and ranging from -10.9 to -14.5 kcal/mol (Fig 3). The estimates for the model parameters of the wt N TAIL /XD pair were found to be in very good agreement with those recently reported [46]. The estimates for binding parameters of variants R489Q, A492T and D493G yielded equilibrium dissociation constant (K D ) very close to that observed for wt N TAIL , indicating that these substitutions poorly affect the interaction (Fig 3). On the other hand, the R490S substitution resulted in a 7-fold decrease in the binding affinity (K D of 20 μM). The decrease in affinity was even further pronounced (K D of 44 μM) in the case of the R497G variant, although the interaction remained measurable (Fig 3). In the case of the S491L variant the interaction strength was below the ITC detection limit and thus K D could not be estimated (Fig 3).
The n values for the A492T/XD and D493G/XD binding pairs were found to deviate from unit, a behaviour that is not unusual and that has been already observed with single-site tryptophan variants [46] and that may arise from relatively poorly defined baselines. In light of all the numerous previous studies [18,19,27,30,31,32,33,34,35,36,37] showing that N TAIL and XD form a 1:1 complex, these deviations were not taken to be significant.
We next focused on how binding affinities obtained by ITC correlate with binding strengths inferred from split-GFP and split-luciferase reassembly assays. In fact, although it has already been established that the higher the fluorescence the higher the interaction strength Notably, if the results obtained by gfp-PCA (see Fig 1B) pointed out similarly low interaction strengths for variants S491L and R497G, ITC studies yielded a different profile. Indeed, while no interaction could be effectively detected for the S491L/XD pair, the K D could be measured for variant R497G (see Fig 3). Using the empirically determined equation relating luminescence and K D values (Fig 4), the K D of the S491L/XD pair was estimated to be 85 ± 33 μM, a value consistent with our inability to detect the interaction by ITC. Indeed, an interaction characterized by a K D of approximately 100 μM could escape detection unless extremely high (and hardly achievable) protein concentrations are used (typically 1 mM N TAIL /10 mM XD) [54].
Altogether, obtained results confirmed that not all Box2 residues are equivalent in terms of their role in N TAIL /XD complex formation. In particular, while substitutions at positions 489, 490, 492 and 493 have a slight to moderate impact, substitutions at positions 497 and 491 drastically affect complex formation without having a strong impact on the overall α-helicity. The role of Box2 residues in complex formation follows the order 491>497>490, reflecting either the orientation of side chains towards the partner (residues 490 and 491) or involvement in stabilizing interactions with XD residue Tyr480 in spite of solvent exposure (residue 497), as already proposed (Fig 2A) [47].
Molecular dynamic simulations of wt, S491L and R497G variants in complex with XD reveal a crucial hydrogen bonding network
In order to further investigate the mechanisms by which residues Ser491 and Arg497 stabilize the N TAIL /XD complex, we performed MD simulations in aqueous solvent using the CHARMM force field [55]. MD simulations were carried out starting from the X-ray structure of the XD/α-MoRE complex [44] or from the in silico generated XD/α-MoRE S491L and XD/ α-MoRE R497G models. In the case of the S491L variant, the three most favorable orientations of the side chains were generated. We first assessed the dynamical stability of the complexes. For this purpose, we analyzed the root-mean square deviation (RMSD) of the Cα atoms with respect to the initial structure as a function of time for the three complexes (i.e. wt, S491L and R497G) (S1 Table). The RMSD values showed very little variations between the different constructs during the time course of the 50 ns simulations (S1 Table). The average RMSD for XD and for the α-MoRE were approximately 0.8 and 0.5 Å, respectively, indicating structural stability of each domain during the simulations (S1 Table). The relative orientation of the α-MoRE compared to XD was also assessed and revealed slightly higher average RMSD values for the two variants due to small local rearrangements of the structures to adapt to the substitutions. However, RMSD fluctuations were in the same order of magnitude. Secondary structure analyses of wt and mutated complexes confirmed that all α-helices are conserved during the whole trajectories. Overall, the different systems were stable during the whole simulation. Since the orientation of the side-chain of L491 showed no impact on the behavior of the complex during the MD simulation, only one conformer was selected in the rest of the study.
Although the association between XD and N TAIL is essentially driven by hydrophobic contacts, the two partners also interact through hydrogen bonds that are thus expected to play a role in the binding affinity. Two intramolecular hydrogen-bond interactions are present in the crystallographic structure of the complex ( Table 1). These interactions involve the side-chain of N TAIL residue Ser491 and side-chain of Asp493 and main-chain of Lys489 from XD. These interactions are preserved in the simulations of the wt and R497G complex ( Table 1 and S2 Fig). Due to the absence of the polar OH group in leucine, hydrogen bonds involving the OH group of Ser491 were lost in the simulations of the S491L complex. Three additional hydrogen bonds that are not present in the X-ray structure were observed in the MD trajectories of the wt complex (Table 1 and S2 Fig). Two of them involve the side-chain of Asp487 from XD and side-chains of either Arg490 or Arg497 of N TAIL . Only the former was also observed in the simulations of both variants (Table 1 and S2 Fig). The third one was formed between the sidechains of Lys489 from XD and Asp487 from N TAIL and was detected in the simulation of the three complexes with the two variants exhibiting even a higher frequency (
Fig).
In addition, a water-mediated hydrogen bond could be identified between the side-chains of Tyr480 of XD and Arg497 of N TAIL , 55 and 41 percent of the time in the wt and S491L complex, respectively. This interaction was not maintained with the same water molecule throughout simulation. However, when a water molecule moved away from this site it was almost immediately replaced by another water molecule. This interaction could not occur in the R497G complex and was not compensated by another interaction. The presence of this watermediated interaction correlates with the stabilization of the aromatic ring of Tyr480. The sidechain of Tyr480 was found in almost only one conformation corresponding to a χ2 angle (CA-CB-CG-CD) of approximately -130˚in both wt and S491L complexes, whereas in the R497G complex, the ring oscillates between 2 conformations (50 and -130˚) corresponding to a 180˚rotation. Although the position of this water molecule in the crystal structure cannot be estimated with precision because the molecule is poorly defined in the electron density, the fact that a water molecule is systematically observed at this position during the simulation argues for its critical role in stabilizing the Arg497-Tyr480 interaction. That water molecules can play crucial roles in stabilizing protein-protein interactions has been widely documented [56].
To further investigate the importance of the effect of the substitutions on the binding affinity, additional MD simulations were carried out using the free energy perturbation (FEP) method (see details of the method in the Materials and Methods section). The calculations were based on the thermodynamic cycle shown in S3 Fig which allowed us to estimate the impact of an amino acid substitution on the binding energy by measuring the ΔΔG between the wt and mutated complexes at 300K. Replacement of Ser491 of N TAIL by Leu led to an average binding free energy change ranging from 3.22 to 3.91 kcal.mol -1 . These ΔΔG values correspond to a 200-fold to 700-fold reduction in binding affinities for the S491L variant which is compatible, although a bit more pronounced, with the K D calculated for this variant using the empirically determined equation between luminescence and K D values (see above and Fig 4). In a similar manner, substitution of R497 with Gly led to ΔΔG values ranging from 1.29 to 1.85 kcal.mol -1 . This corresponds to a 10 to 20-fold reduction of binding affinity which nicely correlates with the K D reduction-fold as measured by ITC.
The dissociation of the XD/N TAIL complex cannot be observed during the time course of free MD simulations. To obtain more insights into the dissociation process, we therefore performed simulations using adaptive biasing force (ABF), a method that allows overcoming barriers of the free-energy landscape [57]. The center of geometry between the two partners was selected as ordering parameter and both proteins were allowed to diffuse reversibly along this reaction coordinate during the different stages of the simulations (no average force was exerted along the ordering parameter). The free energy profiles of the wt and mutated complexes are shown Fig 5A. The global minimum corresponds to a distance around 11.3 Å, very close to the distance observed in the X-ray structure (11.03 Å). Analysis of the wt complex reveals that the dissociation between the two partners proceeds from the C-terminal part of N TAIL corresponding to the more hydrophobic residues (Fig 5B and S2 Fig). The final step of the dissociation corresponds to the disruption of hydrogen bonds between Ser491 of N TAIL and Lys489 and Asp493 of XD. The R497G complex exhibits an energy profile similar to that of the wt complex with slightly lower energy values indicating a lower resistance against disruption. In the case of the S49IL complex, the disruption can occur from either end of the α-helix of N TAIL depending on the trajectory. This behavior can be explained by the loss of hydrogen bonding with Lys489 and Asp493 of XD. As a consequence, the energy profile is profoundly affected and this variant shows less resistance toward disruption.
Altogether, these data provide a mechanistic basis illuminating the critical role played by N TAIL residues Ser491 and Arg497 in stabilizing the N TAIL -XD complex.
Ability of N variants to support re-initiation of transcription at intergenic regions as a function of N TAIL /XD binding strength
In order to investigate the functional consequences of attenuating the interaction between N TAIL and XD we tested the ability of each N variant to support the expression of a reporter gene from a minigenome rescued into a functional nucleocapsid by cotransfecting a plasmid coding for the minigenome under the T7 promoter together with P and L expression plasmid [58]. To take into account the transcription re-initiation at IGRs, we conceived and built new dual-luciferase minigenomes coding for Firefly and Oplophorus gracilirostris (NanoLuc) luciferase as first and second reporter gene respectively separated by each of the five IGRs of MeV genome (Fig 6A). To this end, the NanoLuc luciferase was chosen because it has a~150-times higher specific activity compared to Firefly luciferase [59]. Like many other paramyxoviruses, MeV polymerase has the ability to edit P mRNA by adding one non-templated G when transcribing the specific sequence termed P editing site (3'-uguggguaauuuuuccc-5') [12,60]. We introduced this editing site just downstream the 3'-UAC-5' START codon of the NanoLuc gene so as to condition the creation of the NanoLuc ORF and the ensuing translation of Nano-Luc to the co-transcriptional insertion of one non-templated G by MeV polymerase. We then verified that these newly built dual-luciferase minigenomes harboring individually one of the five IGR faithfully reproduce the expected re-initiation strength gradient. Indeed, when normalized to the NanoLuc/Firefly signal ratio observed with a minigenome carrying the N-P IGR, the ratios observed for the minigenomes harboring the downstream IGRs decrease with their remoteness from the genome 3'end with P-M being equivalent to N-P, M-F and F-H being significantly lower and H-L being the lowest of all (Fig 6B, wt N). These results are in agreement with the transcription gradient observed in MeV infected cells [12,64,65,66] and with the efficacy of Sendai virus re-initiation at each IGR as determined using recombinant viruses [67]. Interestingly, this trend was absolutely conserved for every N TAIL variant upon normalization to the ratio observed with N-P IGR minigenome (Fig 6B) indicating that the observed re-initiation strength gradient is an intrinsic property of each IGR region. When NanoLuc/Firefly ratios observed for each N variant were plotted without normalization as a function of N TAIL /XD binding strength for each of the five MeV IGR minigenome, the NanoLuc/Firefly signal ratio was found to decrease with decreasing binding strength, with the correlation being significant at p~0.05 or below for N-P, P-M, M-F and F-H IGR minigenomes (Fig 6C-6E), and the trend conserved for the minigenomes bearing the remotest H-L IGR (Fig 6F and 6G). Since in the natural situation MeV polymerase has to travel through every IGR, we estimated for each individual variant a mean re-initiation rate through all MeV IGRs by calculating the mean NanoLuc/Firefly ratio of the 5 IGR regions for each N variant. Remarkably this mean re-initiation rate correlates with the N TAIL /XD binding strength (Fig 6H, p = 0.021).
Ability of N variants to misrecognize IGR and generate read-through transcripts
In few percent cases, the viral polymerase fails to recognize an intergenic region. This results in read-through transcripts. To investigate the possible impact of N TAIL /XD binding on read- Ability of N variants to support transcription re-initiation at every MeV intergenic region (IGR) as determined using dual-luciferase 2-gene minigenomes coding for Firefly and NanoLuc luciferases. (a) Schematic structure of the minigenomes encoding the Firefly luciferase gene at the 3' end of the genomic sequence just downstream the leader and N gene UTR as a first gene and NanoLuc luciferase as a second reporter gene. NanoLuc luciferase is conditionally expressed by MeV polymerase mediated-edition of the transcript thanks to an editing site grafted just after the AUG codon in such a way that, without the non-templated addition of one G, the downstream coding sequence is out of frame because of the presence of an in-frame stop codon. The two genes are separated by either the N 5'UTR and the P 3'UTR separated by the natural N-P un-through generation, a 3-gene minigenome was built as follows: the first gene code for the Firefly luciferase, the second gene codes for an irrelevant inactive protein (here the C-terminal half of the Gaussia luciferase (Glu2)) followed by a linker that remains in the same coding phase throughout the second downstream N-P IGR and the third gene which contains the NanoLuc luciferase coding sequence devoid of a start codon and out of frame by one missing nucleotide that can be restored by the editing signal. Consequently, among all possible viral transcripts, only the edited read-through mRNA over gene 2 and gene 3 can give rise to a NanoLuc luciferase activity (Fig 7A and 7B). Therefore, with the 3-gene minigenome, the NanoLuc/Firefly ratio is dependent on two IGR-related effects: the re-initiation of the transcription at the first IGR and the failure to recognize the second. As expected, the NanoLuc/Firefly signal ratios obtained with this 3-gene minigenome were found to be of a much lower level (i.e. few percent) than those observed with the 2-gene minigenome shown in Fig 6C. We normalized the NanoLuc/Firefly signal obtained with the 3-gene minigenome by the signal obtained with the 2-gene minigenome in order to cancel out the effect on the re-initiation at the first IGR and to focus on the generation of read-through transcripts at the second IGR. The resulting ratios are similar for all the variants, thus indicating they all roughly produce the same amount of readthough transcripts (Fig 7C). We conclude that the N TAIL /XD binding strength does not significantly impact the failure of the viral polymerase to recognize the N-P intergenic region.
Ability of N variants to support re-initiation of transcription during polymerase scanning over an elongated un-transcribed IGR Upon crossing an IGR, the polymerase from Mononegavirales having ceased RNA synthesis at the GE is able to scan forward and backward the genome template until it recognizes the transcription re-initiation site GS of the next downstream gene. This search for next GS had been initially observed as measurable temporal pause in transcription [68] (see viral transcription scheme in S6 Fig and [ 13,69] for reviews). Since the frequency of re-initiation decreases with the length of the un-transcribed IGR (UTIGR) [70,71] dual-luciferase Firefly/NanoLuc 2-gene minigenomes with elongated UTIGR based on MeV N-P IGR were also built (see scheme Fig 8A) according to previous work based on the related Sendai virus that has served as the reference study model for Paramyxoviridae [70]. The Homogenous and comparable decrease of the efficiency of the transcription re-initiation mediated by every MeV IGR from 3' to 5' gene position observed with every N variant when the NanoLuc/Firefly signal ratio is normalized as a function of that observed with the first N-P IGR. MeV IGRs can be grouped into three subsets of re-initiation efficiency, high for N-P and P-M, medium for M-F and F-H and low for H-L. (c-g) Variation in the efficiency of re-initiation as a function of the 5 different MeV IGRs as determined from the NanoLuc/Firefly signal ratios (i.e. same data as in (b) but without normalization to the N-P IGR) and expressed as a function of K D of the N TAIL /XD pair. Note the progressive loss of correlation from N-P to H-L IGRs. (h) Correlation of the mean re-initiation rate through the five MeV IGRs (estimated as mean NanoLuc/Firefly signal ratio observed over the five IGR regions) with N TAIL /XD K D . Minigenome data are expressed as the mean +/-SD of at least 3 independent experiments, with each combination being done in triplicate. See also S5 Fig As observed in the previous set of experiments, the NanoLuc/Firefly signal ratios obtained with the N-P minigenome (i.e. UTIGR "+0") nicely correlate with the N TAIL /XD binding strengths (Fig 8B, compare also with Fig 6C for data reproducibility). When N variants were tested with elongated UTIGR minigenomes, the NanoLuc/Firefly signal ratio exponentially and compare the percentage of unpriming per UTIGR nt (%unpriming/ UTIGR nt). The D493G variant exhibits a significantly lower %unpriming/ UTIGR nt compared to wt N, whereas that of R490S, R497G and S491L variant was significantly higher (Fig 8D). Furthermore, the %unpriming/ UTIGR nt of N variants tends to vary according to the log of the N TAIL /XD K D , (Fig 8E, p = 0.062). Remarkably, the %unpriming/ UTIGR nt and mean re-initiation rate through the five MeV IGR regions significantly correlate to each other (Fig 8F, p = 0.0032). Overall these data reveal that lowering the N TAIL /XD binding strength significantly increases the unpriming rate of MeV polymerase during transcription re-initiation and its scanning over un-transcribed genomic sequences, i.e. over each UTIGR.
Impact of N TAIL amino acid substitutions introduced into recombinant unigene and biG-biS viruses on virus production
Since even N TAIL variants with the highest K D for XD were able to reconstitute functional dual-luciferase minigenomes, we sought at evaluating the impact of substitutions in the viral context by expressing N variants into two types of recombinant viruses, namely unigene and biG-biS viruses. Unigene viruses possess only one copy of the N gene and thus express solely the mutated N protein. By contrast, biG-biS viruses contain a duplicated viral gene, here the N gene, one encoding the wt N protein (wt Flag-N1) and one encoding the mutated N protein with a HA tag (HA-N2), the expression of which can be independently silenced thanks to the use of two cell lines expressing shRNA that selectively target one of the two N genes (S8 Fig) [48]. Unigene viruses harboring N TAIL variants were all rescued. The biG-biS viruses were also all rescued in cells allowing the selective expression of the wt Flag-N1 gene copy, although the too low virus production by the R489Q and R490S viruses prevented further analysis. Virus production by recombinant viruses at 3 d.p.i. were determined for unigene viruses in Vero cells, while that of biG-biS viruses was measured in three host cells allowing selective expression of either the wt Flag-N1 gene copy, the HA-N2 gene variant, or both of the N gene copies simultaneously (Fig 9A). Virus production was found to be very low (at least 2 log reduction with respect to the wt counterpart) in the case of unigene and biG-biS S491L viruses. Note that the possibility that the observed differences in virus production of unigene viruses could be ascribed to a defect in N variant expression (S1 and S9A Figs) or to a significant contamination by defective interfering (DI) mini-replicons was checked (S9B- S9D Fig) and ruled out. When plotted against the N TAIL /XD binding strength as determined by glu-PCA, the virus production of unigene N TAIL variants does not significantly correlate with binding strength (Fig 9B). However, the virus titer of biG-biS viruses under the selective expression of HA-N2 variant and under the combined expression of both N copies were found to correlate with N TAIL /XD binding strength (p = 0.04 and p = 0.008, respectively) (Fig 9C and 9D), while no such a correlation was found upon selective expression of the wt Flag-N1 copy as expected (Fig 9E). We noticed that the coexpression of N wt with D493G variant appears deleterious for virus production (Fig 9D). However, in a minigenome assay such a mixture of N was as efficient as N wt alone (S10 Fig), thus ruling out the possibility that N TAIL heterogeneity could directly impact the polymerase activity. Overall these data indicate that the N TAIL /XD binding strength may control the virus production to some extent.
Viral transcription gradient correlates with XD-binding affinity of Box2 variants
We then took advantage of unigene viruses expressing the single-site Box2 variants to determine which activity of the viral polymerase could be affected by a change in the N TAIL /XD binding affinity. Vero cells were infected with wt, R489G, R490S, A492T, D493G and R497G When RNA synthesis parameters were plotted along with N TAIL /XD K D , it appeared that both (+) RNA transcript accumulation rate and ratios between P (or F) and N transcripts could be roughly predicted from the interaction strength between the N TAIL variant and XD as measured by either method (Fig 10). The correlations were statistically significant between the accumulation rate of P (+) transcripts and N TAIL /XD K D (Fig 10A) and between the F/N transcript ratios measured at 24 h.p.i. and the K D (Fig 10B). In further support of the coherence of the results, a good correlation was found between the accumulated levels of N and P (+) RNAs during primary transcription and at 24 h.p.i. (S11A and S11B Fig), and between both N (+) and P (+) RNA transcripts and (-) genomic RNA (S11C and S11D Fig).
When the F/N mRNA ratios at 24 h.p.i. observed with unigene viruses were plotted against the calculated mean re-initiation rate of the 5 IGRs and the %unpriming/ UTIGR nt a significant positive and a negative correlation were found, respectively (Fig 11). Altogether, these data support that the N TAIL /XD binding strength controls, at least in part, the steepness of the viral transcription gradient.
Discussion
By combining in vitro biophysical and biochemical studies, in silico analyses (i.e. MD simulations) and in cellula polymerase functional investigations using recombinant viruses and dualluciferase editing-dependent minigenome assays, we deciphered key molecular parameters that govern the N TAIL /XD interaction. Specifically, we uncovered a correlation between interaction strength and efficiency of transcription re-initiation at intergenic regions.
Impact of preconfiguration of the α-MoRE on the interaction with XD
For most of the N TAIL variants the observed variations in binding affinities cannot be ascribed to differences in the extent of α-helical sampling of the free form of the α-MoRE, nor to differences in the ability of the latter to undergo induced α-helical folding. However, the R489Q substitution represents an exception in this respect: indeed, it has a reduced extent of α-helicity and a slightly increased K D towards XD. The reduced α-helical content of this variant is in line with secondary structure predictions, as obtained using the Psipred server (http://bioinf.cs.ucl. ac.uk/psipred/) [72], that predicts a slightly lower helical propensity. Whether the experimentally observed reduction in affinity towards XD arises from this lower helicity or from other attributes, including charge-related ones, remains to be established. This variant also displays a reduced accumulation rate of primary transcripts. The subtle molecular mechanisms underlying the peculiar behavior of this variant remain however to be elucidated.
N TAIL binding to XD involves a hydrogen bonding network including three α-MoRE and three XD residues
The complex hydrogen bonding revealed by MD simulations of N TAIL /XD complexes allows the drops in binding affinities experimentally observed for the S491L, R497G and R490S variants to be rationalized. Interestingly, these substitutions, which have the most dramatic effects in terms of binding affinities, are also the ones that have the strongest effect on virus replication, with the S491L substitution being very poorly tolerated even in biG-biS viruses.
The lower the N TAIL /XD binding strength the lower the efficiency of transcription re-initiation at intergenic regions
We provide here compelling evidence indicating that the strength of the N TAIL /XD interaction controls, at least in part, the ability of the P+L polymerase complex to re-initiate at IGRs: data obtained using our highly sensitive and reproducible dual-luciferase minigenome assay reveal a significant correlation between the N TAIL /XD binding strength and the efficiency of the transcription re-initiation. Since our minigenome assays rely on the edition of the second reporter gene, we cannot formally exclude that the editing may be also impacted by the N TAIL / XD binding strength. However, the calculated %unpriming/ UTIGR nt only depends on the decrease of the NanoLuc/Firefly signals ratios with the length of the UTIGR. The observed effect is therefore independent of any potential effect on the edition (i.e. if N mutations only had an effect on editing, then this effect should be the same irrespective of the IGR under study and of its length, which is not the phenotype we observed). Moreover, the correlation in the viral context between the P/N and F/N mRNA ratio and the K D , supports a role for the XD/NTAIL interaction strength in the re-initiation at IGRs. A N protein truncated of its last 86 C-terminal amino acids, i.e. truncated of most of N TAIL including the XD binding site, had been shown to be active in transcription and replication both in a minigenome assay and when introduced into a recombinant virus [49]. We confirmed that the N1-439 truncated protein is as good as, if not better than, the wt N in transcribing the Firefly gene from our N-P 2-gene minigenome construct (S12A Fig). However, its ability to support transcription re-initiation over the N-P junction was significantly reduced, with the extent of reduction being comparable with that observed with the low affinity R497G variant (S12B Fig, UTIGR 0 Assuming a very slow degradation of viral mRNA [65,66,73], the transcripts accumulation rate in cells infected with unigene viruses reflects the RNA synthesis rate by the polymerase, the number of active polymerases (and their recruitment onto the nucleocapsid template), and the number of polymerases that are recruited per time unit on a given gene. For the same reason, the transcript ratios between the different genes are likely mostly governed by the efficiency with which the polymerase re-initiates the transcription at each IGR. Assuming this being a conserved feature for every N variant, we can reasonably interpret the inverse correlation we observed between multiple transcript ratios and K D as reflecting a direct control of the N TAIL /XD binding strength on the efficiency of the re-initiation at each IGR. A lower binding strength leads to lower levels of downstream transcripts. After completion of the polyadenylation of the messenger encoded by the upstream gene, the polymerase may remain firmly in contact with its genomic RNA template embedded into the nucleocapsid only if maintained by the anchoring of its P subunit via a dynamic binding of its X domain to the TAIL domain of N subunits located at the IGR (Fig 12). Therefore, a decrease in the XD/N TAIL affinity may favour the unpriming of the polymerase. Whether unprimed polymerases can detach from the nucleocapsid or stay on the template and move forward to the end of the nucleocapsid remain to be established. Hence, XD to N TAIL anchoring would tightly control the re-initiation level of the RNA synthesis by the polymerase in the transcription mode, thus determining the steepness of the transcription gradient (Fig 12, see also S6B Fig). The N TAIL /XD affinity affects the processivity of the polymerase both on the "transcription" and on the "scanning" modes What could be the functional significance of the relationship between the accumulation rate of primary N and P transcripts and the XD/N TAIL binding strength? As speculated, the dynamics of XD/N TAIL binding and release may also affect the polymerase processivity on the nucleocapsid [48]. The XD/N TAIL interaction may act as a brake and slows down the polymerase: the weaker is the interaction, the weaker is the brake. Also, because of the efficient recycling of the polymerases on the promoter [65], if, in the absence of transcription re-initiation, the polymerase detaches from the RNA template, a steeper gradient would release more polymerases available for transcription of the first genes. With weaker N TAIL /XD interactions, the viral production by unigene viruses tends to be negatively affected although the correlation was not statistically significant likely because of the small number of available virus variants and the too high variability of the result due to the multiple intervening parameters (see S6A Fig and the complete scheme of virus replication dynamics in [65]). However with biG-biS viruses, we did observe a significant correlation between virus production and N TAIL /XD binding strength in conditions where the N variant was selectively expressed. This significance may reflect both the higher number of available virus variants and/or the higher impact of the modulation of the transcription re-initiation process in viruses possessing an additional transcription unit (i.e. where the polymerase has to go through one additional IGR). The similar correlation observed upon the co-expression of both wt Flag-N1 and variant HA-N2 copies may indicate similar impact on transcription re-initiation because of the tetrameric valence of the P anchoring on (contiguous?) heterogeneous N TAIL appendages. Alternatively, it is possible that the heterogeneity of N TAIL within a given nucleocapsid template may have a negative impact on other mechanisms such as nucleocapsids packaging into particles since N TAIL also recruits the M protein [74], a key virion assembly factor [75]. The discrepancy we observed between virus production from biG-biS viruses and minigenome data with mixed N TAILs argues for this later hypothesis.
Using minigenomes with elongated UTIGR, we were able to measure the unpriming rate of the polymerase in the "scanning mode" and we show that a decrease in the N TAIL /XD affinity induces an increase of the unpriming rate. In this situation, without the stabilization and the active motion of the polymerase due to the RNA synthesis, the role of the N TAIL /XD interaction in maintaining the polymerase on the nucleocapsid may overcome the "brake" effect. Alternatively, as suggested by Krumm et al [49], the N TAIL may need to be rearranged by P to allow an efficient RNA synthesis. In this case, a too low N TAIL /XD affinity may weaken the efficiency of P in rearranging N TAIL and would favor the unpriming of the polymerases. The fact that the N1-439 variant, that lacks most of N TAIL , has the lowest unpriming rate on UTIGR supports this second hypothesis (0.6 vs 0.81%unpriming/ UTIGR nt for N1-439 and wt N respectively) (S12C Fig). A mechanism among others to control both scanning and re-initiation by MeV polymerase In conclusion, the XD/N TAIL interaction may play a critical role in the polymerase processivity, in maintaining the polymerase anchored to the nucleocapsid during its scanning upon crossing the intergenic regions, and/or in the transcription re-initiation at each intergenic region. Since both increasing [48] or decreasing (this study) the XD/N TAIL affinity negatively affect the viral growth, the wild type XD/N TAIL binding strength seems to have been selected to mediate an optimal equilibrium between polymerase recruitment, polymerase processivity and transcription re-initiation efficiency. A corollary of this is that substitutions that strongly affect affinity towards XD are poorly tolerated. Consistent with this, the substitutions with the most dramatic impact herein investigated (i.e. R490S, S491L and R497G) do not naturally occur in any of the 1,218 non-redundant MeV sequences, while those that have a less drastic impact (i.e. R489Q, A492T and D493G) are found in circulating measles strains [47]. Interestingly, in the case of Ebola virus (EBOV), an additional protein, i.e. VP30, serves as an anti-terminator transcription factor, and mutations that either decrease or increase the binding affinity between N and VP30, decrease RNA synthesis [76] thus arguing for a similarly tightly regulated interaction. According to our work, the N TAIL to XD binding strength tightly controls the transcription gradient. However, this does not rule out the possibility that other mechanisms may be at work in controlling the steepness of the gradient. Indeed, in the brain of three patients suffering from subacute sclerosis encephalitis (SSPE) or measles inclusion bodies encephalitis (MIBE) the transcription gradient was found to be steeper than the one measured in in vitro infected cells [77] although the amino acid sequences of N TAIL and XD were found to be unvaried [78]. Furthermore, in the absence of the C protein, a steeper transcription gradient is also observed [79]. These two lines of evidence advocate for a multiparametric control of the transcription gradient.
The conserved bipartite P to N interaction of Paramyxoviridae members is also shared by other families of the Mononegavirales order
The major role of the N binding site on the C-terminus of P has been postulated to mediate L anchoring to the nucleocapsid without understanding the implication of such anchoring on the polymerase and/or on the nucleocapsid dynamics. The need for an optimized interaction between the P and N proteins might be one of the major evolution constraints to which the polymerase machinery of MeV, and possibly of paramyxoviruses in general, is subjected. Our findings raise also the question as to whether binding of the C-terminus of P to the globular moiety of N, as observed in other Mononegavirales members, needs to be similarly controlled reflecting a similar functional role.
The bipartite nature of P to N binding (see scheme Fig 1A) is remarkably conserved throughout the Mononegavirales order [80]. An α-MoRE located at N-terminus of P binds to the C-terminal globular domain of the N CORE to form the so-called N 0 P complex that is used by the polymerase as the encapsidation substrate. Solved N 0 P structures from members of the Rhabdoviridae family (vesicular stomatitis virus, VSV) [8], Filoviridae family (VP35, of EBOV) [81,82], Pneumoviridae family (human metapneumovirus, HMPV) [9] and Paramyxoviridae family (Nipah virus, NiV a Henipavirus member) [7], MeV a Morbillivirus member [5], mumps virus, MuV a Rubulavirus member [83] revealed a common mechanism whereby the N terminus of P competes out with N arms that stabilize the oligomeric form of N and directly or indirectly prevents RNA binding. Structural and functional evidences indicate that, via its N-terminus, P can transiently uncover the genome at its 3'end from the first N subunits to give L access to its genomic RNA template (see [83] and [84] for review).
An additional N-binding site is located at the C-terminus of P (or VP35 for EBOV) (see scheme Fig 1A) [85] and allows binding to the assembled form of N. While this secondary binding site is required for the polymerase activity in minigenome experiments from several viruses [3,83,85,86,87], the structures of the reciprocal N-binding and P-binding site on P and N, respectively, look less conserved. In the case of NiV [42], Hendra virus [88], SeV [38,89] and MeV [27,30,44,89] the C-terminal domain of P (XD) is structurally conserved and consists of a bundle of 3 α-helices that are structurally analogous, and that dynamically binds to a α-MoRE located near the C-terminus of N TAIL ( [90,91], see [92] for reviews). This N TAIL -XD interaction is commonly characterized by a rather low affinity (K D within the 3-50 μM range, [39,46,89] and this work). In Rubulavirus members, the C-terminal region of P spans in solution a structural continuum ranging from stable triple α-helical bundles to largely disordered, with crystal packing stabilizing the folded form [93,94]. In MuV, this triple α-helical bundle analogous to XD binds directly to the core of N subunits of the nucleocapsid [24] without excluding a complementary binding to the extremity of N TAIL [83]. By analogy with MuV XD, MeV XD might also bind to another binding site located on N CORE . This would explain how transcription and replication can still be observed in the presence of the N1-439 truncated where interaction of P relies only on N CORE ([49] and this paper). Indeed in other Mononegavirales members, the C-terminus of P binds to the core of N. In the case of RSV, the minimal nucleocapsid-binding region of P, which encompasses the last nine P residues, is disordered [95] and remains predominantly disordered even upon binding to the N-terminal lobe of N CORE [96].
The C-terminal domain of P from Rabies virus (RABV) [97], Mokola virus [98,99] and VSV [100] share a fold made of a bundle of α-helices that binds to the core of two adjacent N proteins of the nucleocapsid [101,102]. The N protein of Rhabdoviridae members, along with the N protein from RSV lacks the disordered N TAIL domain that characterizes N proteins from Paramyxoviridae members. In contrast to the XD-N TAIL interaction, the C-terminal domain of RABV P binds to the nucleocapsid with a high affinity (K D in the nanomolar range) [101]. In spite of the diversities of both structural features and binding modes within Mononegavirales members, does the binding of C-terminus of P to the assembled form of N fulfill common functions, namely ensuring the proper efficiency in polymerase scanning and re-initiation at intergenic regions? Further works will unveil to which extent our present findings are relevant for other members of the Paramyxoviridae or other families of the Mononegavirales order.
Plasmid construction
The pDEST17O/I vector [103], allowing the bacterial expression of N-terminally hexahistidine tagged recombinant proteins under the control of the T7 promoter, was used for the expression of all N TAIL variants. The pDEST17 derivatives encoding single-site N TAIL variants bearing substitutions within Box2 were obtained either by Gateway recombination cloning technology (variants R489Q, R490S, S491L and R497G) using the previously described pNGG derivatives [47] as the donor vectors, or by site-directed mutagenesis (variants A492T and D493G). In the latter case, we used a pair of complementary mutagenic primers (Operon) designed to introduce the desired mutation, Turbo-Pfu polymerase (Stratagene), and the pDEST17O/I construct encoding wt MeV (Edmonston B) N TAIL as template [47]. After digestion with DpnI to remove the methylated DNA template, CaCl 2 -competent E. coli TAM1 cells (Active Motif) were transformed with the amplified PCR product.
The pNGG derivative encoding the MXSF N TAIL variant N-terminally fused to the N-terminal fragment of GFP was obtained in four steps using pNGG/N TAIL as template [47,104] and site-directed mutagenesis PCR. In the first step, the pair of mutagenic primers was designed to introduce the first amino acid substitution. After PCR and DpnI digestion, CaCl 2 -competent E. coli TAM1 cells (Active Motif) were transformed with the amplified PCR product. After having sequenced the construct to ensure that the desired mutation had been introduced, a second PCR was carried out using another pair of mutagenic primers designed to introduce the second substitution. Repeating this procedure four times led to the final construct bearing the four desired substitutions (i.e. D437V, R439S, P456S and P485L).
The sequences of the coding regions of all constructs generated in this study were checked by sequencing (GATC Biotech) and found to conform to expectations.
The pDEST17/N TAIL construct encoding wt N TAIL has already been described [47], as is the pDEST14 construct encoding C-terminally hexahistidine tagged MeV XD [30].
The plasmid p(+)MVNSe previously described in [48] was used as the MeV genome backbone. MeV genomic plasmids were built by direct recombination of one or two PCR fragments according to the InFusion user manual (Clontech). To build biG-biS recombinant viruses, the N gene was duplicated in N 1 and N 2 in gene positions 1 and 2, respectively. N 1 was tagged with an N-terminal Flag peptide and three copies of the GFP RNAi target sequence (GAACGGCA TCAAGGTGAA) in the 3'UTR of its mRNA. N 2 was tagged with an N-terminal hemagglutinin (HA) peptide and three copies of the P RNAi target sequence (GGACACCTCTCAAGC ATCAT) in the 3'UTR. Mutations into the N TAIL domain of N, R489Q, R490S, S491L, A492T, D493G, R497G, MXSF (D437V/R439S/P456S/P485L) and MX208 (D437V/P485L/L524R), were introduced by subcloning PCR-amplified fragments from the pDEST17/N TAIL vectors.
Full length wt and mutated N, wt and mutated N TAIL , P PMD-XD and P 376-507 fragments were subcloned downstream Gaussia glu1 and/or glu2 domains by InFusion recombination of PCR-amplified fragments as previously described [48]. All plasmids and viruses (N 1 , N 2 , P, M, and L gene) were verified by sequencing the subcloned PCR fragments or cDNA obtained by reverse transcription-PCR (RT-PCR) performed on virus stocks.
Plasmids encoding dual-luciferase editing-dependent 2-gene minigenomes were built by InFusion subcloning of PCR amplicons encompassing Firefly and NanoLuc coding sequences flanked by N UTR and L 3'UTR. The two luciferase coding sequences are separated by the N-P IGR either unmodified or exchanged with P-M, M-F, F-H and H-L IGRs (i.e. untranscribed 3'-GAA-5' (or 3'-GCA-5' for H-L) triplet flanked by canonical upstream and downstream gene end and gene start sequences arbitrarily fixed to 15 nt) or elongated by 12, 36, 108 or 324 nt (see sequences in S2 and S3 Tables) into the p107(+) MeV minigenome construct that drives the synthesis of (+) genomic strand under the control of the T7 promoter [62]. According to the rule of six that governs the strictly conserved hexameric length of measles virus genome [62,105], all minigenomes share identical phasing of the last U of the polyadenylation signal of the firefly gene (phase 6, i.e. the last nucleotide covered by the N subunit) and of the editing site with the C being in phase 6 as defined in [106]. A 3-gene minigenome coding for Firefly and chimeric Glu2-linker-NanoLuc luciferase as a results of read-through between gene 2 and gene 3 and RNA editing was built by modifying the N-P 2-gene minigenome. As a second gene, the ORF of the C-terminal domain of Glu (glu2) was inserted downstream to a START codon but without a STOP codon. This ORF is followed by a second N-P IGR and by the NanoLuc ORF without its own START codon, in frame "-1nt" to the upstream Glu2 ORF. Following addition of a G thanks to the presence of the P editing site, the NanoLuc ORF becomes in frame with the upstream Glu2 ORF. Consequently, the full-length chimeric Glu2linker-NanoLuc can be uniquely translated from a read-through transcript over the second N-P IGR that is also edited (see sequence in S4 Table).
All plasmids will be deposited in the Addgene plasmid repository service except the glu1 and glu2 constructs that Addgene cannot accept. Those constructs are available upon request.
Production and purification of N TAIL and XD variants
The E. coli strain Rosetta [DE3] pLysS (Novagen) was used for the expression of all recombinant proteins. Transformants were selected on ampicillin and chloramphenicol plates. 50 mL of Luria-Bertani (LB) medium supplemented with 100 μg/mL ampicilin and 34 μg/mL chloramphenicol were seeded with the selected colonies, and grown overnight to saturation. An aliquot of the overnight culture was diluted 1/25 in LB medium containing ampicillin and chloramphenicol and grown at 37˚C. When the optical density at 600 nm (OD 600 ) reached 0.6-0.8, isopropyl ß-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.2 mM, and the cells were grown at 37˚C for 4 additional hours. The induced cells were harvested, washed and collected by centrifugation (5,000 g, 12 min). The resulting pellets were frozen at -80˚C.
All the N TAIL and XD proteins were purified to homogeneity (> 95%) from the soluble fraction of bacterial lysates in two steps: Immobilized Metal Affinity Chromatography (IMAC), and size exclusion chromatography (SEC). Cellular pellets of bacteria transformed with the different expression plasmids were resuspended in 5 volumes (v/w) of buffer A (50 mM Tris/HCl pH 8, 300 mM NaCl, 20 mM imidazole, 1 mM phenyl-methyl-sulphonyl-fluoride (PMSF)) supplemented with lysozyme (0.1 mg/mL), DNAse I (10 μg/mL), 20 mM MgSO 4 and protease inhibitor cocktail (Sigma). After a 30-min incubation with gentle agitation, the cells were disrupted by sonication. The lysate was clarified by centrifugation at 20,000 g for 30 min. The clarified supernatant, as obtained from a one-liter culture, was incubated for 1 h with 5 ml (50%) Chelating Sepharose Fast Flow Resin preloaded with Ni 2+ ions (GE, Healthcare), previously equilibrated in buffer A. The resin was washed with buffer A supplemented with 1 M NaCl to remove contaminating DNA, and the proteins were eluted in buffer A containing 1 M NaCl and 250 mM imidazole. Eluents were analyzed by SDS-PAGE. Fractions containing the recombinant product were concentrated using centrifugal filtration (Centricon Plus-20, 5000 Da molecular cutoff, Millipore). The proteins were then loaded onto a Superdex 200 (N TAIL ) or Superdex 75 (XD) 16/60 column (GE, Healthcare) and eluted in 10 mM Tris/HCl pH 8, 150 mM NaCl.
Protein concentrations were calculated using the theoretical absorption coefficients at 280 nm as obtained using the program ProtParam at the EXPASY server.
Mass spectrometry
Mass analysis of the purified mutated N TAIL proteins was performed using an Autoflex II ToF/ToF (Bruker Daltonics). Spectra were acquired in a linear mode. 15 pmol of samples were mixed with an equal volume (0.7 μL) of sinapinic acid matrix solution, spotted on the target and dried at room temperature.
The identity of the purified N TAIL proteins was confirmed by mass spectral analysis of tryptic fragments obtained by digesting (0.25 μg trypsin) 1 μg of purified recombinant protein isolated onto SDS-PAGE. The tryptic peptides were analyzed as described above and peptide fingerprints were obtained and compared with in-silico protein digest (Biotools, Bruker Daltonics). The mass standards were either autolytic peptides or peptide standards (Bruker Daltonics).
Far-UV circular dichroism (CD)
The CD spectra of N TAIL proteins were recorded on a Jasco 810 dichrograph using 1-mm thick quartz cells in 10 mM sodium phosphate pH 7 at 20˚C. CD spectra were measured between 190 and 260 nm, at 0. The experimental data in the 190-260 nm range were analyzed using the DICHROWEB website which was supported by grants to the BBSRC Centre for Protein and Membrane Structure and Dynamics [107,108]. The CDSSTR deconvolution method was used to estimate the content in α-helical and disordered structure using the reference protein set 7.
Isothermal titration calorimetry
ITC experiments were carried out on an ITC200 isothermal titration calorimeter (Microcal) at 20˚C. Protein pairs used in the binding analyses were dialyzed against the same buffer (10 mM Tris/HCl pH 8, 150 mM NaCl) to minimize undesirable buffer-related effects. The dialysis buffer was used in all preliminary equilibration and washing steps.
The concentrations of purified wt and mutated N TAIL proteins in the microcalorimeter cell (0.2 mL) ranged from 25 μM to 180 μM. XD was added from a computer-controlled 40-μL microsyringe via a total of 19 injections of 2 μL each at intervals of 180 s. Its concentration in the microsyringe ranged from 300 μM to 960 μM.
A theoretical titration curve was fitted to the experimental data using the ORIGIN software (Microcal). This software uses the relationship between the heat generated by each injection and ΔH˚(enthalpy change in kcal mole -1 ), K A (association binding constant in M -1 ), n (number of binding sites per monomer), total protein concentration and free and total ligand concentrations. The variation in the entropy (ΔS˚in cal mol -1 deg -1 ) of each binding reaction was inferred from the variation in the free energy (ΔG˚), where this latter was calculated from the following relation: ΔG˚= -RT ln 1/K A .
Molecular dynamics simulations
All MD simulations were performed in explicit solvent with periodic conditions with CHARMM and NAMD software packages and CHARMM force field version 27 with CMAP corrections. The initial coordinates of the XD/α-MoRE complex were taken from the crystal structure (PDB code 1T6O) [44]. The two XD/α-MoRE mutated models bearing either the S491L or the R497G N TAIL substitution, were built with VMD plugin 'mutator' starting from the X-ray structure of the wt complex (PDB code 1T6O). In the case of the S491L variant, the three most favourable orientations of the leucine side chain were generated with Sybyl. Nonprotein derivatives were discarded. Orientation of the side chains of Asn, Gln, and His residues were checked using in-house VMD plugin and the WHAT IF web interface (http://swift.cmbi. kun.nl/). Residue His498 of XD was assigned HSD type and all other titratable groups were assigned standard protonation state at pH 7.0. Coordinates of missing hydrogen atoms were added using the hbuild algorithm in CHARMM. To improve conformational sampling, three independent simulations were carried out using different initial velocities. The system was solvated with a pre-equilibrated solvation box (edge length around 60 Å) consisting of TIP3P water molecules. Crystallographic water molecules were included in the initial model. Chloride and sodium ions were added to achieve neutralization of the whole system. Periodic boundary conditions were applied. Unfavorable contacts were removed by a short energy minimization with conjugate gradient and ABNR. Electrostatic interactions were treated using the particlemesh Ewald summation method, and we used the switch function for the van der Waals energy interactions with cuton, cutoff and cutnb values of 10, 12 and 14 Å respectively. Vibration of the bonds containing hydrogen atoms were constrained with the Shake algorithm and a 1-fs integration step was used. The system was heated gradually to 300K, followed by an equilibration step (500 ps). During these two early steps, harmonic constraints were applied to protein heavy atoms. The constraint harmonic constant (k) was equal to 1 and 0.1 kcal/mol/Å 2 for the backbone and side chains, respectively, and was removed after 250 ps equilibration. The production phase of 50 ns was performed without any constraints. Snapshots of the coordinates were saved every 0.5 ps. Trajectories were analyzed using a combination of in-house and VMD scripts.
Analysis of molecular dynamics
Overall <RMSD> variations were computed with VMD after superimposition of the Cα atoms of each conformation generated onto the initial structure (last structure of the equilibration step). Flexible N-and C-terminal residues were not included in the calculation. Three types of RMSD were computed as it follows. For each frame, the XD protein was superimposed onto the initial XD model and RMSD was computed over XD Cα atoms only. For each frame the α-MoRE was superimposed onto the corresponding region of the initial structure and RMSD was computed over N TAIL Cα atoms only. For each frame, the XD protein was superimposed onto the initial XD protein and RMSD was computed over N TAIL Cα atoms only.
Free energy perturbation
Free-energy perturbation (FEP) module implemented in NAMD was used to perform alchemical transformation of Ser491 to Leu and Arg497 to Gly. Free energies differences resulting from the Ser to Leu or Arg to Gly substitution were computed using the thermodynamic cycle shown in S3 Fig. The free form of the α-MoRE in solution was taken from the XD/N TAIL complex.
Free energy profile
The free energy profile for the dissociation of the XD/α-MoRE complex (wt and mutated forms) was computed using the adaptive biasing force (ABF) method, implemented in NAMD [109]. This method relies upon the integration of the average force acting on a selected reaction coordinate (here, the center of mass between the two partners). A biasing force is applied to the system in such a way that no average force acts along the reaction coordinate thus allowing overcoming free energy barriers. For a complete description of the method please refer to http://www.edam.uhp-nancy.fr/ABF/theory.html and references therein shown.
The distance separating the centers of mass of the two proteins was selected as reaction coordinate. The distance was calculated on the Cα atoms not taking into account the three atoms at each end (N-term and C-term end) of each protein partner due to their high flexibility. This distance is about 11 Å in the associated form and the partners are considered dissociated after a 10 Å increase in this distance. The reaction coordinate was subdivided into sections of 0.5 Å and each one was successively explored during 5 ns. Bin width was kept at 0.02 Å, the number of samples prior to force application was 500 and the wall force constant is 100 kcal.mol -1 .Å 2 . Once a section is sampled, the conformation in which COM distance is the nearest to the upper boundary is selected as the starting point of the following 0.5 Å section. A post-processing step merges the sampling counts and the PMF of each part and generates the whole profile of PMF along the dissociation process. The trajectories were generated using the same protocol as described for free MD.
Cell lines and viruses
Cells were cultured in DMEM medium (Life Technologies) supplemented with 10% of heatinactivated (30 min at 56˚C) fetal bovine serum, 1% L-glutamine, gentamicin (10 μg/ml) at 37˚C and 5% CO 2 . Medium of 293-3-46 helper cells was supplemented with G418 at 1.2 mg/ ml. Vero (si2) and Vero-SLAM (si1) cells stably expressing shRNA targeting the P and GFP mRNAs, respectively, were previously described [48]. To rescue recombinant viruses, the helper cell line 293-3-46 stably expressing T7 polymerase, MeV N, and P was transfected by using the ProFection kit with two plasmids coding for the MeV genome and MeV-L protein (pEMC-La) [110]. Three days after transfection, the cells were overlaid on either Vero (single N gene virus) or Vero-si2 cells (bi-N virus). Upon appearance, isolated syncytia were picked and individually propagated on relevant Vero (from CelluloNet BioBank BB-0033-00072, SFR BioSciences, Lyon France) (single N virus) or Vero-si2 (bi-N virus) cells. Virus stock was produced after a second passage at a multiplicity of infection (MOI) of 0.03 in the relevant cell line. This stock was checked to rule out mycoplasma contamination, has its N 1 , N 2 , P, M, and L genes sequenced, and was titrated on the relevant host cell before use.
Split-luciferase reassembly assay
Gaussia princeps luciferase-based complementation assay and data analysis (normalized luminescent ratio, NLR) were performed according to [50]. Human 293T cells (from CelluloNet BioBank BB-0033-00072, SFR BioSciences, Lyon France) were cultured in Dulbecco's Medium Eagle's Modified (DMEM) (Life Technologies) supplemented with 10% of heat inactivated (30 min at 56˚C) fetal bovine serum, 1% L-Glutamine and 10 μg/ml gentamycin at 37˚C and 5% CO2. Cells were transfected using the jetPRIME reagent (Polyplus transfection). NLR was calculated by dividing the luciferase value of the two chimeric partners by the sum of the luciferase value of every chimeric partner mixed with the other "empty" glu domain. Results were expressed as fold increase with respect to the reference N TAIL /XD, which was set to 1.
Analysis of viral protein accumulation and virus replication
Parental Vero, si1 and si2 cells were infected at MOI 1 with recombinant viruses with or without addition of 10 μg/ml of fusion inhibitor peptide z-fFG to prevent syncytium formation. Virus production was measured after freeze-thaw cycles of infected cells using a 50% tissue culture infective dose (TCID50) titration assay. Contamination of virus stock with internal deletion and copyback defective interfering (DI) minigenomes were assessed according to the method of [111].
Detection of the expression of viral N, Flag-N1, HA-N2, P and cellular GAPDH proteins was performed by Western blotting. Infected cells were lysed in NP40 buffer (20 mM Tris/HCl pH 8, 150 mM NaCl, 0.6% NP-40, 2 mM EDTA, protease cocktail inhibitor Complete 1 x (Roche)) for 20 minutes on ice. The proteins were then separated from the cell debris by centrifugation at 15,000 g during 10 minutes. The proteins were denatured by the addition of Laemmli 1 x loading buffer before analysis by SDS-PAGE and immunoblotting using anti-N (cl25 antibody), anti-Flag (Sigma), anti-HA (Sigma), anti-P (49.21 antibody) and anti-GAPDH (Mab374, Chemicon) monoclonal antibodies. Western blotting was revealed by chemiluminescence as detailed previously [48].
Minigenome assay
The assay was performed essentially as described in [62,112] with minor modifications. 2.10 4 BSRT7 cells that constitutively express the T7 phage DNA-dependent RNA polymerase [113] were seeded in 96-well plates and transfected the day after with 66 ng of pEMC-N (either wt or mutated) 44 ng of pEMC-(Flag/L+P) (a home-made T7-driven bicistronic construct) [58] and 90 ng of plasmid encoding for the different minigenomes mixed with the transfection reagent as indicated in the manufacturer protocol (jetPRIME Polyplus-transfection). Two days after transfection, Firefly and NanoLuc activity were measured using the Nano-Glo Dual-Luciferase Reporter Assay (Promega). The background luciferase activity from of both luciferases observed in the absence of active L protein was subtracted from the signal measured in the presence of L, and data obtained from three independent experiments were normalized to each other to level the mean signal observed for all combinations tested at the same time. The percentage of unpriming per nucleotide of the un-transcribed intergenic (UTIGR) region (% unpriming/ UTIGR nt) was calculated as follow. The luciferase signals ratios were plotted in relation to the length of the UTIGR region and the equation of the exponential regression curve was calculated (y = b à e a ). The %unpriming/ UTIGR nt = 1-e a . | v3-fos-license |
2018-04-03T04:10:29.301Z | 2017-05-17T00:00:00.000 | 23312083 | {
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} | pes2o/s2orc | Nonenzymatic glycosylation of human serum albumin and its effect on antibodies profile in patients with diabetes mellitus
Background Albumin glycation and subsequent formation of advanced glycation end products (AGEs) correlate with diabetes and associated complications. Methods Human Serum Albumin (HSA) was modified with D-glucose for a 40 day period under sterile conditions at 37°C. Modified samples along with native HSA (unmodified) were analyzed for structural modifications by UV and fluorescence, FTIR, Liquid chromatography mass spectrometry (LCMS) and X–ray crystallography. New-Zealand white female rabbits immunized with AGEs, represent auto-antibodies formation as assessed by competitive and direct binding enzyme-linked immunosorbent assay (ELISA). Neo-epitopesagainst In-vitro formed AGEs were characterized in patients with diabetes mellitus type 2 (n = 50), type 1 (n = 50), gestational diabetes (n = 50) and type 2 with chronic kidney disease (CKD) with eGFR level 60–89 mL/min (n = 50) from serum direct binding ELISA. Results Glycated-HSA showed amarked increase in hyperchromicity of 65.82%,71.98%, 73.62% and 76.63% at λ280 nm along with anincreasein fluorescence intensity of 65.82%, 71.98%, 73.62% and 76.63% in glycated-HSA compared to native. FTIR results showed theshifting of Amide I peak from 1656 cm_1 to 1659 cm_1 and Amide II peak from 1554 cm_1 to 1564 cm_1 in glycated-HSA, with anew peak appearance of carbonyl group at 1737 cm-1. LCMS chromatogram of glycated-HSA showed thepresence of carboxymethyl lysine (CML) at 279.1 m/z. Immunological analysis showed high antibody titre>1:12,800 in theserum of rabbits immunized with glycated-HSA (modified with 400 mg/dL glucose) and inhibition of 84.65% at anantigen concentration of 20μg/mL. Maximum serum auto-antibody titre was found in T2DM (0.517±0.086), T1DM (0.108±0.092), GDM (0.611±0.041) and T2DM+CKD (0.096±0.25) patients immunized with glycated-HSA (modified with 400 mg/dL glucose). Conclusions Non-enzymatic glycosylation of HSA manifests immunological complications in diabetes mellitus due to change in its structure that enhances neo-epitopes generation.
Introduction
Glucose is a prime dietary component and energy exchange of the cell. Glucose, being a biomolecule of great importance, promotes adverse metabolic alterations in biological systems that include hypoglycemia, glucose intolerance, glucotoxicity, and hypertension [1][2][3][4][5]. Diabetes mellitus is a hub of metabolic disorders represented by an imbalance in glucose homeostasis leading to impairment in carbohydrate, lipids, and protein metabolism [6]. It is wellestablished fact that prolonged exposure to proteins with sugars under hyperglycemia generates advanced glycation end products (AGEs) [7]. The non-enzymatic addition of sugar with protein initiates the cascade of pathological mechanism of diabetes and its associated microand macro-vascular complications [8]. Previous literature of immunohistochemical investigations demonstrates AGEs-altered proteins in various human tissues under pathological conditions, including the patients with diabetic nephropathy [9], cardiovascular disease [10] and diabetic retinopathy [11]. Few scientists also describe the role of In-vivo AGEs as immunological epitopes starting the generation of auto-antibodies that plays a role in the progression of immunological complications in diabetes mellitus and its associated complications [12]. AGEs formed in-vivo or in-vitro binds to their receptor known as a receptor for advanced glycation end product (RAGE) that belongs to transmembrane 35kDa protein IgG super-family that acts as a key player in immunological response [13]. RAGE is distributed in many cell types such as monocytes, endothelial cells, smooth muscle cells, hepatocytes, and neurons and their expression may significantly impair in diseased state [14][15][16]. Recent literature has shown that diabetic animal model expresses more RAGE compared to control animals [17].Glycation of protein impairs the secondary and tertiary structure leads to the development of neo-epitopes that have potential to bind with antibodies showing animmunological response.
With the onset of diabetes mellitus, persistent and prolonged chronic hyperglycemia enhances free radicals formation through auto-oxidation of aldehyde group of glucose via non-enzymatic glycation of proteins leading to increasing flux of glucose through polyol pathway [18]. There is aninterconnection between protein glycation and free radicals [19]. Reactive oxygen species (ROS) causes extensive deterioration in human serum albumin (HSA) structure along with other biomacromolecules which formneo-epitopes contributing to its immunogenic potential showed by several animal experiments and clinical studies in patients with diabetes mellitus and its associated complications [20][21]. Covalent attachment of glucose or small residues in autologous proteins and other bio-molecules can generate conjugates that are efficient in inducing an immune response in the host cells. Fabrication of specific glucosederived adducts on biomacromolecules could function in a fashion to form auto-antibodies in diabetic patients. Qualitative studies have shown that chemically produced hexitolamino derivative of collagen Amadori adduct produces such Immunogenic response [22].Recent published literature demonstrated that non enzymation glycation of serum albumin leads to deterioration of albumin properties both structural and functional. Recently it is proved that human serum albumin during the course of non enzymatic glycation leads to development of advanced glycation end products that impairs its biochemical, electrochemical, spectroscopic, optical and fluidity properties [23]. Another study done with glycated HSA showed that presentation of neo-epitopes formed during the course of non enzymatic glycation due to refolding and structural impairments [24 -25].
The aim of this work was to investigate the immunological properties of AGEs formed at thephysiological glycemic and euglycemic range. Induced antibodies against in-vitro formed AGEs were implicated as a probe to detect antibody titer and impairment in homeostasis of thebiological system arising in diabetes and its associated complications.
Test materials and reagents
Human serum albumin (HSA), anti-rabbit IgG alkaline phosphatase conjugates, anti-human IgG alkaline phosphatase conjugates, pnitrophenyl phosphate, ethidium bromide, Tween-20, Protein A-agarose (2.5 mL pre-packed column), Freund's complete and incomplete adjuvants, was purchased from Sigma chemical company (St. Louis, USA), D-glucose, sodium chloride (NaCl) Sodium mono phosphate, diphosphate was purchased from SRL chemicals, India. All others chemicals and reagents used were of highest analytical grade available.
Ethics statement
Healthy female New Zealand white rabbits (1.5 ± 5 Kgs) were obtained from central animal facility and study was prospectively approved by Institutional animal ethics committee, Aligarh Muslim University, India (Certificate approval No. 401/RO/C/2001/CPCSEA) J.N Medical College, Aligarh Muslim University, India as per guideline laid down by CPCSEA (Ministry of Environment and Forests, Govt. of India). The study protocol does not include any anesthesia, euthanasia, or any kind of animal sacrifice. The protocol only involved exsanguination of blood from marginal ear vein at stipulated time intervals.
All subjects were informed about the study procedure and they provided written informed consent. The study was approved by the local Institutional Ethics Committee, Faculty of Medicine, J.N Medical College, Aligarh Muslim University, India (Certificate approval No. 1894/ FM) (Govt. of India). Exclusion criteria [1] Subjects with any immunological complications.
Preparation of advanced glycation end products (AGEs)
AGEs were prepared as previously mentioned protocol [26]. HSA (20 μM) was incubated under sterile conditions with varying concentrations of D-glucose (100, 200, 300 and 400 mg/dL) in phosphate buffer saline (20 mM, pH = 7.5) in the presence of 0.01% sodium azide solution at 37˚C under sterile conditions in capped sterile tubes for 40 days. At the termination of incubation, the samples were extensively dialyzed against sterile PBS buffer (10mM, pH = 7.4) with consecutively two changes of PBS buffer overnight at 4˚C to remove excess glucose. The samples were stored at -20˚C for further analysis.
Spectroscopic analysis
The ultraviolet absorption profile of native and modified HSA was scanned in the wavelength range of 190-400 nm on Shimadzu UV-1700 spectrophotometer with quartz cuvette having 1 cm path length. AGEs specific fluorescence of native and modified HSA sample was done on Shimadzu (RF-5301-PC) spectrofluorophotometer. The samples were excited at a wavelength of 370 nm, and the emission intensities were recorded at 400-600 nm range. To demonstrate the change in secondary structure upon glycation, Fourier transform-infra red spectroscopy analysis of native and HSA modified with 400 mg/dL concentration glucose was recorded. Briefly, 10 μl of protein sample was placed on ATR accessory on FT-IR spectrophotometer (8201 PC) with a resolution of 4 cm-1. FT-IR measurements of native and glycated samples were carried on Shimadzu FT-IR spectrophotometer (8201-PC) in the spectral range of 400-4000 cm -1 .
Liquid chromatography mass spectroscopic analysis
Native and glycated-HSA were subjected to LCMS analysis for identification of AGEs related adduct upon modifications along with carboxymethyl-lysine (CML), as the standard for AGEs. Samples were hydrolyzed with previously described protocol [27]. The digested hydrolyzate followed by filtration loaded into reverse phase separation assembly coupled with the capillary HPLC system having a C 18 analytical column. Briefly 0.4% acetic acid (solvent A), 0.2% acetonitrile (solvent B) comprising 2% formic acid were used as chromatographic conditions. The mass spectroscopic analysis was then performed on Micromass Castro Ultima Triple Quadrupole Mass spectrometer operated in positive ion mode with a full scan range from 0-400 m/z range.
Immunization schedule
The immunization of random, female New Zealand white rabbits (1.5±5 Kgs n = 03/each group) randomly selected and acclimatizedfor one week, performed as in previously described protocol [28]. Immunization was performed intramuscularly at multiple sites with 100 μg each of native and modified protein with 400 mg/dL glucose emulsified with Freund's adjuvant complete. The rabbits were boosted weekly for 6 weeks with Freund's incomplete adjuvant with the same amount of protein (immunogen) (Fig 1). Blood was collected from a marginal ear vein, and serum was separated. To inactivate the complement protein serum was given heat treatment at 56˚C for 30 min, followed by storage at -20˚C in the presence of 0.1% sodium azide as a preservative.
Sera of rabbits (pre-immune and immune) were used for determination of serum biochemical parameters with Randox biochemical automated analyzer (Randox Laboratories, BT29 4QY, United Kingdom).
Rectal temperature measurement
The rectal temperature of animals of both groups was recorded with high capacity sensitive digital thermometer at the termination (6 th week after immunization) of the experiment.
Direct binding enzyme-linked immunosorbent assay
ELISA was performed on polystyrene plates with slight modifications [29]. Briefly, 96 wells microtitre plates were coated with 100 μl of native and glucose-modified HSA (10 μg/mL in protein coating buffer) and incubated for 2 hrs at 37˚C followed by overnight incubation at 4˚C. Each sample was coated in duplicates, leaving half plate devoid of antigen coating that would serve as a control. Unbound antigen was washed thrice with TBS-T followed by blocking with 2.5% fat-free milk in TBS for 4-6 hrs incubation at 37˚C. Subsequentlyafter washing thrice with TBS-T, test serum diluted in TBS (100μl/each well) were added. Plates were incubated for 2 hrs at 37˚C followed by washing thrice with TBS-T. The bound antibodies were assayed with anti-rabbit/anti-human alkaline phosphatase conjugate followed by incubation at 37˚C for 2 hrs. The plates were read at 410 nm after addition of p-nitrophenyl phosphate as substrate. Results were expressed as themean of A test -C ontrol . Similarly, ELISA of T2DM, T1DM, GDM and T2DM+CKD patients were performed according to previously describe protocol [29].
Inhibition ELISA
The immunogenic specificity of the antibody was measured by inhibition ELISA as described previously [30][31]. Briefly, varying amount of inhibitors (native and glucose modified HSA with concentration ranging from 0-20 μg/mL) were incubated with antiserum at 37˚C for 2 hrs followed by overnight incubation at 4˚C. The immune complex formed thus coated in the wells of polystyrene plate. The steps after the coating were performed same as in direct binding ELISA. The results were expressed using the following equation.
Where A inhibited is the absorbance of inhibited wells and A uninhibited is the absorbance of uninhibited wells.
Isolation of IgG
Serum immunoglobulin G (IgG) from the sera of animals (pre-immune and post-immune) was isolated by affinity chromatography Protein A Agarose column as described by the previous protocol [32]. Briefly, 500μl of serum diluted with an equal volume of PBS (pH = 7. 4, 20 mm) was loaded into the column. The washing cycle of the column was performed 2-3 times to remove unbound IgG. The bound IgG was eluted with previously described protocol [30] followed by neutralization with 1 mL of 1 M Tris-HCl (pH = 8.5). The fractions eluted and read at 251nm and 278 nm. IgG concentration was determined (1.40 OD280 = 1.0 mg/mL) and dialyzed against PBS (pH = 7.4) and stored -20˚C with 0.1% sodium azide.
Direct binding and inhibition ELISA of purified IgG from an animal model
The direct binding and inhibition ELISA of isolated IgG were performed in a microtitre plate as described previously [30][31].
Spectroscopy of antigen-antibody complexes
The ultraviolet absorption spectra of serum-antigen (HSA native and glucose modified) and isolated rabbit IgG-antigen (HSA native and glucose modified) in the form of the immune complex were recorded in the range of 233-450 nm. Briefly, the immune complex was formed upon incubation of antigen (native and glucose modified HSA with concentration ranging from 0-20 μg/mL) with isolated IgG for 2 hrs at 37˚C followed by overnight incubation at 4˚C. Fluorescence profile of immune complexes formed was scanned with an excitation wavelength of 278 nm and emission range of 450-550 nm.
X-ray diffraction of immune complex to determine epitopes
To determine the immune complex formation, i.e. presence of epitopes to bind to the antigen (both native and glucose modified HSA), the glycated albumin (antigen) with a concentration of 10μg incubated with isolated IgG (20μM) from animal sera (immunized with native HSA and glucose modified HSA) for 2 hrs at 37˚C. An x-ray diffraction pattern of the film was recorded with Siemens D 5000 diffractometer installed with a flat monochromator. The divergence of the primary beam was enough to permit the low, glancing angles of incidence.
Stepscan mode with increments of 0.020 2θ with the count for 1s at each step was selected for recording the data. The angle of incidence α was set at a range of 0.20 to 100 to vary the interaction diameter and length in each depth area/region with the final scanning of the exit angle.
Statistical analysis
Data are given as mean ± SD. The statistical significance of the data was determined by student's t-test (stat graphics, origin 6.1). A p value of <0.05 was considered statistically significant.
Results and discussion
Spectroscopic analysis HSA (20μM) was incubated with varying concentrations (100, 200, 300 and 400 mg/dL) of glucose under sterile conditions for 40 days at 37˚C and UV absorption spectra were recorded. Native HSA gave a characteristic peak at 280 nm, whereas glucose modified samples of HSA showed an increasing % hyperchromicity. Marked increase in % hyperchromicity at λ 280 nm was recorded with highest modified HSA sample as compared to native HSA. Glucose modified HSA showed 65.82%, 71.98%, 73.62% and 76.63% hyperchromicity with increasing glucose concentration at λ 280 nm. The change in absorbance may be attributed to the formation of AGEs and their aggregates resulting from cross-linking [32][33]. Glycation of HSA protein enables the aromatic amino acids to expose their cyclic ring structure electrons that contribute to hyperchromicity phenomenon. Apart from aromatic amino acids, the unfolding of globular structure and reshuffling of bonds and their electrons also contribute to this effect [34][35].
Possible formation of fluorogenic AGEs was measured from the specific emission fluorescence intensity observed at 370 nm excitation wavelength. Native HSA at this excitation wavelength showed negligible changes fluorescence. Under identical and controlled experimental conditions, the gradual increase in fluorescence intensity of glycated-HSA samples indicates the formation of pentosidine like fluorogenic AGEs [36]. The marked increase in fluorescence intensities observed in glycated samples were 23.82%, 35.69%, 42.87%, and 68.22% compared to native HSA.
FTIR spectra of both native and glycated-HSA with 400mg/dLwere recorded as shown in Fig 2a and 2b respectively to demonstrate the alterations in thesecondary structure on the basis of frequency and shapes of amide I and amide II bands.Amide I showed a characteristic peak at 1656 cm -1 in native HSA that showed shifting to 1659 cm -1 in glycated-HSA that contributed in theabsorption peak of α-helix. Similarly, amide II band (N-H bend vibrations of peptide bonds) showed shifting at 1554 cm -1 in glycated-HSAcompared to 1564 cm -1 of native. A new peak at 1737 cm -1 corresponding to thealdehyde group of carbonyl compound was observed inglycated-HSA. Moreover, in native HSA this peak was absent.
FT-IR spectral results clearly demonstrated the changes in the secondary structure of HSA on post glucose modifications that contributed to band intensity and position displacement. Previous literature showed the presence of variable vibrational peptide moieties in amide bands of proteins [36]. Amide I and II of protein primarily contribute as two major signature bonds in theinfra-red region. Amide I band intensity corresponds to α-helix associated C = O vibrational stretching, while amide II relates with N-H and C-N [29]. The results of the present study also favoured the findings of previous literature on glycation induced alterations in HSA secondary structures [36].
Liquid chromatography mass spectroscopic analysis
Carboxymethyl-lysine (CML) a, known gold standard of advanced glycation end product (AGEs) was used to confer the formation of AGEs in glycated-HSA samples with the LCMS coupled with reverse phase UPLC system. Fig 3a-3f shows themass spectroscopic profile of standard CML, acid hydrolyzed native HSA (unmodified) along with acid hydrolyzed glycated-HSA (modified with D-glucose). As shown in Fig 3a-3f showed an m/z value of 279.1 in all glycated samples matching with the standard CML. No such species were observed in hydrolyzed native HSA mass spectra.
Haematological and biochemical analysis
Hematologic values along with serum biochemical parameters were shown in Tables 1 and 2 respectively.
Rectal temperature measurement
Rectal temperature of rabbits immunized with native HSA (unmodified) and glycated-HSA (modified with D-glucose 400 mg/dL) were recorded (Table 1) and showed arise in temperature in rabbits immunized with glycated-HSA compared to native. The immunogenicity and inflammatory response of AGEs have been studied extensively in the present study. The inflammatory response is mediated by receptor for AGE (RAGE), a prime molecule in the activation and initiation of differentiation of monocytes on the cell surface upon binding with AGEs that mediate the release of pro-inflammatory cytokines along with chemokines. Macrophages are present in varied tissues that upon activation with pathogen recognition or proinflammatory molecules represent with extreme phagocytic mechanisms. Literature has supported that macrophage expresses RAGE receptors similar to circulating monocytes [37]. The immunization of rabbits with in-vitro formed AGEs triggers the pro-inflammatory immune responses mediated by expression of immune cells (WBCs, Eosinophils, Basophils, and Neutrophils) as shown clearly from the results of the present study (Table 1). A recent literature favors the results of this study by demonstrating the role of RAGE in diabetes mellitus and the immune response [38]. Ann Marie Schmidtproposed "two-hit" model for vascular perturbation for RAGE upon binding with its ligands [39]. The "first hit" devotes to enhanced RAGE expression in the presence of its associated ligands,whilst "the second hit" occurs in the presence of diverse stress actions along with external factors that exacerbatepro-inflammatory responses. The Inflammatory potential of injected immunogen is not perturbing the serum biochemistry of animals, thereby showing no significant changes ( Table 2).
Serum direct binding and inhibition ELISA
Native HSA (antigen) immunized rabbits showed moderately antibody immune response with a classical antibody titre of <1:6400 as demonstrated by serum direct binding ELISA (Fig 4a). On the other hand,glycated-HSA was proved to be a potent immunogen with potential to provoke antibodies generation with ahightitre of >1:12,800 (Fig 4b). Under identical conditions The data are represented as mean±S.D and p value <0.05 considered to be significant shown by *. pre-immune serum of animals immunized with both native and glucose modified antigen showed negligible binding titre. The specificity of induced antibody titre in the serum of immunized rabbits was evaluated by competition inhibition ELISA. Immunized rabbits with glycated-HSA (modified with 400 mg/dL D-glucose) showeda classical inhibition of 84.65% with an antigen concentration of 20μg/mL (Fig 5a). Induced antibodies raised against native-HSA (immunogen) showed a maximum inhibition of 66.6% at same immunogen concentration (Fig 5b). Under identical Auto-antibodies against glycated adduct in diabetes mellitus conditions pre-immune serum of animals immunized with both native and glucose modified antigen showed negligible binding titre.
In the present study, immunogenicity of native and glycated-HSA was examined by inducing antibodies in female rabbits. A rabbit immunized with glucose modified-HSA possesses high serum antibody titer in direct binding ELISA compared to native. The results of thepresent study demonstrate that high antibodies titer in rabbits immunized with glycated-HSA represents structural impairments on HSA structure, leading to the generation of neoepitopes, rendering them with more immunogenic potential. Similarly, high percent inhibition was observed in sera of rabbits immunized with glucose modified antigen. Prolonged Auto-antibodies against glycated adduct in diabetes mellitus incubation of HSA with D-glucose exhibits more gluco-oxidation and formed neo-epitopes due to its folding and unfolding of theglobular structure. Antigenicity of affinity purified antiglycated-HSA IgG repeatedly states that purified IgG preferentially recognizes the neoepitopes formed due to glucose-induced modifications thereby showing maximum percent inhibition. Native HSA upon recognition with affinity purified IgG shows less percentinhibition. A previous interpretation was publishedthat demonstrated the formation of autoantibodies against gluco-oxidatively modified HSA [39]. The results of the present study are in full agreement with earlier reported results of ageneration of auto-antibodies against glycated-HSA at higher concentration of glucose [40].
Direct binding and inhibition ELISA of rabbit isolated IgG
IgG purified from the serum of pre-immune and immune rabbits were subjected to direct binding and competitive inhibition ELISA on microtitre plates coated with native and glycated-HSA. Direct binding ELISA of purified IgG showed strong binding with their respective immunogens (Fig 6a and 6b). The saturation of native and glycated-HSA was obtained at 40 and 60 μg/mL of IgG concentration respectively. However, pre-immune IgG showed negligible binding under identical conditions. The specificity of epitopes present at native and glycated-HSA towards their respective antibody was characterized by inhibition immunoassay. A value of 91.23% inhibition was achieved upon binding with glucose modified albumin (Fig 7a). Inhibition of 65.23% in antibody binding at 20μg/mL was achieved with native HSA (Fig 7b).
Serum direct binding ELISA in patients with diabetes mellitus
Auto-antibodies against glycated-HSA in diabetic sera were detected by ELISA on microtitre plate coated with native and glycated-HSA (modified with 400 mg/dL glucose). The bindingtitre of T2DM, T1DM, GDM and T2DM+CKD diabetic sera with native and glycated-HSA havebeen shown in Fig 8. Auto-antibodies in diabetic subjects showed more binding affinity with glycated-HSA (p<0.05) compared to native HSA. Sera of healthy subjects included as acontrol did not show significant binding with the coated antigens (10μg/mL).In thecontext of theimmunogenicbehaviour of AGEs, the present study was aimed to suggest the antibodies generation against AGEs formed at even low glucose concentration (400 mg/dL) that may be reported in diabetic subjects with complications. The in-vivo generation of auto-antibodies against glycated-HSA can be explainedregardingprolonged poor glycemic control that may induce the structural modifications in HSA generating neo-epitopes against which antibodies are raised in serum. In previous studies association between glycated-HSA and immunoglobulin heavy chain, constant regions havebeen described several times [41][42][43]. An elevated level of auto-antibodies in T1DM subjects due to increased content of fructosamine was found compared to T2DM patients [44]. A recent study focused on the role of AGEs in diabetes-associated complications [45]. Sakai et al. studied the production of auto-antibodies in subjects with nephropathy against glycated-HSA [46]. The present work showed the generation of auto-antibodies in diabetic subjects with type 2, type 1, gestational diabetes and type diabetes subjects with chronic kidney disease and found increased binding of antibodies against glycated-HSA coated wells compared to native HSA due to neo-epitopes formed as a resultant of structural modifications upon glycation. Similar results havebeen shown with ananimal model in thepresent study. Normal human sera showed negligible binding with anantigen.
Spectroscopy of antigen-antibody complexes
The UV-absorption profile of isolated IgG and immune complexes (i.e., native HSA-IgG and glycated-HSA-IgG) were recorded in a wavelength range of 200-400 nm as shown in Fig 9a. The Decrease in absorption was reported in immune complexes as compared to UV spectra of IgG alone. Intrinsic fluorescence of immune complexes along with isolated IgG alone was recorded as shown in Fig 9b, demonstrated that immune complexes show quenching in fluorescence intensity compared to thefluorescence intensity of isolated IgG alone. The reason behind the maximum absorption of IgG alone is the availability of all bonding and aromatic amino acids constituting epitopes showing absorbance in UV region. Immune complex with native HSA-IgG showed less absorbance compared to IgG alone due to structural changes and engagement of epitopic residues and bonding electrons in binding. Immune complex formed between glycated-HSA-IgG showed maximum hypochromicity due to extensive structural modifications that lead to thegeneration of neo-epitopes, which upon bindingwith antigen decreases UV absorption mediated by stearic hindrance.
Fluorescence of immune complex also clearly demonstrates the formation of neo-epitopes in an immune complex that exhibits intrinsic quenching of tyrosine and tryptophan compared to IgG alone and native immune complex. Neo-epitopes in glycated-HSA-immune complex showed stearic hindrance and change in microenvironments of these aromatic amino acids present in the vicinity of epitopes, contribute in this quenching and demonstrate the pattern as n = 50). The plate was coated with the respective antigens (10μg/mL). A p value <0.05 considered to be significant. ns = non-significant IgG alone>HSA-IgG>HSA glucose modified-IgG (Fig 9b). The glucose concentration used in this experiment is low and therefore, no correction of inner filter effect inner filter effect in fluorescence spectra measurement. A work performed on molten globule states of cytochrome C showed that low concentration of N-alkyl sulphates alters its electrochemical behaviour [47]. The same property has also been studies in HSA upon non enzymatic glycation [23]. Another study done by Ahmadi et al. In 2014, uses fluorescence, CD and UV spectroscopic techniques to demonstrates the complex formation between β-lactoglobulin and retinol at interplay of hydrophobic interactions [48]. Previous study uses spectroscopic approach to demonstrate the Auto-antibodies against glycated adduct in diabetes mellitus interaction between human serum albumin and carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) [49]. In another study done on HSA interaction, fluorescence spectroscopy and zeta potential was used to decipher complex formation [50].
X-ray diffraction of immune complex to determine epitopes X-ray angular diffraction pattern of affinity purified IgG along with immune complexes (i.e. native HSA-IgG and glycated-HSA-IgG) were recorded with flat monochromator on a slide containing afilm of the same . Fig 10a-10c clearly demonstrates the alteration patterns in the immune complexes upon compared to isolated IgG alone. Fig 10a shows the Braggs peaks of affinity purified IgG film with an angular value of 9.97E+00. Fig 10b and 10c show the distortion in the structure of the isolated IgG due to binding of epitopes with the native and glycated-HSA antigen thereby showing Braggs peak shifting in the diffractogram with a value of 1.93E+00 and 1.35E+01 respectively.Several published literature revealed the structure of HSA by small-angle X-ray scattering [51], quasi-elastic light scattering [52], hydrodynamic technique [53], neutron scattering [54] and 1-H NMR [55] but surprisingly X-ray diffraction pattern of the immune complex formed between affinity purified IgG with their respective antigens has not yet solved. Here, thefirst time we describe the x-ray diffraction pattern of affinity purified IgG along with immune complex to investigate the formation of the new epitopes generated as a resultant ofglucose modifications on HSA. The thin film diffractogram of IgG alone showed the Braggs angular diffraction of 9.97E+00 while theimmune complex with native and glucose modified antigen showed angular diffraction of 1.93E+00 and 1.35E+01 respectively, therebyconferring the structural alterations of these bio-molecules rendering generation of neo-epitopes due to glycation (Fig 9a-9c). The attemptto identify and characterize the immune complex with these novel techniques to prove the formation of glycation induced neo-epitopeshas been implicated well and furthermore extensive studies are required in this approach.
Conclusion
Our study revealed extensive binding of circulating auto-antibodies with D-glycated-HSA in comparison with its native form indicating the fact that glucose-induced structural modifications in HSA molecule represent the structural compactness and generation of neo-epitopes on the protein. The neo-epitopes render the HSA antigenic potential, due to the distortion of HSA structure upon glycation that formed auto-antibodies against self-protein. The direct binding ELISA and inhibition ELISA also validate the formation of neo-epitopes. Furthermore, the immune complex formed between induced antibodies and protein complex thereby represents the formation of neo-epitopes formed that havebeen further revealed with spectroscopic and X-ray diffraction pattern.
Limitation of the study
The obvious limitation of the present study is the range of glucose selected for the modifications. Due to near similar range ofD-glucose concentrations used in this study (i.e 100 mg/dL to 400 mg/dL), the experiments were performed with only native (unmodified) and glycated-HSA (modified with 400 mg/dL glucose). | v3-fos-license |
2019-03-31T13:02:57.949Z | 2019-03-28T00:00:00.000 | 88481372 | {
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} | pes2o/s2orc | Efficient Conversion of Cane Molasses Towards High-Purity Isomaltulose and Cellular Lipid Using an Engineered Yarrowia lipolytica Strain in Fed-Batch Fermentation
Cane molasses is one of the main by-products of sugar refineries, which is rich in sucrose. In this work, low-cost cane molasses was introduced as an alternative substrate for isomaltulose production. Using the engineered Yarrowia lipolytica, the isomaltulose production reached the highest (102.6 g L−1) at flask level with pretreated cane molasses of 350 g L−1 and corn steep liquor of 1.0 g L−1. During fed-batch fermentation, the maximal isomaltulose concentration (161.2 g L−1) was achieved with 0.96 g g−1 yield within 80 h. Simultaneously, monosaccharides were completely depleted, harvesting the high isomaltulose purity (97.4%) and high lipid level (12.2 g L−1). Additionally, the lipids comprised of 94.29% C16 and C18 fatty acids, were proved suitable for biodiesel production. Therefore, the bioprocess employed using cane molasses in this study was low-cost and eco-friendly for high-purity isomaltulose production, coupling with valuable lipids.
Introduction
Cane molasses is one of the main by-products of sugar refineries, which contains saccharides (primarily sucrose, glucose and fructose) and a small amount of nitrogenous compounds, vitamins, and trace metal elements as well as colloids [1,2]. Sugarcane grows worldwide and mainly distributes in Brazil, India, and China. The molasses production in China is around 400 million tons per year, yet, large volumes of waste molasses are simply discharged, contributing to severe environmental pollution [3]. Cane molasses can be used as an available source of quick energy for animal feed or feed supplement [4]. However, livestock frequently suffers from some nervous symptoms and blindness caused by molasses toxicity [5]. Given that available ingredients enrich in cane molasses, nowadays, it is increasingly utilized as an alternative feedstock for microbial fermentation after a pretreatment or as the raw material. Different high value-added metabolites have been harvested via microbial fermentation from molasses, such as astaxanthin [6], 5-hydroxymethylfurfural [7], ethanol [8,9], organic acids [3,[10][11][12], and enzymes [13].
As a functional sweetener from microbial fermentation, isomaltulose has attracted extensive attention. Isomaltulose (or palatinose) is a structural isomer of sucrose, sharing similar sweet sense and fine taste with sucrose. It has been approved that a safer sucrose substitute with some advantages, including higher stability, lower digestibility, lower glycemic index and more tooth-friendly [14]. Isomaltulose can be converted from sucrose by sucrose isomerase (SIase) without any cofactors due to the less free-energy of the process [15]. Several SIase-producing microbes were adopted for isomaltulose production, such as Pantoea dispersa [16], Erwinia spp. [17], Enterobacter spp. [18], and Serratia plymuthica [19]. However, they have not been considered to synthesize isomaltulose commercially for lack of genetic background in food-grade level [2,20].
Recently, heterologous expression of SIases in non-pathogenic or food-grade strains, such as Saccharomyces cerevisiae (S. cerevisiae), Yarrowia lipolytica (Y. lipolytica), Bacillus subtili (B. subtili), and Lactococcus lactis (L. lactis), has been proved effective for isomaltulose production [2,18,21,22]. Noteworthily, the recombinant Y. lipolytica expressing SIase gene has exhibited a prominent isomaltulose yield advantage over other hosts, which meets the requirement of isomaltulose commercialization [22,23]. Since sucrose substrate takes a substantial part of the total production cost, reusing low-cost materials rich in sucrose can be a feasible way to reduce the cost.
In traditional cane molasses fermentations using sucrose as the carbon source, sucrose was hydrolyzed into monosaccharides and utilized by microorganisms. However, the strain used in this study can just utilize monosaccharides, leaving sucrose not hydrolyzed for no sucrase generating. The high sucrose content in cane molasses makes it a promising substrate for isomaltulose production. On the other hand, the synthesis of isomaltulose from cane molasses can greatly improve the value of cane molasses. In this study, pretreated cane molasses (PCM) was used as the only carbon source, and corn steep liquor (CSL) was selected as substrate to replace yeast extract for the recombinant Y. lipolytica S47, which is capable of expressing SIase and transforming monosaccharides to lipids as shown. To further develop an economical fermentation and recycling efficiently by-products using strain S47, fed-batch fermentation was also conducted.
Isomaltulose Production Using PCM as Sole Carbon Source
Sucrose in cane molasses can be hydrolyzed into monosaccharides by microbial enzymes, which is believed to be suitable for microbial growth and metabolite production [8,13]. In this study, to develop a cost-effective fermentation, PCM was used for cells growth and isomaltose synthesis. As shown in Table 1, isomaltulose production increased as the activity of SIase secreted from strain S47 improved, based on the increase of PCM concentration. A maximal isomaltulose concentration (96.7 g L −1 ) was obtained with the SIase activity of 3.6 U mL −1 at 350 g L −1 PCM, and the yield achieved 0.96 (g g −1 ). Although the SIase activity (3.6 U mL −1 ) was limited, which is lower than 5.2, 7.4 and 49.3 U mL −1 [2,23,24], this yield is identical to the same strain using sucrose as a substrate [23]. The results proved cane molasses as a suitable substrate for isomaltulose synthesis. In addition, as strain S47 cells grew to the peak, intracellular lipids continuously accumulated and reached 5.3 g L −1 , which accounted for 43.1% (w/w) of cell dry weight, respectively (Table 1). When PCM concentration exceeded 350 g L −1 , isomaltulose and lipid productions both declined significantly, so did isomaltulose yield and others, except for residual sugar content (Table 1). This might be the result of cell dehydration and growth inhibition triggered by high PCM concentration, leading to suppressed expression of SIase and several crucial lipid-synthesizing enzymes, such as delta-9 stearoyl-CoA desaturase and acetyl-CoA carboxylase [2,25]. Generally, all the components in cane molasses can be consumed by the yeast. The flow of sucrose was directed into SIase-catalyzing isomaltulose synthesis, the byproducts of the SIase-catalyzing reaction were utilized by the yeast; monosaccharides in cane molasses were consumed for cell growth and lipid production ( Figure 1). However, the residual sugar concentration (8.6 g L −1 ) presented might infer that yeast extract is not desirable nitrogen source in the medium. Therefore, an alternative nitrogen source will be needed to solve the residual sugar.
Effect of CSL on Enhancement of Isomaltulose and Lipid Production
CSL generally contains abundant nutritional ingredients. It has been evaluated to be favorable for microbial growth and synthesis of natural products [26,27]. To further facilitate the conversion of PCM effectively to isomaltulose and lipid production, CSL was utilized as organic nitrogen to avoid the expensive yeast extract. Cell growth and fermentation at different concentrations of CSL media are shown in Table 2. It showed that most sucrose in PCM was converted to isomaltulose (102.6 g L −1 ) with 0.96 g g −1 yield, followed with enhanced SIase activity by 20.5% and biomass by 11.8% at the optimal CSL concentration (1.0 g L −1 ) compared with those in control (Table 2). Meanwhile, a distinct consumption of residual sugar was reduced by 73.3%. As a result, sugar reduction contributed to improving isomaltulose production by 5.5%, and lipids (7.2 g L −1 ) by 35.8% with 44.7% (w/w) content ( Table 2). Enhancements of CSL for isomaltulose and lipid co-production indicated that CSL is more suitable for strain S47 growth to produce target products than yeast extract, and it could act as an economic alternative for yeast extract.
To date, CSL has been also employed to produce diverse metabolites [26,28,29]. The result in table 2 showed that CSL played important roles in enhancing biomass generation and isomaltulose production due to the high secreted SIase activity, which is consistent with their positive changes in CSL-added media [2,27]. Nitrogen sources affect cell growth and metabolites formation during microbial fermentation. The components of amino acids, vitamins and trace elements in CSL may help boost initial biomass formation, while amino acids and/or biotin could also start releases of related enzymes for synthesizing metabolites [29]. The reports would probably account for improvements of biomass, SIase activity and isomaltulose production in this study. Besides, during CSL fermentation, intermediates (e.g., citric acid and poly malic acid) required for lipid accumulation have been enhanced in previous studies resulting from overexpression of crucial enzymes, i.e., citric acid synthetase (CS), malic enzymes (ME) and pyruvate carboxylase (PYC) [26]. We deduce that these enzymes (CS, ME and PYC) would perform at higher levels and synthesize more citric acid and poly malic acid in this study, further facilitating intracellular lipid accumulation. Moreover, significant sugar consumption and transformation during CSL fermentation suggest monosaccharides might flow mainly to lipid accumulation and cell growth, and a small share for isomaltulose production.
Fed-Batch Fermentation for Isomaltulose and Lipid
High osmotic pressure strongly affects product biosynthesis of Y. lipolytica cells [30]. To reduce high osmotic pressure in the optimized medium and to improve isomaltulose production, fed-batch fermentation was conducted using a two-stage bioprocess in a 10-L bioreactor. Figure 2 shows that cell growth, SIase activity and isomaltulose production were all detected to increase slowly when the process was close to 32 h in the first stage. Remarkably, during the second stage after 200 g L −1 PCM supplemented, cell growth and isomaltulose concentration were both switched to increase and achieved the maximal values of 21.3 g L −1 and 161.2 g L −1 at 80 h accompanied by higher SIase activity (5.7 U mL −1 ), respectively ( Figure 2). Generally, the higher yield and higher production are the key factors of saving cost. The yield derived from PCM and CSL in the bioreactor maintained 0.96 g g −1 , which reaches the same level from engineered Y. lipolytica strains both expressing SIase using sucrose as the substrate [23,24]. The isomaltulose concentration (161.2 g L −1 ) was also evidently higher compared with those (36, <4, <33.5 g L −1 ) from strains L. lactis, S. cerevisiae and S. plymuthica using sucrose or molasses as the substrate, respectively (Table 3). It indicates that sucrose in PCM was almost converted completely at the end of the fermentation. In fact, Li et al. (2013) revealed that high SIase activity (48 U mL −1 ) was achieved by a recombinant Escherichia coli expressing SIase using cane molasses and CSL substrates, while it lacks isomaltulose concentration and yield [31]. "-" represented that no other products produced or the products did not get detected. In addition, lipids accumulated to 12.2 g L −1 and the content reached 57.3% (w/w) during the two-stage fermentation (Figure 2), which is enhanced markedly than 7.2 g L −1 production at flask level above and that (8.1 g L −1 ) produced by the same strain from sucrose [23]. The lipid content (57.3%) obtained is much higher than those from Rhodotorula kratochvilovae (25%, w/w) and engineered Ashbya gossypii (38.25%, w/w) using cane molasses, respectively [32,33]. Strain S47 can be regarded as an oleaginous yeast due to the lipid content over 20% [26]. It also exhibits a significant advantage over liquid contents (23.4-49.6%) of other specially engineered Y. lipolytica strains and Aureobasidium melanogenum, and even co-cultured strains using other substrates [25,26,34,35]. Consequently, lipid accumulation coupled with isomaltulose production by strain S47 was demonstrated to be economical and valuable.
It is noteworthy that original monosaccharides and bits of them produced by SIase catalysis from strain S47 were depleted and only small amounts of trehalulose were detected, leading to produce a high isomaltulose purity of 97.4%. This purity is comparable with that (97.8%) by strain S47 using sucrose [23]. In fact, isomaltulose is difficult to separate completely from a fermented mixture including trehalulose, glucose, fructose and residual sucrose [22,27]. However, high-value lipid accumulation in this work was testified to benefit isomaltulose purity due to the consumption of undesirable by-products. The results suggest that high-purity isomaltulose can be achieved successfully via coupling with considerable lipid accumulation by the food-grade strain Y. lipolytica S47 using PCM and CSL substrates in fed-batch fermentation, thus, avoiding the effect of residual sugar on isomaltulose crystal.
Fatty Acids Composition of Intracellular Lipids and Biodiesel Production
After transmethylated fatty acids were detected, the main fatty acids of intracellular lipids were C 14:0 , C 16:0 , C 16:1 , C 18:0 , C 18:1 and C 18:2 , especially C 18:1 (45.14%) ( Table 4). This is similar to those produced by Rhodotorula glutinis TR29 cultivated in molasses medium, with producing 63.5% C 18:1 fatty acid [36]. Moreover, many microbes such as A. melanogenum, Rhodosporidium toruloides, and other engineered Y. lipolytica strains are capable of generating fatty acids of intracellular lipids that primarily contain C 16 -C 18 and the largest share of C 18:1 using other substances [25,26]. Indeed, 94.29% of fatty acids of lipids from strain S47 cells in this study were C 16 and C 18 . It reveals that lipids accumulated in Y. lipolytica S47 cells from cane molasses are suitable for biodiesel production that requires C 16 and C 18 fatty acids [36,37]. Biodiesel was prepared from the transesterification of obtained intracellular lipids. In this work, the conversion rate of lipids into biodiesel reached near 83.2% (data not shown), which is comparable with others (85-86.7%) [25,38], indicating that intracellular lipids produced from Y. lipolytica S47 cells is a promising feedstock for biodiesel production.
Strain and Cane Molasses Fermentation
The recombinant Y. lipolytica S47 was previously constructed and cultivated as seed culture in YPD medium (yeast extract 1%, peptone 2%, and dextrose 2%) [23]. The promoter for expression of SIase gene was constitutive, thus, recombinant SIase can be secreted extracellularly without any induction [23]. Furthermore, strain S47 was derived from the typical oleaginous yeast Y. lipolytica ACA-DC 50109, which can transform extracellular sugars into cellular lipid [23]. Cane molasses was obtained from a sugar refinery in Guangxi and it was pretreated as described in the methods [24]. The composition of pretreated cane molasses (PCM) consisted of sucrose (30.55%, w/v), glucose (5.71%) and fructose (7.78%). Glucose potassium phosphate buffer (GPPB) medium with added different concentrations of PCM (200, 250, 300, 350, 400 g L −1 ) instead of glucose was optimized to improve isomaltulose production by strain S47 at 30 • C for 96 h, pH was adjusted to 6.0 [23]. By the action of phosphate buffer in the medium, pH can be stable.
CSL Optimization for Enhancement of Isomaltulose and Lipid Co-Production
Corn steep liquor (CSL) was derived from a local corn processing facility, which was used as a substitute for yeast extract in the fermentation medium. The fermentation was performed at different concentrations of CSL (0.5, 1.0, 1.5 g L −1 ) to enhance isomaltulose production and purity, together with lipid synthesis compared to those from strain S47 fermentation using 0.5 g L −1 yeast extract. pH was adjusted to 6.0.
Fed-Batch Fermentation in a 10-L Fermentor
Fermentation was scaled up in a 10-L Biostat fermentor (B. Braun Biotech International, Melsungen, Germany) with the obtained medium (6.0 L), which contained optimized PCM (350 g L −1 ) and CSL (1.0 g L −1 ). After inoculation (5.0%, v/v), the strain fermented to secrete SIase for isomaltulose synthesis and express some enzymes like ATP citrate lyase to accumulate intracellular lipids under the conditions of aeration rate (50 L min −1 ), rotate speed (300 rpm), and temperature (30 • C) [23]. Fed-batch fermentation was carried out by adding 200 g L −1 PCM at the initial 32 h of fermentation. During bioprocess, samples (50 mL) were taken at intervals of 7 h to detect isomaltulose and lipid contents, SIase activity and monosaccharides as well as biomass. pH was controlled at 6.0.
Enzyme Assay and Determination for Isomaltulose and Lipid Contents, Residual Sugar
The fermented broth from strain S47 was centrifuged at 5000× g to obtain the supernatant, and SIase activity was measured according to previous methods [16,22]. Briefly, the supernatant was added to a sucrose solution (100 g L −1 ) that dissolved in phosphate buffer (50 mM and pH 6.0) in a ratio of 1:1; the mixture was then incubated at 30 • C for 10 min. The process was terminated through boiling for 10 min and centrifuged (10,000× g, 20 min) to eliminate denatured proteins. Finally, the new supernatant was filtered through 0.22 µm membrane and properly diluted for high-performance liquid chromatography (HPLC) analysis using Agilent 1200 system (Agilent Technologies, Palo Alto, CA, USA). One unit (U) of enzyme activity was calculated as the SIase amount responsible for the release of 1.0 µmol isomaltulose per min at 30 • C and pH 6.0 [23]. The fermented supernatant was boiled before mixing as the control.
The contents of isomaltulose, trehalulose, residual glucose and fructose in fermentation supernatant were also determined using HPLC after membrane (0.22 µm) filtration. Biomass and quantification of intracellular lipids were gravimetrically detected [25]. All tests above were performed in triplicate.
Determination of Lipid Composition and Preparation for Biodiesel
Lipid was extracted and fatty acid esters were determined using gas chromatography (GC) [26]. 1 µL of sample was injected in a 10:1 split mode at 275 • C, using helium as the carrier gas at a flow rate of 1 mL/min. The GC oven temperature was held at 150 1C for 1 min and ramped to 230 • C (rate: 15 • C /min, hold: 2 min) for a total run time of approximate 13.5 min. Different fatty acid methyl esters were identified and characterized using the authentic fatty acid methyl ester (FAME) standards. Biodiesel was prepared and detected using H 2 SO 4 solution (in methanol) through thermocatalytic treatment as described in previous research [25].
Statistical Analysis
The results obtained above were subjected to a one-way analysis of variance (ANOVA) using SPSS 22.0 software (SPSS Inc., Chicago, MI, USA), and presented as the mean ± standard deviation. Statistically significant differences between groups were showed as p < 0.05 (*) and p < 0.01 (**).
Conclusions
An efficient, economical strategy to produce isomaltulose from cane molasses was established. During the fed-batch fermentation, the maximal isomaltulose concentration achieved 161.2 g L −1 and yielded 0.96 g g −1 . Monosaccharides were completely consumed and transformed into intracellular lipids and biomass, which resulted in a high isomaltulose purity of 97.4% and 12.2 g L −1 lipids. The lipids produced from Y. lipolytica S47 cells have potential as a candidate for biodiesel production. | v3-fos-license |
2018-04-03T00:17:03.922Z | 2010-12-22T00:00:00.000 | 205305905 | {
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} | pes2o/s2orc | Activation Loop Phosphorylation of ERK3/ERK4 by Group I p21-activated Kinases (PAKs) Defines a Novel PAK-ERK3/4-MAPK-activated Protein Kinase 5 Signaling Pathway*
Classical mitogen-activated protein (MAP) kinases are activated by dual phosphorylation of the Thr-Xxx-Tyr motif in their activation loop, which is catalyzed by members of the MAP kinase kinase family. The atypical MAP kinases extracellular signal-regulated kinase 3 (ERK3) and ERK4 contain a single phospho-acceptor site in this segment and are not substrates of MAP kinase kinases. Previous studies have shown that ERK3 and ERK4 are phosphorylated on activation loop residue Ser-189/Ser-186, resulting in their catalytic activation. However, the identity of the protein kinase mediating this regulatory event has remained elusive. We have used an unbiased biochemical purification approach to isolate the kinase activity responsible for ERK3 Ser-189 phosphorylation. Here, we report the identification of group I p21-activated kinases (PAKs) as ERK3/ERK4 activation loop kinases. We show that group I PAKs phosphorylate ERK3 and ERK4 on Ser-189 and Ser-186, respectively, both in vitro and in vivo, and that expression of activated Rac1 augments this response. Reciprocally, silencing of PAK1/2/3 expression by RNA interference (RNAi) completely abolishes Rac1-induced Ser-189 phosphorylation of ERK3. Importantly, we demonstrate that PAK-mediated phosphorylation of ERK3/ERK4 results in their enzymatic activation and in downstream activation of MAP kinase-activated protein kinase 5 (MK5) in vivo. Our results reveal that group I PAKs act as upstream activators of ERK3 and ERK4 and unravel a novel PAK-ERK3/ERK4-MK5 signaling pathway.
ERK3 and ERK4 define a distinct subfamily of atypical MAP kinases that display structural and functional differ-ences with conventional MAP kinases (1). First, they possess a single phospho-acceptor site (Ser-Glu-Gly) in their activation loop instead of the classical dual phosphorylation Thr-Xxx-Tyr motif (2,3). Accordingly, they are not phosphorylated and activated by dual-specificity MAP kinase kinase family members (4,5). Second, ERK3 and ERK4 bear the sequence Ser-Pro-Arg instead of Ala-Pro-Glu in subdomain VIII of the kinase domain, being the only kinases in the human kinome to have an arginine at this position. Third, contrary to conventional MAP kinases like ERK1/ERK2, which are multifunctional Ser/Thr kinases capable of phosphorylating a vast array of substrates, ERK3 and ERK4 appear to have a very narrow substrate specificity (6). Their only known physiological substrate is the protein kinase MK5 5 (7)(8)(9)(10).
The regulation of ERK3 and ERK4 activity remains poorly understood. We and others have shown that the serine residue within the activation loop of ERK3/ERK4 (Ser-189/Ser-186) is phosphorylated in vivo (6,(11)(12)(13). Catalytically inactive forms of the kinases are similarly phosphorylated on this motif, indicating that activation loop phosphorylation is mediated by an upstream cellular kinase (12,13). Phosphorylation of Ser-189/Ser-186 leads to enzymatic activation of ERK3/ERK4 and is required for binding to and for cytoplasmic relocalization of the substrate MK5 (12,13). Recently, we have shown that phosphorylation of ERK3 in the C-terminal extension by CDK1 stabilizes the protein and leads to its accumulation in mitosis (14).
The lack of information on the stimuli and upstream regulatory events that control the activity of ERK3 and ERK4 has hampered the comprehensive study of these atypical MAP kinase signaling pathways. To get new insights into the regulation of ERK3/ERK4 activity, we have used a classical biochemical purification approach to isolate the kinase(s) responsible for ERK3 Ser-189 phosphorylation. Here, we identify group I PAKs as ERK3 and ERK4 activation loop kinases. We demonstrate that PAKs phosphorylate ERK3 and ERK4 in vitro and in vivo, leading to their catalytic activation and to downstream MK5 phosphorylation and activation.
Recombinant Protein Purification-Recombinant Hsp27 and His 6 -MK5 were produced as described (12). Other recombinant proteins were expressed in BL21 pLys(DE3) strain and purified as described (16). Protein purity and yield were evaluated by SDS-PAGE and Coomassie staining using BSA as standard.
Cell Culture and Transfections-HEK 293 cells were cultured in minimal essential medium supplemented with 10% fetal bovine serum plus antibiotics. Cells were transiently transfected by the calcium phosphate precipitation method.
Purification of ERK3 Ser-189 Kinase-Exponentially proliferating HEK 293 cells were lysed with a Dounce homogenizer in hypotonic buffer B (50 mM -glycerophosphate (pH 7.3), 1.5 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol (DTT), 100 M sodium orthovanadate, 1 M pepstatin A, 1 M leupeptin, 100 M PMSF). The lysate was clarified by centrifugation, and 50 mg of protein was fractionated on a Mono Q 10/100 GL column (GE Healthcare) using an AKTA Explorer FPLC system (GE Healthcare). Fractions were collected and assayed for ERK3 Ser-189 kinase activity as described below. Fractions 43-53 were pooled and further fractionated on a gel filtration Superdex 16/60 200 column (GE Healthcare). Superdex fraction 36 containing high Ser-189 kinase activity was then incubated with 1 g of His 6 -ERK3(1-365)KA-GST recombinant protein bound on glutathione-agarose beads for 2 h at 4°C. The beads were washed four times with buffer B, and bound proteins were further separated by SDS-PAGE. The gel was stained with Coomassie Blue, and seven bands were excised, reduced with DTT, and alkylated with iodoacetamide prior to trypsin digestion. Peptides were extracted three times with 90% acetonitrile/0.5 M urea. Combined extracts were dried and resuspended in 5% acetonitrile, 0.1% trifluoroacetic acid. Tryptic peptides were analyzed by nanoLC-MS/MS using an Eksigent nano2D system coupled to a LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific). Peptides were separated on a 150-m ϫ 10-cm C 18 analytical column using a gradient of 5-40% acetonitrile (in 0.2% formic acid) over 65 min. The mass spectrometer was operated in a data-dependent acquisition mode with a 1-s survey scan at 60,000 resolution, followed by three ion trap product ion spectra (MS/MS) of the most abundant precursors. The centroided data were merged into single peak-list files and searched with the Mascot search engine version 2.10 (Matrix Science) against the combined forward and reverse human IPI protein data base version 3.24 containing 133,842 protein sequences.
Immunoblotting, Immunoprecipitation, and Kinase Assays-Cell lysis, immunoprecipitation, and immunoblot analysis were performed as described (11,18,19). For immunoprecipitation experiments, 750 g of lysate proteins were incubated with the indicated antibodies for 2-4 h at 4°C. For the detection of endogenous ERK3 phosphorylation on Ser-189, HEK 293 cell lysate (5 mg of protein) was incubated for 4 h at 4°C with monoclonal anti-ERK3 antibody precoupled to protein A-agarose beads. Immunoprecipitated proteins were analyzed by immunoblotting with the indicated antibodies.
For in vitro kinase assay of Ser-189 phosphorylation, cell lysate (50 g of protein) or recombinant PAK2 (1 g) was incubated with 1 g of the indicated purified recombinant ERK3(1-365) protein in 50 l of buffer A (50 mM Tris-HCl (pH 7.5), 1.5 mM KCl, 25 mM MgCl 2 , 1 mM DTT, 200 M ATP) at 37°C for 45 min. The reaction was stopped with Laemmli buffer, and Ser-189 phosphorylation was analyzed by immunoblotting with a phospho-ERK3(Ser-189)-specific antibody. For analysis of FPLC fractions, 8% of each fraction was assayed in a final volume of 100 l of buffer A. For radioactive assays, 10 Ci of [␥-32 P]ATP was added to buffer A, and the reaction was analyzed by autoradiography with a FLA5000 PhosphorImager (Fuji).
For in vivo assay of ERK3/ERK4 activity, immunoprecipitated proteins were incubated with 0.5 g of recombinant His 6 -MK5 and Hsp27 in kinase assay buffer (25 mM Hepes (pH 7.5), 25 mM MgCl 2 , 1 mM DTT) with 50 M ATP and 20 Ci of [␥-32 P]ATP. Proteins were separated by SDS-PAGE, transferred to nitrocellulose membrane, and analyzed by autoradiography. Expression of transfected proteins was analyzed by immunoblotting. The same conditions were used to monitor MK5 activity, except that FLAG-MK5 protein was immunoprecipitated from cells, and no recombinant MK5 was added to the assay.
Mass Spectrometry Analysis of MK5 Phosphorylation-For the in vivo quantitative analysis of MK5 Thr-182 phosphorylation, FLAG-MK5 was immunoprecipitated from HEK 293 cell lysates using anti-FLAG M2 agarose beads (Sigma). After elution in 0.1 M glycine (pH 3.5), proteins were dialyzed in 8 M urea, 50 mM NH 4 HCO 3 , 0.5 mM TCEP using 10-kDa cut-off microcon devices (Millipore) as published (20). Prior to digestion, the urea concentration was diluted to 1.5 M in 50 mM NH 4 HCO 3 , 0.5 mM TCEP. Proteins were digested with sequence-grade trypsin (Promega) for 3 h and the eluted peptides dried in a SpeedVac. Peptides were analyzed on an Orbitrap mass spectrometer using similar settings as above. Peptide intensities were extracted from raw data, clustered with Mascot results, and validated using the in-house software ProteoProfile. Specifically, for the quantification of MK5 Thr-182 phosphopeptides, we detected four isoforms by MS/MS with peptide MOWSE scores of 57-114: double-and triple-charged IDQGDLMpTPQFTPYYVAPQVLEAQR (m/z 1480.705 and 987.4684, respectively); triple-charged form of the methionine-oxidized IDQGDLMpTPQFTPYYVAPQV-LEAQR (m/z 992.8018), and the low abundant, triple-charged missed cleavage form IDQGDLMpTPQFTPYYVAPQV-LEAQRR (m/z 1044.84). We extracted ion intensities of the four detected peptide isoforms containing the phosphorylated Thr-182 site from three independent replicates. The average intensities of each peptide isoform and the corresponding standard deviations were calculated. The summed peptide intensity of the MK5 control experiment (without ERK4) was set to 1, and the relative increase in MK5 phosphorylation was calculated from the ratio of peptide intensities over that of the control condition.
RESULTS
Purification of ERK3 Activation Loop Kinase-To purify the ERK3 activation loop kinase(s), we developed a robust in vitro assay to monitor Ser-189 kinase activity using recombinant catalytically inactive His 6 -ERK3(1-365)KA protein (produced in Escherichia coli) as substrate and a phospho-ERK3(Ser-189)-specific antibody for detection. This assay readily detects the phosphorylation of wild-type and kinase-dead ERK3, but not that of the S189A mutant from a HEK 293 cell lysate (Fig. 1A). The strategy used for the purification of ERK3 Ser-189 kinase activity is summarized in Fig. 1B. Extracts of proliferating HEK 293 cells were sequentially fractionated on anion exchange and gel filtration columns (Fig. 1, C and D). Then, fraction GF1 of the Superdex column was incubated with recombinant His 6 -ERK3(1-365)KA-GST immobilized on glutathione-agarose beads. After washing, bound proteins were further separated by SDS-PAGE (Fig. 1E). The gel was stained with Coomassie Blue and cut into seven bands that were subjected to in-gel digestion with trypsin and analyzed by LC-MS/MS. Among 142 proteins detected in the samples, two protein kinases were identified with a high Mascot score: PAK2 and PAK1 (Fig. 1F and supplemental Table S1). Three peptides shared by PAK1 and PAK3 were also identified from this analysis.
To determine whether PAK2 phosphorylates ERK3 in vivo, we co-transfected HEK 293 cells with ERK3, PAK2, and an activated form of Rac1 (Rac1CA). Co-transfection of PAK2 increased the phosphorylation of ERK3 on Ser-189 (Fig. 2E, lane 3). The phosphorylation signal was further increased when activated Rac1 was transfected at the same time (Fig. 2E, lane 4). It should be noted that cotransfection of ERK3 with Rac1CA is sufficient to increase the Ser-189 phosphorylation signal compared with ERK3 alone (Fig. 2, E, lane 5, and G). This is likely due to the activation of endogenous PAK by Rac1. Similar results were obtained with the other group I PAK activator Cdc42 (Fig. 2G).
MS analysis identified both PAK2 and PAK1, and possibly PAK3, as candidate ERK3 Ser-189 kinases (Fig. 1F). We therefore wished to determine whether PAK1 and PAK3 are also able to phosphorylate ERK3 on Ser-189 in intact cells. Both PAK2 and PAK3 markedly increased the activation loop phosphorylation of ERK3, whereas PAK1 had a weaker albeit measurable effect (Fig. 2F). We conclude from these results that group I PAKs display ERK3/ERK4 activation loop kinase activity.
Silencing of Group I PAKs Inhibits Rac1-Induced ERK3 Ser-189 Phosphorylation-To address directly the contribution of endogenous group I PAKs to the activation loop phosphorylation of ERK3, we silenced the expression of PAK1, PAK2, and PAK3 by RNAi using SMARTpool siRNAs targeting each individual kinase. Silencing of a single PAK isoform partially reduced Rac1-stimulated ERK3 Ser-189 phosphorylation, with PAK2 and PAK3 siRNAs showing greater effects (Fig. 3, A and B). Importantly, a combination of siRNAs to all three group I PAKs completely abolished the effect of Rac1 on both ectopic and endogenous ERK3 phosphorylation (Figs. 3 and 4). These results confirm that group I PAKs are bona fide activation loop kinases for ERK3 and mediate the stimulatory effect of the small GTPases Rac1/Cdc42 on ERK3 phosphorylation.
Group I PAKs Stimulate the Enzymatic Activity of ERK3 and ERK4-We wanted to verify whether PAK-induced phosphorylation of ERK3 or ERK4 leads to their enzymatic activation. HEK 293 cells were co-transfected with ERK3/ ERK4 and Rac1CA, in the absence or presence of PAK2 or a mixture of siRNAs targeting PAK1/PAK2/PAK3. Ectopi-cally expressed ERK3 or ERK4 was immunoprecipitated, and its phosphotransferase activity was measured in a coupled kinase assay using recombinant His 6 -MK5 and its substrate Hsp27 in the presence of [␥-32 P]ATP. Expression of activated Rac1 clearly induced the catalytic activation of ERK3 and ERK4 as demonstrated by the increased phosphorylation of Hsp27 (Fig. 5, A and B, lane 3). This activation was significantly attenuated in cells transfected with
PAKs Are Activation Loop Kinases for ERK3/ERK4
FEBRUARY 25, 2011 • VOLUME 286 • NUMBER 8 siRNAs to group I PAKs. These data indicate that PAKmediated phosphorylation of ERK3 and ERK4 leads to their catalytic activation in vivo.
Activation of ERK3/ERK4 by PAK3 Results in Downstream Activation of MK5 in Vivo-We next wished to determine whether the activation of ERK3/ERK4 by Rac1-PAK signaling translates into enzymatic activation of the downstream substrate MK5 in vivo. Because all of the antiphospho-MK5(Thr-182) antibodies tested were found unsatisfactory, we analyzed the extent of MK5 Thr-182 (activation loop) phosphorylation by quantitative MS. HEK 293 cells were transfected with FLAG-MK5 in the absence or presence of ERK4, activated Rac1, and PAK3. MK5 was immunoprecipitated with anti-FLAG M2 beads, eluted, digested with trypsin, and subjected to quantitative LC-MS/MS analysis. Activating Thr-182 phosphorylation of MK5 was increased by 26-fold following ERK4 overexpression and by 46-fold when ERK4 was co-expressed with activated Rac1 and PAK3 (Fig. 6A and supplemental Fig. S1). A comparison of the intensity of nonphosphorylated and phosphorylated peptide species around MK5 Thr-182 indicated that MK5 is phosphorylated at high stoi- To confirm that increased Thr-182 phosphorylation results in MK5 catalytic activation in vivo, the phosphotransferase activity of MK5 was measured by immune-complex kinase assay using recombinant Hsp27 as substrate. Expression of ERK3 or ERK4 augmented MK5 activity, and this activation was further potentiated by co-expression of Rac1CA and PAK3 (Fig. 6, B and C). These results confirm that activation of the Rac1-PAK-ERK3/4 signaling pathway leads to downstream activation of MK5 in cells.
DISCUSSION
A full understanding of the physiological functions of the atypical MAP kinases ERK3 and ERK4 will be greatly facilitated by the characterization of the upstream regulatory signals and pathways that control their activity. ERK3 and ERK4 are not components of classical MAP kinase modules, and their activator(s) has remained elusive so far. Of note, Cobb and colleagues have previously reported the partial characterization of a protein kinase immunologically distinct from MEK1 and MEK2 that phosphorylates ERK3 on Ser-189 (6). Here, we report the successful purifi-
PAKs Are Activation Loop Kinases for ERK3/ERK4
FEBRUARY 25, 2011 • VOLUME 286 • NUMBER 8 cation and identification of group I PAKs as ERK3 activation loop kinases. The PAK family is composed of six members divided in two subgroups of three members each based on their mechanism of regulation (21). All six members share a similar architecture with an N-terminal regulatory domain containing the Rac1/Cdc42 binding site and a C-terminal kinase domain. Group I PAKs include PAK1, PAK2, and PAK3, which are enzymatically activated by binding of Rac1 and Cdc42. PAKs belong to the STE (homologs of yeast Ste7, Ste11, Ste20 kinases) group of protein kinases, which also includes MAP kinase kinases.
We have used a three-step chromatographic procedure including an affinity step on ERK3(1-365)KA-agarose beads to isolate the ERK3 Ser-189 kinase. The affinity step was inspired by the observation that a candidate ERK3 Ser-189 kinase binds tightly to the catalytic domain of ERK3 (6). ERK3-binding proteins were further separated by SDS-PAGE and analyzed by LC-MS/MS, leading to the identification of PAK1 (and possibly PAK3) and PAK2. Overexpression and loss-of-function experiments confirmed that PAK1, PAK2, and PAK3 efficiently phosphorylate ectopic and endogenous ERK3 on Ser-189 and that the three protein kinases are responsible for all the Ser-189 kinase activity induced by activated Rac1 in cells. We conclude from these results that group I PAKs are bona fide ERK3 activation loop kinases. However, we cannot exclude the possibility that other kinases can phosphorylate ERK3 on Ser-189 under different cellular conditions. All ERK3 activation loop (from Leu-174 to Trp-216) amino acids but three are conserved in human ERK4 sequence. These residues are also highly conserved from zebrafish to human (12). In agreement with this observation, we found that group I PAKs similarly phosphorylate ERK4 on activation loop residue Ser-186, indicating a common mechanism of activation of ERK3 and ERK4 MAP kinases.
Rennefahrt et al. have used a degenerate peptide library to analyze the optimal phosphorylation sequence of group I and II PAKs (22). According to this work, the ERK3/ERK4 activation loop contains several favorable residues for PAK1 and PAK2 phosphorylation (Fig. 7). Notably, these FIGURE 5. PAKs stimulate the phosphotransferase activity of ERK3 and ERK4. HEK 293 cells were transfected with the indicated constructs in combination or not with SMARTpool siRNAs to PAK1/2/3. After 36 h, the cells were lysed, and Myc 6 -ERK3 WT or SA (A) or FLAG-ERK4 (B) was immunoprecipitated with anti-Myc or anti FLAG-antibody, respectively. Phosphotransferase activity was measured in a coupled kinase assay by incubating immunoprecipitated ERK3 or ERK4 with recombinant His 6 -MK5 and Hsp27 proteins in the presence of [␥-32 P]ATP. The reaction products were analyzed by SDS-PAGE and autoradiography (upper panel). Ponceau staining was used to control for equal His 6 -MK5 and Hsp27 protein loading. The activation loop phosphorylation of ERK3/ ERK4 and the expression of ERK3/ERK4, Rac1CA, and PAKs were analyzed by immunoblotting of cell lysates. The bar graph represents the mean Ϯ S.E. (error bars) of three independent experiments. kinases clearly favor serine residues over threonine as substrates (22). It is noteworthy that the ERK3/ERK4 sequence appears to fit more closely the PAK2 consensus motif than PAK1, consistent with our observation that PAK2 is apparently a better activation loop kinase than PAK1.
Importantly, we have demonstrated that PAK-mediated phosphorylation of ERK3/ERK4 leads to their enzymatic activation and to downstream activation of MK5 in vivo. Group I PAKs have been implicated in many cellular processes, including transcription, apoptosis, cell proliferation, and cell motility (23)(24)(25)(26). Specifically, multiple studies have documented the important role of PAKs in the regulation of actin dynamics and cytoskeletal remodeling (27,28). In the past years, MK5 has also emerged as a regulator of F-actin polymerization and cell migration (29 -31). In this study, we uncover a hitherto unrecognized link between Rac/Cdc42-PAK signaling and ERK3/4-MK5 activation, potentially explaining in part the common effect of group I PAKs and MK5 on the actin cytoskeleton. Future studies will investigate how this new signaling branch of PAK signaling contributes to physiological responses such as cell motility, apoptosis, and senescence that can eventually impact cancer progression. | v3-fos-license |
2017-10-18T11:42:14.330Z | 2001-11-25T00:00:00.000 | 42552672 | {
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} | pes2o/s2orc | Competing pathways in the reaction of the pesticide Fenitrothion [ O,O -dimethyl O -(3-methyl-4-nitrophenyl) phosphorothioate] with some nitrogen nucleophiles in aqueous solution†
The reaction of the title compound, 1 , with the nitrogen-based nucleophiles n-butylamine, ethanolamine
Introduction
2][3][4] The reaction of the broad spectrum insecticide fenitrothion, 1, with a variety of oxygen-based nucleophiles has engaged our recent attention. 3,4In aqueous solution, 1 reacts with a variety of oxygen nucleophiles, to include some alpha-nucleophiles and a series of structurally related phenoxides as well as the highly basic nucleophiles OH -and CF 3 CH 2 O -, exclusively at the P center. 3In other words, products of nucleophilic attack at the alkyl and aromatic carbon centers were not detected.Analysis of the kinetic data showed that the thiophosphoryl group transfer proceeds by a concerted mechanism in which bond formation with the incoming nucleophile is slightly ahead of leaving group departure.On the other hand, ethanolysis of 1 by alkali metal alkoxides (M + EtO -, M + = Li + , K + , and Na + ) in anhydrous ethanol proceeds by nucleophilic attack at both P and aliphatic carbon centers; a minor SNAr route was also detected. 4Hence changing the nucleophile/solvent system has clearly introduced additional reaction pathways for 1.An earlier report on the hydrolysis of 1 reveals that CH 3 -O bond fission occurs at low pH while P-OAr fission is the exclusive reaction at high pH. 5To date we have found no report of the reaction of 1. with nitrogen nucleophiles, which is the subject of the present paper.Such a study would be important not only from the point of view of structurereactivity considerations, but also in contributing to the design of possible decontamination strategies for ameliorating the effects of pesticide overload in general.A recent study of phosphoryl transfer from ATP to amine nucleophiles has sought to provide a basis for understanding the analogous enzymatic reactions. 6he pathways available to 1 in its reaction with nucleophiles are shown in Scheme 1.In this paper, we report that 1 reacts with some amine nucleophiles to give mainly products of substitution at P and aromatic carbon centers.An attempt is made to explain site preferences in the reaction of 1 with oxygen and nitrogen nucleophiles by invoking the Hard Soft Acid Base (HSAB) theory and transition state stabilization by hydrogen bonding.The relative nucleophilicities of both nucleophile types towards aromatic carbon and the stability of σcomplex intermediates formed by them in SNAr reactions 7 are additional factors which should also be considered in discussing site preferences by oxygen and nitrogen nucleophiles in their reactions with 1 and similar substrates.
Results and Discussion
Reaction pathways 3-Methyl-4-nitrophenoxide, the product of nucleophilic attack at the P center of 1, absorbs at 398 nm.Quantitative thiophosphoryl group transfer should yield experimental absorbances at the end of the reaction which are identical to the calculated values for this nucleofuge, within experimental error.A definitive check for reactions monitored by conventional uv-visible spectrophotometry would be the measurement of absorbances at the end of the reaction at both high and low pH.Such experiments with n-butylamine, ethanolamine, and glycyl ethyl ester revealed the presence of substituted anilines, in addition to 3-methyl-4-nitrophenoxide. The former are products of attack at the aromatic carbon center of 1.
Product analysis of the reaction of n-butylamine with 1 by GC-MS showed unambiguously the presence of N-butyl-3-methyl-4-nitrophenylaniline, a product of nucleophilic attack at the aromatic carbon center.Although the same chromatogram suggested the presence of minor quantities of products of S N 2(C) attack, these were not taken into consideration in the calculations of product distributions given below.For reactions involving ethanolamine and glycyl ethyl ester as nucleophiles, the presence of products of attack at the aromatic carbon center was inferred from spectrophotometric measurements.Values of the pK a of the amines employed in this study are listed in Table 1.The approximate % SNAr product in each case was calculated according to equation (i), where A ∞ and A obs are the theoretical and observed infinity absorbances for the substituted aniline, respectively, on the assumption that the amounts of products formed by nucleophilic attack at the aliphatic carbon are negligible.The dependence of the extent of S N Ar and S N 2(P) products formation on amine concentration is given in Table 2.It is clear that while % S N Ar product increases with amine concentration for n-butylamine and ethanolamine, the amount of S N Ar product remains independent of glycyl ethyl ester concentration.This is a clear indication that the S N Ar reactions of n-butylamine and ethanolamine are base catalyzed while the corresponding reaction of glycyl ethyl ester is not.Plots of % S N Ar product vs.
[amine], using the data of Table 2, are linear for both n-butylamine and ethanolamine (not shown).
Base catalysis and the mechanism of the S N Ar reaction
The gross mechanism for S N Ar reactions is given in Scheme 2, in which either the formation of the Meisenheimer-type intermediate, PH, or its decomposition to products could be rate determining.According to this mechanism, the observed second-order rate constant, k A , is related to the microscopic rate constants for the constituent steps through equation (ii).
The three nucleophiles investigated in this study are all primary amines; there is therefore Kirby and Jencks, 17 have reported that primary and secondary amines react with pnitrophenyl phosphate dianion in aqueous solutions at both P and aromatic carbon sites; at the latter reaction site, the primary amines investigated did not show any susceptibility to base catalysis while secondary amines participated in reactions which showed varying degrees of response to base catalysis.Such dichotomy in the response of primary and secondary amines to base catalysis in SNAr reactions involving moderately to strongly activated substrates is well documented and the causative factors have also been discussed. 18,25 ernasconi's review 26 provides several examples of SNAr reactions of less activated substrates which are catalyzed by strongly and moderately basic amines, as found in the present study.
Site preferences in the reaction of 1 with oxygen and nitrogen nucleophiles
8][29][30][31][32][33][34][35][36] Khan and Kirby 37 have published on the multiple structure-reactivity correlations for reactions of phosphate triesters with a variety of nucleophiles.What is clear from the literature is that reactivities are sensitive to substrate structure, nucleophile type and basicity, nature of the leaving group, solvent type and solvation factors, etc. 27-31, 39, 40 Our intention in this section is to keep the substrate constant while examining how change of nucleophile type, from oxygen to nitrogen nucleophiles, influences reaction site preferences especially in alkaline media.In this way, it is hoped that an appreciation of some of the factors that determine site preferences will emerge.It is noted that existing literature comparisons do not incorporate significant information on P=S substrates such as 1, the subject of the present investigation.
To summarize, the following are the results so far obtained in the reactions of 1 with oxygen and nitrogen nucleophiles in water and ethanol as solvents.At pH ≤ 7.5, hydrolysis of 1 in aqueous solutions proceeds by way of alkyl-O bond fission; at higher pH (> 9), reaction occurs exclusively by displacement of ArO -through attack at the P center. 5,6In ethanol, the predominant products are those of alkyl-O (ca.43 %) and P-OAr (ca.50%) bond fission; a minor S N Ar pathway, accounting for ca.7% of the reaction products, was observed. 4With a series of structurally related phenoxides as nucleophiles, reaction in water occurs exclusively at the P center. 3Changing the nucleophile to the primary amines used in the present study sets up a competitive process in which attack at the aromatic carbon center is favored to varying extents over the corresponding reaction at P. The behavior of these nucleophiles in alkaline media in their reaction with 1 is amenable to an explanation that is based on the HSAB theory, 41 in which case the "hard" oxygen bases react mainly with the "hard" P center.The fact that "hard" amine nucleophiles, whose basicities are comparable to those of the oxyanions studied previously, show considerable affinity for the aromatic carbon center as well could be attributed to two factors.Firstly, Terrier 42 has shown that amines are intrinsically more reactive than alkoxides of similar basicities towards aromatic carbon centers.This may not be unconnected, in part, with the need to desolvate alkoxide reagents in an activated process which precedes the actual step involving nucleophilic attack.Jencks' results 43,44 demonstrate that amines and thiolates generally tend to be more nucleophilic than oxyanions, essentially as a consequence of the decreased demand for solvation of these nucleophiles as well as the high carbon basicity of thiolates, in comparison to oxyanions.6][47][48] Primary and secondary amine nucleophiles form σcomplexes quite readily, in part because the deprotonation of the first-formed intermediate (see Scheme 2) provides the thermodynamic driving force for the forward reaction, and this effect is lacking when O-based nucleophiles are involved.This factor disfavoring the reaction of O-based nucleophiles should assume greater prominence when the aromatic moiety is much less activated, as in 1.On this score, the observation of SNAr products with amines but not with oxygen nucleophiles can be accounted for.
The formation of significant amounts of S N 2(C) products in the ethanolysis of 1 by alkali metal ethoxide in ethanol recently reported by Buncel and co-workers, 4 can be understood if the bulk solvent stabilizes the transition state for alkyl-O bond cleavage through hydrogen bonding to the departing -OP(S)(OCH 3 )OAr anion.These authors showed that free ethoxide is more reactive than either ion paired or dimerized alkali metal ethoxide.Attack by ethoxide at the P center is catalyzed by alkali metal ethoxides.In addition to the products of attack at the P center, which are explicable by the HSAB theory, minor products of ethoxide ion attack at the aromatic carbon center were also observed.The latter products conceivably result from the nucleophilic activity of the significantly more basic EtO -, which promotes attack at the aromatic center in a reaction that is considerably less favored than the processes at the P and alkyl carbon centers.Kirby and Younas 49 have shown that changing oxyanion nucleophiles to more basic ones in the reaction of methyl 2,4-dinitrophenyl phosphate enhances the prospects of SNAr product formation, in addition to attack at the P center.
Experimental Section
General Procedures.Materials.Reagents were commercial products which were either used as received or purified according to literature procedures.Stock solutions of the substrate were prepared under nitrogen and stored in volumetric flasks which were sealed with rubber septa, protected from light by wrapping with aluminum foil and stored in a refrigerator.pH was measured with an Accumet 825 pH meter with a glass electrode calibrated with standard buffer solutions.NMR spectra of the substrate were obtained on a Bruker 400 spectrometer operating at 400 MHz.Product analysis by GC-MS was performed using a Fisons 8000 Gas Chromatography instrument equipped with a MD800 Mass Spectrometer.The GC was equipped with a DB-5 capillary column, and used helium as the carrier gas.The qualitative experiments for the determination of reaction products for processes involving the three amines investigated were performed on a Perkin-Elmer Lambda 5 uv-visible spectrophotometer.Water was distilled, deionized, degassed, and filtered through 0.22 µm filter.
O,O-Dimethyl-O-(3-methyl-4-nitrophenyl)phosphorothioate, 1, was a gift from Sumitomo Chemical Co. and was purified by column chromatography; 50 its purity was checked by 1 H and 31 P NMR as well as by GC-MS.
Determination of reaction products
Reaction was initiated by injecting the required amount of a stock solution (9.54 x 10 -3 M) of 1 into a capped glass vial containing the appropriate concentration of the relevant amine at room temperature.In all cases, the amine concentration was in large excess of the substrate.The pH of the reaction mixture was measured.Aliquots of the reaction mixture were then withdrawn, its pH was adjusted to < 5 with dilute HCl and the absorbance measured at 400 nm.At this pH, the absorbances recorded were those due to substituted anilines, i.e. the S N Ar products, since the phenolate products resulting from displacement at the P center would exist in their protonated forms , i.e. the phenols, which would not absorb at pH < 5.The absorbance of the reaction mixture was monitored until no further changes were observed.In all cases, experimental absorbances at "infinite" time were lower than calculated ones, indicating the existence of competing reaction(s).The observed and theoretical infinity absorbances were used to calculate % S N Ar reaction products according to equation (i).The % S N 2(P) products recorded in Table 2 were obtained by difference, on the assumption that the amounts of S N 2(C) products formed by nucleophilic attack at the aliphatic carbon are negligible.
structural similarity, in a sense, among them.The results presented above show that while the S N Ar reactions of n-butylamine and ethanolamine proceed by way of rate-determining decomposition of PH (Scheme 2), its formation is rate-limiting for glycyl ethyl ester.With reference to equation (ii), what these results indicate is that for the reactions involving nbutylamine and ethanolamine, the kinetic condition k -1 >> k 2 + k 3 [B] holds, while for the reaction of glycyl ethyl ester the kinetic condition k -1 << k 2 + k 3[B] obtains.The factors determining the incidence of base catalysis or otherwise in SNAr reactions are now well known and have been discussed extensively.[7][8][9][10][11][12][13][14][15][16]
Table 1
pK a Values of amines employed in this study a a Data taken from D.D Perrin, "Dissociation Constants of Organic Bases in Aqueous Solution",IUPAC, Butterworths, London, 1965.Issue in Honor of Prof. O. S. Tee ARKIVOC 2001 (xii) 134-142 ISSN 1424-6376 Page 137 © ARKAT USA, Inc
Table 2 %
S N Ar and S N 2(P) products obtained in the reaction of 1 with amines in water as a function of amine concentration a a Calculated by neglecting the minor amounts of S N 2(C) products formed in some of the reactions. | v3-fos-license |
2017-03-31T00:19:57.990Z | 2015-02-10T00:00:00.000 | 31283040 | {
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} | pes2o/s2orc | Correction: Using Hansen Solubility Parameters to Study the Encapsulation of Caffeine in Mofs
a Correction for 'Using Hansen solubility parameters to study the encapsulation of caffeine in MOFs' by Lorena Paseta et al.
Introduction
Encapsulation is a process in which an active material or guest is entrapped within another material or system known also as host, carrier or encapsulant. 1Although there is strong modern interest in both industry and academia, the concept is not so new.This technique was used in the 50s when capsules containing dyes were incorporated into paper for copying purposes and replaced carbon paper. 2 Besides, the food industry has been using encapsulation for over 60 years as a way to protect the active material from environment degradation. 3Interest in encapsulation techniques remains high due to the numerous advantages that it provides, among others: • Thermal enhancement.Encapsulation may enhance the thermal stability of the encapsulated substance, an important effect in some process in which the working temperature is near the degradation temperature of the additive. 4
•
Chemical protection.This aspect is very useful in the food industry, above all in the encapsulation of lipid ingredients, where this method prevents their oxidation and therefore reduces undesirable flavors and odors. 5
•
Controlled release.One important problem of the pharmaceutical industry is how to control the release of medication into the body to maximize its efficacy.Encapsulation provides such controlled release, increasing patient comfort and compliance. 6
•
Compatibility improvement.Nowadays, most drugs used in pharmaceutical industry are poorly water-soluble, which is a problem for the absorption in the body, because first the drug must be dissolved in the gastrointestinal fluids.A solution to this problem is the encapsulation into other material that improves the drug dissolution rate. 7 Homogeneous catalyst heterogenization.Host-guest interactions have use in catalysis to heterogenize homogeneous catalysts such as metal complexes that once encapsulated in a porous inorganic matrix (e.g. a zeolite) can be handled as a common solid catalyst. 8Porous materials allow the encapsulation of a wide range of chemical compounds thanks to their high specific surface area and tunable micro-or mesoporosity.Some examples of these materials are porous silicas 9 and zeolites. 10In this context, metal-organic frameworks (MOFs) are a new class of porous materials that combine the properties of organic and inorganic substances, 11 with remarkable features linked to their regular crystallinity and
ENCAPSULATION
permanent porosity.MOFs are porous materials formed by the coordination of metal ions or clusters with organic ligands to form 1D, 2D and 3D crystal lattices.These materials are characterized by having large pore volumes and the highest surface areas known to date. 12Another important characteristic that makes MOFs so attractive is the possibility to modify the chemical functionality and the shape and pore size changing the connectivity of the metal ion or cluster and the nature of the organic ligands. 13s said, encapsulation in porous materials applies to a wide range of fields (Figure 1).In catalysis, for example, zeolites have been used as a support for complexes of Co(II), Cu(II) and Zn(II) giving rise to catalysts for the phenol hydroxylation, 14 while cobalt encapsulated into ordered mesoporous silica SBA-15 has been applied to the selective oxidation of cyclohexene. 15Silica microparticles have been used to encapsulate and control the release of biocides in wood paints. 16Vitamin E, known for its antioxidants properties, has been encapsulated in zeolite Y in order to obtain special fibers of polyamide. 17Dyes have been encapsulated into zeolite L searching for artificial photonic antenna systems. 18Nevertheless, the encapsulation field that nowadays attracts more attention is medicine, where there are a number of reviews that summarize the use of silicas 19 and MOFs 20 for this purpose.In the case of drug encapsulation, the substance most used as a model molecule is caffeine.The interest in caffeine can be divided into two kinds.The first is related to the chemical field and the other to health.First of all, caffeine is modestly soluble in water (its solubility is comparable to that in acetone but ten times lower than that in aniline), which is very useful during the encapsulation experiment, simplifying the experiments and allowing the use of ecofriendly solvents.The other interesting chemical property of caffeine deals with its amphiphilic nature, which may help the creation of chemical interactions with both organic and inorganic moieties in case of MOFs.Moreover, caffeine is used as an active drug in both cosmetics and pharmaceuticals.Caffeine is a liporeductor, 21 it has positive effects on psychological systems, 22 analgesic properties, 23 etc.Hansen solubility parameters (HSP) have classically been applied to the evaluation of solvent-polymer chemical interactions 24 but also to study barrier properties and chemical resistance of protective clothing, 24 prediction of cytotoxic drug interactions with DNA 25 , optimization of the extraction of bioactive compounds from biomass with subcritical water, 26 identification of an alternative, less toxic solvent used in a microencapsulation process, 27 and preparation of stable dispersions of TiO 2 and hydroxyapatite nanoparticles in organic solvents, 28 among other prominent examples.Regarding the application of HSP to metal-organic framework (MOF) materials, to the best of our knowledge, there is only a single report dealing with the formation of composites between MOF HKUST-1 (with 5 wt% loading) and poly(L-lactic acid) (PLA). 29In this case, the solubility distance parameter (see below) between PLA and MOF was found to be 5.9, judged by the authors relatively high, thus explaining the poor affinity between MOF particles and PLA observed by SEM.As suggested by the authors the filler was not able to establish dipolar and hydrogen bonding interactions as happened when the more hydrophilic zeolite NaA was used as filler. 30Thanks to HSP the basic principle of "like dissolves like", i.e. the qualitative idea behind most of previous examples, realized in numbers easy to handle and compare, as will be shown along the next narrative.Nevertheless, a limitation of HSP application is related to insufficient availability of HSP data for systems of interest (MOFs in particular).Table 1 summarizes the examples of HSP applications described above.Prediction of cytotoxic drug interactions with DNA Hansen 25 Extraction of bioactive compounds from biomass with subcritical water Srinivas et al. 26
Microencapsulation of therapeutic proteins
Bordes et al. 27 Preparation of stable dispersions of inorganic nanoparticles in organic solvents Wieneke et al. 28 Formation of composites between MOF HKUST-1 and poly(L-lactic acid) Elangovan et al. 29 In this work we propose the application of HSP to evaluate host-guest interactions involving MOFs and different compounds.In particular, the residual solvents and ligands after certain MOF synthesis (those using 2-methylimidazole and NH 2 -benzenedicarboxylate ligands) and the one-step encapsulation of caffeine into ZIF-8 31 and NH 2 -MIL-88B(Fe) 4 will be revisited in light of HSP.Finally, Figure 2 shows the structures of MOFs NH 2 -MIL-88B, ZIF-8 and HKUST-1, whose HSP parameters will be discussed below.Oxygen, nitrogen and carbon atoms are in red, blue and white, respectively.These structures were made with Diamond 3.2.[34]
Application of Hansen solubility parameters to the encapsulation of caffeine
The host-guest interactions established between MOF and solvent or ligand can be of different nature, i.e. dispersion, polar or hydrogen bonds.In case of solvents these interactions can be discussed in terms of the so-called Hansen solubility parameters (HSP). 24These parameters (δ D , δ P and δ H for dispersion or London interaction, polar interaction and hydrogen bonds, respectively) are given in Table 2 for some selected solvents, common in the synthesis and activation of MOFs.However, besides these parameters which account for the possible solvent interactions with MOF, guest molecular dimensions and MOF pore sizes have to be considered, even though they may not be as important as in the case of rigid materials such as zeolites and other microporous silicates due to the intrinsic flexibility of MOFs revealed as breathing 35 and gate-opening 36 phenomena.In any event, the interaction between two substances 1 (in this case the solvent or the ligand used to synthesize the MOF) and 2 (the MOF itself) can be obtained calculating the parameter Ra 24 with the following equation (1): In our case δ D1 , δ P1 and δ H1 and δ D2 , δ P2 and δ H2 sets of parameters correspond to MOF synthesis solvent, MOF ligand or caffeine (when this molecule is added to the synthesis solution, as shown below), and MOF ligand, respectively.Since HSP are not available for MOFs (with the exception of HKUST-1 29 ) we have simplified the approach for the forthcoming discussion by attributing to the ligand the solubility properties of the MOF, or, equivalently, by making the assumption that the solvent/ligand and encapsulant/ligand interactions dominate the encapsulation process.This approach is similar to that established by Hansen when used δ D , δ P and δ H of DNA base segments (i.e.guanine, cytosine, adenine and thymine) to estimate affinity between cytotoxic drugs and DNA itself. 25ble 2. Hansen solubility parameters (HSP) for some common solvents in the synthesis and activation of MOFs, some common ligands, MOF HKUST-1 and caffeine (CAF).THF, DMF, 2MI, BDC, NH2-BDC and BTC correspond to tetrahydrofurane, dimethylformamide, 2-methylimidazole, benzene-1,4-dicarboxylic acid, 2-aminobenzene-1,4-dicarboxylic acid and benzene-1,3,5-tricarboxylic acid, respectively.Distances between materials obtained from Ra calculations with equation (1).In general, values were obtained from literature (solvents, ligands and caffeine from, 37 and HKUST-1 from 29 ) with the exception of HSPs for 2MI, BDC, NH2-BDC and BTC, calculated using Y-MB technique with the commercial package Hansen Solubility Parameters in Practice. 38SP [MPa 44 etc.) and benzenetricarboxylate (BTC, for HKUST-1, 34 MIL-96, 45 etc.).Although "large" HSP distances imply poorer interaction there are no general rules for specifying a minimum Ra for strong interactions.In case of polymer-solvent, Ra values below about 7.5 meet the Flory-Huggins criterion for compatibility 37 which at least gives us a starting scale for looking at MOF Ra values.The relative discrepancy between BTC-HKUST-1 pair (Ra in Table 2 is 7.9), is basically due to the fact that estimated value for the BTC δ H (17.0) is higher than expected because most probably all the three OH groups in BTC would not be available for H-bonding, as the experimental HKUST-1 δ H (10.7) obtained by Auras et al. 29 suggests.Table 2 confirms that common ligands are not very soluble in usual solvents; however, chloroform, THF and especially DMF are preferred over small alcohols and water.This is particularly true when, for instance, MIL-53(Al) was synthesized in water and the BDC ligand remained occluded in the MOF microporosity 46 so that the activation of the material was achieved by either calcination at about 330 ºC 46 or DMF extraction at 70 ºC with final drying at 150 ºC. 47The conclusion is that MIL-53(Al) would prefer the ligand BDC and the activation solvent DMF more than the water used as synthesis solvent.
An even more interesting conclusion from Table 2 is that caffeine exhibits good interaction with all the four ligands (2MI, BDC, NH 2 -BDC and BTC, see Figure 3) with Ra values in the 3.1-4.5 range.Furthermore, the caffeine-ligand interaction predictions are better than those corresponding to any solvent-ligand pairs.This is in agreement with the fact that caffeine has been encapsulated in onestep (i.e.adding caffeine directly to the synthesis solution of the MOF) in MOFs ZIF-8 31 and NH 2 -MIL-88B(Fe) 4 including 2MI and NH 2 -BDC ligands in their respective formulations.However, even using the same ligand to synthesize both MIL-53(Fe) and UiO-66(Zr) MOFs, the different structure and metal center may give rise to different amounts of encapsulated caffeine depending on the working solvent (multi-step encapsulation, see below): 48 2.2 and 29.3 wt% for MIL-53(Fe) in water and ethanol, and 22.4 and 2.5 wt% for UiO-66(Zr) in water and ethanol as well.In the case of MIL-53(Fe) the presence of ethanol would lead to a full pore opening form of the MOF favoring caffeine insertion, while in the case of UiO-66(Zr) ethanol could be coordinated to the zirconium unsaturated Lewis sites decreasing the accessibility of caffeine. 48It is obvious from this discussion that the availability of HSP for MOFs of interest would open a new world of drug-MOF encapsulation not only in one-step but also through the conventional methodology of synthesis-activation-encapsulation (multi-step encapsulation). 49The comparison of both one-step and multi-step methodologies will be done in a next section.A comment must be done to the fact that the HSP of caffeine generally used in the HSP community is that estimated many years ago by Hansen to be [19.5, 10.1, 13.0]. 37The most powerful recent estimation tool using automated functional group contributions within the HSPiP package gives [20.0, 12.7, 8.3]. 38Furthermore, it is fair to point out that HSP theory confidently predicts that caffeine will be insoluble in water; the actual modest solubility in water is variously explained by hydrogen bonding and complex formation.Indeed, HSP for drugs and MOFs, or in general polymers or porous inorganic materials 17 able to act as capsules, would help to select the best encapsulating material for every drug, rationalizing part of the host-guest practical approach.In fact Table 3 lists a series of polymeric materials, from polypropylene to cellulose acetate which are compared with caffeine.Thus caffeine-polymer distances (Ra values) were calculated and the polymers arranged from highest to lowest values.A quick general behavior can be drawn from the Ra calculations, hydrophobic polymers (PP, PE, N66, PTFE, PS) would not be suitable for caffeine interaction, while hydrophilic polymers (PLA, PSF, CA) could be used as systems in which caffeine would be "dissolved".Moreover, HKUST-1 presents a Ra value (3.9) similar to that of the best polymer CA (4.1).This predicts good caffeine-HKUST-1 interaction and the encapsulation would be in addition enhanced by the microporosity of the MOF (BET surface area of 692 m 2 /g, pore size of 1 nm 34 ).As mentioned above, the availability of HSP for MOFs is almost zero.The same can be stated for traditional materials such as amorphous silica and zeolites. 50This is why inorganic TiO 2 and hydroxyapatite particles, for which data are available, 28 were added, with relatively good result in terms of Ra calculation with caffeine, to Table 3.It is worth emphasizing that different versions of the "same" material are expected to show different HSP values because the measured values will depend strongly on the chemical nature of the surface.For example, the HSP of "silica" will depend on the percentage of Si-OH groups at the surface, on partitioning of additives to the surface and on the porosity and, therefore, the relative amounts of "surface" and "interior".In addition, some of these surface properties may change with particle size suggesting that HSP for nano-sized and micro-sized particles of the same material would present relatively different values.This means that access to high-throughput measurements of the HSP of such systems will be important for progress in this field.
Solvent and ligand encapsulation as a precedent of one-step encapsulation
During the synthesis of MOFs both solvent and ligand molecules can be converted into guest species in the as-synthesized MOF.This means that it would be possible to make a relation between HSP and the synthesis of MOFs considering the possibility of having as guest in the as made MOF solvent, ligand or other molecule (e.g.caffeine in its one-step encapsulation) present in the synthesis solution.Thus a small Ra between ligand and any other species (e.g.solvent) would be expected to produce a good interaction.This is obviously a simplification that takes into account neither the nature of the metal cluster nor the structure of the MOF (porosity, flexibility, breathing 51 ).In any event, after the synthesis certain molecules (solvent, ligand, etc.) may become guest molecules into a MOF, as there is a good interaction between them and appropriate matching between guest molecular size and host porosity.
As shown in Table 4, water (coming from the hydrated zinc salt) and DMF remain encapsulated in the ZIF-8 structure after its synthesis in DMF.In case of water as solvent, ligand (2MI) can remain as well.
When the solvent is methanol or a mixture of water and methanol, ZIF-8 is obtained as an activated porous solid.Ra distances for 2MI (as mentioned above, somehow representing ZIF-8) and DMF, methanol and water are 4.4, 14.7 and 14.0 (Table 2), respectively.This is in agreement with the fact that DMF is retained by ZIF-8 after synthesis.The ligand 2MI would remain in the solid when using water and no ethanol as synthesis solvent, this is explained by a better preference of the ligand for the MOF than for water.When caffeine is incorporated into the molar composition it is retained by ZIF-8 better than any other molecule due to the good chemical compatibility between caffeine and 2MI (Ra 3.6, see Table 2).
In contrast to the ZIF-8 synthesis, in the case of NH 2 -MIL-88B(Fe) (Table 5), a MOF where caffeine was also encapsulated in one-step, 4 there is not a clear relationship between the solvent used in the synthesis and the guest molecule retained by the structure, in general water coming from the hydrated iron salt, and DMF as usual solvent or cosolvent.In terms of HSP, the one step encapsulation of caffeine would be favored given the small Ra between caffeine and the organic ligand NH 2 -BDC (4.5, see Table 2), explaining the high loading of caffeine in the MOF (38.5%).
One-step encapsulation (OSE) versus multi-step encapsulation (MSE)
To encapsulate caffeine or another drug into a porous material there are two possible methodologies.The one-step encapsulation (OSE) corresponds to build the MOF around the guest-molecule which is chosen to encapsulate (Figure 4a).Generally, this experiment consists in putting all reagents and the additive in solvent and stirring and heating for a short time. 31The other method is the conventional multi-step encapsulation (MSE) (Figure 4b). 49t least for caffeine, OSE 4,31 is superior to the conventional MSE 48,57 which requires: 1) the MOF host synthesis, 2) the subsequent activation of the host, and 3) the encapsulation itself (i.e. the liquid phase adsorption) of the drug to reach at the end a similar value of encapsulated guest.Thus, at least for caffeine, both processes may lead to similar quantitative results. 31The activation stage is not always an easy process, and a certain amount of solvent may remain in the material affecting its performance.Besides, caffeine was not lost during the procedure and could be recovered as the other compounds used in OSE or MSE methods.In general, MOFs can be prepared in milder conditions than other materials such as zeolites making more likely that expensive and sensitive drugs can survive the encapsulation process.So far, the caffeine OSE has been applied to MOFs ZIF-8 31 and NH 2 -MIL-88B(Fe). 4The one-step encapsulation of caffeine in ZIF-8 produced high guest loading, i.e. ca.28 wt% in only 2 h at 25 ºC, when the multi-step methodology required at least 3 days working at 80 ºC to achieve similar encapsulated amount.In the case of NH 2 -MIL-88B(Fe) the caffeine loading was 38.5 wt%.It is worth mentioning that the absence of caffeine from the synthesis solution led to a different framework (NH 2 -MIL-53(Fe)).Thus caffeine played the role of a structure directing agent or template.In both cases, controlled release of caffeine in both water and PBS (phosphate-buffered saline) was achieved.Although there are potentially thousands of drugs to be encapsulated, the interest for caffeine is not only related to its properties but to its role as model substance 57 (as ibuprofen 58 is often studied by many other researchers) used several times to evaluate different materials and strategies.Table 6 summarizes the advantages and disadvantages of the two encapsulation strategies.OSE is simpler than MSE, not requiring of activation, since caffeine would be favored in the synthesis conditions over solvents and ligands.Of course a low guest chemical stability may limit the applicability of OSE but most MOFs are prepared in milder conditions or their synthesis routes can be adapted by searching for low temperature reactions in presence of environmentally friendly solvents.In both strategies of encapsulation excess reactants would be reused giving rise to a simple and scalable process.From the point of view of the industrial operation, it would be more difficult to scale up three different stages than a single one.
In any event, it is expected that HSP can facilitate a more rational optimization approach than the trial and error system.First, HSP can be used to find a solvent (or solvent blend) with similar solubility properties (a solvent like DMF is unusably toxic in the context of drug delivery).Second, there will be a balance between solvents that are too good (they will preferentially be encapsulated in the MOF) and those that are too bad (insufficient solubility to allow the process to take place).There is an interesting parallel with the use of HSP in the development of low molecular weight organo-gelators (MOGS) which similarly must be in a narrow optimal zone. 59Another field of possible application of HSP would be related to the development of mixed matrix membranes (MMMs), 60 in which a porous filler (e.g.silica, zeolite, MOF, etc.) is dispersed into a membrane polymer to enhance the membrane performance in terms of permeability and separation selectivity.In these MMMs the filler-polymer interaction is of paramount importance to achieve homogeneous filler dispersion and enhanced performance.The availability of HSP for fillers and polymers of interest would be of practical interest to obtain optimum MMMs.As mentioned above, the caffeine-ligand interaction predictions in terms of HPS were better than those corresponding to any solventligand pairs.This points to the fact that during the one-step encapsulation process of caffeine this molecule can be favored over solvent molecules even though these are smaller.
Conclusion
Even though for the same ligand the MOF texture, structure and composition (different metal) affect drug encapsulation in MOFs, Hansen solubility parameters (HSP) appear as a new tool which may help to predict the best encapsulating material and solvent system for a target drug, helping to convert the traditional trial and error approach into a rational one to be applied to a wider library of possible hosts and guests.The availability of HSP for MOFs is almost zero, so it is important to measure values for common MOFs, preferably by using highthroughput techniques that will make such measurements scalable.In case of having HSP for MOFs their especial features (breathing and pore opening maximum flexibility effects; richness of isostructures, i.e. phases having different chemical composition but the same crystallographic structure), predictions should be carefully considered in combination with existing drug encapsulation experimental results.In the particular case of the one-step encapsulation of caffeine, the caffeine-ligand interaction predictions in terms of HPS were better than those corresponding to any solvent-ligand pairs.This is in agreement with the fact than caffeine is favored over solvent molecules, even though these are smaller, and is efficiently encapsulated.Finally, beyond the classical HSP applications (mainly dealing with solvent-polymer chemical interactions) and besides the new use of HSP proposed here for caffeine encapsulation in MOFs, HSP could help the development of different systems in which the interaction of relatively heterogeneous materials to be combined (as is the case of low molecular weight organo-gelators and mixed matrix membranes) would have to be taken into account.In the context of this paper, HSP could as well help to predict the release in the body of the drugs encapsulated in a certain porous material.In any event HSP parameters for the possible involved substances would need to become available.
Figure 2 .
Figure 2. (a) Building blocks of NH2-MIL-88B (Fe) with the trimers of µ3-O-bridged FeO6 octahedra (see inset for detail) in green; (b) building blocks of ZIF-8 with the ZnN4 tetahedra in green; and (c) building blocks of HKUST-1 with dimmers of CuO4.Oxygen, nitrogen and carbon atoms are in red, blue and white, respectively.These structures were made with Diamond 3.2.using the corresponding CIF files.[32][33][34]
Table 3 .
Hansen solubility parameters (HSP) for some common polymers and materials, MOF HKUST-1 and caffeine (CAF).Distances from caffeine obtained from Ra calculations with equation(1).HSP
Table 4 .
Synthesis of ZIF-8 under different conditions. | v3-fos-license |
2019-04-05T03:31:43.949Z | 2010-09-30T00:00:00.000 | 95228638 | {
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} | pes2o/s2orc | DETERMINATION OF OIL AND GREASE, TOTAL PETROLEUM HYDROCARBONS AND VOLATILE AROMATIC COMPOUNDS IN SOIL AND SEDIMENT SAMPLES
. This paper describes a case study of petroleum-contaminated soil/sediment samples which were analyzed using gas chromatography-flame ionization detector (GC-FID) for total petroleum hydrocarbons (TPH), volatile aromatic compounds: benzene, toluene, ethylbenzene, and xylenes (BTEX) and naphthalene by GC-MS, and oil and grease (O/G) content by sonication in hexane. The ratio of (TPH) / (O/G) shows that the hydrocarbon fraction is between 7% and 87%. The content of volatile organic fraction BTEX accounts for only a small proportion of total TPH, and the ratio of (BTEX) / (TPH) ranges from 0.1% to 0.6%. It should be stressed that the use of TPH methods as against gas chromatography must be done with care because the potential risk posed by BTEX compounds may not be adequately addressed.
Introduction
When petroleum products are released to the environment, physical, chemical, and biological processes change the contaminated site. Petroleum hydrocarbons released to the ground may move through the soil to the groundwater (Riccardi et al. 2008;Pawlak et al. 2008;Wang et al. 2002). Individual contaminant compounds at the site may separate from the original mixture, depending on the chemical properties of the compounds. Some of these compounds evaporate into the air (Laškova et al. 2007;Paulauskienė et al. 2009) and others will dissolve into groundwater and move away from the release area. Other compounds will attach to particles in the soil and may stay in the soil for a long time, while others will be broken down by the organisms found in the soil (Research Triangle Institute 1999).
Petroleum hydrocarbons (PHCs) are common site contaminants, but they are not generally regarded, and hence, regulated as hazardous wastes. PHCs indicate degradable and biodegradable properties in soil, water and sediment environment (Fedorak, Westlake 1981;Mills 1994;Prince et al. 1994;Leahy, Colwell 1990). The hydrocarbon analyses can be used for environmental assessment of remediation (Douglas et al. 1991) or soil bioremediation (Korda et al. 1997;Jørgensen et al. 2000). The term total petroleum hydrocarbons (TPH) is used to describe a broad family of several hundred chemical compounds that originally come from crude oil. It is useful to measure the total amount of all hydrocarbons found together in a particular sample of water, soil, or air. TPH is defined as the measurable amount of petroleum-based hydrocarbon in environmental media (Research Triangle Institute 1999;Gustafson 1997).
Re-refined petroleum products, e.g., gasoline, diesel fuel, used engine oil constitute a major class of contaminants for environmental investigators. Volatile aromatic hydrocarbons BTEX in unleaded gasoline contain benzene, toluene, ethylbenzene and xylenes in concentration above 10,000 ppm for each compound in the ratio (1:5:1:5). In diesel fuel, BTEX compounds are present in concentration of about two orders of magnitude lower than gasoline in the ratio (1:3:2:14) for benzene, toluene, ethylbenzene and xylenes (Romeu 1990). The approximate carbon numbers for individual hydrocarbon products present in petroleum products are as follows: gasoline (C 6 -C 12 ), diesel (C 8 -C 26 ), kerosene (C 8 -C 18 ), fuel oil (C 17 -C 26 ), and lubricating oils (C 21 -C 50 ) (George 1994).
Lubricating engine oil is a petroleum product that is a complex mixture of low and high molecular weight aliphatic and aromatic hydrocarbons (C 21 -C 50 ), metals, and additives. Additives (which can account for up to 20% of the weight of oil formulations) include detergents, metallic salts (e.g., molybdenum and zinc salts), and organometallic compounds. Metals (e.g., cadmium, lead and zinc) and polyaromatic hydrocarbons (PAHs) have been demonstrated to increase in oil with continued use in an engine (IARC 1984).
An oil and grease (O/G) contaminants are defined as any material recovered as a substance extracted in the form of organic solvent from a sample, and are composed primarily of fatty matter from animal and vegetable sources, hydrocarbons of petroleum origin, sulfur compounds, certain organic dyes, and chlorophyll. Many solvents are used in the determination of O/G: petroleum ether, trichlorotrifluoroethane, a mixture 80% n-hexane + 20% methyltert-butyl ether, and currently n-hexane (Standard methods 1995). Methods developed for monitoring O/G included EPA 413.1, 1664, 9071A, and Standard Method 5520E. If O/G is present in excessive amount, it may interfere with aerobic and anaerobic biological processes, leading to a decrease in wastewater treatment efficiency. The knowledge of the present O/G quantity is helpful in the proper design and operation of wastewater systems and also for identifying treatment difficulties.
The TPH value represents a mixture of compounds and can be obtained from one of several analytical methods, some of which have been used for decades and others developed more recently, namely, EPA 418.1, 8015B, 1664 and 8020B. EPA method 418.1 provides a "one number" value of TPH in an environmental medium; it does not provide information on the composition (e.g., individual constituents of the hydrocarbon mixture). The amount of TPH measured by this method depends on the ability of the solvent used to extract the hydrocarbons from the environmental medium and the absorption of infrared (IR) light by the hydrocarbons in the solvent extract. At this juncture, it should be noted that the EPA method 418.1 is not specific to hydrocarbons and does not always indicate petroleum contamination. Method 418.1 has been one of the most widely used methods for determining TPH in soil analysis. Freon-extractable material has also been reported as O/G. Polar components may be removed by treatment with silica gel, and material remaining, as determined by IR-spectrometry, is defined as TPH. Therefore, EPA method 1664, "n-Hexane Extractable Material (HEM) and Silica Gel Treated n-Hexane Extractable Material (SGT-HEM) by Extraction and Gravimetry (Oil and Grease and Total Petroleum Hydrocarbons)", will replace the methods using Freon-113 (US EPA method 1664, 2002. Another analytical method commonly used for TPH is gas chromatography-flame ionization detector (GC/FID), a modified EPA method 8015B. The underground storage tank programs started in the mid-1980s, and they are capable of addressing groundwater contamination problems precisely. This gas chromatography (GC) method, coupled with specific extraction techniques, can provide information on the product type relative to values from chromatogram with benchmarks. Hence, there is a growing trend in the use of GC techniques in the analysis of soils and sediments. One aspect of this is that "volatiles" and "semivolatiles" are determined separately. The volatile or the gasoline range organic (GRO) components are recovered using purge-and-trap or stripping techniques. The semivolatile range is determined by the analysis of an extract by GC/FID and is referred to as diesel range organic (DRO). The results are most frequently reported as single numbers for purgeable and extractable hydrocarbon (US EPA method 8015B, 1986). Analytical methods that can provide more detailed information on the components of petroleum than traditional/standard methods are developed (Wang, Fingas 1997;Mills et al. 1999;Mc Kenzie et al. 1983;Roques et al. 1994).
The focus of this study was to identify and characterize O/G, TPH and BTEX and naphthalene in soil/ sediment samples. It continues an earlier publication concerning the determination of heavy metals, volatile aromatic compounds in used engine oils and sludges (Rauckyte et al. 2006).
Methods
The starting materials were obtained from "naturally" contaminated sites in a petroleum refinery at 5-10 cm deep. Soil samples 1-4 were taken from the area of so called petrochemical complex, and samples 5-9 from the refinery complex. Sediments for samples 10-11 were taken from retention containers storing groundwater and rain water from the refinery complex and samples 12-13 from the same containers but storing water from the petrochemical complex. In case of samples 14 and 15, they were taken from water pumps containers: 14 from the refinery complex and 15 from the petrochemical complex. Samples from this refinery were prepared and analyzed in accordance with EPA procedures and methods: 9071A, 8015B, and 8020B.
The oil/grease EPA method 9071A is a procedure for extracting nonvolatile and semivolatile organic compounds from solids such as soil, sediments, sludges, and industrial and domestic wastes. The sonication process ensures the intimate contact of the matrix with n-hexane during the extraction. The method is not applicable to measure light hydrocarbons that volatilize at temperature below 70°C (US EPA method 9071A, 1994), with the method reported limit (MRL) of 50 ppm.
The TPH were measured by EPA method 8015B gas chromatography/flame ionization detection (GC/FID) with reported method of limit 10 ppm for soil/sediment (US EPA method 8015B).
The gas chromatography-mass spectrometry (GC/MS) method 8020B was used for analysis of benzene, toluene, ethylbenzene, xylenes, and naphthalene (BTEX) in soil/sediment samples. The reporting limit was 0.025 ppm for benzene, toluene ethylbenzene, m-xylene and 0.050 ppm for o-and p-xylene and naphthalene. The standards were prepared as specified in EPA method 8020B (US EPA method 8020B, 1992).
Results and discussion
The results of the oil/grease, total petroleum hydrocarbon and volatile organic compounds are presented in Table 1. The oil/grease in soil samples (n = 9) ranged from 100 ppm to 2400 ppm, and in sediment samples (n = 6) ranged from 3400 ppm to 13800 ppm. A similar trend was observed for total petroleum hydrocarbon (TPH) levels which in soil samples (n = 9) ranged from 20 to 400 ppm, and in sediment samples (n = 6) ranged from 850 to 1250 ppm. The mean ratio of (TPH) / (O/G) in soil samples ranged from 7% to 87%, and in sediment samples ranged from 8% to 27%. a Accuracy (% recovery) and precision (relative % difference) of O/G, TPH and BTEX analysis: Accuracy was determined as a ratio of LSB (found) to LSB (true) (85% to 115%), or as a ratio of LSM (found) to LSM (true) (80% to 120%); the precision was determined by calculating the difference between the results found for the LSB and LSBD, and then dividing the difference by the average of the two results (3% to 8%).
A linear relation between TPH and total O/G was observed (Fig. 1). This can be expressed: TPH = 0.153x + 37.7, R 2 = 0.897 for soil samples (n = 9), and the relation: TPH = 0.032x + 781.0, R 2 = 0.861 for sediments samples (n = 6). The total petroleum hydrocarbons from soil samples were effectively five times more concentrated than for sediment samples. The BTEX results in Table 1 indicate that the volatile fraction of aromatic hydrocarbons accounted for only a small part of the total TPH for contaminated samples and the ratios of (BTEX)/(TPH) ranged from 0.1% to 0.6% .
A good correlation between measured BTEX and TPH was obtained (Fig. 2). This yielded the relation: BTEX (ppm) = 0.0011 TPH (ppm) + 0.129; R 2 = 0.955, n = 15. Based on this correlation, the background BTEX (TPH = 0 ppm) was further estimated to be ~ 0.115 ppm for this contaminated site. The most contaminated area by BTEX was 1.8 ppm at the same site (TPH was 1300 ppm).
Until recently, routine TPH analyses in soil samples have been performed by means of IR spectrometry method 418.1 (Standard methods 1995; ISO.TR 11046, 1992). However, this method is no longer supported by international standardization since the ban of 1,1,2trichlorotrifluoroethane as an extraction agent, and was replaced by gas chromatography/flame ionization detection (GC/FID) after extraction with a halogen-free solvent (US EPA method 8015B, 1986;ISO/DIS 16703, 2001).
The results of TPH obtained from 155 participants laboratories in three proficiency-testing rounds were compared in soil by IR -spectrometry and gas chromatography (Becker et al. 2002). Participants were supplied with soil samples with different levels of mineral oil content. Two methods: 418.1 IR -spectrometry and 8015B GC/FID-gas chromatography were compared with soil samples for analyzing TPH.
The results obtained with both methods were compared using 1,1,2 -trichlorotrifluoroethane (with IR quantification) and n-hexane (with GC/FID quantification) as the extraction solvents. The agreed value of the means obtained with (GC/FID) were typically 10% to 20% (ranging between 0% and 25%) higher than those found with IR-spectroscopy (Becker et al. 2002). The obtained results are comparable with data obtained by other authors who made analyses of similar cases, for instance Taylor, Viraraghaven 1999 determined TPH in soil within the range from 2500 to 10000 ppm, and Paavo et al. 2008 in the extended range from 100 to 10000 ppm with the use of GC/FID technique. Data from Park's (San Juan 2000) paper remained at the same level. In literature, it is difficult to find data recording (TPH) / (O/G) and (BTEX) / (TPH) relations which were determined in our paper. So, the mentioned above relations in case of soils greatly depend on the intensity of biological transformations (Seo et al. 2009;Peng et al. 2008). However, the processes which are the causes of these phenomena can be used in practice for instance to eliminate VOC (Baltrėnas, Zagorskis 2009;Del'Arco, De Franҫa 2001).
Despite the large number of hydrocarbons found in petroleum products, only a relatively small number of them were characterized for toxicity. The health effects of some fractions can be adequately characterized with respect to their components or representative compounds (e.g., light aromatic BTEX fraction (benzene, toluene, ethylbenzene, and xylenes) and naphthalene. The Agency for Toxic Substances and Disease Registry (ATSDR) does not assess cancer potency for TPH components, and toxicological information is only provided for some components, e.g., minimal risk level (MRL) (Research Triangle Institute 1999).
It is noteworthy that the health effects that are common to the BTEX compounds are of neurological nature (Mumtaz et al. 2004;Robinson, Mc Donell 2004;Pohl et al. 2003). Benzene has hematological effect and is classified in EPA Group A (human carcinogen). Minimal risk levels (MRL) and cancer classification for BTEX compounds and whole products are summarized in Table 2. The inhalation minimal risk level for each of the BTEX compounds (acute MRL) was determined with the results indicating quantities of the components as: benzene 0.05 ppm, toluene 0.02 ppm, ethylbenzene 0.2 ppm, o-, m-, and p-xylene 1.0 ppm, and naphthalene 0.002 ppm (Research Triangle Institute 1999).
Conclusions
This paper describes a case study in which three analytical techniques including GC/MS, GC/FID, and sonication were used to identify and characterize oil/grease, total petroleum hydrocarbons and volatile aromatic compounds BTEX in soil and sediment samples. Our results and data interpretation clearly indicate that: 1. The contamination of soil/sediment samples by gasoline/diesel fuel is real and evident. The detected volatile aromatic hydrocarbons in soil and sediment samples were shown to have originated from gasoline/diesel fuels. 2. Samples of sediments were contaminated by TPH compounds, but to a much lesser degree (five times less) in comparison with soil sample. 3. The significant part of total petroleum hydrocarbons (TPH) was heavy petroleum product. When a sediment is released onto the environment, PHCs cause serious damage to the water system, and their persistent toxic effect may last for over a long time. When treated appropriately by optimizing their biodegradation potential, hydrocarbons degrade occurs naturally, and with this, the main part of a PHCs-pollution can be eliminated more rapidly.
Teresa RAUCKYTE. Dr, Dept of Technology and Chemical Engineering, University of Technology and Life Sciences, Bydgoszcz, Poland. Her research expertise is heavy metal ions in used engine oils, sludges, soil and environmental pollution. She has published about 50 scientific papers in international journals and conferences proceedings, parts of monographs.
Sławomir ŻAK. Dr, Dept of Technology and Chemical Engineering, University of Technology and Life Sciences, Bydgoszcz, Poland. His research expertise is environmental technology and analysis of environmental pollution. He is a fellow of Polish Hydrology Society and Ecological Chemistry and Engineering Society. He has published about 75 scientific and technical papers in international journals and conferences proceedings, also is an author of parts of monographs and 18 patents.
Zenon PAWLAK. Prof., Dr Habil, University of Economics, Bydgoszcz, Poland. His research expertise is in lubrication of engine oils, lubrication of human joints, environmental pollution and radiochemistry. He is a fellow of American Chemical Society and Polish Chemical Society. He has published about 190 papers in international journals and conferences, edited a number of special issues of international journals and conference proceedings.
Adekunle OLOYEDE. Prof., Queensland University of Technology, Brisbane, Australia. He is an established and wordacknowledged expert in the area of connective tissue research and in particular articular cartilage biomechanics. He has published notable concepts contributing to fundamental thoughts in the field. His research has led to 7 patents, 30 scientific papers in international journals. Also is an author or co-author of numerous conferences papers and parts of monographs. | v3-fos-license |
2019-04-07T13:09:47.969Z | 2014-04-22T00:00:00.000 | 52024679 | {
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} | pes2o/s2orc | Analytical & Bioanalytical Techniques A Validated High Performance Liquid Chromatography Method for the Determination of Saxagliptin and Metformin in Bulk, a Stability Indicating Study
In this paper a simple, precise and rapid high performance liquid chromatography method with UV detection has been developed for the determination of saxagliptin and metformin in bulk. An Agilent, Zorbax CN (250 × 4.6 mm I.D., 5 µm) column was used with a mobile phase mixture of methanol-50 mM phosphate buffer (pH 2.7) in a gradient elution mode at a flow rate of 1.0 ml min -1 . The analytes were detected at 225 nm and total run time for the method was 7 min. The calibration graphs were linear in the range of 5.00-125.00 µg ml -1 for saxagliptin and 2.50-62.50 µg ml -1 for metformin. For stability indicating study, saxagliptin was subjected to acid, neutral and alkali hydrolysis, oxidation and heat stress. The developed method could be used for quality control assay for SAX in tablets and for stability studies as the method separates SAX from its degradation products and tablet excipients.
Introduction
Saxagliptin (SAX) (1S,3S,5S)-2-[(2S)-2-amino-2-(3-hydroxy-1adamantyl)acetyl]-2 azabicyclo[3.1.0]hexane-3-carbonitrile) ( Figure 1), with C 18 H 25 N 3 O 2 chemical formula and 315.41 g/mol molecular weight, is an orally active, potent and selective inhibitor of dipeptidyl peptidase-IV (DPP-4) for the treatment of Type 2 diabetes, is marketed as Onglyza TM tablet, and also in a fixed dose combination SAX/ metformin (MET) as Kombiglyze TM tablet in USA, Europe and some other countries [1]. DPP-4 inhibitors enhance the body's own ability to control blood glucose by increasing the active levels of incretin hormones in the body. Their mechanism of action is distinct from any existing class of oral glucose-lowering agents. They control elevated blood glucose by triggering pancreatic insulin secretion, suppressing pancreatic glucagon secretion, and signaling the liver to reduce glucose production [2].
In literature two spectrophotometric methods have been reported for the determination of SAX in pharmaceutical preparations [3,4] and three other high performance liquid chromatography (HPLC) methods were developed for analysis of SAX and MET simultaneously [5][6][7][8].
In this study a new stability indicating HPLC method was developed for determination of SAX and MET in bulk drug and validated. The developed method could be used for quality control assay for SAX in tablets and for stability studies as the method separates SAX from its degradation products and tablet excipients.
Experimental Chemicals
Saxagliptin was purchased from Richem International Co., Ltd. (Shanghai, China). Metformin was kindly supplied by Abdi Ibrahim Ilac (Istanbul, Turkey). Commercially available Onglyza ® tablets were purchased from local drug store.
Sodium dihydrogen phosphate (NaH 2 PO 4 ), analytical grade ortho-phosphoric acid, HPLC grade acetonitrile and methanol were purchased from Merck (Darmstadt, Germany). Deionized water up to a resistivity of 18.2 MΩ was purified with an Elga water purification system (London, UK).
Instrumentation and analytical conditions
A Shimadzu (Kyoto, Japan) LC 20A liquid chromatograph, consisting of a model LC 20 AT solvent delivery system, a DGU-20A5 degasser and a SIL-20AC auto sampler (set at ambient temperature) and a diode array detector SPD-M20A was used. During method development different wavelengths were scanned with PDA detector in one run and 225nm was found to be the ideal for this study. Data acquisition was performed using LC Solution 1.21 SP1 system software. Separations were performed on an Agilent, Zorbax CN (250 × 4.6 mm I.D., 5 µm) column with an Agilent guard column (4 × 3 mm I.D.) packed with the same material. Chromatographic separation was achieved at room temperature by using methanol-50 mM phosphate buffer (pH 2.7) mixture as mobile phase in gradient elution mode at a (Table 1) was run for 7 min. A typical injection volume was 20 µL.
Preparation of solutions
Stock solutions were prepared by dissolving SAX and MET in acetonitrile and water respectively to give a concentration of 1 mg ml -1 . Standard solutions with a concentration of 100 µg ml -1 were obtained by diluting of the stock solutions with mobile phase mixture (10% methanol, 90% buffer). The final concentrations for SAX and MET solutions were 5.00-125.00 µg ml -1 and 2.50-62.50 µg ml -1 , respectively. The chromatograms were evaluated on the basis of the peak areas of SAX and MET. Buffer solution was prepared by dissolving 6.0 g NaH 2 PO 4 in 1000 mL of water, pH is adjusted to 2.7 by adding orthophosphoric acid drop wise. The prepared solution was filtrated through 45µm filter and sonicated prior to HPLC analysis.
Stress testing
Stress testing was carried out according to the ICH stability testing guidance [9]. SAX was stressed under various conditions until to facilitate approximate 5-20 % degradation [10].
Acidic, neutral and basic hydrolysis:
A solution of SAX 10 mg ml -1 concentration was prepared in acetonitrile, 1 ml of this solution was transferred to three different 10 mL volumetric flasks and diluted up to volume with water, 0.1 HCl and 0.1 NaOH solutions to give a concentration of 1 mg ml -1 . Samples of 0.25 mL were transferred to 5 mL volumetric flask, neutralized and diluted to the volume with mobile phase to a final concentration of 50.00 µg ml -1 . The samples were kept at room temperature and analyzed starting from first hour up to 6 hours. According to the degradation amount, the samples were further subjected to heat 70°C for an hour.
Oxidation: A solution of SAX 10 mg ml -1 concentration was prepared in acetonitrile, 1 ml of this solution was transferred to a 10 mL volumetric flask and diluted up to volume with 6 % H 2 O 2 . The prepared solution was kept at room temperature and was analyzed as the hydrolysis samples.
Thermal degradation: Bulk drug of SAX was exposed to 50°C dry heat for 1 h, 70°C for 1 h and 105°C for 6 hours.
Validation of the method
The methods were validated according to the ICH guidelines.
The calibration curves were obtained between the concentrations of 5.00-125.00 µg ml -1 and 2.50-62.50 µg ml -1 , for SAX and MET respectively. The solutions were prepared in triplicate. Linear regression equations (intercepts and slopes) for mixtures of SAX and MET were established and calculated by the least square regression method.
Precision of the analytical method was tested by analyzing six replicate determinations of three different concentrations (high, medium and low) of SAX and MET both on within-day and day-to-day.
The accuracy was determined by recovery test using standard addition method. This experiment was performed at three different concentration levels, in which sample stock solutions (25.00 µg ml -1 and 12.50 µg ml -1 for SAX and MET, respectively) were spiked with standard drug solutions of 25.00, 50.00 and 75.00 µg ml -1 for SAX and 12.50, 25.00 and 37.50 µg ml -1 for MET. The mixtures were then analyzed by the proposed methods. Five replicate samples of each concentration level were prepared. The recovery tests were performed using tablet solution containing various amounts of SAX and MET.
Sensitivity of the method was decided by determination of limit of detection (LOD) and limit of quantification (LOQ). LOD is the lowest detectable concentration of the analytes by the method while LOQ is the minimum quantifiable concentration. LOD and LOQ were calculated by using the values of slopes and intercepts of the calibration curves for both of the drugs.
Assay for the tablets
Onglyza tablet which contains 5 mg saxagliptin was used for the tablet assay. Ten tablets were separately weighed and finely powdered. About 25 ml of methanol and 50 mL of buffer solution was added to accurately weighed amount of the powder equivalent to the median mass of one tablet in a 100 ml calibrated flask. The mixture was shaken mechanically and sonicated in ultrasonic bath totally 30 min and diluted to volume with buffer solution and then filtered. The filtrate was diluted with the mobile phase (90% buffer: 10% methanol) to give a final concentration of 20.00 µg ml -1 of SAX and analysed with HPLC. The quantity of the SAX was calculated from the regression equation constructed for the SAX assay.
Method development and optimization
To optimize the chromatographic conditions a reversed phase cyano HPLC column and a phosphate buffer at a pH of 2.7 and methanol mixture were found to be the best stationary phases and mobile phase combinations to have two symmetrical and well-resolved peaks for SAX and MET. An optimized gradient program provided sufficient selectivity in a short separation time. The total runtime for the analysis is 7 min and the retention time for MET and SAX 3.47 and 5.83 min, respectively. Blank chromatogram overlapped with mixture of SAX and MET was presented in Figure 2 and single chromatograms of SAX and MET could be found in Figures 3-5, respectively. The system suitability parameters were found to be within the suitable range Rs (resolution) > 15 between two peaks (range Rs ≥ 2); T (peak tailing factor) 1.21
Stress testing
SAX was forced to degradation acid and alkali hydrolysis. The rate of acid hydrolysis was slower as compared to that alkali. Acid hydrolysis has almost no degradation product but just decreases in amount. The solution was stable up to 6 hours at room temperature, after this the solution was subjected to heat of 70°C for an hour and 17.03% of the solution was decomposed ( Figure 5). In alkali hydrolysis there was degradation product in the beginning and the drug was decomposed of 14.36 % (Figure 6). The solution was stable in water first two hours but it was decomposed up to 15.41% without any degradation by-product (Figure 7). As per the oxidative hydrolysis the drug was decomposed immediately 24.75%, degradation products were shown in Figure 8. The drug powder was exposed to 50°C for 1 h and found to be stable, the temperature was increased to 70°C and the drug is degraded 16.23% in 1 h. SAX exposed to the heat for 6 hours at 105°C and the bulk drug substance was melted completely and decomposed at the end of this treatment. The percent amount of drug degraded after degradation studies are given in Table 2.
The results obtained from the stress testing showed that SAX is unstable under hydrolysis, oxidation and thermal stress. Therefore, care should be taken in the manufacturing process and during the storage of the product and the developed method could be used for stability studies of SAX.
Validation of the method
The equations of the calibration curves were y= 14859x+103450, r 2 =0.9976 and y=69798x-70654, r 2 =0.994 for SAX and MET, respectively. The result shows a good correlation existed between peak area and concentration of each drug within the concentration range tested. The analytic and chromatographic data for the developed methods were given in Table 3.
The precision of the methods were assessed by carrying out six replicate determinations of three different concentrations of SAX and MET both on within-day and day-to-day (Table 4). RSD values were less than 1.07 and 1.52% indicating good precision and there was no significant difference between the assays tested on the same day or different days.
The accuracy of the developed method was determined by the standard addition technique which was done by adding three different concentrations of the standard drug solution to the tablet solution. The results obtained from the standard addition method were given in Table 5.
LOD and LOQ were calculated as 3σ/S and 10σ/S, respectively, where S is the slope of the calibration curve and σ is the standard deviation of y-intercept of regression equation [11].
Assay for the tablets
The proposed method was successfully applied to the analysis of marketed product (Onglyza Tablet ® ). The obtained results are satisfactorily accurate and precise as indicated by the good % recovery and SD<2 ( Table 6). The related chromatograms were given in Figures 9 and 10.
Conclusion
The developed and validated HPLC method is selective, rapid and precise for the determination saxagliptin and metformin from bulk and it could be applied to the tablets. Since the method separates SAX from its degradation products and tablet excipients it could also be used for quality control assay and for stability studies. The results obtained from the stress testing showed that saxagliptin is unstable under hydrolysis, oxidation and thermal stress. Therefore, care should be taken in the manufacturing process and during the storage of the product. | v3-fos-license |
2018-11-15T18:38:58.456Z | 2018-11-08T00:00:00.000 | 53248256 | {
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} | pes2o/s2orc | Targeting Groups Employed in Selective Dendrons and Dendrimers
The design of compounds with directed action to a defined organ or tissue is a very promising approach, since it can decrease considerably the toxicity of the drug/bioactive compound. For this reason, this kind of strategy has been greatly important in the scientific community. Dendrimers, on the other hand, comprise extremely organized macromolecules with many peripheral functionalities, stepwise controlled synthesis, and defined size. These nanocomposites present several biological applications, demonstrating their efficiency to act in the pharmaceutical field. Considering that, the main purpose of this review was describing the potential of dendrons and dendrimers as drug targeting, applying different targeting groups. This application has been demonstrated through interesting examples from the literature considering the last ten years of publications.
Introduction
The drug targeting of cells, tissues or specific diseases is a powerful tool in the treatment of pathological disorders, since it may increase the chemotherapeutic effect and decrease the toxicity in normal tissues [1]. Dendrimers, on the other hand, have been extensively applied in this field, once drugs can be encapsulated inside them or conjugated in their surfaces through covalent bonds [1][2][3].
Dendrimers represent an emerging class of low polydispersity hyperbranched macromolecules, which confer unique features such as: significant control over the molecular size, high branching density, nanoscale size and great surface functionality [4][5][6][7][8][9]. Those structures are composed of multifunctional core, which allows branches coupling, repeated branches layers from core named as dendrons and functional surface groups [4,10,11] (Figure 1). The first unity containing a core substituted with dendrons results in the first dendrimer generation. According to the increase of branches number in the dendrimer structure, higher dendrimer generations can be obtained. Therefore, the second layer of repeated units leads to the second dendrimer generation and thus subsequently [12]. Regarding to the dendrimer synthesis, these compounds can be, mainly, synthesized by convergent or divergent approaches [3].
Two features contribute to the dendrimer complexity, as generation number and surface terminal groups. In relation to generations, there are dendrimers from the first up to the tenth generation, although in the seventh-generation steric hindrance between the branches occurs, decreasing the synthetic yield of these compounds. Also, high generations may have influence in dendrimer toxicity [4,16,17].
decreasing the synthetic yield of these compounds. Also, high generations may have influence in dendrimer toxicity [4,16,17]. In general, surface groups are anionic, cationic, or neutral and the toxicity studies have shown the cationic dendrimers as the most cytotoxic [14,18,19]. To overcome the cytotoxicity induced by dendrimers, diverse approaches have been developed based on suppression of the cationic surface through PEGylation, acetylation and chemical modification with anionic or neutral molecules [12].
It is also important to emphasize the multifunctional character of the dendrimers, which allow the linkage of different ligands of multiple receptors, achieving the selectivity and even synergistic effect [40].
Based on the foregoing interesting characteristics, this review aimed at describing the targeting groups employed in dendrimer and dendrons to obtain targeted drug delivery systems, which showed to be important in the field of pharmaceutical research. Table 1 reflects the targeting groups diversity related to the disease and the dendrimer architectures. The RGD modified dendrimer showed a higher therapeutic effect on melanoma cells and a higher accumulation in tumor regions [41] Cancer PAMAM The modified PAMAM dendrimer showed a selective intake in melanoma cells. However, showed a low tumor intake [42] Cancer Janus The modified dendrimer showed an increased targeting property and optimized release property [43] Cancer PPI dendron The modified dendron showed a significantly higher cellular uptake and selectivity for lysosomes [44] Cancer PAMAM Higher in vitro uptake and in vivo accumulation [45] Cancer PEG The dendrimer showed an excellent load capacity and synergic effect of both substituents in vivo and in vitro [46] Cancer PAMAM The modified dendrimer showed a greater cellular uptake of 5-FU [47] Cancer PLL The dendrimer showed a high cellular uptake and could be carried into lysosomal compartments [48] Cancer Substance P dendron The SP dendron showed a higher cellular uptake and decreased tumor cell viability [49] Cancer PAMAM The (GFLG) dendrimer-DOX was more accumulated in tumor area than in liver and other organs [50] Cancer PLL dendron The dendritic drug delivery system showed better biosafety and higher in vitro cytotoxicity [51] Cancer PLL dendron The modified dendrimer demonstrated targeting ability at both in vitro and in vivo assays, also it exhibited tumor growth inhibition [52] Cancer DendGDP The conjugate dendrimers presented superior cell uptake than free DOX in vitro trials [3] Folate Cancer PPI The modified dendrimer showed lower toxicity and higher cellular uptake [53] Cancer PAMAM The modified dendrimer showed higher tumor cell cytotoxicity [54] Cancer PAMAM The FA modified dendrimer showed a lower healthy cell toxicity and higher cancer cell accumulation [55] Cancer PAMAM The FA modified dendrimer showed a better activity against tumor cells [56] Cancer PAMAM The designed G5 PAMAM coupled to MTX and FA was more efficient and presented higher action in tumor cells [57] Cancer PAMAM The modified dendrimer showed a lower toxicity and increased half-life [58] Cancer PAMAM There was not a difference in the activity between the G3 and G5 dendrimer. Both showed a good delivery system for the drug [59] Cancer PAMAM The dendrimer improved the solubility of the flavonoid and showed a high selectivity for HeLa cells [60] Cancer PAMAM The dendrimer showed a high accumulation on tumor sites, which indicates a promising use as drug delivery and diagnostics [61] Arthritis PAMAM The dendrimer showed a higher plasma concentration, higher selectivity, and lower gastric toxicity [62] Arthritis PAMAM The indomethacin-FA-dendrimer showed a more controlled release than other dendrimers [63] Cancer PAMAM These dendrimers showed high loading capacity, low cytotoxicity, and redox-driven cleavage through disulfide bridges [64] Carbohydrates Cancer PAMAM The conjugated dendrimers showed a much higher HepG2 uptake than the non-conjugated [65] Malaria PPI The galactose conjugated dendrimer was able to decrease the hemolytic property of the primaquine [66] HIV PPI Dendrimers were able to decrease the drug toxicity. However, the mannose derivative presented 12-times-higher cellular uptake when compared with that free drug [67] HIV TPPI Both dendrimers showed good results in cell uptake assays, since mannose interacted with lectin receptor and TPPI was absorbed via phagocytosis [67] Cancer Arginine dendron In vitro assays exhibited excellent biocompatibility. LP-g-G3P/DOX was internalized into the hepatoma carcinoma cells, inhibiting cell proliferation [68] Cancer PPI The dendrimer exhibited lower hemolytic property than free drug and a better accumulation in the brain than in other organs, such as liver and kidney [69] Monoclonal antibodies Cancer PAMAM The modified dendrimer was capable of selectively bind to the prostate specific membrane antigen receptor [70] Cancer PAMAM This molecule presented high affinity for HER, which resulted in significant internalization of IL-6-G5 PAMAM dendrimers into HeLa cells [1] Pharmaceutics 2018, 10, 219 5 of 27
Peptides as Targeting Groups
Some overexpressed enzymes in cells/tissues have been interesting by means of their specific peptides used as targeting groups to achieve selectivity.
Cathepsin B, a lysosomal cysteine protease overexpressed in several cancer tissues, is known to degrade extracellular matrix during invasion and metastasis. Additionally, it is commonly observed as a prognostic factor in breast tumor. The tetrapeptide Gly Phe-Leu-Gly (GFLG) is a substrate of cathepsin B, which demonstrated good blood stability during delivery and allows intralysosomal drug release after endocytosis. PEGylated PAMAM dendrimers conjugated to DOX and GFLG spacer were described as an enzyme-responsive drug delivery system for breast tumor therapy. Peptide dendrimer-DOX compounds improved in vivo antitumor activity over commercial DOX formulation at the same dose. Additionally, the dendrimer provided lower toxicity as analyzed by acute changes in body weight, blood cell counts and histological analysis [81].
With the purpose of decreasing cytotoxicity and clearance, increasing selectivity and accumulating drug in breast tumor area, Li and coworkers [50] synthesized a dendrimer with two different dendrons ( Figure 2). One of them, mPEGylated, was used to enhance molecular weight and size, decreasing renal filtration, thus accumulating it by enhanced permeability and retention (EPR) effect. Other PEGylated dendron, glycylphenylalanyl-leucylglycine conjugated with DOX (mPEGylated dendrimer-GLFG-DOX) is considered substrate for cathepsin. Through ex vivo studies, free DOX presented lower accumulation in tumor area as well as higher amount in other tissues such as in liver and kidneys. Meanwhile, the dendrimer was more accumulated in tumor area than in liver and other organs. In addition, they described this dendrimer as a possible control for tumor metastasis and in tumor growth inhibition, showing better effects than free DOX. The same group also used GFLG dendrimer, linking a targeting group conjugated to DOX (GFLG-DOX) on the surface. Applying the same strategy showed previously, the authors analyzed the dendrimers for ovarian tumor treatment, observing less activity than free DOX. Notwithstanding, a significant cytotoxicity against normal cell line was not observed at in vitro assays. On the contrary, at in vivo test, the dendrimer demonstrated higher anticancer efficacy than free DOX and could induce higher apoptosis levels. Histological analysis showed no toxicity and dendrimer presented more accumulation in tumor tissue [81].
Peptides as Targeting Groups
Some overexpressed enzymes in cells/tissues have been interesting by means of their specific peptides used as targeting groups to achieve selectivity.
Cathepsin B, a lysosomal cysteine protease overexpressed in several cancer tissues, is known to degrade extracellular matrix during invasion and metastasis. Additionally, it is commonly observed as a prognostic factor in breast tumor. The tetrapeptide Gly Phe-Leu-Gly (GFLG) is a substrate of cathepsin B, which demonstrated good blood stability during delivery and allows intralysosomal drug release after endocytosis. PEGylated PAMAM dendrimers conjugated to DOX and GFLG spacer were described as an enzyme-responsive drug delivery system for breast tumor therapy. Peptide dendrimer-DOX compounds improved in vivo antitumor activity over commercial DOX formulation at the same dose. Additionally, the dendrimer provided lower toxicity as analyzed by acute changes in body weight, blood cell counts and histological analysis [81].
With the purpose of decreasing cytotoxicity and clearance, increasing selectivity and accumulating drug in breast tumor area, Li and coworkers [50] synthesized a dendrimer with two different dendrons ( Figure 2). One of them, mPEGylated, was used to enhance molecular weight and size, decreasing renal filtration, thus accumulating it by enhanced permeability and retention (EPR) effect. Other PEGylated dendron, glycylphenylalanyl-leucylglycine conjugated with DOX (mPEGylated dendrimer-GLFG-DOX) is considered substrate for cathepsin. Through ex vivo studies, free DOX presented lower accumulation in tumor area as well as higher amount in other tissues such as in liver and kidneys. Meanwhile, the dendrimer was more accumulated in tumor area than in liver and other organs. In addition, they described this dendrimer as a possible control for tumor metastasis and in tumor growth inhibition, showing better effects than free DOX. The same group also used GFLG dendrimer, linking a targeting group conjugated to DOX (GFLG-DOX) on the surface. Applying the same strategy showed previously, the authors analyzed the dendrimers for ovarian tumor treatment, observing less activity than free DOX. Notwithstanding, a significant cytotoxicity against normal cell line was not observed at in vitro assays. On the contrary, at in vivo test, the dendrimer demonstrated higher anticancer efficacy than free DOX and could induce higher apoptosis levels. Histological analysis showed no toxicity and dendrimer presented more accumulation in tumor tissue [81]. GFLG dendrons conjugated to DOX were developed to self-assemble into nanoparticles. PEGylated dendron-GFLG-DOX demonstrated responsive-enzyme ability and, according to the in vitro assays, nanoparticles could kill breast cancer cells. Moreover, conjugated dendrimer was safer and showed higher antitumor activity than free DOX, due to enzyme-sensitive linker employed to compose PEGylated peptide dendron [50]. The same research group synthetized PEGylated PLL dendron, using Pro-Val-Gly-Leu-Ile-Gly (PVGLIG) peptide as spacer group, which is sensitive to matrix metalloprotease-2/metalloprotease-9, and was functionalized with DOX. This conjugated drug was evaluated for delivery in breast cancer cells through two mechanisms: increase of EPR factor due to PEG and via substrate enzyme-sensitive. The dendritic drug delivery system showed better biosafety, comparatively to free DOX, through in vivo assays. This compound exhibited higher in vitro cytotoxicity in breast tumor cells, in the presence of metalloprotease-2 enzyme. The association of peptide dendron PEGylation and enzyme-sensitive property revealed to be an efficient and safe possibility for drug delivery system.
As reported in another work, a Janus peptide dendron-drug, composed by a sequence of PVGLIG peptide, sensitive to metalloproteases 2 and 9, and PEG, was synthesized [82]. Janus dendrimer, also called surface-block codendrimer, is a kind of compound containing a double-faced head with different properties [83]. DOX side effects with the dendrimer were lower comparatively to free drug administration. In addition, DOX activity was the same as the free drug after the complex administration Transferrin (Tf) is a targeting group for brain action, due to Tf receptor overexpression on the brain capillaries endothelial surfaces. Besides that, several cancer tissues overexpress Tf receptor on the surface of tumor cells, which provides iron to cells growth and participates in their survival [52,84]. HAIYPRH is a peptide with high affinity to Tf receptor and PEGylated PAMAM dendrimers were functionalized with it to deliver loaded DOX. The complex showed a high rate of internalization and selectivity, compared to free drug.
Based on the same approach, wheat germ agglutinin (WGA) showed high affinity for the brain capillary endothelium, with high binding for tumor cells and reduced toxicity for normal cells. It was used so that PEGylated dendrimers containing both directing groups (Tf and WGA) and encapsulated DOX were designed to obtain targeting systems ( Figure 3). Those dendrimers could cross blood-brain barrier and improve the cell uptake by brain tumor. The authors related the decrease of DOX cytotoxicity to the normal tissues, while it inhibited the growth of C6 glioma cells. Additionally, if there was DOX accumulation in tumor tissue, the inhibition rate to the C6 glioma cells increased, resulting in the blood-brain barrier transport improvement and synergistic effect of both endocytosis mechanisms (Tf and WGA) [84].
With the purpose of improving blood-brain barrier transport and drug accumulation in the glioma cells Li and colleagues [52] designed two dendrimer types based on G4 PAMAM dendrimer. One of them has DOX, PEG and Tf (G4-DOX-PEG-Tf) added on G4 PAMAM surface and the other has dual-targeting composed of DOX, PEG, Tf and tamoxifen added in G4 PAMAM surface (G4-DOX-PEG-Tf-tamoxifen). Dual-targeting dendrimer presented better transport ability in vitro blood-brain barrier trials. In addition, G4-DOX-PEG-Tf-tamoxifen had enhanced cytotoxicity against tumor cells and improved the drug delivery.
The association of chemotherapy and gene therapy is a promising strategy for treatment of cancer as, together, these techniques can provide synergic actions. Liu and colleagues [85] synthetized a copolymer with β-cyclodextrin (CD) core and poly(L-lysine) dendron (PLLD) to co-deliver docetaxel antitumor drug and MMP-9 siRNA plasmid for nasopharyngeal carcinoma therapy. MMP-9 siRNA was more effective for nasopharyngeal carcinoma therapy, when a folate modified (FA-CD-PLLD) was employed as targeting moiety. In addition, FA-CD-PLLD showed good blood compatibility and non-toxicity. According to the authors, this is a promising strategy for nasopharyngeal carcinoma therapy. . PEGylated dendrimers containing Tf and WGA (targeting groups) and encapsulated DOX for drug delivery to brain tumor [84].
Lee and coworkers [3] also applied the tetrapeptide GFLG for anticancer drug targeting, aiming to evaluate if the tetrapeptide can deliver DOX to the site of action. The conjugate dendrimers presented superior cell uptake than free DOX in vitro trials, although the tumor growth inhibition was lower than the DOX alone. This might have happened due to the drug delayed release from the dendrimer. In vivo studies showed positive results such as high dendrimer concentration on the induced tumor, prolonged accumulation on the tumor site and low deposition in other organs.
The protein αvβ3 integrin is overexpressed in melanomas, glioblastomas, and ovary cancer and, most importantly, it presents low expression in normal cells. The tripeptide arginine-glycineaspartate (RGD) presents high interaction with cancer-related integrin and it is one of the most studied targeting group for cancer therapy. PAMAM (poly(amidoamine)) dendrimers coupled to the RGD peptide ensured the release of cytotoxic agents into the illness cells, showing no toxicity to normal ones [42]. The respective dendrimers were also covalently conjugated to RGD and they were applied as imaging agent for angiogenesis.
Using the same approach, Ma and coworkers [45] conjugated PAMAM with RGD and encapsulated methotrexate (MTX) and observed this conjugate could reduce MTX toxicity mostly due to the slow drug release from the carrier. In vivo assays showed higher accumulation in tumor site, when compared to free MTX and non-functionalized dendrimer. Other examples comprehended Alexa Fluor 488, biotin or MTX connected to the branches. According to the authors, these findings could be a breakthrough development to delivery systems of multiple drugs and imaging agents.
Jiang and coworkers [43] developed a dual-targeting Janus dendrimer based on peptide dendrons for bone cancer. The branch was designed by peptide functionalized 5-fluorouracil (5-FU) and RGD (bone targeting group due to interaction with αvβ3 integrin receptor overexpressed in bone metastatic cells and osteoclasts). Four different dendrons that demonstrated binding ability to HAP (hydroxyapatite-inorganic component in hard tissues as bone and tooth) were synthesized. These target compounds could reduce toxicity in normal tissues and sustain the release.
Other strategy to target bone tissue was to use poly aspartic acid (Aspn). In 2009, Ouyanga and coworkers [86] developed dendritic compounds with two or three fragments of Asp(4-6) to increase the delivery of naproxen and improve its therapeutic index. All compounds presented good pharmacokinetic and pharmacodynamic properties, although the trimer had a slower binding rate than the dimer, due to steric hindrance. Other Janus dendrimers changed sequences of aspartic and glutamic acid aiming to deliver naproxen to the bone tissue with no significant differences [87].
PPI (poly(propyleneimine)) dendrons were planned based on octa-guanidine residues as a molecular carrier. These compounds contained DOX, as well as lysosomal peptide to mimic cellpenetrating peptide features. The nanocarrier named as G8-PPI showed to be non-toxic and higher cellular uptake ability compared to arginine-octamer. It also exhibited excellent selectivity towards Lee and coworkers [3] also applied the tetrapeptide GFLG for anticancer drug targeting, aiming to evaluate if the tetrapeptide can deliver DOX to the site of action. The conjugate dendrimers presented superior cell uptake than free DOX in vitro trials, although the tumor growth inhibition was lower than the DOX alone. This might have happened due to the drug delayed release from the dendrimer. In vivo studies showed positive results such as high dendrimer concentration on the induced tumor, prolonged accumulation on the tumor site and low deposition in other organs.
The protein αvβ3 integrin is overexpressed in melanomas, glioblastomas, and ovary cancer and, most importantly, it presents low expression in normal cells. The tripeptide arginine-glycine-aspartate (RGD) presents high interaction with cancer-related integrin and it is one of the most studied targeting group for cancer therapy. PAMAM (poly(amidoamine)) dendrimers coupled to the RGD peptide ensured the release of cytotoxic agents into the illness cells, showing no toxicity to normal ones [42]. The respective dendrimers were also covalently conjugated to RGD and they were applied as imaging agent for angiogenesis.
Using the same approach, Ma and coworkers [45] conjugated PAMAM with RGD and encapsulated methotrexate (MTX) and observed this conjugate could reduce MTX toxicity mostly due to the slow drug release from the carrier. In vivo assays showed higher accumulation in tumor site, when compared to free MTX and non-functionalized dendrimer. Other examples comprehended Alexa Fluor 488, biotin or MTX connected to the branches. According to the authors, these findings could be a breakthrough development to delivery systems of multiple drugs and imaging agents.
Jiang and coworkers [43] developed a dual-targeting Janus dendrimer based on peptide dendrons for bone cancer. The branch was designed by peptide functionalized 5-fluorouracil (5-FU) and RGD (bone targeting group due to interaction with αvβ3 integrin receptor overexpressed in bone metastatic cells and osteoclasts). Four different dendrons that demonstrated binding ability to HAP (hydroxyapatite-inorganic component in hard tissues as bone and tooth) were synthesized. These target compounds could reduce toxicity in normal tissues and sustain the release.
Other strategy to target bone tissue was to use poly aspartic acid (Asp n ). In 2009, Ouyanga and coworkers [86] developed dendritic compounds with two or three fragments of Asp (4)(5)(6) to increase the delivery of naproxen and improve its therapeutic index. All compounds presented good pharmacokinetic and pharmacodynamic properties, although the trimer had a slower binding rate than the dimer, due to steric hindrance. Other Janus dendrimers changed sequences of aspartic and glutamic acid aiming to deliver naproxen to the bone tissue with no significant differences [87]. PPI (poly(propyleneimine)) dendrons were planned based on octa-guanidine residues as a molecular carrier. These compounds contained DOX, as well as lysosomal peptide to mimic cellpenetrating peptide features. The nanocarrier named as G8-PPI showed to be non-toxic and higher cellular uptake ability compared to arginine-octamer. It also exhibited excellent selectivity towards lysosomes in HeLa cells, being considered, therefore, an important candidate for targeting cancer therapy [46]. The same research group has developed dendron (G8-PPI) with FA, targeting group to folate receptor) and peptide FKE (Phe-Lys-Glu-substrate for cathepsin B overexpressed on neoplastic cells). G8-FKE-FA-DOX ( Figure 4) demonstrated an excellent response to folate receptor-targeting, as well as increased cellular uptake and intracellular lysosome-mediated DOX delivery. In addition, G8-FKE-FA-DOX triggered the programmed cell death through extrinsic and intrinsic pathways, without affecting normal and folate receptor-negative cells [44]. lysosomes in HeLa cells, being considered, therefore, an important candidate for targeting cancer therapy [46]. The same research group has developed dendron (G8-PPI) with FA, targeting group to folate receptor) and peptide FKE (Phe-Lys-Glu-substrate for cathepsin B overexpressed on neoplastic cells). G8-FKE-FA-DOX ( Figure 4) demonstrated an excellent response to folate receptor-targeting, as well as increased cellular uptake and intracellular lysosome-mediated DOX delivery. In addition, G8-FKE-FA-DOX triggered the programmed cell death through extrinsic and intrinsic pathways, without affecting normal and folate receptor-negative cells [44]. Tat peptide, GRKKRRQRRRPQ, a transactivator of human immunodeficiency virus and a cellpenetrating peptide, can also be used to target cancer cells. G4 PAMAM dendrimer was conjugated to it to increase its internalization. Assays in heart, lung and spleen tissues showed that it presented low accumulation in healthy organs [88].
HER (Human Epidermal Growth Factor Receptor) is an important target for diverse types of cancer, being peptide H6 one of its ligands. In this context, PEGylated G4 PAMAM dendrimers were functionalized with peptide H6 to carry DOX for specific action in breast cancer cells. They did not show toxicity, while the DOX-encapsulated dendrimers presented high cytotoxicity [89]. Directed drug delivery system for pancreatic cancer was designed using tumor target peptide plectin-1, which is a biomarker for this kind of cancer, and siRNA. Nuclear receptor siRNA reduces the expression of antiapoptotic proteins, such as Bcl-2 and survivin. Consequently, tumor growth is not expected due to induction of apoptosis. The hydrophobic drug conjugated was paclitaxel and a synergistic effect was observed using siRNA. In vitro assays showed high dendrimers accumulation because of receptor-mediated endocytosis. Additionally, there was an increase in cell uptake and high transfection in Panc-1 cell lines [90].
The aptamer AS1411 is a selective oligonucleotide that binds to nucleolin, a nucleus membrane protein, which is overexpressed in some tumor cells, as gastric cancer. In general, aptamers are small single stranded RNA that can recognize and link, with high affinity, to other molecules by tridimensional folding [91]. Behrooz and colleagues [47] designed targeted polymers composed of PEGylated PAMAM dendrimers functionalized with aptamer AS1411 to deliver 5-FU and they succeeded. Based on the role of mucin on tumor growth and metastases [48] and in the importance of aptamers as targeting group, Masuda and coworkers [92] designed a sixth-generation glutamic Tat peptide, GRKKRRQRRRPQ, a transactivator of human immunodeficiency virus and a cell-penetrating peptide, can also be used to target cancer cells. G4 PAMAM dendrimer was conjugated to it to increase its internalization. Assays in heart, lung and spleen tissues showed that it presented low accumulation in healthy organs [88].
HER (Human Epidermal Growth Factor Receptor) is an important target for diverse types of cancer, being peptide H6 one of its ligands. In this context, PEGylated G4 PAMAM dendrimers were functionalized with peptide H6 to carry DOX for specific action in breast cancer cells. They did not show toxicity, while the DOX-encapsulated dendrimers presented high cytotoxicity [89]. Directed drug delivery system for pancreatic cancer was designed using tumor target peptide plectin-1, which is a biomarker for this kind of cancer, and siRNA. Nuclear receptor siRNA reduces the expression of antiapoptotic proteins, such as Bcl-2 and survivin. Consequently, tumor growth is not expected due to induction of apoptosis. The hydrophobic drug conjugated was paclitaxel and a synergistic effect was observed using siRNA. In vitro assays showed high dendrimers accumulation because of receptor-mediated endocytosis. Additionally, there was an increase in cell uptake and high transfection in Panc-1 cell lines [90].
The aptamer AS1411 is a selective oligonucleotide that binds to nucleolin, a nucleus membrane protein, which is overexpressed in some tumor cells, as gastric cancer. In general, aptamers are small single stranded RNA that can recognize and link, with high affinity, to other molecules by tridimensional folding [91]. Behrooz and colleagues [47] designed targeted polymers composed of PEGylated PAMAM dendrimers functionalized with aptamer AS1411 to deliver 5-FU and they succeeded. Based on the role of mucin on tumor growth and metastases [48] and in the importance of aptamers as targeting group, Masuda and coworkers [92] designed a sixth-generation glutamic acid modified with PLL dendrimer coupled to anti-MUC1 aptamer responsible for targeting several epithelial tumors. They observed that the dendrimer presented high cellular uptake and could be carried into the lysosomal and endosomal compartments. On the other hand, Taghdisi and coworkers [93] developed a polymer based on DNA dendrimer composed of MUC1 and AS1411 aptamers, employing the anticancer drug epirubicin, which shows cardiotoxicity and brown marrow suppression. Selectivity was achieved as the cellular viability assay demonstrated that normal cells were not affected, while tumor cells were destroyed after administration.
Neurokinin-1 receptors, overexpressed in some cancer cells, are part of a family of undecapeptides of tachykinin neuropeptides. The substance P (SP) is rapidly internalized due to neurokinin-1 interaction. Therefore, Wu and coworkers designed a SP dendron with branches containing 5-FU and near-infrared labeled ( Figure 5). SP dendron showed effectiveness in decreasing cell viability of tumor cells when compared to normal cells, which suggests an effective targeting feature [49].
Pharmaceutics 2018, 10, x FOR PEER REVIEW 9 of 27 epithelial tumors. They observed that the dendrimer presented high cellular uptake and could be carried into the lysosomal and endosomal compartments. On the other hand, Taghdisi and coworkers [93] developed a polymer based on DNA dendrimer composed of MUC1 and AS1411 aptamers, employing the anticancer drug epirubicin, which shows cardiotoxicity and brown marrow suppression. Selectivity was achieved as the cellular viability assay demonstrated that normal cells were not affected, while tumor cells were destroyed after administration. Neurokinin-1 receptors, overexpressed in some cancer cells, are part of a family of undecapeptides of tachykinin neuropeptides. The substance P (SP) is rapidly internalized due to neurokinin-1 interaction. Therefore, Wu and coworkers designed a SP dendron with branches containing 5-FU and near-infrared labeled ( Figure 5). SP dendron showed effectiveness in decreasing cell viability of tumor cells when compared to normal cells, which suggests an effective targeting feature [49]. . SP dendron containing branches conjugated to 5-FU for brain targeting [49].
The use of peptides as directing group for dendrimer nanocarriers for drugs has arouse increasing interest despite of their instability. The approaches herein discussed gave a panel of what can be done for achieving selectivity with these groups.
FA as Targeting Group
FA is an important targeting group once folate receptor is overexpressed in diverse types of human carcinomas, such as ovary, colon, lung, and breast, being one of the most studied ligands. Folate receptor is a tumor marker that binds to folate-drug conjugates with high affinity, which can provide drug delivery by receptor-mediated endocytosis ( Figure 6) [56,94,95]. The use of peptides as directing group for dendrimer nanocarriers for drugs has arouse increasing interest despite of their instability. The approaches herein discussed gave a panel of what can be done for achieving selectivity with these groups.
FA as Targeting Group
FA is an important targeting group once folate receptor is overexpressed in diverse types of human carcinomas, such as ovary, colon, lung, and breast, being one of the most studied ligands. Folate receptor is a tumor marker that binds to folate-drug conjugates with high affinity, which can provide drug delivery by receptor-mediated endocytosis ( Figure 6) FA-conjugated G5 PPI (poly(propylenimine) dendrimers were designed by loading DOX in the interior of dendrimer [53]. Other report showed DOX encapsulated in FA-G5 dendrimer and the DOX was released via photocleavable action [96]. Wang and colleagues [54], conversely, described G5 PAMAM dendrimers conjugated to FA, labeled with fluorescein isothiocyanate carrying 2methoxyestradiol (2-ME), which led to cell death. Dendrimer release rate was more controlled than free drug in two pH conditions (7.4 and 5.0). No toxicity was observed in non-drug dendrimer and only 2-ME dendrimer was able to lower cell viability in KB cells.
Experiments employing KB cells with overexpressed folate receptor, as well as KB cells containing normal FA receptor, demonstrated that 2-ME dendrimer was more recognized by cells with overexpressed FA receptor. This research group applied the same strategy for DOX, which showed sustained release without pH influence [97]. Similar approaches were used by Majoros and coworkers [55] and Shukla and coworkers [98]. Both studies used FA to deliver MTX to cancer tissues and the conclusions were the same: the conjugate was not toxic to healthy cells and caused cell growth inhibition. Other study performed by Singh and his group [99] aimed to synthesize PAMAM dendrimers with FA and PEG as targeting moieties. These dendrimers were further loaded with 5-FU to evaluate their capacity to specifically deliver this drug to cancer cells. PEG moiety increases the circulation time of the drug and the folate moiety delivers the 5-FU in a site-specific way in both receptor-mediated endocytosis and through EPR due to reduced lymphatic drainage. This effect occurs in most solid tumors, to increase the vascular permeability to provide nutrients and oxygen in tumor area for their growth [100,101].
Thomas and coworkers (64) studied FA as a directing group covalently coupled to G5 PAMAM dendrimers as a selective delivery system to the MTX. Another research synthesized G5 PAMAM conjugated to MTX (Figure 7) with the purpose of enhancing affinity by folate receptor [102]. MTX was employed for its dual activity, as a targeting and cytotoxic agent. Therefore, G5-MTX displayed better activity against tumor cells and promoted more effectively their death in contrast with free drug in in vitro tests [103]. Myc and colleagues [57] also designed G5 PAMAM coupled to MTX and FA to confirm their specificity and efficacy. In cytotoxicity assays, dendrimer was more efficient and presented higher action in cells with overexpressed FA receptor comparatively to normal cells, inhibiting the tumor growth. FA-conjugated G5 PPI (poly(propylenimine) dendrimers were designed by loading DOX in the interior of dendrimer [53]. Other report showed DOX encapsulated in FA-G5 dendrimer and the DOX was released via photocleavable action [96]. Wang and colleagues [54], conversely, described G5 PAMAM dendrimers conjugated to FA, labeled with fluorescein isothiocyanate carrying 2-methoxyestradiol (2-ME), which led to cell death. Dendrimer release rate was more controlled than free drug in two pH conditions (7.4 and 5.0). No toxicity was observed in non-drug dendrimer and only 2-ME dendrimer was able to lower cell viability in KB cells.
Experiments employing KB cells with overexpressed folate receptor, as well as KB cells containing normal FA receptor, demonstrated that 2-ME dendrimer was more recognized by cells with overexpressed FA receptor. This research group applied the same strategy for DOX, which showed sustained release without pH influence [97]. Similar approaches were used by Majoros and coworkers [55] and Shukla and coworkers [98]. Both studies used FA to deliver MTX to cancer tissues and the conclusions were the same: the conjugate was not toxic to healthy cells and caused cell growth inhibition. Other study performed by Singh and his group [99] aimed to synthesize PAMAM dendrimers with FA and PEG as targeting moieties. These dendrimers were further loaded with 5-FU to evaluate their capacity to specifically deliver this drug to cancer cells. PEG moiety increases the circulation time of the drug and the folate moiety delivers the 5-FU in a site-specific way in both receptor-mediated endocytosis and through EPR due to reduced lymphatic drainage. This effect occurs in most solid tumors, to increase the vascular permeability to provide nutrients and oxygen in tumor area for their growth [100,101].
Thomas and coworkers (64) studied FA as a directing group covalently coupled to G5 PAMAM dendrimers as a selective delivery system to the MTX. Another research synthesized G5 PAMAM conjugated to MTX (Figure 7) with the purpose of enhancing affinity by folate receptor [102]. MTX was employed for its dual activity, as a targeting and cytotoxic agent. Therefore, G5-MTX displayed better activity against tumor cells and promoted more effectively their death in contrast with free drug in in vitro tests [103]. Myc and colleagues [57] also designed G5 PAMAM coupled to MTX and FA to confirm their specificity and efficacy. In cytotoxicity assays, dendrimer was more efficient and presented higher action in cells with overexpressed FA receptor comparatively to normal cells, inhibiting the tumor growth. It is important to notice that the conjugation of PAMAM with FA reduces the cationic toxicity of the dendrimer, as shown by Kersharwani and coworkers [58] in anticancer formulations in the drug targeting.
PAMAM G3 and G5 containing FA and ursolic acid (UA-anticancer agent) were developed to overcome UA pharmacokinetic problems and provide the selectivity towards cancer cells. There was no difference regarding the release rate between G3 and G5 dendrimers. The findings suggested these compounds as good delivery systems [104].
FA was also conjugated to PAMAM dendrimers to load baicalin to improve water solubility and tumor selectivity. Even though this flavonoid presents anticancer effects, it displays low water solubility and bioavailability [105]. In another work, PAMAM dendrimers functionalized with FA were designed to deliver a highly hydrophobic flavonoid derivative, the 3,4-difluorobenzylidene diferuloylmethane. This study aimed to improve the water solubility and achieve the transport selectivity to overexpressed FA receptors in HeLa and ovarian cancer cells. Targeted dendrimers exhibited remarkable antitumor activity with greater accumulation in FA receptor-overexpressing cells, larger apoptosis rate, high expression of tumor suppressor phosphatase and tensin homolog, and inhibition of nuclear factor kappa B. All findings indicated the selective ability of this system [60]. The same research group developed PAMAM dendrimers composed of superparamagnetic iron oxide nanoparticle core (SPION), ornamented with FA on surface (FA-PAMAM) and containing 3,4difluorobenzylidene diferuloylmethane via encapsulation to increase solubility and selectivity for ovarian and HeLa cancer cells (Figure 8). The compounds displayed a better anticancer action in targeted dendrimers than in non-targeted derivatives. Also, a larger population of cells suffering apoptosis due to upregulation of tumor suppressor phosphatase and tensin homolog, caspase 3, and inhibition of NF-κB were shown. In addition, these compounds have been studied as imaging agent in diagnostic, enhancing Magnetic Resonance contrast and fluorescence microscopy [61]. It is important to notice that the conjugation of PAMAM with FA reduces the cationic toxicity of the dendrimer, as shown by Kersharwani and coworkers [58] in anticancer formulations in the drug targeting.
PAMAM G3 and G5 containing FA and ursolic acid (UA-anticancer agent) were developed to overcome UA pharmacokinetic problems and provide the selectivity towards cancer cells. There was no difference regarding the release rate between G3 and G5 dendrimers. The findings suggested these compounds as good delivery systems [104].
FA was also conjugated to PAMAM dendrimers to load baicalin to improve water solubility and tumor selectivity. Even though this flavonoid presents anticancer effects, it displays low water solubility and bioavailability [105]. In another work, PAMAM dendrimers functionalized with FA were designed to deliver a highly hydrophobic flavonoid derivative, the 3,4-difluorobenzylidene diferuloylmethane. This study aimed to improve the water solubility and achieve the transport selectivity to overexpressed FA receptors in HeLa and ovarian cancer cells. Targeted dendrimers exhibited remarkable antitumor activity with greater accumulation in FA receptor-overexpressing cells, larger apoptosis rate, high expression of tumor suppressor phosphatase and tensin homolog, and inhibition of nuclear factor kappa B. All findings indicated the selective ability of this system [60]. The same research group developed PAMAM dendrimers composed of superparamagnetic iron oxide nanoparticle core (SPION), ornamented with FA on surface (FA-PAMAM) and containing 3,4-difluorobenzylidene diferuloylmethane via encapsulation to increase solubility and selectivity for ovarian and HeLa cancer cells (Figure 8). The compounds displayed a better anticancer action in targeted dendrimers than in non-targeted derivatives. Also, a larger population of cells suffering apoptosis due to upregulation of tumor suppressor phosphatase and tensin homolog, caspase 3, and inhibition of NF-κB were shown. In addition, these compounds have been studied as imaging agent in diagnostic, enhancing Magnetic Resonance contrast and fluorescence microscopy [61]. Pharmaceutics 2018, 10, x FOR PEER REVIEW 12 of 27 FA can also be used as directing group for inflammatory tissues. Indomethacin (anti-arthritis drug) was encapsulated in a G3.5 PAMAM dendrimer functionalized with PEG and FA. The study demonstrated the increase of plasma residence time of the complexes, as well as their higher concentration in inflamed tissue, reducing the stomach bleeding [62]. In other study four types of dendrimers were proposed contained different composition in terms of FA. The results suggested the folate amount provides an enhancement of the controlled delivery system. Indomethacin-FAdendrimers increased plasma circulation time and reduced the cellular uptake by reticuloendothelial system [63].
PAMAM dendrimer and dendron have provided high ability for drug and gene delivery, exhibiting stability, and creating complexes with DNA. Dendron coated mesoporous particles have also been used for intracellular plasmid-DNA delivery. Mesoporous silica nanoparticles have attracted interest due to their multifunctional properties and have been studied as a template for drug delivery. Weiss and colleagues [64] investigated the application of mesoporous silica nanoparticles coated with PAMAM dendrons and FA for drug targeting to cancer cells. These dendrimers showed high loading capacity, low cytotoxicity, and redox-driven cleavage through disulfide bridges. Their targeting potential were able to enhance cellular uptake.
Dendron micelles were developed for a drug delivery platform based on nanoparticles able to carry the drug into polyethylene glycol corona. The compounds were developed using various PEGs molecular weight to build the dendrons. Moreover, the conjugated constituents were varied to dendrons-FA and incorporated into dendron micelles, obtaining self-assembled nanostructure based on copolymers, containing an amphiphilic triblock. According to the authors, these compounds may FA can also be used as directing group for inflammatory tissues. Indomethacin (anti-arthritis drug) was encapsulated in a G3.5 PAMAM dendrimer functionalized with PEG and FA. The study demonstrated the increase of plasma residence time of the complexes, as well as their higher concentration in inflamed tissue, reducing the stomach bleeding [62]. In other study four types of dendrimers were proposed contained different composition in terms of FA. The results suggested the folate amount provides an enhancement of the controlled delivery system. Indomethacin-FA-dendrimers increased plasma circulation time and reduced the cellular uptake by reticuloendothelial system [63].
PAMAM dendrimer and dendron have provided high ability for drug and gene delivery, exhibiting stability, and creating complexes with DNA. Dendron coated mesoporous particles have also been used for intracellular plasmid-DNA delivery. Mesoporous silica nanoparticles have attracted interest due to their multifunctional properties and have been studied as a template for drug delivery. Weiss and colleagues [64] investigated the application of mesoporous silica nanoparticles coated with PAMAM dendrons and FA for drug targeting to cancer cells. These dendrimers showed high loading capacity, low cytotoxicity, and redox-driven cleavage through disulfide bridges. Their targeting potential were able to enhance cellular uptake.
Dendron micelles were developed for a drug delivery platform based on nanoparticles able to carry the drug into polyethylene glycol corona. The compounds were developed using various PEGs molecular weight to build the dendrons. Moreover, the conjugated constituents were varied to dendrons-FA and incorporated into dendron micelles, obtaining self-assembled nanostructure based on copolymers, containing an amphiphilic triblock. According to the authors, these compounds may be employed further to design efficient targeted nanocarriers for the treatment of several diseases [108].
Considering the number of examples of FA-conjugate dendrimers, briefly presented herein, it is clear the importance of using this directing group either in therapeutic agents or as imaging agents.
Carbohydrates as Targeting Groups
The use of carbohydrates is largely widespread in the research of drug targeting, considering the variety of receptors that can recognize them. As the kind of receptors changes from tissue to tissue, the targeting dendrimer containing carbohydrate may be more efficient [109]. The interaction with the carbohydrates in the membrane leads to selective internalization providing the carbohydrate receptor is specifically identified.
With this purpose, the asialoglycoprotein receptors (ASGPR) are highly employed as target. ASGPR are present on the surface of hepatic tumor cells, which allows the use of glycosylated nanocarriers for development of targeted drug delivery systems. N-acetylgalactosamine (NAcGal) is a selective sugar, substrate for ASGPR. Considering that, NacGal has been coupled to the G5 PAMAM dendrimers through peptide and thiourea bonds to act on ASGPR, being responsible for receptor-mediated endocytosis. These dendrimers functionalized with NAcGal were planned for drug targeting in hepatic cancer, aiming to compare the cell uptake with functionalized or non-functionalized dendrimer. According to the authors, NAcGal application is a promising strategy in drug targeting [65].
Another report showed the conjugation of galactose and DOX in PAMAM dendrimers to obtain a targeted drug for hepatoma cells [1]. Bhadra and coworkers [66] used galactose and primaquine conjugated with PPI dendrimer for malaria ( Figure 9). The galactose conjugated dendrimer was able to decrease the hemolytic property of the primaquine and target the erythrocytes better than other evaluated nanoparticles.
Dutta and coworkers [67] designed dendrimers composed of mannosylated-PPI (MPPI) containing efavirenz and PPI-efavirenz to reach macrophages. Once HIV virus is inside these immune system cells, it is expected that the dendrimer cited above can be more efficient to combat it. Both dendrimers were able to decrease the drug toxicity. However, the mannose derivative presented 12-times-higher cellular uptake when compared with that of free drug and the dendrimer conjugate without the target carbohydrate. In another study, applying the same antivirus agent, MPPI and T-Boc-glycine-PPI (TPPI) dendrimers were described to decrease serum concentrations and drug side effects. Both dendrimers showed good results in cell uptake assays, since mannose interacted with lectin receptor and TPPI was absorbed via phagocytosis. The same group designed other two types of G5 PPI dendrimers as carriers with and without mannose (MPPI and PPI, respectively). MPPI showed more prolonged release ratio than PPI and, in in vitro cellular uptake assays, MPPI was more effective than free lamivudine and free dendrimer. Furthermore, significant improvement of anti-HIV activity was observed by MPPI when compared to the free drug, which could be related to the cellular uptake enhancement [109]. Figure 9. PPI G5 dendrimer conjugated with galactose and encapsulated with primaquine [66].
In other study, mannosylated PEGtide dendrons, G1 to G5, were synthesized containing amino acids, PEG and functionalized with mannose to reach macrophage. These compounds demonstrated good water solubility and potential biocompatibility due to high PEG in dendritic structure. Mannosylated dendrons presented higher uptake than non-mannose derivatives in murine models. Therefore, dendrons were mannose-dependent receptor for cell uptake. PEGtide dendrons could be an efficient platform to drug delivery and imaging applications [59].
Potential targeted drug delivery system was developed with arginine G3 dendron covalently attached to a hydrophilic polysaccharide (pullulan), which is a neutral linear compound. The LP-g-G3P is composed of lactosylated pullulan-graft-G3arginine dendrons, which showed self-assemble ability, as well as small size particles, low polydispertion and higher affinity to lectin receptor ( Figure 10). DOX was encapsulated in LP-g-G3P through multiple interactions, was internalized into the hepatoma carcinoma cells, inhibiting cell proliferation. This type of targeted dendrimer showed to be a promising directed drug delivery system [68]. Figure 10. Chemical structure of LP-g-G3P an amphiphilic dendrimer [68]. In other study, mannosylated PEGtide dendrons, G1 to G5, were synthesized containing amino acids, PEG and functionalized with mannose to reach macrophage. These compounds demonstrated good water solubility and potential biocompatibility due to high PEG in dendritic structure. Mannosylated dendrons presented higher uptake than non-mannose derivatives in murine models. Therefore, dendrons were mannose-dependent receptor for cell uptake. PEGtide dendrons could be an efficient platform to drug delivery and imaging applications [59].
Potential targeted drug delivery system was developed with arginine G3 dendron covalently attached to a hydrophilic polysaccharide (pullulan), which is a neutral linear compound. The LP-g-G3P is composed of lactosylated pullulan-graft-G3arginine dendrons, which showed self-assemble ability, as well as small size particles, low polydispertion and higher affinity to lectin receptor ( Figure 10). DOX was encapsulated in LP-g-G3P through multiple interactions, was internalized into the hepatoma carcinoma cells, inhibiting cell proliferation. This type of targeted dendrimer showed to be a promising directed drug delivery system [68]. In other study, mannosylated PEGtide dendrons, G1 to G5, were synthesized containing amino acids, PEG and functionalized with mannose to reach macrophage. These compounds demonstrated good water solubility and potential biocompatibility due to high PEG in dendritic structure. Mannosylated dendrons presented higher uptake than non-mannose derivatives in murine models. Therefore, dendrons were mannose-dependent receptor for cell uptake. PEGtide dendrons could be an efficient platform to drug delivery and imaging applications [59].
Potential targeted drug delivery system was developed with arginine G3 dendron covalently attached to a hydrophilic polysaccharide (pullulan), which is a neutral linear compound. The LP-g-G3P is composed of lactosylated pullulan-graft-G3arginine dendrons, which showed self-assemble ability, as well as small size particles, low polydispertion and higher affinity to lectin receptor ( Figure 10). DOX was encapsulated in LP-g-G3P through multiple interactions, was internalized into the hepatoma carcinoma cells, inhibiting cell proliferation. This type of targeted dendrimer showed to be a promising directed drug delivery system [68]. Figure 10. Chemical structure of LP-g-G3P an amphiphilic dendrimer [68]. Figure 10. Chemical structure of LP-g-G3P an amphiphilic dendrimer [68]. Blood-brain barrier membrane can overexpress several types of receptors and proteins responsible for transportation to the brain, as for example sialic acid receptors and glucose transporters. Patel and coworkers compared the efficiency in drug targeting of PPI dendrimers functionalized with sialic acid (SPPI), glucosamine (GPPI) and concanavalin A (CPPI). Paclitaxel was entrapped in dendrimer cavities. All derivatives exhibited lower hemolytic property than free drug. Moreover, the dendrimers presented a better accumulation in the brain than in other organs, such as liver and kidney when compared to free paclitaxel and PPI. For targeting potential, SPPI demonstrated the best results, implying the sialic acid receptor as a good strategy for drug delivery in central nervous system [69].
Monoclonal Antibodies as Targeting Group
Monoclonal antibodies developed against specific antigens may aid to target drug delivery to the site of action [70]. However, few examples have been found, considering the profile of these compounds, which could lead to many unwanted reactions in the body.
Prostate specific membrane antigen J591 antibody was conjugated to the G5 PAMAM dendrimer, being capable of selectively bind to the prostate specific membrane antigen receptor [70].
Interleukins have been employed in dendrimers functionalization for drug delivery of some diseases such as cancer, which can overexpress receptors for these molecules. Interleukin-6 (IL-6) is a crucial cytokine, which acts in angiogenesis, owing to fast tumor neovascularization. IL-6 was coupled to PAMAM dendrimer and the internalization and competitive assays indicated its fast and efficient cellular uptake. This molecule presented high affinity for HER, which resulted in significant internalization of IL-6-G5 PAMAM dendrimers into HeLa cells via receptor-mediated endocytosis. The same research group compared IL-6 and RGD functionalized PAMAM dendrimer to target HeLa cells. DOX was encapsulated inside the dendrimer and, then, its cellular uptake and in vitro toxicity were compared to free drug. Both functionalized dendrimers were less toxic than free DOX due to slow release of the drug from dendrimer, demonstrating better cellular uptake when compared to free drug [1].
Other Targeting Groups
Besides those groups described before, whose action has been evidenced by several studies, many different types of targeting group were found as potentially interesting with the purpose of selectively directing the drug action to specific cells/tissues.
Biotin is a micronutrient, which participates in fatty acid biosynthesis, gluconeogenesis, cell growth and catabolism. In addition, biotin level is rapidly increased in tumor cells, proving to be an interesting approach [1,71]. PAMAM dendrimers were functionalized with RGD peptide and then, biotinylated [110]. In another study, biotinylated G4 and G5 PAMAM were planned to overcome the blood-brain barrier. The uptake and selectivity in HeLa cells were more appropriate for biotinylated dendrimers and more selective for cancer cells, without toxicity [111]. Sodium-dependent multivitamin transporter has been indicated as responsible for biotin uptake. All findings imply that this is an interesting approach to improve therapeutic efficacy and decrease side effects of anticancer agents [1,110].
Other potential targeting group is the follicle stimulating hormone receptor, which is overexpressed by ovarian cancer cells. Taking this into account, Modi and colleagues [72] designed G5 PAMAM labeled with fluorescein and follicle stimulating hormone 33, since it presents high affinity to follicle stimulating hormone receptor. The dendrimer showed better cellular uptake profile than labeled dendrimer, mainly by respective receptors.
Wen and colleagues [112] designed a nanoparticle conjugated with dendrons to deliver a photosensitizer. Natural nanoparticle Cowpea mosaic virus (CPMV) was used due to its target property. Additionally, CPMV has been shown to be selective for subpopulation of macrophages in cancer cells. The photosensitizer can react under light, resulting in reactive oxygen species, killing cells. Porphyrin is widely employed as photosensitizer, because it leads to electrostatic interaction with CPMV-dendron surface. The study succeeded in deliver the photosensitizer in the proper site.
It is important to consider the tendency of designing theranostic agents, that aggregates drugs and photosensitizers [113].
Jin and colleagues [73] synthesized a PAMAM dendrimer derivative, poly(2-(N,Ndiethylamino)ethyl methacrylate), with methoxy-poly(ethylene glycol)-poly(amido amine) loaded with 5-FU ( Figure 11). The poly (2- (N,N-diethylamino)ethyl methacrylate derivative is a nanostructure sensitive to pH, from which 5-FU release is favored in the tumor acidic medium. This does not happen in the blood, due to the neutral/basic environmental characteristics. According to the authors, this system is a promising nanocarrier because it provides great drug encapsulation, high targeting, and fast drug release in tumor. It is important to consider the tendency of designing theranostic agents, that aggregates drugs and photosensitizers [113].
Polyethylene glycol (PEG) has been widely used in dendrimers with many purposes, as, for example, to confer biocompatibility through cytotoxicity and hemolytic toxicity reduction, improvement in water solubility, decreased particle aggregation and opsonization by the reticuloendothelial system and tumor accumulation increase by EPR as well [41]. The examples that follow present some of those applications.
Acid-sensitive bindings between drugs and PEGylated PAMAM dendrimers allowed drug release from polymer-drugs into the acidic cellular environment after tumor cell internalization, preserving the stable compounds in the bloodstream [114]. The first acid-sensitive bond polymer proposal was the cis-aconityl linkage in G4 PEGylated dendrimers, developed to obtain a selective drug delivery system for tumor action containing DOX. Therefore, the cis-aconityl acid-sensitive binding was introduced between DOX and the polymer carrier, resulting in PPCD (PEG-PAMAMcis-aconityl-DOX conjugates). In addition, the researchers synthesized the acid-insensitive derivative composed by succinic bond, producing PPSD (PEG-PAMAM-succinic-DOX conjugates) for comparison. PPCD increased cytotoxicity in murine model of B16 melanoma cells, due to drug release in lysosomes after cellular uptake. PPSD derivatives released DOX in any pH condition showing low cytotoxicity in tumor cells. This evidenced the importance of acid-sensitive bindings as a targeted group.
Super stealth liposomes with PEG-dendron-phospholipid using a β-glutamic acid dendron as an anchor to PEG attachment and several distearoyl phosphoethanolamine lipids were synthesized [74]. The liposomal composition demonstrated higher stability, lower toxicity, greater intracellular uptake, prolonged half-life time, improved biodistribution profile and enhanced DOX anticancer potency.
In the same way, a micellar drug delivery system was designed, containing dendrons conjugated to a hydrophilic PEG linear polymer of well-defined structure. Their biodegradable polyester dendrons were coupled to an antiangiogenic drug, combretastatin-A4, aiming to obtain proper sized flower-like hydrosoluble micelles for passive tumor targeting, enhancing the permeability and retention. The drug release from this conjugate occurred in acidic conditions, which is an interesting profile. In assays to evaluate the antiangiogenic efficacy, this dendrimer reduced the cell viability and uptake, showing efficient inhibition [75].
Another approach was based on the difference between physiological and tumor pH to lead a smart drug delivery system. In this context, Qi and coworkers [76] designed a dendrimer with Polyethylene glycol (PEG) has been widely used in dendrimers with many purposes, as, for example, to confer biocompatibility through cytotoxicity and hemolytic toxicity reduction, improvement in water solubility, decreased particle aggregation and opsonization by the reticuloendothelial system and tumor accumulation increase by EPR as well [41]. The examples that follow present some of those applications.
Acid-sensitive bindings between drugs and PEGylated PAMAM dendrimers allowed drug release from polymer-drugs into the acidic cellular environment after tumor cell internalization, preserving the stable compounds in the bloodstream [114]. The first acid-sensitive bond polymer proposal was the cis-aconityl linkage in G4 PEGylated dendrimers, developed to obtain a selective drug delivery system for tumor action containing DOX. Therefore, the cis-aconityl acid-sensitive binding was introduced between DOX and the polymer carrier, resulting in PPCD (PEG-PAMAM-cis-aconityl-DOX conjugates). In addition, the researchers synthesized the acid-insensitive derivative composed by succinic bond, producing PPSD (PEG-PAMAM-succinic-DOX conjugates) for comparison. PPCD increased cytotoxicity in murine model of B16 melanoma cells, due to drug release in lysosomes after cellular uptake. PPSD derivatives released DOX in any pH condition showing low cytotoxicity in tumor cells. This evidenced the importance of acid-sensitive bindings as a targeted group.
Super stealth liposomes with PEG-dendron-phospholipid using a β-glutamic acid dendron as an anchor to PEG attachment and several distearoyl phosphoethanolamine lipids were synthesized [74]. The liposomal composition demonstrated higher stability, lower toxicity, greater intracellular uptake, prolonged half-life time, improved biodistribution profile and enhanced DOX anticancer potency.
In the same way, a micellar drug delivery system was designed, containing dendrons conjugated to a hydrophilic PEG linear polymer of well-defined structure. Their biodegradable polyester dendrons were coupled to an antiangiogenic drug, combretastatin-A4, aiming to obtain proper sized flower-like hydrosoluble micelles for passive tumor targeting, enhancing the permeability and retention. The drug release from this conjugate occurred in acidic conditions, which is an interesting profile. In assays to evaluate the antiangiogenic efficacy, this dendrimer reduced the cell viability and uptake, showing efficient inhibition [75].
Another approach was based on the difference between physiological and tumor pH to lead a smart drug delivery system. In this context, Qi and coworkers [76] designed a dendrimer with carboxymethyl chitosan (CMCS) as shell and PAMAM as core, responsible for interacting via electrostatic adsorption. There was high drug release due to the positive charge in PAMAM surface masked by negative CMCS charge, decreasing dendrimer clearance and toxicity. Moreover, when dendrimer reached tumor area, CMCS became positively charged, leaving PAMAM surface, owing to pH difference. Through this approach, DOX was encapsulated in PAMAM (PAMAM-DOX-CMCS) and its rate release was correlated with the conjugate (Figure 12) increase, when pH dropped from 7.4 to 6.5 in 48 h, while free DOX was insensitive to pH. PAMAM-DOX-CMCS exhibited greater uptake than free DOX, indicating that CMCS was releasing from PAMAM surface at pH 6.5 and afterwards, through positive surface charge. carboxymethyl chitosan (CMCS) as shell and PAMAM as core, responsible for interacting via electrostatic adsorption. There was high drug release due to the positive charge in PAMAM surface masked by negative CMCS charge, decreasing dendrimer clearance and toxicity. Moreover, when dendrimer reached tumor area, CMCS became positively charged, leaving PAMAM surface, owing to pH difference. Through this approach, DOX was encapsulated in PAMAM (PAMAM-DOX-CMCS) and its rate release was correlated with the conjugate (Figure 12) increase, when pH dropped from 7.4 to 6.5 in 48 h, while free DOX was insensitive to pH. PAMAM-DOX-CMCS exhibited greater uptake than free DOX, indicating that CMCS was releasing from PAMAM surface at pH 6.5 and afterwards, through positive surface charge. Figure 12. Nanocapsule of chitosan containing PAMAM G5 encapsulated DOX [76].
In previous studies, Li and coworkers [51] showed that supramolecular hybrid dendrimers exhibit 50,000 times more gene transfection efficiency to tumor cells than single dendrons. Taking this into account, they synthesized a supramolecular dendritic system composed of PLL and poly(Lleucine), which interacts through non-covalent bonds, leading to an amphiphilic self-assembly structure, mimicking a virus capsid. DOX was encapsulated in hydrophobic supramolecular dendritic pocket (D-CLNs) and at in vitro test, this molecular architecture disassembled, and drug was released.
Another way to target tumor tissue is through pH-sensitive compounds, using a group that links to the dendrimer via pH-sensitive bond, as, for instance, the hydrazone bond [115] or conjugating the respective drug in a G5 PAMAM with succinimydilpropylamine on the dendrimer surface [116]. They showed an interesting profile of selectively deliver DOX. In both cases, DOX release was dependent of pH, as proposed.
Also using a pH-sensitive hydrazine bond, a novel amphiphilic fluorinated peptide dendron functionalized with dextran was successfully synthetized. This conjugate has demonstrated selfassembly ability in carrying hydrophobic drugs. In in vitro assays, this dendron showed an excellent biocompatibility for normal and tumor cells, exhibiting a stimulus-induced self-disassembled endo/lysosome pH-responsive, providing their disassembly, and controlling encapsulated DOX [77].
Heparin, an inhibitor of serine proteases in blood coagulation, is also used in antitumor chemotherapy due to its ability of inhibiting tumor growth and metastasis. Based on those properties a novel drug delivery system to carry DOX containing heparin dendronized was designed and synthesized via click chemistry. An acid-labile hydrazone bond was employed for breast tumor therapy. Dendronized heparin-DOX conjugate was not toxic, comparatively to free DOX in In previous studies, Li and coworkers [51] showed that supramolecular hybrid dendrimers exhibit 50,000 times more gene transfection efficiency to tumor cells than single dendrons. Taking this into account, they synthesized a supramolecular dendritic system composed of PLL and poly(L-leucine), which interacts through non-covalent bonds, leading to an amphiphilic self-assembly structure, mimicking a virus capsid. DOX was encapsulated in hydrophobic supramolecular dendritic pocket (D-CLNs) and at in vitro test, this molecular architecture disassembled, and drug was released.
Another way to target tumor tissue is through pH-sensitive compounds, using a group that links to the dendrimer via pH-sensitive bond, as, for instance, the hydrazone bond [115] or conjugating the respective drug in a G5 PAMAM with succinimydilpropylamine on the dendrimer surface [116]. They showed an interesting profile of selectively deliver DOX. In both cases, DOX release was dependent of pH, as proposed.
Also using a pH-sensitive hydrazine bond, a novel amphiphilic fluorinated peptide dendron functionalized with dextran was successfully synthetized. This conjugate has demonstrated self-assembly ability in carrying hydrophobic drugs. In in vitro assays, this dendron showed an excellent biocompatibility for normal and tumor cells, exhibiting a stimulus-induced self-disassembled endo/lysosome pH-responsive, providing their disassembly, and controlling encapsulated DOX [77].
Heparin, an inhibitor of serine proteases in blood coagulation, is also used in antitumor chemotherapy due to its ability of inhibiting tumor growth and metastasis. Based on those properties a novel drug delivery system to carry DOX containing heparin dendronized was designed and synthesized via click chemistry. An acid-labile hydrazone bond was employed for breast tumor therapy. Dendronized heparin-DOX conjugate was not toxic, comparatively to free DOX in histological analysis. Additionally, the dendronized derivative demonstrated high antitumor activity on breast cancer cell line, as well as antiangiogenics effects and apoptosis induction. According to the She and colleagues work [78], this conjugate may not only be a background for safe nanoparticles design but also an efficient carrier for drug delivery.
An alternative to PAMAM dendrimers for drug delivery via encapsulation was developed using other polymeric structures. Although PAMAM dendrimers with antimalarial drugs exhibited specific binding, their IC 50 were modest against Plasmodium-infected cells. Taking this into account, a Janus dendrimer (with two different generations GA and GB- Figure 13 and hybrid dendritic-linear-dendritic block copolymers (with two different generations GC and GD- Figure 13) were synthesized, with three encapsulated drugs (chloroquine-CQ, primaquine-PQ and rhodamine B) against Plasmodium falciparum. In vitro tests showed better efficacy of the conjugate when compared with free CQ. However, in vivo assays have shown no drug efficacy improvement when GD-CQ is compared with CQ. In both cases, the mice survival was slightly better for GD-CQ and GC-PQ dendrimers ( Figure 13) [79].
Dendrimers composed of fluocinolone acetonide were designed to treat neuroinflammation in the outer retina, when coupled to G4-OH PAMAM through the spacer glutaric acid. Conjugated dendrimer labeled to fluorescein isothiocyanate (label to cell uptake visualization) presented higher uptake than free-fluorescein isothiocyanate according to Royal College of Surgeons retinal degeneration rat models. Iezzi and coworkers [80] used dendrimers conjugated to Cy5.5-mono-NHS ester (another labeled non-susceptible to tissue autofluorescence) to explain that effect and observed the same profile mentioned above, in which, after 35 days of administration, the dendrimers were still present in target cells. Additionally, the dendrimer containing fluocinolone acetonide showed better performance than the free drug in attenuation neuroinflammation and neuroprotection. According to the authors, dendrimer cell uptake was enhanced, increasing the drug residence time, delivering specific retinal area, and reducing side effects due to PAMAM dendrimers intrinsic ability to their localization within activated microglia. Pharmaceutics 2018, 10, x FOR PEER REVIEW 19 of 27 Figure 13. Janus dendrimer (with two different generations GA and GB) and hybrid dendritic-linear-dendritic block copolymers (with two different generations GC and GD) to of CQ, PQ and rhodamine B encapsulation [79]. Figure 13. Janus dendrimer (with two different generations GA and GB) and hybrid dendritic-linear-dendritic block copolymers (with two different generations GC and GD) to of CQ, PQ and rhodamine B encapsulation [79].
Concluding Remarks
Selectivity has been the goal of chemotherapeutic agents, mainly for cancer. That is why most papers herein presented are related to tumor targeting. It is interesting that most of the examples are referred to doxorubicin (DOX) conjugation, probably because it has been used for many kinds of tumors and its severe side effects not rarely compromise its therapeutic application. Besides DOX, methotrexate (MTX) is the prototype in the design of selective conjugate compounds. On the other hand, dendrons and dendrimers, as well, are interesting kind of polymers whose properties favor their use either to attach covalently or to encapsulate bioactive compounds giving prodrugs and delivery forms of drugs, respectively. Those carriers are very flexible, in terms of positions they furnish for bonding different molecules, including target groups. Using dendrons and dendrimers it is possible to achieve selective delivery, provide the specific and proper target group is chosen. Different kinds of targeting groups have been used and this is possible due to the advance in the study of molecular biology and genetics, which allows the discovery of cell receptors and selective mechanisms of drug release. Obtaining selective dendrimer conjugate drug compounds has been a complex goal, but the interesting properties the matrix imparts in terms of toxicity, solubility, bioavailability, and effectiveness, among others, compensates for the complexity mentioned before.
Although cancer has shown to be one of the main cases of death worldwide, it is important to think about other classes of diseases, as the neglected ones. This review shows few examples of application of the approach of targeting drugs by means of dendrons or dendrimers for those diseases. The reason probably is the low interest the research on this kind of drugs arouses in general, which leads to the thinking that the complexity, besides the costs, do not compensate the low revenues for pharmaceutical industries.
Our team has been using dendrons and dendrimers and some selective groups based on specific cell receptors to obtain target drugs (data not published). We have been working on neglected diseases and mainly for Chagas disease, leishmaniasis and malaria our goal is to obtain dendrons and/or dendrimers through prodrug design using drugs and/or bioactive compounds. We intend to stimulate research groups working either on target dendrons or dendrimers to apply their ideas to obtain specific conjugate compounds for neglected diseases. It is worth noting that the 17 diseases that the World Health Organization (WHO) considers neglected ones are responsible for 1 billion people infected worldwide [117].
Conflicts of Interest:
The authors confirm that this article content has no conflict of interest. | v3-fos-license |
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} | pes2o/s2orc | Smad2/3 Upregulates the Expression of Vimentin and Affects Its Distribution in DBP-Exposed Sertoli Cells
Sertoli cells (SCs) in the testes provide physical and nutritional support to germ cells. The vimentin cytoskeleton in SCs is disrupted by dibutyl phthalate (DBP), which leads to SCs dysfunction. In a previous study, we found that peroxisome proliferator-activated receptor alpha (PPARα) influenced the distribution of vimentin by affecting its phosphorylation in DBP-exposed SCs. In the present study, we investigated the role of Smad2/3 in regulating the expression of vimentin in DBP-exposed SCs. We hypothesized that Smad2/3 affects the distribution of vimentin by regulating its expression and that there is cross talk between Smad2/3 and PPARα. The real-time PCR and ChIP-qPCR results showed that SB431542 (an inhibitor of Smad2/3) could significantly attenuate the expression of vimentin induced by DBP in SCs. Phosphorylated and soluble vimentin were both downregulated by SB431542 pretreatment. WY14643 (an agonist of PPARα) pretreatment stimulated, while GW6471 (an antagonist of PPARα) inhibited, the activity of Smad2/3; SB431542 pretreatment also inhibited the activity of PPARα, but it did not rescue the DBP-induced collapse in vimentin. Our results suggest that, in addition to promoting the phosphorylation of vimentin, DBP also stimulates the expression of vimentin by activating Smad2/3 in SCs and thereby induces irregular vimentin distribution.
Introduction
Spermatogenesis is an essential process for male reproductive function. In one report, subfertility or infertility was generally due to defects in spermatogenesis [1]. Dibutyl phthalate (DBP) is a plasticizer that is widely distributed in the environment and frequently contacted by human. Previous epidemiological, animal, and molecular studies have shown that DBP disrupted spermatogenesis [2][3][4][5][6]. Spermatogenesis occurs in the seminiferous tubules; SCs are unique somatic cells in the seminiferous tubules, which provide physical and nutritional support for spermatocyte maturation [7,8]. Periodically "open" and "closed" junctions between SCs and germ cells mediate the maturation process of spermatocyte [9]. Vimentin is tightly connected to the desmosome structure linking germ cells and SCs. The distribution of vimentin filaments changes dynamically during the seminiferous epithelium cycle [10]. Previous studies have shown that the vimentin cytoskeleton was disrupted in DBP-exposed SCs [11]. However, the mechanism of this process is still unknown.
In our previous study, we found that levels of phosphorylated and soluble vimentin were higher in DBP-exposed SCs compared to GW6471 + DBP-treated SCs [12]. We did not investigate vimentin mRNA expression, but the expression of vimentin may also affect the concentration of soluble vimentin. According to a previous study, vimentin mRNA was upregulated in DBP-exposed SCs [13].
The expression of vimentin mRNA was regulated by Smad2/3 in several other cell lines, such as esophageal squamous cell carcinoma cell lines (e.g., Eca109, EC9706, and KYSE150), squamous cell carcinoma of the head and neck cell line Tu686, skeletal myogenic cell line C2C12, and normal human epidermal keratinocytes cells. Smad2/3 binds to the Smad binding element in the promoter region of vimentin to regulate its expression [14][15][16][17]. In this study, we investigated the role of Smad2/3 in the regulation of vimentin mRNA expression in DBP-exposed SCs.
We hypothesized that Smad2/3 could affect the distribution of vimentin by regulating its mRNA expression in DBPexposed SCs, possibly through cross talk with PPAR ; these 2 PPAR Research two factors cooperate to affect the distribution of vimentin. Chromatin immunoprecipitation and quantitative real-time PCR (ChIP-qPCR) analysis was performed to identify the regulatory region in the promoter of vimentin bound by phosphorylated Smad2/3. The distribution of vimentin in SCs after different treatments was determined with immunocytochemistry and immunofluorescence, and treatments with various agonists or antagonists were used to investigate the relationship between Smad2/3 and PPAR .
Cell Culture and
Treatments. Approximately 2 × 10 6 SCs were seeded into 10 cm dishes. DBP was diluted in DMSO (dimethyl sulfoxide) to 100 mM. Ten microliters of DBP was added to each 10 cm dish, for a final concentration of 100 M. Equal volumes of DMSO were added to control dishes. Agonists or antagonists (GW6471, WY14643, and SB431542) were added two hours before DBP treatment at a concentration of 10 M, according to previous studies [14,22,23]. SCs were treated with DMSO, DBP, SB431542 + DBP, and SB431542 for 24 h to extract RNA. SCs were treated with DBP, WY14643 + DBP, GW6471 + DBP, or SB431542 + DBP for 0 h, 1 h, 3 h, 6 h, 12 h, and 24 h, respectively, to extract protein. The dosage of DBP was based on the CCK8 and immunofluorescence experiments. In the CCK8 experiment, we added 0, 20, 40, 60, 80, 100, and 200 M of DBP to detect its toxicity in SCs. The results showed that DBP promoted Sertoli cell proliferation in a dose-dependent manner. We selected 100 M because its adverse effect on vimentin in SCs at the 24 h time point was apparent but not severe enough to interfere with western blotting. We selected a series of time points within 24 h because the damage induced by DBP to SCs was too severe at 48 h or 72 h. Moreover, we also took into account the dosage and time points used by other researchers [24].
Real-Time PCR.
RNA was extracted with TRIzol (Invitrogen, USA) from the treated SCs. Concentrations and purity of RNA were quantified by a Nanodrop 2000 (Thermo Fisher Scientific, Rockford, IL, USA). Approximately 1 g total RNA was used to generate the cDNA using the PrimeScript RT Reagent Kit with gDNA Eraser (Perfect Real Time). For the PCR reaction, 7 L nuclease-free water, 10 L GoTaq Probe qPCR Master Mix (2x), and 1 L cDNA and primers were used, respectively, in a 20 L total reaction volume. Real-time PCR was performed using a Bio-Rad IQ5 (Bio-Rad, USA) with a preheated lid up to 105 ∘ C, initial denaturation at 95 ∘ C for 5 min, and 40 repeats of (95 ∘ C for 10 s, 60 ∘ C for 30 s), followed by extension at 72 ∘ C for 5 min, and the data were analyzed using the 2 (−ΔΔCt) method. Primers used in this experiment are listed in Table 1.
2.5.
ChIP-qPCR. The ChIP assay kit (P2078) used in this experiment was purchased from Beyotime Institute of Biotechnology (Haimen, China). Detailed procedures for ChIP-qPCR were previously described [12]. An antip-Smad2/3 antibody was used in this experiment (1 g). Primers used in this experiment were targeted to Smad binding elements (SBE) in promoter regions of vimentin [25]. We designed four pairs of primers flanking SBE-containing sequences, 2000 bp upstream of the transcription start site of vimentin. Primer sequences are listed in Table 1. After the preliminary experiment, only the primers targeting region −837 to −924 showed high amplification efficiency, so we chose this region for further experiments. The product size was 88 bp.
Role of Smad2/3 in Regulating Vimentin mRNA
Expression. Real-time PCR was used to measure vimentin mRNA expression in control and DBP-, Smad2/3 inhibitor-(SB431542-), and DBP + SB431542-treated SCs. DBP-exposed SCs presented significant vimentin mRNA levels increase to 1.22 ± 0.009-fold variation relative to cells cultured in control conditions (Figure 1 0.52 ± 0.002or 0.44 ± 0.004-fold variation relative to SCs cultured in control conditions, respectively. We designed four pairs of primers to amplify SBE-containing sequences in the promoter region of vimentin. The most efficient pair of primers was selected to perform the ChIP-qPCR experiment. As showed in Figure 1(b), binding of p-Smad2/3 to the SBE of vimentin in DBP-treated SCs significantly increased to 3.77 ± 0.009-fold variation relative to SCs cultured in control conditions, and the inhibition of Smad2/3 by SB431542 could significantly decrease the binding of p-Smad2/3 to the SBE of vimentin to 1.70 ± 0.01-fold variation in SB431542 + DBPtreated SCs and to 0.63 ± 0.003-fold variation in SB431542treated SCs relative to SCs cultured in control conditions. ChIP-qPCR results were consistent with the real-time PCR results. In summary, these findings indicate that Smad2/3 upregulated vimentin mRNA expression in DBP-exposed SCs.
Role of Smad2/3 in Regulating Vimentin
Protein Expression. Western blotting was used to detect the activity of p-Smad2/3 in DBP-treated SCs, and phosphorylated and soluble vimentin were detected in the DBP-and SB431542 + DBP-treated SCs. As showed in Figures 2(a) and 2(d), relative concentrations of total Smad2/3 protein compared to actin remained stable within 24 h in DBP-treated SCs; relative concentration of p-Smad2/3 compared to actin in DBP-treated SCs was significantly increased to 1.53 ± 0.002-fold variation at 1 h, 2.69 ± 0.0005-fold variation at 3 h, 1.87 ± 0.0009fold variation at 6 h, 1.94 ± 0.006-fold variation at 12 h, and 2.37 ± 0.0002-fold variation at 24 h relative to SCs cultured in control conditions, respectively. To determine the effect of Smad2/3 on phosphorylated and soluble vimentin, western blotting was performed. As showed in Figures 2(b) and 2(e), phosphorylated vimentin levels in DBP-treated SCs increased to 1.28 ± 0.009-fold variation at 1 h, to 1.30 ± 0.03-fold variation at 3 h, to 1.21 ± 0.003-fold variation at 6 h, and to 1.11 ± 0.01-fold variation at 12 h and then decreased to 0.71 ± 0.03-fold variation at 24 h relative to SCs at 0 h. As showed in Figures 2(c) and 2(e), phosphorylated vimentin levels increased in SB431542 + DBP-treated cells to 1.21 ± 0.002-fold variation at 1 h and then decreased to 0.83 ± 0.001-fold variation at 3 h, to 0.70 ± 0.004-fold variation at 6 h, to 0.56 ± 0.007-fold variation at 12 h, and to 0.21 ± 0.005fold variation at 24 h relative to SCs at 0 h. Phosphorylated vimentin levels were significantly lower at each time point in SB431542 + DBP-treated than DBP-treated cells ( < 0.05). The relative quantity of soluble vimentin to actin in DBPtreated SCs increased to 1.76 ± 0.006-fold variation at 1 h, to 1.75±0.008-fold variation at 3 h, to 1.77±0.004-fold variation at 6 h, to 2.20 ± 0.01-fold variation at 12 h, and to 1.67 ± 0.02-fold variation at 24 h relative to SCs at 0 h. Soluble vimentin in SB431542 + DBP-treated SCs decreased to 1.04 ± 0.003-fold variation at 1 h, to 0.84±0.002-fold variation at 3 h, to 0.89 ± 0.001-fold variation at 6 h, to 0.77 ± 0.002-fold variation at 12 h, and to 0.80 ± 0.001-fold variation at 24 h, relative to SCs at 0 h. Soluble vimentin levels were significantly higher in the DBP-treated SCs than the SB431542 + DBP-treated cells at each time point ( < 0.05). In summary, Smad2/3 affected the phosphorylation and solubility of vimentin in DBP-exposed SCs.
Relationship between PPAR and Smad2/3 in DBP-Treated
SCs. An antagonist (GW6471) and an agonist (WY14643) of PPAR were used to investigate the effect of PPAR on the activity of Smad2/3. As shown in Figures 3(a) and 3(b), the relative quantity of p-Smad2/3 to actin in WY14643 + DBP-treated SCs increased to 1.16 ± 0.003fold variation at 1 h, to 1.03 ± 0.008-fold variation at 3 h, to 1.36±0.003-fold variation at 6 h, to 1.04±0.004-fold variation at 12 h, and to 1.35 ± 0.002-fold variation at 24 h relative to SCs at 0 h. The relative quantity of p-Smad2/3 to actin in GW6471 + DBP-treated SCs increased to 1.11 ± 0.01-fold variation at 1 h and then decreased to 0.63 ± 0.004-fold variation at 3 h, to 0.63 ± 0.007-fold variation at 6 h, to 0.85 ± 0.003-fold variation at 12 h, and to 0.39 ± 0.004-fold variation at 24 h relative to SCs at 0 h. The relative quantity of p-Smad2/3 was significantly higher in WY14643 + DBPtreated SCs than GW6471 + DBP-treated cells at each time point ( < 0.05). Smad2/3 levels in the WY14643 + DBPtreated SCs increased to 1.30 ± 0.1-fold variation at 1 h, to 1.09 ± 0.006-fold variation at 3 h, and to 1.02 ± 0.003-fold variation at 6 h and decreased to 0.91 ± 0.004-fold variation at 12 h and to 0.56 ± 0.003-fold variation at 24 h relative to SCs at 0 h. Smad2/3 levels in the GW6471 + DBP-treated SCs increased to 1.16±0.01-fold variation at 1 h, to 1.18±0.01fold variation at 6 h, and to 1.02 ± 0.008-fold variation at 12 h and decreased to 0.88 ± 0.01-fold variation at 3 h and to 0.94 ± 0.02-fold variation at 24 h relative to SCs at 0 h. There were no significant differences in levels of Smad2/3 between WY14643 + DBP-treated and GW6471 + DBP-treated SCs at 1 h, 6 h, and 12 h ( < 0.05). The level of Smad2/3 was significantly ( < 0.05) higher in WY14643 + DBP-treated than GW6471 + DBP-treated SCs at 3 h and less at 24 h. In addition, an inhibitor of p-Smad2/3 (SB431542) was used to assess the influence of Smad2/3 on the activity of PPAR . As shown in Figures 3(c) and 3(d), PPAR levels in DBP-treated SCs increased to 1.16 ± 0.002-fold variation at 1 h, to 2.54 ± 0.002-fold variation at 3 h, to 1.76 ± 0.003-fold variation at 6 h, and to 1.81 ± 0.005-fold variation at 12 h and then decreased to 0.62 ± 0.001-fold variation at 24 h relative to SCs at 0 h; the relative quantity of PPAR in SB431542 + DBP-treated cells increased to 1.17 ± 0.006-fold variation at 1 h and then decreased to 0.77 ± 0.003-fold variation at 3 h, to 0.79 ± 0.004-fold variation at 6 h, to 0.48 ± 0.004-fold variation at 12 h, and to 0.50 ± 0.002-fold variation at 24 h relative to SCs at 0 h. PPAR levels were significantly ( < 0.05) higher in DBP-treated SCs than SB431542 + DBPtreated cells at 3 h, 6 h, 12 h, and 24 h. This result suggested that DBP could activate PPAR , and the activation of PPAR in turn activated p-Smad2/3, initiating a positive feedback loop with PPAR . In summary, WY14643 induced the activity of Smad2/3, GW6471 inhibited the activity of Smad2/3, and SB431542 inhibited the activity of PPAR in DBP-exposed SCs.
Discussion
Spermatogenesis occurs in the seminiferous tubules, and SCs are unique somatic cells in the seminiferous tubules [26] that provide structural and nutritional support during the process of germ cell maturation [27]. Vimentin is an important component of the desmosome [28], and disruption of the vimentin cytoskeleton by DBP leads to dysfunction of the desmosomes [24]. Without the structural support and nutritional supply from SCs, germ cells are prone to apoptosis [29].
Our previous results demonstrated that the increased phosphorylation of vimentin by PPAR was the main mechanism of the disruption of vimentin in SCs [12]. However, whether the expression of vimentin is also regulated by PPAR is still not clear. In this study, we investigated the role of Smad2/3 in regulating vimentin mRNA expression and the relationship of Smad2/3 and PPAR in vimentin distribution in SCs.
Our qRT-PCR results showed that vimentin mRNA expression was upregulated in DBP-exposed SCs at 24 h. A previous microarray study showed that vimentin was upregulated in DBP-treated rat testes at gestational day 20 (GEO accession number: GSE13550). A previous proteomics study also showed that vimentin was upregulated in DBP-exposed rat testes [13]. These results indicated that vimentin expression was stimulated by DBP. Previous studies have indicated that Smad2/3 regulates the expression of vimentin, and the targets were bound by p-Smad2/3 at SBE-specific sequence GTCTG or CAGAC [30,31]. In this study, we found an SBE sequence in the promoter of vimentin which was located at −837 to −924 bp relative to the transcriptional start site by ChIP-qPCR. Moreover, 12 bp downstream of our selected target region for the ChIP-qPCR assay is an AP1 target site. The AP1 site may facilitate the binding of p-Smad2/3 to its target sequence [17]. We hypothesized that Smad2/3 interacts with other transcriptional factors in regulating the expression of vimentin.
Next, we investigated the influence of Smad2/3 on the phosphorylation and solubility of vimentin. The results (as shown in Figures 2(b), 2(c), 2(e), and 2(f)) demonstrated that the inhibition of Smad2/3 had an inhibitory effect on the phosphorylation of vimentin compared with DBP-treated SCs. The effect of Smad2/3 on the solubility of vimentin was apparent as the relative concentration of soluble vimentin decreased significantly ( < 0.05) in a time-dependent manner in SB431542 + DBP-treated SCs. This was consistent with previous studies [14,15,32,33]. In these studies, the upregulation of vimentin was closely related to the stimulation of Smad2/3, and SB431542 treatment directly inhibited the expression of vimentin. These results suggested that the regulation of vimentin expression by Smad2/3 may be a universal mechanism.
To illuminate the relationship between PPAR and Smad2/3, GW6471, WY14643, and SB431542 were used. The results showed that the activity of Smad2/3 was promoted by WY14643 and inhibited by GW6471. The relationship between PPAR and Smad2/3 is similar to a previous study, where TGF 1-induced -SMA was elevated in the WY14643 pretreated group, while it was inhibited in the GW6471 pretreated group [34]. Another study showed that WY14643 could upregulate the mRNA expression of TGF in lung allografts and spleens [35]. Additionally, SB431542 was used to inhibit the activity of p-Smad2/3, and the results showed that activity of PPAR was not affected by SB431542 at 1 h but decreased after 3 h. These results suggested that PPAR stimulates the activation of p-Smad2/3 in DBP-exposed SCs, and Smad2/3 may initiate a positive feedback loop with PPAR .
The disrupted vimentin cytoskeleton in DBP-treated SCs was not recovered by SB431542 but showed a new abnormal distribution, mainly consisting of abundant irregular long filamentous structures. Phosphorylation of vimentin in SB431542 + DBP-treated SCs was inhibited compared to the DBP-treated group. SB431542 inhibited the expression of vimentin. Abnormally high and low levels of phosphorylation both affected the dynamic reassembly of vimentin [36]. These two factors may be reasons for the abnormal distribution of vimentin in SB431542 + DBP-treated SCs. Studying the interaction of Smad2/3 with other kinases which affected the posttranslational modifications of vimentin in DBP-treated SCs would be our next objective. The mechanism behind the irregular long filamentous structures of vimentin may involve signaling transduction along these filaments [37].
We determined that abnormal stimulation of the activity of Smad2/3 in SCs by DBP disrupted the dynamic assembly of connections between SCs and the developing spermatids, and the effect of Smad2/3 on vimentin was one aspect of a cascade of responses induced by DBP. Proliferation of SCs is mediated by activin through Smad3; Smad3 knockout mice exhibited delayed SCs maturation [38,39]. Sertoli epithelium separates germ cells from testis interstitium and cooperates with peritubular myoid cells to facilitate the spermatogenesis [40]. DBP, as a phthalate, could induce Leydig cells hyperplasia and then deprive the space belonging to SCs [41]. According to a recent study, SCs could be reprogrammed to Leydig cells by Wt1 ablation [42]. Two previous gene array studies showed that Wt1 was downregulated in testes induced by DBP (GSE25196, GSE13550). Upregulation of Vimentin, as a marker of epithelial-to-mesenchyme transformation [43][44][45], by Smad2/3 induced by DBP in SCs may also trigger the epithelial-to-mesenchyme transformation, disturb the epithelial layer, and affect the stability of blood testes barrier. Disruption of blood testes barrier is one of the major adverse effects induced by DBP [46]. Besides, cross talk of TGF 1/Smad3 with orphan nuclear receptor Nur77 was reported to repress the steroidogenesis in Leydig cells [47]. Vimentin participates in the transportation of cholesterol during steroidogenesis [48][49][50]. Steroidogenesis is disrupted by DBP according to several previous studies [51,52]. PPAR was reported to be an indirect transrepressor of SF1 on steroidogenic genes in testes [53]. To some extent, PPAR may also affect the steroidogenesis by affecting vimentin in DBP-exposed testes cells. Although we performed our experiment in SCs here, there is vimentin in Leydig cells too [54]. So mechanism deduced here may also shed some light on DBP-exposed Leydig cells studies.
A limitation of this research is that we only studied the expression and phosphorylation of vimentin in DBP-exposed : Schematic diagram of the relationship between PPAR and Smad2/3 in regulation of vimentin in DBP-exposed SCs. DBP stimulated the activity of PPAR , activated PPAR promoted the activity of Smad2/3, and activated Smad2/3 upregulated the vimentin mRNA expression. As SB431542 inhibited Smad2/3 and could inhibit PPAR to some extent, we deduced that Smad2/3 may have a positive feedback response to PPAR .
SCs. In physiological situations, ubiquitination, glycosylation, acetylation, and many other modifications affect the distribution and functions of vimentin [55]. In future studies, other modifications should be taken into consideration. In vivo studies are also needed to confirm the conclusions deduced from these in vitro studies.
Conclusion
In this study, we found that Smad2/3 upregulated the expression of vimentin in DBP-exposed SCs. The inhibition of PPAR could attenuate the activity of Smad2/3. Smad2/3 may have a positive feedback effect on PPAR . Smad2/3 and PPAR cooperated in regulating the expression, phosphorylation, and distribution of vimentin in DBP-exposed SCs (as shown in the schematic diagram in Figure 5). | v3-fos-license |
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} | pes2o/s2orc | Screening of Suitable Ionic Liquids as Green Solvents for Extraction of Eicosapentaenoic Acid (EPA) from Microalgae Biomass Using COSMO-RS Model
Omega-3 poly unsaturated fatty acids (PUFA) particularly eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), have many health benefits including reducing the risk of cancer and cardiovascular disease. Recently, the use of ionic liquids (ILs) in lipid extraction from microalgae provides the potential to overcome common drawbacks, offers several other benefits. To date, very limited researches are available to focus on extracting microalgae lipid and PUFA in particular by using ILs. The objective of current work is to screen the potential ILs that can be applied in EPA extraction. In this study, fast ILs screening was performed with the help of a conductor like screening model for real solvents (COSMO-RS) and the ILs with higher capacity values for use in extraction of EPA were compared. According to the results, the highest capacity for EPA extraction among 352 screened cation/anion combinations belongs to [TMAm][SO4]. It is expected to achieve a higher yield of EPA once applying this combination as the solvent in the process of extraction. ILs with small anions were observed to have higher capacities, as well possessing higher charge density compared to larger ones, and therefore, they are more preferable for extraction purposes. Moreover, shorter alkyl chain cations are preferred when using imidazolium-based IL, which agrees with experimental data.
Introduction
Health benefits provided by omega-3 polyunsaturated fatty acids (PUFAs), including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in particular are noteworthy to the society as it reduces the risk of cancer, autoimmune, cardiovascular disease, inflammatory disorders, cystic fibrosis, disrupted neurological function, bowel disease, and mental illness [1][2][3][4][5][6]. The use of PUFA as health prospects has gained great attention in last years. Current dietary health organization recommends a daily intake of 250-500 mg of EPA + DHA for primary and secondary prevention of coronary heart disease [7,8]. Additionally, according to Shahidi and Ambigaipalan human bodies requires the supplementary sources of EPA and DHA as they are vulnerable to shortages of necessary from n-hexane. Additionally, the study reported by Zeeshan et al. [25] COSMO-RS software used for the calculation of activity coefficient at infinite dilution and then selectivity, capacity, and performance index to predict different types of ILs potential for asphaltene extraction as a part of liquid-liquid extraction. Moreover, in a recent study by Xing et al. [26], it has been used COSMO-RS for prediction and exploring the potential separation mechanism of long chain fatty acids (EPA, DHA, CLAs, OA, and SA) through the calculation of different solvents capacity, selectivity, and performance index values. In another study, adjustable parameters were re-optimized in COSMO-RS to fit for the systems containing ionic liquids (ILs), whereby a vast numbers of activity coefficients and at infinite dilution and CO 2 solubility were collected from references and used as a training set. The results showed that the predicted results by COSMO-RS model with the new optimized parameters are in agreement with experimental data [27]. As pointed out by previous literatures, COSMO-RS only require the structural information of interacting species in order to calculate their related thermodynamic properties through a set of defined mathematical equations and hence one does not need to perform any experimentations in advance [28,29]. It is noteworthy to mention that, although COSMO-RS is a powerful screening tool, this software has been addressed by a number of authors as a mean field model that does not account for solvent molecular structure and correlations. Therefore, it is suggested that when using COSMO-RS, the important effects of explicit interactions between organic molecules and water, which can lead to charge transfer between the solute and water, must also be considered [30][31][32][33][34].
While the ILs screening potential seems crucial for extraction of omega-3 (EPA, DHA and ALA) compounds from biomass, however to the best of the author's knowledge, no significant report has been published yet to concern the application of COSMO-RS in that particular regard. Therefore, this study strictly help and open a new horizon about ILs interaction toward valuable omega-3 compound for researchers who seek to replace the conventional solvent with proper green solvent (ILs) without any requirement to perform expensive and time consuming lab experimentations. Moreover, what makes the COSMO-RS screening even more valuable is that it provides the facility to study over the variety of 352 combination of cations and anions as ILs to act as solvents in interaction with EPA compounds.
In this study, COSMO-RS has been used as the screening methodology to select the most suitable ILs as a part of solid-liquid extraction of EPA from microalgae. This work aims to predict infinite capacity values of ILs for EPA extraction and without the use of any experimental data.
Results and Discussion
The principle of COSMO-RS as a physical model considers the interaction energy of polarization charge densities between the sigma bonds. Net surface charge of EPA is calculated in terms of Sigma profile (σ-profile), which is related to the charge density profiles of molecular surface and Sigma potential (σ-potential) in order to clear its chemical nature. The polarity of the molecule is typically determined by the help of molecular electrostatic potentials. The best approach to investigate the molecular similarity is through comparing the electron density or electrostatic potential that is created by molecules, considering all space within the van der Waals surfaces of the molecules or within all regions outside the molecular cores [35]. The interactions of the electrostatic (polarity) among IL systems will integrate the hydrogen-bonding and hydrophobicity of a structure in the method of COSMO-RS. Therefore, it demonstrates multiple solvation interactions of ILs and presents the quantity and clear visual of polarity distribution on molecular surfaces with the help of sigma (σ) profiles [36]. According to the σ-profile, higher absolute values of σ indicates the stronger compound as a hydrogen bond donor or a hydrogen bond acceptor [37].
The chemical structure of EPA and its sigma surface are illustrated in Figure 1. The results of σ-profile and σ-potential are indicated in Figures 2 and 3, respectively. Significant terms related to qualitative description of EPA molecules including hydrogen bonding, polarity, and lipophilicity/hydrophilicity can be visualized with the help of COSMO-RS 3D screening charge distribution (sigma surface). Charge distribution is coded in colors such as red color, which represents the negative (−ve) charge, whereas blue and green represents the positive (+ve) and neutral (0) charge on the molecular surface, respectively. The screening charge density surface (s-surface) contains all relevant information for COSMO-RS to calculate the chemical potential. The capacity values related to five types of cation-base ILs including imidazolium, pyridinium, pyrrolidinium, piperidinium, and tetra-methyl ammonium with 22 types of anions were screened at 298.15 K and the results are summarized in Figure 4a-e. The results are followed by recommendations for the selection of suitable ILs to be applied in EPA extraction. The capacity values related to five types of cation-base ILs including imidazolium, pyridinium, pyrrolidinium, piperidinium, and tetra-methyl ammonium with 22 types of anions were screened at 298.15 K and the results are summarized in Figures 4 (a-e). The results are followed by recommendations for the selection of suitable ILs to be applied in EPA extraction. Generally, sigma profile divided into three important parts. The first part, where σ < −0.01 e/nm 2 , shows the hydrogen bond donor part. The second part extending between −0.01 < σ < +0.01 e/nm 2 , which belongs to the non-polarized area and the third part, where σ > +0.01 e/nm 2 , shows the hydrogen bond acceptor part. It is remarkable that there are a series of sharp a peak in the nonpolarized area and there is also a slight peak in hydrogen bond acceptor and donor. Consequently, EPA is highly non-polar and is due to all of the peaks merely crowded in the non-polar region.
Chemical potential or sigma potential (μ(σ)) demonstrate the deep understanding about which ILs structures have a high impact on interaction with EPA molecules. Sigma potential (μ(σ)) like sigma profile is divided into three important areas. The first zone where σ < −0.01 e/nm −2 belongs to the interaction with hydrogen bond donors. The second zone where −0.01< σ < 0.01 e/nm −2 is related to non-polar area and the third zone, where σ > 0.01 e/nm −2 belongs to the hydrogen bond acceptor part. Sigma potential shows that EPA can interact with a polar compound due to the peaks located in negative values on the y axis from 0 < (μ(σ)) < −0.7. Additionally it has high attraction toward both hydrogen-bond donor and acceptor molecules.
Investigation of the Effect of Cations Alkyl Chain Length on EPA Extraction:
In this study, the capacity values represent the interaction between the chemical structure of ILs and EPA molecules. Further, the COSMO-RS method is used to predict the behaviour of 22 types of anions through the investigation of the effect of increasing cation alkyl chain length base ILs towards EPA in the case of imidazolium, pyridinium, pyrrolidinium, piperidinium, and tetraalkyl ammonium. From the screening, it can be seen that increasing the alkyl chain length in the case of imidazolium cations, had a negative impact on the extraction capacity of the IL (Figure 4 and Appendix).
This seems true as the capacity could be arranged in the order [ Generally, sigma profile divided into three important parts. The first part, where σ < −0.01 e/nm 2 , shows the hydrogen bond donor part. The second part extending between −0.01 < σ < +0.01 e/nm 2 , which belongs to the non-polarized area and the third part, where σ > +0.01 e/nm 2 , shows the hydrogen bond acceptor part. It is remarkable that there are a series of sharp a peak in the non-polarized area and there is also a slight peak in hydrogen bond acceptor and donor. Consequently, EPA is highly non-polar and is due to all of the peaks merely crowded in the non-polar region.
Chemical potential or sigma potential (µ(σ)) demonstrate the deep understanding about which ILs structures have a high impact on interaction with EPA molecules. Sigma potential (µ(σ)) like sigma profile is divided into three important areas. The first zone where σ < −0.01 e/nm −2 belongs to the interaction with hydrogen bond donors. The second zone where −0.01< σ < 0.01 e/nm −2 is related to non-polar area and the third zone, where σ > 0.01 e/nm −2 belongs to the hydrogen bond acceptor part. Sigma potential shows that EPA can interact with a polar compound due to the peaks located in negative values on the y axis from 0 < (µ(σ)) < −0.7. Additionally it has high attraction toward both hydrogen-bond donor and acceptor molecules.
Investigation of the Effect of Cations Alkyl Chain Length on EPA Extraction
In this study, the capacity values represent the interaction between the chemical structure of ILs and EPA molecules. Further, the COSMO-RS method is used to predict the behaviour of 22 types of anions through the investigation of the effect of increasing cation alkyl chain length base ILs towards EPA in the case of imidazolium, pyridinium, pyrrolidinium, piperidinium, and tetraalkyl ammonium. From the screening, it can be seen that increasing the alkyl chain length in the case of imidazolium cations, had a negative impact on the extraction capacity of the IL (Figure 4 . However, the IL capacity is remarkably high when combined with the anions, SO 4 2− > Cl − > Br − . It is speculated that the interaction between the cation and the anion in these four exceptions is due to the absence of any alky chain in the anion, which makes the interaction (cation-anion) stronger. In addition, the charge density on the cation decreases and the molar volume increases according to the increase in alkyl chain length. The flexible alkyl chain may avoid the ions to pack effectively and cause a decrease in density as the result. It is expected that the increase in alkyl chain length and higher flexibility as the result, may interrupt the movement of IL component and avoid them to pass by each other. Furthermore it may increase the dispersion forces due to the enlarged molecular volume [38]. The inorganic ion's influence seems to be well described by the Hofmeister series. The Hofmeister effect has been studied for decades and in several aspects related to interfacial phenomena such as the behaviour of colloids, the denaturation of proteins, and the structure of microemulsions. The mechanism of the Hofmeister effect is not yet clear, however the notable fact has been proven that the interface within the hydrophilic and hydrophobic environments can be stabilized with presence of ions in the solution and their connections with the charged or polar head groups of amphiphilic molecules [39]. It is also noteworthy that the small anions can easily be replaced by the EPA hydroxyl group.
On the other hand, increasing the alkyl chain length attached to the cation, 3-methyl-pyridinium, contributed to the reduction of the extraction capacity in general, regardless of the anion used. Together, chloride followed by sulfate was the best among other anions tested in the screening. While the capacity value for the pair 1-ethyl-3-methyl-pyridinium and SO 4 was the highest, a general conclusion cannot be drawn based on the simulation. However, it can be accepted that according to properties of S=O as a good proton acceptor in forming H-bonds with OH from EPA ( Figure 5), therefore the hydrogen bond (S=O . . . H) between the cation and anion pair is strong. The regarded fact is even more emphasized while dealing with imidazolium-based IL with SO 4 2− as the anion. The different changing patterns in hydrogen bond strength suggest the possible existence of selective interactions among water, anion, and acidic protons on the imidazolium ring [40]. due to the absence of any alky chain in the anion, which makes the interaction (cation-anion) stronger. In addition, the charge density on the cation decreases and the molar volume increases according to the increase in alkyl chain length. The flexible alkyl chain may avoid the ions to pack effectively and cause a decrease in density as the result. It is expected that the increase in alkyl chain length and higher flexibility as the result, may interrupt the movement of IL component and avoid them to pass by each other. Furthermore it may increase the dispersion forces due to the enlarged molecular volume [38].
The inorganic ion's influence seems to be well described by the Hofmeister series. The Hofmeister effect has been studied for decades and in several aspects related to interfacial phenomena such as the behaviour of colloids, the denaturation of proteins, and the structure of microemulsions. The mechanism of the Hofmeister effect is not yet clear, however the notable fact has been proven that the interface within the hydrophilic and hydrophobic environments can be stabilized with presence of ions in the solution and their connections with the charged or polar head groups of amphiphilic molecules [39]. It is also noteworthy that the small anions can easily be replaced by the EPA hydroxyl group.
On the other hand, increasing the alkyl chain length attached to the cation, 3-methyl-pyridinium, contributed to the reduction of the extraction capacity in general, regardless of the anion used. Together, chloride followed by sulfate was the best among other anions tested in the screening. While the capacity value for the pair 1-ethyl-3-methyl-pyridinium and SO4 was the highest, a general conclusion cannot be drawn based on the simulation. However, it can be accepted that according to properties of S=O as a good proton acceptor in forming H-bonds with OH from EPA ( Figure 5), therefore the hydrogen bond (S=O … H) between the cation and anion pair is strong. The regarded fact is even more emphasized while dealing with imidazolium-based IL with SO4 2− as the anion. The different changing patterns in hydrogen bond strength suggest the possible existence of selective interactions among water, anion, and acidic protons on the imidazolium ring [40]. EPA has two hydrogen bond acceptors and one hydrogen bond donor, and therefore, possibilities to form H-bonding with another EPA molecule is likely to occur during extraction, thus one molecule could attract another molecule, as a chain reaction. The cation, in this case, has no protonated hydrogen, so no hydrogen bond could be from between the alkyl group and the fatty acid. However, it is possible to form H-bond with the nitrogen due to the lone pair. This makes the proposed scheme a likelihood during extraction.
While poor capacity was observed in case of Brwith [EMPyr], higher values were recorded when paired with [EMPyrro] in the order: EPA has two hydrogen bond acceptors and one hydrogen bond donor, and therefore, possibilities to form H-bonding with another EPA molecule is likely to occur during extraction, thus one molecule could attract another molecule, as a chain reaction. The cation, in this case, has no protonated hydrogen, so no hydrogen bond could be from between the alkyl group and the fatty acid. However, it is possible to form H-bond with the nitrogen due to the lone pair. This makes the proposed scheme a likelihood during extraction.
While has a different trend from previous cases. The nitrate anion has an electron to donate as well as the lone pair on the nitrogen, which makes it into a potential electron donor (able to bind). Moreover, looking into the tetra-methyl ammonium cation, it has positively charged nitrogen as all electrons are occupied with methyl groups. This may explain the low capacity of the pair (tetra-methyl ammonium nitrate) in comparison to Cl − , Br − which possess higher electronegativity. It may be predicted that in case larger alkyl groups, the anion can be at a more stable position, which could hinder its capacity for fatty acids extraction. Another suggestion is that ILs with hydrophobic anions (such as NTF 2 ) and long alkyl chain are a better choice for the extraction of non-polar substances, such as EPA, as the long carbon chain reduced the polarity of the FFA. On the other hand, ILs such as [EMIM]Cl are highly hydrophilic.
In this situation, the correlation between n-3 PUFA extraction and ionic liquids structures (aromatic or delocalized cation) for more enhanced PUFA extraction was suggested by Cheong et al. (2011) [41]. The hydrophilic or hydrophobic characteristics of ILs is significantly dependent on the structures of cations and anions, however the water miscibility of ILs are highly associated with the anion portion. The IL based on [PF 6 ] − and [Tf 2 N] − are typically water-immiscible and are therefore desirable for forming biphasic systems. The regarded ILs are widely used for the target of extraction [42]. was the first to report a study on the recovery of n-3 PUFA methyl esters from fish oil in which the ILs were combined with silver salts, and as the result, the hydrophobic ILs with large anions of low lattice energy were selected as the most desirable output [43,44]. Viscosity plays a crucial role, in the extraction of which, lower viscosity extensively improves the process. Moreover from the results, it was observed that the extraction capability of pyrrolidinium and tetra ammonium-based ILs were considerably lower than imidazolium and pyridinium-based ILs as it was below 20% for n-3 PUFAs and lower than 6% for n-3 PUFAEEs.
It can be noticed from these findings that the enrichment efficiency of n-3 PUFAs are strongly correlated to the aromatic structure of ILs. The correlation can be explained as the IL with aromatic structure is able to form temporary weak π-π interactions with polyunsaturated bonds in n-3 PUFA and enriches the component in the IL phase. It is found that ILs with cations containing aromatic rings, namely, the N,N -dialkylimidazolium ( 4 ) types have higher selectivity and extraction capability for n-3 PUFA and n-3 PUFAEE, as compared to those without aromatic structure. It can be concluded that the selectivity increases and hence the extraction capability improves with alkyl chain length from C 4 to C 8. However, it is not yet accurate to select the IL with higher selectivity merely by looking through cations. For example, in a study by Reference [41], same cation was used with different anions in screening ILs and it was observed that smaller anions have better extraction capability for PUFA. Fluoride (F − ) has the highest electronegative value and has the smallest anion size among the other anions in the comparison, [F − = Ne = 1s 2 2s 2 2p 6 ], which might be one of the reasons of its extraction capability, besides the stable electron configuration (the corresponding noble gas), although the COMSO-RS analysis demonstrated the opposite. Table 1 summarize Figure 4a-e for the ILs with high potential of EPA extraction in terms of infinite dilution activity coefficient and capacity values. In the following table, there is shortlist of the ILs with high capacity and lower activity coefficient at infinite dilution. Activity coefficient at infinite dilution is a term firstly introduced and discussed by Reference [45]. In this study, the activity coefficient is a term to account for the effective concentration of a solute in a solution of which its chemical potential can be expressed by the activity of the substances present in the solution. When the concentration of the solute tends to zero, the terms are hence called the activity coefficient at infinite dilution [46,47]. Activity coefficient at infinite dilution provides a deep vision on the degree of non-ideal behaviour in ILs-EPA compounds mixtures. Low activity coefficient values show high solubility of the ILs with weak solute-ILs interaction and vice versa, as described by References [48,49]. According to the listed ILs in Table 1, mono-cations were the selected cations since the anion (SO 4 2− ) with the charge of (−2) is outstanding and hence the overall charge for the IL molecule will be (−1). The presence of free charge is expected to increase the polarity and therefore increase the solvent extractive particularly. The final conclusion about the rate of mass transfer, the solubility of all components of the model solution, the distribution coefficients, and selectivity must be determined in order to decide the suitable IL for extraction [50].
Computational Details of COSMO-RS
The molecules geometries were optimized by turbo mole 6.3 program package (TURBOMOLE GmbH (ltd), Univerisity of Karlsruhe Karlsruhe, Germany) that follows the density functional theory (DFT), to generate COSMO files using Becke and Perdew (BP) functional B88-86 with a triple-zeta valence with polarization (TZVP), and the resolution of identity standard (RI) approximation as a basis set [51].
COSMO-RS is a theory with the purpose of quantum theory, surface interactions, dielectric continuum models, and statistical thermodynamic properties that has been described for the first time in a work by Reference [23]. At first, in 1998 the application of COSMO-RS was limited by calculation of the activity coefficient in infinite dilution for vapour-liquid equilibria of binary mixtures. Then, the usage of COSMO-RS go further to calculate all kinds of phase equilibrium such as vapour-liquid, liquid-liquid, and solid-liquid predictions [52,53].
In this study, the COSMO-RS approach has been used to screen 352 ILs (22 anions-16 cations combination) and to calculate their related capacity values. Selected ILs (with higher capacity values) are intended to be applied as solvents in EPA extraction. Figure 6 illustrates the following steps for screening, and therefore, the selection of an appropriate IL to be used in the extraction of the total EPA compound.
Data Collection and Screening Process:
The chemical structure of EPA which is illustrated in Table 2 is first optimized by TZVP according to thermodynamic properties and inserted to the data bank of COSMO-RS. Next, cations were selected from imidazolium, pyridinium, pyrrolidinium, piperidinium, and tetra-methyl ammonium based-ILs and hydrophilic and hydrophobic anions. The screened anions and cations based ILs were referred from the lipid extraction literature as a basis, as shown in Tables 3 and 4. Then, the software estimates the sigma profile and sigma potential of EPA and calculates the related capacity values for each IL toward EPA compound.
Data Collection and Screening Process
The chemical structure of EPA which is illustrated in Table 2 is first optimized by TZVP according to thermodynamic properties and inserted to the data bank of COSMO-RS. Next, cations were selected from imidazolium, pyridinium, pyrrolidinium, piperidinium, and tetra-methyl ammonium based-ILs and hydrophilic and hydrophobic anions. The screened anions and cations based ILs were referred from the lipid extraction literature as a basis, as shown in Tables 3 and 4. Then, the software estimates the sigma profile and sigma potential of EPA and calculates the related capacity values for each IL toward EPA compound.
Data Collection and Screening Process:
The chemical structure of EPA which is illustrated in Table 2 is first optimized by TZVP according to thermodynamic properties and inserted to the data bank of COSMO-RS. Next, cations were selected from imidazolium, pyridinium, pyrrolidinium, piperidinium, and tetra-methyl ammonium based-ILs and hydrophilic and hydrophobic anions. The screened anions and cations based ILs were referred from the lipid extraction literature as a basis, as shown in Tables 3 and 4. Then, the software estimates the sigma profile and sigma potential of EPA and calculates the related capacity values for each IL toward EPA compound.
Data Collection and Screening Process:
The chemical structure of EPA which is illustrated in Table 2 is first optimized by TZVP according to thermodynamic properties and inserted to the data bank of COSMO-RS. Next, cations were selected from imidazolium, pyridinium, pyrrolidinium, piperidinium, and tetra-methyl ammonium based-ILs and hydrophilic and hydrophobic anions. The screened anions and cations based ILs were referred from the lipid extraction literature as a basis, as shown in Tables 3 and 4. Then, the software estimates the sigma profile and sigma potential of EPA and calculates the related capacity values for each IL toward EPA compound.
Data Collection and Screening Process:
The chemical structure of EPA which is illustrated in Table 2 is first optimized by TZVP according to thermodynamic properties and inserted to the data bank of COSMO-RS. Next, cations were selected from imidazolium, pyridinium, pyrrolidinium, piperidinium, and tetra-methyl ammonium based-ILs and hydrophilic and hydrophobic anions. The screened anions and cations based ILs were referred from the lipid extraction literature as a basis, as shown in Tables 3 and 4. Then, the software estimates the sigma profile and sigma potential of EPA and calculates the related capacity values for each IL toward EPA compound.
Data Collection and Screening Process:
The chemical structure of EPA which is illustrated in Table 2 is first optimized by TZVP according to thermodynamic properties and inserted to the data bank of COSMO-RS. Next, cations were selected from imidazolium, pyridinium, pyrrolidinium, piperidinium, and tetra-methyl ammonium based-ILs and hydrophilic and hydrophobic anions. The screened anions and cations based ILs were referred from the lipid extraction literature as a basis, as shown in Tables 3 and 4. Then, the software estimates the sigma profile and sigma potential of EPA and calculates the related capacity values for each IL toward EPA compound.
Data Collection and Screening Process:
The chemical structure of EPA which is illustrated in Table 2 is first optimized by TZVP according to thermodynamic properties and inserted to the data bank of COSMO-RS. Next, cations were selected from imidazolium, pyridinium, pyrrolidinium, piperidinium, and tetra-methyl ammonium based-ILs and hydrophilic and hydrophobic anions. The screened anions and cations based ILs were referred from the lipid extraction literature as a basis, as shown in Tables 3 and 4. Then, the software estimates the sigma profile and sigma potential of EPA and calculates the related capacity values for each IL toward EPA compound.
COSMO-RS Assumptions
Solvent extraction process is a robust and economical methodology to be applied for extraction of EPA. There are three assumptions that were made during the screening process including: (a) The liquid state is incompressible, (b) all parts of the molecular surfaces can be in contact with each other, and (c) only pairwise interactions of molecular surface patches are allowed.
With respect to the stated assumptions, screening of charge density and capacity value at infinite dilution were performed by COSMO-RS. The significance of capacity value at infinite dilution is where it defined whether the targeted solvent has adequate strength in extraction. The screening charge density is defined as sigma profile of mixture ( ( )) and it can be calculated by Equations (1) and (2) [23,54,55]: In Equation (1), X i is the mole fraction of compound X, and p X i (σ) is the sigma profile of compound X. Additionally, in Equation (2), n i (σ) is the number of the segment with the surface charge density σ, A i (σ) is the total surface area of all segment with particular charge density σ.
COSMO-RS Assumptions
Solvent extraction process is a robust and economical methodology to be applied for extraction of EPA. There are three assumptions that were made during the screening process including: (a) The liquid state is incompressible, (b) all parts of the molecular surfaces can be in contact with each other, and (c) only pairwise interactions of molecular surface patches are allowed.
With respect to the stated assumptions, screening of charge density and capacity value at infinite dilution were performed by COSMO-RS. The significance of capacity value at infinite dilution is where it defined whether the targeted solvent has adequate strength in extraction. The screening charge density is defined as sigma profile of mixture ( ( )) and it can be calculated by Equations (1) and (2) [23,54,55]: In Equation (1), X i is the mole fraction of compound X, and p X i (σ) is the sigma profile of compound X. Additionally, in Equation (2), n i (σ) is the number of the segment with the surface charge density σ, A i (σ) is the total surface area of all segment with particular charge density σ.
COSMO-RS Assumptions
Solvent extraction process is a robust and economical methodology to be applied for extraction of EPA. There are three assumptions that were made during the screening process including: (a) The liquid state is incompressible, (b) all parts of the molecular surfaces can be in contact with each other, and (c) only pairwise interactions of molecular surface patches are allowed.
With respect to the stated assumptions, screening of charge density and capacity value at infinite dilution were performed by COSMO-RS. The significance of capacity value at infinite dilution is where it defined whether the targeted solvent has adequate strength in extraction. The screening charge density is defined as sigma profile of mixture ( ( )) and it can be calculated by Equations (1) and (2) [23,54,55]: In Equation (1), X i is the mole fraction of compound X, and p X i (σ) is the sigma profile of compound X. Additionally, in Equation (2), n i (σ) is the number of the segment with the surface charge density σ, A i (σ) is the total surface area of all segment with particular charge density σ.
COSMO-RS Assumptions
Solvent extraction process is a robust and economical methodology to be applied for extraction of EPA. There are three assumptions that were made during the screening process including: (a) The liquid state is incompressible, (b) all parts of the molecular surfaces can be in contact with each other, and (c) only pairwise interactions of molecular surface patches are allowed.
With respect to the stated assumptions, screening of charge density and capacity value at infinite dilution were performed by COSMO-RS. The significance of capacity value at infinite dilution is where it defined whether the targeted solvent has adequate strength in extraction. The screening charge density is defined as sigma profile of mixture ( ( )) and it can be calculated by Equations (1) and (2) [23,54,55]: In Equation (1), X i is the mole fraction of compound X, and p X i (σ) is the sigma profile of compound X. Additionally, in Equation (2), n i (σ) is the number of the segment with the surface charge density σ, A i (σ) is the total surface area of all segment with particular charge density σ.
COSMO-RS Assumptions
Solvent extraction process is a robust and economical methodology to be applied for extraction of EPA. There are three assumptions that were made during the screening process including: (a) The liquid state is incompressible, (b) all parts of the molecular surfaces can be in contact with each other, and (c) only pairwise interactions of molecular surface patches are allowed.
With respect to the stated assumptions, screening of charge density and capacity value at infinite dilution were performed by COSMO-RS. The significance of capacity value at infinite dilution is where it defined whether the targeted solvent has adequate strength in extraction. The screening charge density is defined as sigma profile of mixture ( ( )) and it can be calculated by Equations (1) and (2) [23,54,55]: In Equation (1), X i is the mole fraction of compound X, and p X i (σ) is the sigma profile of compound X. Additionally, in Equation (2), n i (σ) is the number of the segment with the surface charge density σ, A i (σ) is the total surface area of all segment with particular charge density σ.
COSMO-RS Assumptions
Solvent extraction process is a robust and economical methodology to be applied for extraction of EPA. There are three assumptions that were made during the screening process including: (a) The liquid state is incompressible, (b) all parts of the molecular surfaces can be in contact with each other, and (c) only pairwise interactions of molecular surface patches are allowed.
With respect to the stated assumptions, screening of charge density and capacity value at infinite dilution were performed by COSMO-RS. The significance of capacity value at infinite dilution is where it defined whether the targeted solvent has adequate strength in extraction. The screening charge density is defined as sigma profile of mixture ( ( )) and it can be calculated by Equations (1) and (2) In Equation (1), X i is the mole fraction of compound X, and p X i (σ) is the sigma profile of compound X. Additionally, in Equation (2), n i (σ) is the number of the segment with the surface charge density σ, A i (σ) is the total surface area of all segment with particular charge density σ.
COSMO-RS Assumptions
Solvent extraction process is a robust and economical methodology to be applied for extraction of EPA. There are three assumptions that were made during the screening process including: (a) The liquid state is incompressible, (b) all parts of the molecular surfaces can be in contact with each other, and (c) only pairwise interactions of molecular surface patches are allowed.
With respect to the stated assumptions, screening of charge density and capacity value at infinite dilution were performed by COSMO-RS. The significance of capacity value at infinite dilution is where it defined whether the targeted solvent has adequate strength in extraction. The screening charge density is defined as sigma profile of mixture (p s (σ)) and it can be calculated by Equations (1) and (2) [23,54,55]: In Equation (1), X i is the mole fraction of compound X, and p X i (σ) is the sigma profile of compound X. Additionally, in Equation (2), n i (σ) is the number of the segment with the surface charge density σ, A i (σ) is the total surface area of all segment with particular charge density σ.
The sigma potential (µ s (σ)) rely on composition, temperature (T), sigma profile (P s ), and electrostatic interaction energies (E mis f it ), which is determined by Equation (3): RT a e f f ln P s σ exp a e f f RT µ s σ − E mis f it σ, σ − E HB σ, σ dσ Equations (4) represent the electrostatic interaction energies [56].
where R is ideal gas constant, T is also absolute temperature, a e f f is the effective area of contact, E HB is hydrogen bonding energy, E mis f it is electrostatic interaction energy of two segments per unit area, α is an interaction parameter, σ and σ are two different screening charge densities for solute molecules that are in contact with each other, and E mis f it is the electrostatic interaction energy of two segments per unit area. The solvent capacity accounts for the quantity of solute that has been removed from the mixture by the extracting solvent [57]. The solvent capacity at infinite dilution, is numerically the maximum amount of solute (EPA) that can be dissolved in the solvent (IL). The capacity values of ILs for the extraction of EPA calculated by Equation (5).
where, γ ∞ is the activity coefficient of solvent at infinite dilution. The value of capacity indicates the amount of solvent that is used during the separation process. The higher solvent capacity results in a lower amount of solvent used. Therefore, the solvent with highest capacity value tends to be more desirable to choose as a separating agent.
Conclusions
Overall, the COSMO-RS method was used to screen the suitable ILs for microalgae lipid extraction based on the findings from this study. 4 ]) to produce a high yield of EPA from microalgae. It is expected to have a better extraction with the small anions that possess high charge density compared to larger ones. Moreover, shorter alkyl chain cations are preferred when using imidazolium-based IL, which agrees with experimental data. Overall, we may conclude that other factors should be addressed to select the IL for extraction such as the rate of mass transfer, the solubility, selectivity, and viscosity. Indeed, screening for the most suitable ILs for extraction reduces the time required and therefore would be more efficient in terms of resources. | v3-fos-license |
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} | pes2o/s2orc | Chromium (VI) in phosphorus fertilizers determined with the diffusive gradients in thin-films (DGT) technique
Phosphorus (P) fertilizers from secondary resources became increasingly important in the last years. However, these novel P-fertilizers can also contain toxic pollutants such as chromium in its hexavalent state (Cr(VI)). This hazardous form of chromium is therefore regulated with low limit values for agricultural products even though the correct determination of Cr(VI) in these fertilizers may be hampered by redox processes, leading to false results. Thus, we applied the novel diffusive gradients in thin-films (DGT) technique for Cr(VI) in fertilizers and compared the results with the standard wet chemical extraction method (German norm DIN EN 15192) and Cr K-edge X-ray absorption near-edge structure (XANES) spectroscopy. We determined an overall good correlation between the wet chemical extraction and the DGT method. DGT was very sensitive and for most tested materials selective for the analysis of Cr(VI) in P-fertilizers. However, hardly soluble Cr(VI) compounds cannot be detected with the DGT method since only mobile Cr(VI) is analyzed. Furthermore, Cr K-edge XANES spectroscopy showed that the DGT binding layer also adsorbs small amounts of mobile Cr(III) so that Cr(VI) values are overestimated. Since certain types of the P-fertilizers contain mobile Cr(III) or partly immobile Cr(VI), it is necessary to optimize the DGT binding layers to avoid aforementioned over- or underestimation. Furthermore, our investigations showed that the Cr K-edge XANES spectroscopy technique is unsuitable to determine small amounts of Cr(VI) in fertilizers (below approx. 1% of Cr(VI) in relation to total Cr). Electronic supplementary material The online version of this article (10.1007/s11356-020-08761-w) contains supplementary material, which is available to authorized users.
Introduction
Phosphorus (P) is essential for all living beings and is supplied by their respective nutrition. It is removed from farmlands in the form of agricultural products and must be replaced by application of fertilizers to allow continuous farming. Since phosphate rock and P became a scarce resource in the European Union (EU 2017), several processes are under development to recover P from wastewater for fertilizer production (Schaum 2018;Kratz et al. 2019). However, these novel P-fertilizers can also contain pollutants that are toxic for plants, animals, and humans.
One potential pollutant in P-fertilizers is chromium (Cr) in the hexavalent oxidation state (Cr(VI)) also called chromate (CrO 4 2− ) or dichromate (Cr 2 O 7 2− ). In contrast to the trivalent form Cr(III), which is an essential trace element, Cr(VI) is a highly mobile, toxic, and carcinogenic compound (Shahid Responsible Editor: Philippe Garrigues Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11356-020-08761-w) contains supplementary material, which is available to authorized users. et al. 2017). Therefore, the German fertilizer ordinance (2012) has a strict limit of 2 mg kg −1 Cr(VI) in fertilizers. Previous research (Krüger et al. 2017) revealed that various kinds of common and novel fertilizers can contain Cr(VI). An established chemical extraction method for the determination of Cr(VI) is the German standard DIN EN ISO 15192. In this method, an alkaline media is used at temperatures above 90°C to extract Cr(VI) and subsequently determine its concentration. However, the use of elevated temperatures and high pH values may hamper the correct determination of Cr(VI) concentrations especially in organic-and/or Fe(II)-rich matrices. The organic matter and Fe(II) in these matrices can reduce Cr(VI) and therefore cause misleading results. To overcome this problem, this study applied the novel diffusive gradients in thin-films (DGT) technique where water is used to extract the Cr(VI) from the solid material at ambient temperature. Consequently, the transformation of Cr species by high pH values at high temperatures is avoided by the DGT method. The DGT approach was subsequently compared with the aforementioned standard extraction method and observations made with Cr K-edge X-ray absorption near-edge structure (XANES) spectroscopy.
Previous DGT studies showed that the amount of nutrients and pollutants adsorbed by the binding layer of the DGT method nicely correlates to their bioavailability (Davison 2016;Vogel et al. 2017). The DGT device consists of a binding layer, a diffusion gel, and a filter (to protect the gel) assembled in a plastic holder (Davison 2016). The dissolved and labile Cr(VI) fraction of the fertilizer from moist fertilizer samples diffuses through the filter and the diffusion gel. During deployment, it subsequently adsorbs on a Cr(VI) selective binding layer (Pan et al. 2015). However, small amounts of Cr(III) may also adsorb to that binding layer. Hence, to evaluate the Cr(VI) selectivity of the DGT method, we applied Cr K-edge XANES spectroscopy because this approach enables a clear differentiation between the two oxidation states. In fact, a compound containing Cr(VI) will exhibit a prominent pre-peak within the XANES spectra, whereas compounds without any hexavalent Cr will not show this feature (Vogel et al. 2014(Vogel et al. , 2015a
Fertilizers origin and treatment
To provide samples with a variety of different Cr(VI) mass fractions, a recycling P-fertilizer was chosen as a model substance for this investigation. The recycling P-fertilizers are produced from sewage sludge ashes by thermochemical treatment with a sodium additive to produce the bioavailable phosphate form CaNaPO 4 (Stemann et al. 2015;Herzel et al. 2016). In contrast to the real thermochemical process, which is generally operated under reducing conditions, the samples here were prepared under oxidizing conditions to trigger Cr(VI) formation. The SSA originated from industrial-fluidized bed incinerators in Germany, where sewage sludge from municipal wastewater treatment plants was incinerated solely. This SSA was thermochemically treated with different amounts of Na 2 CO 3 to prepare the Cr(VI) containing model substances for the investigation. Therefore, 40 g of SSA was mixed with 1 g (SSA-Na1), 4 g (SSA-Na2), 7 g (SSA-Na3), 10 g (SSA-Na4), 11 g (SSA-Na5; similar to Pfertilizer in Vogel et al. 2014 andKrüger et al. 2017), and 20 g (SSA-Na6) of Na 2 CO 3 (Fisher Chemicals Loughborough, UK), respectively. Afterwards, it was thermochemically treated at 950°C for 30 min, under o x i d i z i n g co n d i t i o n s ( a i r ) i n a m u ff l e fu r n a c e (Nabertherm LH 15/14, Lillenthal, Germany).
Furthermore, a sedimentary phosphate rock (PR) from Morocco, a triple super phosphate (TSP) and a converter lime (CL) were used as potentially Cr(VI) bearing fertilizers for the investigation.
All fertilizer samples were air-dried, divided representatively by a dividing cross, and grinded with a tungsten carbide vibratory disc mill (see also Krüger et al. 2017). The total Cr amount of the fertilizers was measured by ICP-OES (Thermo iCAP 7000 Series, Dreieich, Germany) after total digestion (HNO 3 /HClO 4 /HF) in a microwave (mikroPrepA, MLS GmbH, Leutkirch, Germany; heating with 1000 W; 20 min isotherm segment at 240°C). These measurements were carried out in triplicate.
Wet chemical chromium(VI) extraction (DIN method)
We performed Cr(VI) wet chemical extraction according to DIN EN 15192 (2006). The procedure consists of an alkaline extraction of the solid sample, followed by filtration for liquid-solid separation and measurement of Cr(VI) by ICP-MS (Thermo iCAP Q, Dreieich, Germany). Therefore, 2.5 g of the sample (weighed in on 0.1 mg) were put in a 250 mL beaker, and the following chemicals were added: 50 mL of the extraction solution containing 0.5 mol L −1 NaOH and 0.28 mol L −1 Na 2 CO 3 (Fisher Chemicals Loughborough, UK), 1 mL of 4.2 mol L − 1 MgCl 2 •6H 2 O (Merck, Darmstadt, Germany) to avoid Cr(III) oxidation by oxygen, and a buffer solution of each 0.5 mol L − 1 K 2 HPO 4 and KH 2 PO 4 (both Carl Roth, Karlsruhe, Germany). We capped the beaker with a watch glass and heated the solution for 1 h at 92.5°C on a magnetic hot plate stirrer with a PTFE-coated magnetic stir bar. The temperature was controlled with a contact thermometer to avoid boiling of the solution as well as evaporation to dryness. The measurements were carried out in triplicate.
Chromium(VI) DGT extraction
The content of mobile Cr(VI) in the various fertilizers was also analyzed by DGT devices (window size, 2.54 cm 2 ) equipped with a 0.8 mm APA (polyacrylamide) diffusion layer and a 0.6 mm Cr(VI) selective N-methyl-D-glucamine (NMDG) binding layer (Pan et al. 2015; purchased from DGT Research, Lancaster, UK). After a 24 h conditioning period of the fertilizer at 60% of the water holding capacity (WHC; ISO 2012), the fertilizers were brought to 100% WHC, transferred onto the DGT devices, and deployed for 24 h at 25°C. Subsequently, the extraction of adsorbed Cr from the DGT binding layer was carried out with 1 M HNO 3 for 24 h. Afterwards, the Cr concentrations of the extracts were analyzed by ICP-MS (Thermo iCAP Q, Dreieich, Germany) and used to calculate the DGT values. The C DGT values of Cr(VI) were calculated using the following equation: where M is the mass of Cr(VI) accumulated on the binding gel, Δg is the diffusion gel thickness, D is the diffusion coefficient of Cr(VI) for the deployment temperature, A is the area of the exposure window of the DGT device, and t the deployment time. The diffusion coefficient for Cr(VI) at 25°C is 8.82 × 10 −6 cm 2 s −1 (Pan et al. 2015). The DGT measurements were carried out in triplicate. Furthermore, DGT devices (window size, 2.54 cm 2 ) with 0.8 mm APA diffusion layer and 0.6 mm NMDG binding layer (DGT Research, Lancaster, UK) were loaded with 200 mL solutions (30 mg Cr L −1 ) of various water-soluble Cr(III) (CrCl 3 ) and Cr(VI) (NaCrO 4 , K 2 CrO 4 , K 2 CrO 7 ) compounds. The DGT devices were deployed for 24 h at 25°C in constantly agitated solutions. After deployment, the binding layers of the DGT devices were washed with distilled water, dried at room temperature, and analyzed by Cr K-edge XANES spectroscopy (see below).
Chromium K-edge XANES spectroscopy of fertilizers
Chromium K-edge XANES spectroscopy measurements were carried out at P64 beamline (Caliebe et al. 2019) at the electron storage ring PETRAIII (DESY, Hamburg, Germany). The incident beam was monochromatized with a Si<111> double crystal monochromator. The scans were acquired at 50 K, to reduce radiation damage in the sample. The Cr K-edge XANES spectra were collected in the range of 5980-6100 eV with a Canberra 100 pixel high-purity germanium detector in fluorescence mode for the fertilizer samples. The Cr references were measured in transmission mode using two ion chambers filled with nitrogen. All spectra were background subtracted and normalized to an edge jump of Δμd = 1.
Afterwards, Cr K-edge XANES spectra of the fertilizers were fitted with linear combination fitting (LCF) of Cr reference compounds (following mentioned as XANES-LCF) with the software Demeter Athena (Ravel and Newville 2005). Therefore, the following XANES spectra were used: CaCrO 4 , Na 2 CrO 4 , K 2 CrO 4 , K 2 Cr 2 O 7 , Cr 2 O 3 , MgCr 2 O 4 , CaCr 2 O 4 , FeCr 2 O 4 , CrCl 3 , Cr(OH) 3 , CrPO 4 , and Cr-sub. FeOOH. The relative proportions of the components, whose number was limited to three, were forced to add up to 100%. From the remaining fits, the best fit was chosen, as seen by the highest R value.
A second method, in the following named XANES-H, was applied to calculate the Cr(VI) amounts from the XANES spectra for comparison. Here, the height of the pre-peak was analyzed in relation to the height of the edge: The values from the reference spectra of Cr 2 O 3 and CaCrO 4 were set to 0% and 100% Cr(VI), respectively.
Chromium and Cr(VI) contents of the investigated fertilizers
In Table 1, the amounts of total Cr and Cr(VI) are listed for the different P-fertilizers. For Cr(VI), the data of the different investigated methods are given, respectively. For the series of thermochemically treated SSA (SSA, SSA-Na1 -SSA-Na6), it can be clearly seen that the total Cr content decreases from 118.3 (SSA) to 86.9 mg/kg (SSA-Na6) due to the dilution with increasing amounts of added Na 2 CO 3 . At the same time, the formation of Cr(VI) is clearly initiated by thermochemical reaction with the Na additive and oxygen starting from 0.2 Cr(VI) in SSA-Na3 and increasing to 68.5 mg/kg Cr(VI) present in SSA-Na6 (data of DIN method) (see also Fig. S1). A thermochemical treatment according to recipe SSA-Na4 would lead to Cr(VI) mass fractions above the limit of 2 mg/kg of the German Fertilizer Ordinance. The tested TSP and the PR have both Cr(VI) mass fractions below the limit of 2 mg/kg (DIN method). The converter lime CL showed with 7.4 mg/kg Cr(VI) a value above the limit for fertilizers in Germany (DIN method).
Applicability of the DGT method for the determination of Cr(VI) in P-fertilizers
The DGT method adsorbs mobile Cr(VI) from the fertilizer/ water mixture for a certain definite time span and is thus not an absolute method. The data from DGT are given in μg L −1 after extraction of the DGT binding layer following a defined procedure (see "Materials and methods" section). Thus, a calibration with standard materials would be required to determine Cr(VI) mass fractions by DGT which was not part of this work. However, we found a good correlation (R 2 = 0.93) between the wet chemical extraction (DIN method) and the data determined by the DGT method for the series of ash-based model fertilizers SSA -SSA-Na6 (see Fig. S2).
However, the conventional fertilizers TSP and CL show no correlation between DIN and DGT results (see red and green circle, respectively, in Fig. 1). The CL fertilizer contains a very high mass fraction of CaO (> 40%) which could support the development of CaCrO 4 (Vogel et al. 2015b). This Cr(VI) compound is, in contrast to the other Cr(VI) compounds, only hardly water-soluble (Vogel et al. 2014) which might be the reason for the low DGT value. In contrast, TSP has a much higher DGT value compared with wet chemical extraction. TSP is made from phosphate rock which is treated with phosphoric acid during production. Here, a formation of mobile Cr(III) species could be possible. In fact, Pan et al. (2015) stated that small amounts of Cr(III) can also be adsorbed to the DGT binding layer. Thus, to determine the Cr(VI) selectivity and robustness of the DGT method, the investigated P- Comparison between DGT method and XANES spectroscopy Figure 2 shows the Cr K-edge XANES spectra of various Pfertilizers. All spectra have a white line at 6008 eV and a postedge feature at 6023 eV (except for SSA-Na6). These features come close to those of Cr(III) reference compounds presented in Fig. 3. The fertilizers SSA-Na4, SSA-Na5, and SSA-Na6 show an additional pre-peak of Cr(VI) at 5992 eV. Linear combination fitting (LCF) of SSA (Fig. 4 top) indicates mainly chromite (MgCr 2 O 4 or FeCr 2 O 4 ) and chromium phosphate as Cr-bearing phases. Very similar results were also detected for the materials SSA-Na1 to SSA-Na3. In contrast, the LCF fit of SSA-Na6 (Fig. 4 bottom) also indicates Cr(VI) compounds in the fertilizer. A similar result was found for SSA-Na4 and SSA-Na5. From the results of the LCF fits and the total mass fractions of Cr (Table 1) in the fertilizers, the Cr(VI) Fig. 2 Chromium K-edge XANES spectra of various fertilizers amount was calculated (see XANES-LCF in Table 1). In contrast to the LCF of the XANES spectra (XANES-LCF), the DGT and wet chemical Cr(VI) analyses indicate also Cr(VI) in SSA-Na3, PR, TSP, and CL (Table 1). Only the results from the SSA-based materials with higher Cr(VI)-contents show a good correlation to the DGT method (R 2 = 0.99; Fig. S3).
In contrast to the XANES-LCF approach, the XANES-H approach indicates Cr(VI) in SSA-Na3 to SSA-Na6 as it was observed by DGT and the wet chemical DIN method. Similarly to the XANES-LCF method, there is a good correlation between the XANES-H and DGT method for SSAbased fertilizers (R 2 = 0.97; Fig. S4) and an even better Fig. 3 Chromium K-edge XANES spectra of various Cr compounds agreement for SSA-Na3. For the RP, TSP, and CL fertilizers, there is no agreement between DGT, wet chemical, and XANES-H method, since the latter indicates no Cr(VI) in these samples. Similarly to our approach, Bajt et al. (1993) and Rinehart et al. (1997) used a method where the area of the pre-peak was calculated. These authors revealed that fractions of 5% and 3% Cr(VI) out of total Cr could barely be detected by Cr K-edge XANES spectroscopy, respectively. It is therefore likely to assume that a fraction of 3% Cr(VI) out of total Cr represents the quantification limit for this technique. However, this XANES Cr(VI) quantification limit is independent from the total Cr amount. RP, TSP, and CL contain only 0.4-0.6% Cr(VI) in relation to total Cr (according to DIN method; Table 1) and thus much less than the proposed quantification limit. Accordingly, these samples could be below the quantification limit of Cr K-edge XANES spectroscopy. In contrast, SSA-Na3 has only a Cr(VI) amount of 0.2% in relation to total Cr (DIN method, Table 1), but with the XANES-H method, a little bit higher amount (0.8%, Table 1) could be detected. Thus, there might be a possibility that a quantification with the XANES-H method might be possible down to approx. 1% Cr(VI) in relation to total Cr.
XANES spectroscopy investigations of deployed DGT binding layers
Figure 5 (top) shows the Cr K-edge XANES spectra of the Cr(VI) selective DGT binding layers saturated with various Cr(III) and Cr(VI) compounds. The XANES spectra of the Cr(VI) saturated binding layers (top) are very similar to those of solid Cr(VI) compounds (see Fig. 3) and also show the characteristic pre-peak. In contrast, the Cr(III) saturated binding layer (DGT-CrCl 3 ) has no pre-peak, and the XANES spectrum features are close to those of solid chromites (see Fig. 3).
Due to the very low amounts of adsorbed Cr on the DGT binding layer, it was not possible to collect Cr K-edge XANES spectra for all deployed binding layers of the various fertilizers. SSA-Na4 and SSA-Na6 (SSA-Na5 was not investigated) show the Cr(VI) pre-peak, and the spectra come close to those of the Cr(VI) DGT references. Due to the high signal-to-noise ratio of the XANES spectrum of SSA-Na3, a detection of a Cr(VI) pre-peak was impossible (see Fig. 5). In contrast to SSA-Na4 and SSA-Na6, the XANES spectrum of the deployed binding layer of TSP Fig. 4 Chromium K-edge XANES spectrum of SSA (top) and SSA-Na6 (bottom) and corresponding linear combination fittings of different Cr references did not show a pre-peak. Furthermore, this XANES spectrum is also very similar to that of the DGT-Cr(III) reference. Therefore, we assume that TSP contains mobile Cr(III) that adsorbs also on the Cr(VI) selective binding layer. Thus, the existence of mobile Cr(III), which probably develops during the production of TSP, where phosphate rock is treated with phosphoric acid, might lead to an overestimation of Cr(VI) with the DGT method.
Conclusion
All three investigated methods for the determination of Cr(VI) in P-fertilizers from secondary or primary raw materials seem to have advantages and drawbacks. Krüger et al. (2017) mentioned earlier that the presence of organic matter and inorganic substances with potential to reduce Cr(VI) during wet chemical extraction according to the DIN method can cause underestimation of Cr(VI) in P-fertilizers. The DGT method was very sensitive and for most tested materials selective for the analysis of Cr(VI) in P-fertilizers made from recycled materials. However, the results of certain types of P-fertilizers containing mobile Cr(III) or hardly soluble Cr(VI) show that some optimization of the method is still required to avoid over-or underestimation of Cr(VI). Therefore, the DGT binding layer should be optimized so that mobile Cr(III) (like from fertilizer TSP) will no longer be adsorbed. Furthermore, the DGT method cannot detect hardly soluble Cr(VI). This, however, is also an advantage of this method because only the mobile and bioavailable Cr(VI), which is an important parameter for plant growth, will be analyzed. Additionally, the Cr Kedge XANES spectroscopy method also shows good results but fails to determine small amounts of Cr(VI) (below approx. 1% Cr(VI) in relation to total Cr) in fertilizers.
Funding information CV thanks the German Research Foundation (VO 1794/4-1) for financial support. VM thanks the German Federal Ministry for Education and Research (05K16PX1) for financial support.
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} | pes2o/s2orc | In Vitro Cultured Melissa officinalis Cells as Effective Ingredient to Protect Skin Against Oxidative Stress, Blue Light, and Infrared Irradiations Damages
: Skin is being increasingly exposed to artificial blue light due to the extensive use of elec ‐ tronic devices, which can induce cell oxidative stress, causing signs of early photo aging. The Melissa officinalis phytocomplex is a new standardized cosmetic ingredient obtained by an in vitro plant cell culture with a high content of rosmarinic acid. In this study, we examine the activity of the Melissa officinalis phytocomplex to protect skin against blue light and infrared damages, evaluating the ROS (Radical Oxygen Species) level in keratinocyte cell line from human skin (HaCaT) and Nrf2 (Nuclear factor erythroid 2 ‐ related factor 2), elastin, and MMP1 (Matrix Metalloproteinase 1) immunostain ‐ ing in living human skin explants ex vivo. This phytocomplex demonstrates antioxidant activity by reducing ROS production and thus the oxidant damage of the skin caused by UV and blue light exposure. In addition, it inhibits blue light ‐ induced Nrf2 transcriptional activity, IR ‐ induced elastin alteration, and IR ‐ induced MMP ‐ 1 release. This Melissa officinalis phytocomplex is a new innovative active ingredient for cosmetic products that is able to protect skin against light and screen exposure damages and oxidative stress. ROS were detected by diacetylated 2 ,7 ′‐ dichlorofluorescein (DCF ‐ DA) staining at basal condition and following the exposure to oxidative stimulus H 2 O 2 . The fluorescence was measured by using a Victor3X multilabel plate counter (Ex 485 nm and Em 535 nm). Results are expressed as fold change of ROS production compared to control. Each bar the mean SD of n=3 experi ‐ ments. $$$
Introduction
Melissa officinalis L. (lemon balm) is an edible perennial herb of the Lamiaceae family. Its name originates from the Greek words for bee (melissa) and honey (meli). There are worldwide records of its medicinal and culinary use that date back to Dioscorides (father of pharmacology) times (40-90 A.D.) and allow its safe exploitation [1]. Melissa officinalis is reputed in folk medicine for memory-enhancing effects, promoting long life, action against gastrointestinal disorders, rheumatism, Alzheimer's, thyroid diseases, colic, anemia, nausea, vertigo, syncope, asthma, bronchitis, amenorrhea, cardiac disorders, epilepsy, insomnia, migraines, nervousness, malaise, depression, psychosis, hysteria, and wounds [2]. Several scientific papers confirm the medicinal effectiveness of Melissa officinalis preparations, as well as its antioxidant and other properties suggesting its use for the prevention of oxidative stress-related diseases [3]. The bioactivity of Melissa officinalis extracts is mainly attributed, as for any other plant formulation, to the qualitative and quantitative composition of secondary metabolites (i.e., phenolic acids, flavonoids, and terpenoids). Rosmarinic acid is a caffeic acid ester with 3,4-dihydroxyphenyllactic acid, and it is a main bioactive component of the Melissa officinalis extracts [1]. The rosmarinic acid and phenolic acids content in the Melissa officinalis extract is highly variable. The variability is associated with multiple factors, which are difficult to control: seasons, plant age, geographical growing areas, and tissues used for the preparation of products [4]. Furthermore, the preparation of standardized Melissa officinalis derivatives with a reproducible content of metabolites is very difficult. The extreme variability in the content of phytoconstituents of plant preparations obtained directly from a plant, or parts thereof, by extraction negatively impacts the effectiveness of the same. An alternative method for obtaining contaminant-free standardized plant preparation in industrial quantities is to use in vitro cell cultures.
Plant cell culture technology is a technique for growing plant cells under strictly controlled environmental conditions [5] that makes it possible to solve the problems tied to the variability of plant extracts, since it provides preparations with a content of active substances that can be reproduced in a standardized manner. The derived extracts can be easily standardized in their primary and secondary metabolites and are compliant with the safety requirements of being contamination-free and phytochemically uniform [6]. In addition, to respect biodiversity and greater environmental sustainability with a drastic reduction in the use of natural resources such as water and soil, this technology makes it possible to overcome seasonal and geographical limitations by guaranteeing a higher safety profile for the consumer (free from heavy metals, pesticides, aflatoxins, bacterial or fungal contamination) and an elevated degree of standardization. The objective of the present study concerns the appraisal of the biological activity of a product rich in rosmarinic acid obtained by a stable and selected cell line of Melissa officinalis [7]. In this work, we demonstrate the efficacy of a Melissa officinalis product obtained by in vitro cell cultures to protect the skin against blue light and IR damages.
The skin is the main defensive barrier in the body against a large variety of environmental factors and is responsible for maintaining body homeostasis, defence, and repair [8]. It is well known that exposure to internal and external factors activates lots of molecular processes that damage skin structure. The sunlight spectrum consists of short, highenergy wavelengths, from ultraviolet radiation (280-400 nm) to visible light (400-700 nm) and infrared radiation (700 nm-1 mm), long and low energy wavelengths [9]. Visible light wavelengths penetrate the deepest parts of the dermis, reaching the different types of skin cells and inducing cell oxidative stress, damage, and dysfunction, causing signs of early photo-aging [10]. Skin is being increasingly exposed to artificial blue light due to the extensive use of electronic devices. This, together with recent observations reporting that blue light can exert cytotoxic effects associated with oxidative stress and promote hyperpigmentation, has sparked interest in blue light and its potential harmful effects on skin [11]. Several studies report that blue light contributes to skin aging and carcinogenesis, mostly during direct sunlight exposure, similar to UVA [12].
Numerous pharmacological activities of rosmarinic acid have been described. Among other activities, it protects cells membranes against oxidative damage [13]. Fernando et al. [14] showed that rosmarinic acid treatment of keratinocyte cell line from human skin (HaCaT) cells damaged from UVB radiation recovered the expression levels of Nrf2 (Nuclear factor erythroid 2-related factor 2) protein from the cytosol into the nucleus. These results indicate that rosmarinic acid may protect cellular environments from freeradical damage and thereby enhance the cellular antioxidant defense system. In this work, we examine the efficacy of an ingredient highly standardized in rosmarinic acid that was obtained by a selected cell line of Melissa officinalis, to protect the skin against blue light and IR damages. Especially, we demonstrated the activity of this new ingredient in countering the harmful action of ROS (Radical Oxygen Species), in inhibiting the expression of Nrf2 and MMP1 (Matrix Metalloproteinase 1), and in protecting the degradation of elastin. The evaluation of these parameters has allowed us to ascertain that the Melissa officinalis phytocomplex obtained by in vitro cell culture, standardized in rosmarinic acid, is able to reduce skin damages caused by ROS, blue light, and IR exposure.
Melissa officinalis Cell Culture
Cell culture of Melissa officinalis L. was obtained from seeds bought and certified from the nursery plant "Le Georgiche", Brescia, Italy.
The sterile plants of Melissa officinalis L. were obtained from seed sanitized by means of a treatment in sequence with 70% (v/v) ethanol (Honeywell, Wunstorfer Straβe 40, D-30926 Seelze, Germany)) in water for about 2 minutes, 2% (v/v) of sodium hypochlorite solution (6-14% active chlorine, (MERCK KGaA, 64271 Darmstadt, Germany) and 0.1% (v/v) Tween 20 (Duchefa, Postbus 809, 2003 RV-Haarlem, The Netherlands) for 15 minutes and, finally, at least 4 washes with sterile distilled water. The sanitized seeds were placed in trays containing nutrient medium Gamborg B5 [15] rendered solid by adding 0.8% w/v of plant agar (Duchefa) and without growth hormones. After a suitable period of incubation under light (15 days) and at 25 °C, the seeds began to sprout. Twenty days after germination, small leaves were collected from the plants grown under sterile conditions. The leaves were fragmented into small pieces (explants) of sub-centimetric dimensions (0.1-0.5 cm). The fragments of plant tissue were deposited in Petri dishes containing solidified Gamborg B5 medium supplemented with 20 g/L sucrose (Sudzucker AG, Manheim, Germany), 1 mg/L of (NAA) naphtalenacetic acid (Duchefa), 1 mg/L of (IAA) indoleacetic acid (Duchefa), 0.5 mg/L of (K) Kinetin (Duchefa), and 0.9% (w/v) of plant agar, final pH 6.5 (MO Medium). The pH was adjusted to 6.5 before autoclaving. Explants were incubated at 25°C in the dark. Calli grown on Melissa officinalis phytocomplex (MO) medium were subjected to subculture for at least 4 months until they became friable and homogeneous, with a constant growth rate (Melissa officinalis stable cell line). The botanical origin of Melissa officinalis L. was confirmed by fingerprint DNA analysis made from Parco Tecnologico Padano, Lodi, Italy [16].
A part of selected calli (10% w/v) was transferred into 250 ml of culture liquid medium (MO medium without Plant Agar) contained in Erlenmeyer flasks of 1 liter volume. The suspension cultures were maintained at 25 °C in the dark in constant agitation on the rotary shaker at 120 rpm, and every 7 days of fermentation, they were subjected to a liquid subculture. The volume of biomass was increased by subculture on flasks of 3l volume containing 1l of fresh liquid medium culture. The amount of suspension culture inoculated into the liquid medium was equal to 6% v/v.
To increase the content of rosmarinic acid, after 7 days of fermentation, the suspension culture was inoculated in a bioreactor of 5L volume containing 3L of MO final liquid medium (Gamborg B5 with the addition of 35 g/L of sucrose, 0.5 mg/L di NAA, 0.5 mg/L of IAA, and 0.25 mg/L of K, final pH 6.5). The suspension culture was grown for a culture cycle of 14 days.
Phytocomplex Preparation from Melissa officinalis Cell Culture
After 14 days of growth, at 25 °C and in the dark, the Melissa officinalis cell suspensions were filtered by 50 μm mesh filter, and the medium cultures were discarded. Cells were washed with a double volume of saline solution (0.9% w/v NaCl in sterile water) and added with 1% (w/w) of citric acid and then homogenized with ultraturrax at 15,000 rpm for 20 minutes. The biomass of homogenated cells was dried to obtain a powder of phytocomplex with a high content of rosmarinic acid.
Quantification of Rosmarinic Acid and the Total Polyphenols in the Melissa officinalis Phytocomplex by UPLC-DAD
First, 100 mg of powder of the Melissa officinalis phytocomplex were weighed into a 15 ml test tube, and 30 volumes of ethanol (Honeywell) and water 60:40 (v/v) were added.
The suspension was mixed for 30 seconds with a vortex mixer and sonicated for 15 minutes in an ice bath; finally, it was centrifuged at 4000 rpm for 15 minutes at 6 °C. At the end of centrifugation, the supernatant was recovered. Then, 15 mL of supernatant were transferred into a new test tube 15 and preserved in ice until loading into the UPLC system. The sample was diluted 1:10 (first 1:5 in a solvent and then 1:2 in water). The diluted sample was filtered over 0.22 μm filters before being loaded into the UPLC system. Five independent replicates of the phytocomplex were extracted and measured. The chromatography system used for the quantification of rosmarinic acid consists of an Acquity UPLC BEH C18 1.7 μm column, size 2.1 mm × 100 mm, coupled to an Acquity UPLC BEH C18 1.7 μm VanGuard Pre-Column 3/Pk, size 2.1 mm × 5 mm. The platform used for the UPLC-DAD (Ultra Performance Liquid Chromatography-Diode Array Detection) analysis comprises a UPLC system (Waters Corporation, Milford, MA 01757, USA) consisting of an eluent management module, Binary Solvent Manager model I Class, and an autosampler, Sample Manager-FTN model I Class, coupled to a PDA (Photo Diode Array) eλ diode array detector. Empower 3 (Waters) software was used to acquire and analyze the data. The chromatography method used was the following: solvent A: water, 0.1% formic acid; solvent B: 100% acetonitrile. The initial condition is 99% solvent A; moreover, the flow remains constant at 0.350 mL/min throughout the duration of the analysis. The chromatography column was temperature controlled at 30 °C. Elution of the molecules was conducted by alternating gradient and isocratic phases, as indicated in Table 1. For quantification of the rosmarinic acid, the chromatogram obtained at the wavelength of 330 nm was used. The rosmarinic acid was quantified thanks to the calibration curve of the authentic commercial standard of rosmarinic acid (CAS 20283-92-5; pu-rity≥95%; Sigma Aldrich). The data analysis was carried out with Empower 3 software.
HaCaT cells (5 × 10 3 ) were seeded into 96-well plates, allowed to adhere overnight, and then treated for 24 hours with the compounds, according to the experimental protocol. ROS levels were measured after the addition of 100 μM DCF-DA, further incubation for 30 min at 37 °C, and wash with phosphate-buffered saline (PBS) [17] as previously described. N-acetyl-L-cysteine 5 mM (Sigma Aldrich) and Rutin at 0.1% w/v were used as positive controls and the Melissa officinalis phytocomplex was used at 0.1% w/v.
Probe fluorescence intensity was measured at excitation 485 nm-emission 535 nm, in the absence or presence of 1.1 μM H2O2 used as oxidative stimulus, using a Multilabel Plate Reader VICTOR X3 (PerkinElmer). Fold increases in ROS production were calculated using the equation: where F is the fluorescence reading.
The statistical analysis was performed using GraphPad Prism version 6 for Windows (GraphPad Software, San Diego, CA, USA). Results are presented as mean ± SD of 3 experiments. An unpaired Studentʹs t-test was used to compare ROS values, and p values <0.05 were considered statistically significant.
Sample Preparation and Explant Distribution
Human living skin explants were prepared from an abdominoplasty (aesthetic surgery) from a 35-year-old healthy Caucasian woman with a phototype II-III (Fitzpatrick classification), after obtaining informed consent. Adipose tissue was removed, and explants of 1 cm² were prepared using a circular biopsy punch. The explants were maintained in survival cell culture conditions at 37° C in a humid atmosphere enriched with 5% CO2 in BIO-EC's explant medium. BIOEC's explant medium is a proprietary culture medium specifically engineered by the BIO-EC Laboratory for the survival of skin explants.
Product Preparation and Application
The Melissa officinalis phytocomplex (MO) has been prepared in sterile distilled water at 0.05% and 0.1% (w/v). Successively, the MO at 0.05% and MO at 0.1% were applied topically based on 2 mg/cm² and spread using a small spatula on day 0 (D0), day 1, day 4, and day 5 (30 min before blue light or infrared exposure). The control explants "C" did not receive any treatment.
Blue Light Irradiations
Skin explants from the batches "BL", "MO at 0.05% + BL", and "MO at 0.1% + BL" were exposed to blue light using the Solarbox® device (SATIE, France), which is a visible light radiation system, designed and produced by the SATIE laboratory (CNRS joint research unit UMR 8029, Cergy Paris University). The light source is ensured by a set of configurable light-emitting diodes able to deliver visible blue radiation with an emission spectrum of 420 to 580 nm, with a peak at 455 nm.
On day 5, the explants of the batches mentioned above were exposed to a blue light dose of 63.75 J/cm 2 . The unirradiated explants were kept in the dark during the whole time of blue light exposure.
Infrared Irradiation
Skin explants from the batches "IR", "MO at 0.05% + IR", and "MO at 0.1% + IR" were irradiated using an infrared lamp (Dr FISCHER 1000W, 235V 2500K; 760-1150 nm) on day 5, with an infrared dose of 720 J/cm 2 . The unirradiated explants were kept in the dark during the whole time of infrared exposure.
Histological Processing
On day 6 (D6), 24 hours after blue light or infrared exposure, the explants from each batch were collected and cut in two parts. Half was fixed in buffered formalin, and the other half was frozen at -80°C.
After fixation in buffered formalin, the samples were dehydrated and impregnated in paraffin using a Leica PEARL dehydration automat. The samples were embedded using a Leica EG 1160 embedding station. Then, 5-μm-thick sections were made using a Leica RM 2125 Minot-type microtome, and the sections were mounted on Superfrost® (Menzel-Gläser, Hungary) histological glass slides.
Staining and Immunostaining
The cell viability of epidermal and dermal structures was observed on formol-fixed paraffine-embedded (FFPE) skin sections after Masson's trichrome staining, Goldner variant. The cellular viability was assessed by microscopical observation. Both immunohistochemistry and immunofluorescence stainings were performed on either 5μm-thick FFPE skin sections or 7μm-thick frozen sections using the following primary antibody: anti- The microscopical observations were realized using an Olympus BX63 microscope. Pictures were digitized with a numeric DP74 Olympus camera with cellSens Dimension storing software (Olympus, Tokyo, Japan). The intensity level of each immunostaining was scored from 1 to 6.
Statistical Analysis
The statistical analysis was performed using GraphPad Prism version 3.03 for Windows (GraphPad Software, San Diego, CA, USA). Results are presented as mean ± SD of 3 experiments. An unpaired Studentʹs t-test was used to compare ROS values and, p values <0.05 were considered statistically significant.
Melissa officinalis Phytocomplex With High Content of Rosmarinic Acid Was Obtained by a Stable and Selected Cell Line
A stable and selected cell line of Melissa officinalis was obtained using the MO solid medium. In this selected solid medium, the Melissa officinalis cell line is beige-colored with brown tinges and has a friable texture and a high rate of growth (subculture in fresh solid medium every 14 days). Figure 1 shows the cell culture maintained in solid MO medium (a) and cells of the line Mo-4AR seen under an AXIO-Imager A2 optical microscope (ZEISS), in the bright field mode (b) and after staining with fluorescein diacetate (c). The content of rosmarinic acid and total polyphenols in the cell line was optimized using a MO final liquid culture medium with a higher content of sucrose (35g/L) and with a lower concentration of growth hormones (0.5 mg/L of NAA, 0.5 mg/l of IAA, and 0.25 mg/L of K). The cells grown into MO final liquid culture medium were used to prepare the Melissa officinalis phytocomplex. The UPLC-DAD analysis was used to estimate the content of rosmarinic acid and total polyphenols content into the Melissa officinalis phytocomplex. The chromatographic profile of the phytocomplex at 330 nm is shown in Figure 2. The data obtained show that the content of rosmarinic acid in the phytocomplex is 7.6 ± 0.1% w/w and the content of total polyphenols expressed as equivalent in rosmarinic acid is 9.2 ± 0.1% w/w. Thus, the main component of total polyphenols in the phytocomplex is represented by rosmarinic acid (83% of the total polyphenols).
Melissa officinalis Phytocomplex Exhibits Strong Inhibition of ROS Production after H2O2 Oxidative Stimulus in HaCaT Cell Line
In order to evaluate the ability of the Melissa officinalis phytocomplex to modulate the oxidative stress, ROS levels in HaCaT cells were evaluated, following 24 hours' treatment. ROS generation was evaluated both in basal conditions and following the oxidative stimulus, which was induced by the addition of H2O2. The obtained results showed a significant decrease in ROS production in HaCaT cells treated with the Melissa officinalis phytocomplex, in the presence of oxidative stimulus, able to increase ROS levels, as shown in the untreated cells ( Figure 3). The positive antioxidant controls, NAC 5 mM and Rutin 0.1% w/v, confirmed the anti-oxidant effect of the Melissa officinalis phytocomplex. ROS were detected by diacetylated 2′,7′-dichlorofluorescein (DCF-DA) staining at basal condition and following the exposure to oxidative stimulus H2O2. The fluorescence was measured by using a Victor3X multilabel plate counter (Ex 485 nm and Em 535 nm). Results are expressed as fold change of ROS production compared to control. Each bar represents the mean ± SD of n=3 experiments. *p < 0.05, **p < 0.01, ***p < 0.001, treatment vs. basal control. $$$ p<0 .001, treatment vs. stimulated control.
Cell Viability
The application of the Melissa officinalis phytocomplex at 0.05% w/v and at 0.1% w/v on human skin explants induces no modification on the cell viability compared to the untreated control on D6, demonstrating that the product is well tolerated by ex vivo human skin.
Neither the blue light nor infrared irradiation induces modification of the cell viability. Upon irradiations, the application of the Melissa officinalis phytocomplex at 0.05% and at 0.1% w/v induces no modification of the cell viability to the batches (Figure 4).
Nrf2 Immunostaining
The results of the immunostaining using the antibody anti-phosphorylated Nrf2 in the living epidermidis are summarized in Figure 5. After 6 days of treatment, the Melissa officinalis phytocomplex 0.05% (P1) or 0.1% w/v (P2) does not modify the basal level of Nrf2, compared to the untreated control batch. Figure 5. Scoring of the Nrf2 immunostaining by microscopical examination on D6, on unexposed or blue light-exposed batches (BL). T is the untreated control, P1 and P2 are the batches treated with the Melissa officinalis phytocomplex at 0.05% and 0.1%, respectively. The blue light irradiation induces a moderate increase of the activated (nuclear) form of Nrf2 in the living epidermis. The Melissa officinalis phytocomplex application at 0.05% w/v induces a slight decrease of the activated form of Nrf2 activation upon blue light exposure, so it partially reduces blue light-induced Nrf2 increase.
In parallel, the phytocomplex application at 0.1% w/v completely inhibits the blue light-induced Nrf2 increase (Figure 6).
Elastin Immunostaining
The results of the immunostaining of elastin in the papillary dermis on all the batches are summarized in Figure 7. After 6 days of treatment, the Melissa officinalis phytocomplex at 0.05 % w/v induces a slight decrease on elastin expression compared to the untreated control batch, while when applied at 0.1% w/v, no modifications are observed.
MMP-1 Immunostaining
The results of the staining of MMP-1 in the papillary dermis on all the batches are summarized in Figure 9. After 6 days of treatment, the Melissa officinalis phytocomplex 0.05% w/v induces a slight increase of MMP-1 level, whereas when applied at 0.1% w/v, the phytocomplex induces a slight decrease of MMP-1 level compared to the untreated control batch. Figure 9. Scoring of the MMP-1 (Matrix Metalloproteinase 1) immunostaining by microscopical examination on day 6 on unexposed or infrared exposed batches (IR). T is the untreated control, P1 and P2 are the batches treated with the Melissa officinalis phytocomplex at 0.05% and 0.1% respectively.
Upon IR irradiation, the Melissa officinalis phytocomplex at 0.05% w/v induces no variation of MMP-1 level in the papillary dermis. On the contrary, the phytocomplex at 0.1% w/v induces a fairly clear decrease, so it totally prevents IR-induced MMP-1 increase in the dermis (Figure 10).
Discussion
The Melissa officinalis phytocomplex with a high and a standardized content of rosmarinic acid (7.6 ± 0.1% w/w) was obtained by using a plant cell culture technology [7]. Traditional plant extracts have extreme variability in the phytocomplex composition, and it depends on a lot of factors such as climate, soil, and cultivation techniques. This variability cannot guarantee the efficacy of the extract in health care applications. Using in vitro cell culture technology, we can obtain standardized products free from pesticides, contaminates, and residual solvents, maintaining similar biological efficacy in all batches [6]. The Melissa officinalis phytocomplex is standardized in the rosmarinic acid and total polyphenols content, which confers highly reproducible biological activities.
The use of the plant secondary metabolite rosmarinic acid has been studied in pharmaceutical and dietary supplements in Alzheimer's disease, atopic dermatitis, and cardiovascular disease [18,19]. Previous research has revealed that the effects of rosmarinic acid in these contexts are mediated by its antioxidant properties [20].
In the present work, we demonstrate the antioxidant properties of the Melissa officinalis phytocomplex using a stress oxidative model in the human cell line HaCaT after stimulus with H2O2. Keratinocytes represent 95% of the epidermal cells. Primarily, they play the structural and barrier function of the epidermis, but their role in the initiation and perpetuation of skin inflammatory and immunological responses, and wound repair, is also well recognized. The spontaneously immortalized human cell line HaCaT exhibits normal morphogenesis and expresses all the major surface markers and functional activities of isolated keratinocytes; for this reason, HaCaT will be chosen as a model for the study of keratinocytes functions and for investigating the effects of the compounds of interest on an epidermal model. Lorrio et al. demonstrated that a natural aqueous extract of Deschampsia antarctica protects skin against artificial ad natural light by reducing the production of ROS and hyperpigmentation in fibroblasts and melanocytes [11]. In this study, the chemical composition of the natural aqueous extract of Deschampsia antarctica was not described, while in our work, we obtained a phytocomplex highly strandardized in polyphenol content using the cell culture technology. The standardization in marker metabolites of the final product is a key point to guarantee the antioxidant efficacy of the Melissa officinalis phytocomplex.
Skin is the essential barrier protecting organisms against environmental insults and minimizing water loss from the body. Skin aging and dysfunctions are determined by several factors, including internal metabolism and environmental toxicant exposure. One of the leading causes of these processes is oxidative stress. Although there are efficient antioxidant systems in skin, the excessive and uncontrolled production of ROS is a major pathogenic factor that causes a range of skin diseases, inflammations, allergic reactions, and also neoplastic processes. To assess the ability of the Melissa officinalis phytocomplex to modulate the oxidative stresses, the general ROS levels in HaCaT cells were evaluated using a chemical fluorescent probe. The results obtained show that among a significant increase in ROS levels induced by oxidative stimulation, 0.1% w/w of the Melissa officinalis phytocomplex is able to prevent ROS formation, showing a potent antioxidant effect. The effect of the Melissa officinalis phytocomplex is comparable to the one observed for the pure compound Rutin (0.1% w/w) and is stronger than NAC (N-Acetyl-L-Cysteine) at 5 mM. This result indicates a potential protective effect of the Melissa Officinalis phytocomplex against ROS-mediated cell impairments.
Probably, the Melissa officinalis phytocomplex with a high content of rosmarinic acid (7.6% w/w) rescued H2O2-mediated inhibition of Nrf2 transcriptional activity and the consequential decrease in Nrf2 target gene expression [20]. Consistent with these findings, a previous study demonstrated that rosmarinic acid inhibits UVB-induced ROS production and the decrease in protein levels encoded by Nrf2 target genes in HaCaT keratinocytes [14].
Nrf2 is a key transcription factor in the cellular response to oxidative stress. Human Nrf2 has a predicted molecular mass of 66 kDa, and it is ubiquitously expressed in a wide range of tissues and cell types. Under oxidative stresses, including UV irradiation, Nrf2 is activated by phosphorylation and translocates from the cytoplasm to the nucleus. So far, different cytosolic kinase, including protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinase (MAPK), andER-localizedpancreatic endoplasmic reticulum kinase (PERK) have been shown to modify Nrf2 and are potentially involved in the dissociation of Nrf2 from its inhibitor, Keap1 [21]. Once in the nucleus, Nrf2 binds to the DNA at the location of the Antioxidant Response Element (ARE) or also called hARE (Human Antioxidant Response Element), which is the master regulator of the total antioxidant system. Nrf2 plays a role in protecting human skin keratinocytes from UVA radiation-induced damage [22] and environmental pollution [23]. The protection effect of the Melissa officinalis phytocomplex against the irradiation of blue light was evaluated on living human skin explants ex vivo by the immunostaining of Nrf2. As shown in Figure 6, the blue light irradiation induces an increase of nuclear translocation of Nrf2 in the living epidermis. The Melissa officinalis phytocomplex at 0.05% w/w presents a fairly good protection activity against blue light irradiations by partially reducing the Nrf2 blue light-induced activation. The higher concentration (0.1% w/w) of the phytocomplex presents a good protection activity against blue light irradiations by totally reducing the Nrf2 blue light-induced activation, suggesting that the Melissa officinalis phytocomplex is able to maintain a healthy balance of anti-oxidant response upon blue light irradiation.
The protection effect of the Melissa officinalis phytocomplex against the irradiation of infrared was evaluated on living human skin explants ex vivo by immunostaining of elastin and MMP-1.
Elastin is a protein of the connective tissue responsible for the elastic properties of the skin. It is rich in hydrophobic amino acids such as glycine and proline, which form mobile hydrophobic regions bounded by crosslinks between lysine residues, and it is localized mainly on the elastic fibers of the papillary dermis [24]. The principal function of elastin is its ability to elastically extend and contract in repetitive motion when hydrated. Elastin is the major component (90%) of the elastic fibers of the skin. Several factors including the formation of methylglyoxal [25] or UV exposure are able to damage the elastic network of dermis, including elastin [26]. In dermo-cosmetic research, elastin is usually used as a bio-marker of the structural state of the extracellular matrix of the dermis.
MMP-1 is an enzyme also known as interstitial collagenase and fibroblast collagenase. MMP-1 is expressed in migrating keratinocytes via ligation of the α2β1 integrin with dermal collagen and is a reliable marker of activated keratinocytes in wounded human skin. Other factors, including smoke [27] and UV exposure [28], induce MMP-1 expression, leading to premature skin aging.
The Melissa officinalis phytocomplex at 0.05% w/w shows a moderate protection activity against infrared irradiations by totally preventing the IR-induced elastin alteration ( Figure 8). The higher concentration (0.1%w/w) of the phytocomplex shows a good protection activity against infrared irradiations by totally inhibiting the elastin alteration and MMP1 release induced by IR exposure (Figure 10). These results indicate that the Melissa officinalis phytocomplex at 0.1% w/w may protect the skin against the damages of oxidative stress and blue light by reduction of Nrf2 activation. In addition, the product protects the skin against the infrared irradiations damage by the inhibition of elastin alteration and MMP-1 release.
Conclusions
In conclusion, the findings of the present study suggested that the Melissa officinalis phytocomplex is a new standardized cosmetic ingredient obtained by an in vitro plant cell culture with a high effectiveness to protect skin against oxidative stress, blue light, and irradiations of infrared damages. The in vitro plant cell culture has allowed us to obtain a phytocomplex with high content of rosmarinic acid and without environmental contaminants using an eco-sustainable process. This Melissa officinalis phytocomplex demonstrate antioxidant activity by reducing ROS production and thus the oxidant damage of the skin caused by UV and blue light exposure. In addition, the efficacy of this phytocomplex is related to the inhibition of blue light-induced Nrf2 transcriptional activity, IR-induced elastin alteration, and IR-induced MMP-1 release. The Melissa officinalis phytocomplex is a new innovative active ingredient for cosmetic products that is able to protect skin against light and screen exposure damages.
Patents
Patent ITA102019000004113-PCT/IB2020/052589: Phytocomplex and selected extract of a meristematic cell line of a plant belonging to the genus Melissa. | v3-fos-license |
2021-07-27T00:05:41.128Z | 2021-05-25T00:00:00.000 | 236371514 | {
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} | pes2o/s2orc | 3D Electrospun Synthetic Extracellular Matrix for Tissue Regeneration
matrices (ECM), which greatly contribute to biomaterials design to stimulate tissue regeneration. Herein, the recent advances in electrospinning technology for 3D production of ECM-mimicking biomaterial scaffolds are systematically summarized and the applications in neural, cardiac, bone, skin, and vascularized tissue regeneration are thoroughly discussed. Challenges and future scopes related to each fi eld of tissue regeneration are discussed after each subsection. A few examples of liver, kidney, esophageal tissue engineering are also discussed. Finally, the key challenge in the cost-effective upscale of the electrospinning technique to mature and prevalent industrial applications is outlined. Herein, a systematic, thorough summary of the recent evolutions in electrospinning and its emerging applications for a broad range of tissue regenerations is provided.
Introduction
Tissue regeneration is a rapidly evolving and interdisciplinary field at the intersection of life science, biology, material science, and engineering. Centrally, it involves functional cell-free or cell-laden constructs or biomaterial scaffolds that are fabricated utilizing various strategies. [1] Cell-free biomaterial scaffolds implanted directly at the sites of injury may mechanically support local cells to promote local tissue repair. [2] Furthermore, they can be functionalized both on the surface or in the interior with bioactive materials to stimulate regeneration of functional tissues. [3] In cell therapy applications, cell-laden constructs may enhance both survival and differentiation of the therapeutic cells compared with cell transplantation alone. The grafted cells may then ideally replace lost tissue and/or exert beneficial effects on the host tissue. [4] Among different biomaterial constructs, 3D fibrous scaffolds recreate a 3D microenvironment, which closely resembles the native extracellular matrix (ECM), including both structural and biochemical properties that guide cell survival and differentiation. [5] Scaffolds with fibrous networks possess unique characteristics, including sufficiently high interconnected porosity, high specific surface area, tunable mechanical properties, as well as optimal morphological features. The high porosity of the fibrous scaffolds facilitates mass transfer for effective nutrient supply, oxygen diffusion, metabolic waste removal, and enhancement of intercellular communications, consequently allowing high cell viability and function throughout the entire scaffold. [6] In addition, compared with 2D culture, the 3D cell culture provides a more realistic biochemical and biomechanical microenvironment, [7] creating an optimal environment for cell migration, proliferation, and differentiation. Hence, nanofibrous scaffolds with appropriate biomechanical properties are highly suitable for tissue engineering. [8] Electrospinning, as a relatively simple and versatile fiber preparation technique, has been developed to create fiber-based constructs in combination with cells, bioactive molecules, proteins, and biocompatible nanomaterials. [9] Many studies have demonstrated that the electrospinning technology has the potential for significant progress within the field of tissue regeneration. Chen et al. [10] summarized the methods for producing electrospun 1D nanofiber bundles, 2D DOI: 10.1002/smsc.202100003 Electrospinning is considered the most versatile micro-/nanofiber fabrication technology. The electrospun fibers hold high surface area, desired mechanical properties, controlled topography, as well as the ease of biochemical functionalization. The 3D electrospun fibrous structures closely mimic the hierarchical architecture and fibrous features of the extracellular matrices (ECM), which greatly contribute to biomaterials design to stimulate tissue regeneration. Herein, the recent advances in electrospinning technology for 3D production of ECMmimicking biomaterial scaffolds are systematically summarized and the applications in neural, cardiac, bone, skin, and vascularized tissue regeneration are thoroughly discussed. Challenges and future scopes related to each field of tissue regeneration are discussed after each subsection. A few examples of liver, kidney, esophageal tissue engineering are also discussed. Finally, the key challenge in the cost-effective upscale of the electrospinning technique to mature and prevalent industrial applications is outlined. Herein, a systematic, thorough summary of the recent evolutions in electrospinning and its emerging applications for a broad range of tissue regenerations is provided.
nanofiber membranes, and 3D nanofiber scaffolds and indicate the possible combinations of electrospinning with 3D printing, flexible electrodes, and microfluidics for biomedical applications. Ogueri et al. [11] reviewed electrospinning in matrix-based regenerative engineering, focusing mainly on musculoskeletal tissues. Sun et al. [12] discussed electrospinning techniques for fabricating 3D nanostructures, and He et al. [13] summarized electrohydrodynamic-based bioprinting techniques, including near-field solution electrospinning, melt electrowriting, and electrospray cell printing. There are recent reports of advances in tissue regeneration of bone, [14] skin, [15] vascular, [16] and cardiac tissue. [17] However, comparatively few reviews extensively discuss in-depth fabrication processes together with bioengineering of diverse tissues. This review aims to update and summarize the most recent advancements in electrospinning technique toward fabricating 3D scaffolds, while also thoroughly reviewing emerging applications in neural, cardiac, bone, skin, vascular, liver, kidney, and esophageal tissue regeneration (Figure 1). After discussing the breadth and depth of electrospun fiber biomaterials, we outline the key challenges and future perspectives of electrospinning for tissue regeneration.
Advancement of Electrospinning in 3D Production
Electrospinning is a highly versatile scaffold fabrication technique that combines an electric field with spinning to draw polymer solutions into micro-or nanofibers. The typical electrospinning setup is composed of four essential elements: 1) a syringe pump with a syringe that supports a stable flow of polymeric fluid through a flat-tipped stainless-steel needle to the initial jet point; 2) a high-voltage power supply that provides a robust electrostatic force drawing the polymeric solutions into polymer jets; 3) a fume hood that can remove solvent evaporation from polymer jets and accelerate the solidification process during spinning; and 4) a metal collector plate for collecting the fibers. In the most commonly used electrospinning configurations, the stainless-steel needle is connected with high voltage, and the collector is grounded. Some modified electrospinning devices use an opposite connection or a dual electrode with both positive and negative voltage. To further improve the electrospinning process, modifications of the spinneret/collector and addition of an assisted magnetic/electric field have been proposed in the literature. A graphical illustration of a simple electrospinning device and different modified electrospinning configurations are shown in Figure 2.
Conventional electrospinning is known for fabricating densely packed submicrometer fibers. Simply increasing the electrospinning time is the easiest way to obtain scaffolds with a certain thickness. However several hours of electrospinning only increases the scaffold thickness by %0.14 mm, [18] due to the loss of electrostatic force after insulating the collector with the collected fibers.
Template-assisted electrospinning is one of the derived electrospinning techniques, which is done by modifying the shape of the collector. Using an insulated poly(methyl methacrylate) (PMMA) mask on the collecting copper plate to focus the collection of fibers, it is possible to produce scaffolds with 3 mm thickness in a relatively short time. [19] The architecture of the fiber collector plates may be modified as well, to yield a template, which makes it possible to create customized 3D structures. Template-assisted collectors are usually designed using either a computer-aided design (CAD) program [20] or conventional textiles. [21] Many shapes such as honeycomb-like, [22] helical spring, [23] metal pin, [24] and micropatterned structures [25] have been designed for various purposes. Furthermore, some biomimetic collectors, including auricle-shaped [26] and vascular-like [27] collectors, have been designed to be used for unique biomimetic tissue applications. The template itself may also be added to or produced in the process of electrospinning. For instance, microice crystals formed during low-temperature electrospinning under high humidity were found to act as removable pore templates between the fibers. This resulted in enlarged distance between fibers, producing a 3D loosely packed architecture. [28] Similarly, Park et al. proposed a method based on NaCl templates. [29] Here they found that NaCl crystals increased the pore size of the electrospun fibers, improving cell proliferation within the scaffold. Template-assisted electrospinning has furthermore been used in combination with microfluidic devices to improve the cell culture conditions under continuous flow. [30] Alternatively, self-assembly electrospinning can be used to increase the scaffold thickness and obtain 3D structures. Due to fast solidification from the polymer jets and the electrostatic attraction between the fibers on the grounded collector and the charged jets, the scaffold structure may grow toward the charged jets, thereby forming 3D structures with a thickness reaching more than 10 cm. [31] Honeycomb-patterned structures fall into the self-assembly electrospinning category due to the electrostatic repulsion between adjacent fibers. [32] This type of structural network has been intensively investigated due to the similarity to the osteon structure. [33] The choice of polymer solution (polymer solutes and solvents) is the dominant effective factor when modifying the scaffold thickness in self-assembly electrospinning. Other parameters, such as applied voltage, polymer concentration, and flow rate, etc., have less effect. So far, polystyrene (PS), [31] polyacrylonitrile (PAN), poly(vinyl alcohol) (PVA), polyethylene oxide (PEO), [32] and their hybrids [34] reportedly exhibit self-assembly properties during electrospinning. It is worth mentioning that surfactant coating (Beycostat A B09) [35] and emulsion [36] likewise promote the self-assembly electrospinning process. As the electrostatic interactions and solidification rate are factors difficult to quantify, the optimization of selfassembly electrospinning is still under investigation.
Generally, the collector type is a significant element in electrospinning and makes a great impact on the surface topography and the 3D geometry of the scaffolds. One type of collector is a coagulation bath in a metal container, which leads to liquidassisted collection, also called wet electrospinning. [37] Ethanol, [38] mixed ethanol/water solution, [39] hexane, [40] or subcritical CO 2 fluid [41] have been used as coagulating agents. In addition, some of the chemicals in the solutions may induce crosslinking [42] or functional surface coating on the fibers. [38a,43] An important aspect to consider for the collector bath is the surface tension of the collector solution. Solutions with low surface tension for target fibers create loosely packed 3D fibrous scaffolds. Conversely, solutions with high surface tension resemble a traditional collector plate, where the fibers are collected on top of the solution, resulting in densely packed fibers. It is noteworthy that electrolyte solutions (e.g., KCl solutions) can act as a ground collector that combines the functions of the metal container and the coagulation bath into one. This yields a more effective way to fabricate free-standing, complex nanofiber architectures according to the designed patterns. [44] High humidity [45] and immersions [46] are variations of the wet electrospinning technique. Wet electrospinning has further been combined with a rotating collector, [39a,42b] layer-by-layer [47] electrospinning, and template electrospinning [44] for various 3D scaffolds. In addition, post-treatment with immersion and freeze drying may be helpful in transforming dense electrospinning membranes into loosely packed 3D structures. [48] Further improvements have been made to the traditional electrospinning technique, such as applying an x-y translational motion collector, similar to the one used in standard 3D printing. [49] Utilizing the jetting initiated prior to the onset of bending stability, high-precision and direct-write deposition of microfibers have been achieved by decreasing the electrospinning distance, a technique called near-field electrospinning (NFE). As an extension to NFE, the atomic force microscope (AFM) system (AFM-based voltage-assisted electrospinning) allows fabrication of nanofibers in a controllable fashion via polymer deposition from an AFM probe. [50] Initially, NFE was mainly applied for polymers dissolved in solvents. [51] As evaporation of solvents may affect the precision of the fiber deposition, [52] solvent-free electrospinning was introduced to the field of NFE in 2008, creating the emerging melt electrospinning writing (MEW) technology. [53] Although MEW makes it possible to achieve high-resolution nanostructures with tunable coil densities [54] and precise deposition of single fibers, [49] it cannot achieve wide-range regulation in height. This challenge must be overcome before MEW is fully applicable in the construction of artificial organs. Combining MEW with template-assisted collectors could be one way of addressing this limitation. [55] Besides using NFE and MEW, controlled deposition of fibers has been achieved using an additional magnetic/electric field to standard electrospinning. Using a cylindrical side-wall electrode and a sharp-pin ground electrode, the electrospinning jet was focused and a patterned nanofibrous mat was fabricated. [56] However, until now, aligned architecture and tunable deposition area have only been achieved for bundles of fibers. [57] Inspired by MEW, polymer melts may possibly be utilized in future fieldassisted electrospinning techniques to potentially yield precise control of single-fiber deposition. [49,58] Field-assisted electrospinning could become the next-generation leading electrospinning technique for single-fiber deposition.
Last but not the least, layer-by-layer electrospinning may be the most flexible strategy. It combines not only different electrospinning methods, [59] but also other technologies, such as cell layer and electrospinning layer, [60] 3D printing layer and electrospinning layer, [61] and electrospraying layer and electrospinning layer. [62] Table 1 shows the six electrospinning techniques covered in this Review with their pertinent features and limitations. It is foreseen in the future that even more techniques could be incorporated into the versatile electrospinning platform.
Tissue Regeneration Based on 3D Electrospun Scaffolds
3D electrospun scaffolds can help fabricate highly porous structures with biomaterials mimicking the natural 3D extracellular microenvironment for improved cell adhesion, proliferation, and differentiation. [54,63] 3D electrospun scaffolds display strong potential in tissue regeneration through their biochemical cues from natural, synthetic, or hybrid biomaterial selections and physical cues from the mechanical, topographical, and geometrical features of the fiber constructs. [38b,64] Most recent studies have focused on producing 3D bioactive and biocompatible scaffolds with potential applications in fields such as neural, cardiac tissue, and bone tissue regeneration. [63c,65] The following sections seek to give an in-depth description of the role of electrospinning as a tool in tissue regeneration applications.
Neural Tissue Regeneration
Millions of people worldwide suffer from peripheral nerve injuries and central nervous system (CNS) damage produced by stroke, traumatic brain injury, neurodegenerative diseases, spinal cord injuries. Though the CNS is highly plastic and to some degree capable of self-regeneration, lost neural tissue is often not Centimeter scale, [167] depending on the template PCL, poly(vinylidene fluoride), poly(ester-urethane), poly(lactic acidco-glycolic acid), polylactide, PEO, collagen, polyaniline (PANI), PS 3D template-assisted collection; patterned collector with accurately controlled geometries The broad range of materials; direct fabricating; good controllability (porosity, fiber organization, and geometry) Depend on the shape and geometry of collector; customized collector Self-assembly electrospinning 10 cm for 30 min [31] PCL, poly(L-lactide), PS, poly(vinylpyrrolidone), PEO, pellethane, poly(lactic acid) (PLA), poly (carboxybetaine-co-methyl methacrylate) co-polymer, poly(vinylidene fluoride-cotrifluoroethylene) fully replaced. Therefore, much hope for the treatment of CNS injury lies in tissue engineering approaches. As the fibers can provide physical guidance and topical stimulation in the repair process of nerves, and fibrous nerve conduits can be introduced at lesion sites by implantation, many in vitro investigations have focused on optimizing electrospun fibrous scaffolds to provide a suitable microenvironment for neural growth. [66] Combining the 3D structures with bioactive cues, inspired by the composition of the ECM, is one way to improve scaffold performance. For example, covalently bonded laminin [66a] and immobilized brain-derived neurotrophic factor [67] combined with electrospun scaffolds were found to promote neurite extension. In addition, basement membrane extract used as a coating material was found to yield increased production of bioactive nerve growth factor (NGF) and vascular endothelial growth factor (VEGF) by rat embryonic stem cells. [68] A PCL/CS/ polypyrrole (PPy) nanofibrous scaffold significantly increased cell proliferation up to 356% in comparison with pure PCL. [69] A key component of the ECM, glycosaminoglycan (GAG), was coated onto the surface of electrospun scaffolds and enhanced Schwann cell activity. [70] GAG mimetic containing cellulose sulfate (CelS) and partially sulfated cellulose sulfate (pCelS) was introduced into gelatin scaffolds, an approach that increased NGF binding to the scaffold and neurite extension of dorsal root ganglion neurons. [71] To sum up, 3D structures combined with bioactive cues have proven a fairly straightforward way of providing a suitable substrate for neural growth and maturation. Interestingly, these benefits have also been observed after transplantation in vivo. 3D fibrous architectures were found to selectively reduce the presence of residual proliferative-induced pluripotent stem (iPS) cells (proliferation marker Ki67) and accelerate maturation in vitro (higher expression of microtubuleassociated protein 2, MAP2) ( Figure 4A a-c). These cell-laden scaffolds were transferred into the mouse striatum in vivo ( Figure 4A d). Three weeks after transplantation, postsynaptic density protein 95 (PSD95, depicted in blue, with downward-pointing arrows) was detected adjacent or colocalized to transplanted iPS-induced neuronal (iN) neurite terminals ( Figure 4A e), indicating synaptic integration with the host tissue. In addition, these 3D electrospun scaffold-supported neuronal networks were found to enhance survival and engraftment of neurons in the murine brain tissue after injection compared with injection of isolated cells ( Figure 4A f ). [63c] As neurons are electroactive, conductive scaffolds recently received considerable attention in the field of neural tissue regeneration due to their potential in improving tissue prosthetics using electrical stimulation (ES) and bioelectronic signal transfer. [72] In vitro, conductive materials have been shown to promote growth and differentiation of neural and neural-like cells. [73] For instance, application of ES using a triboelectric nanogenerator device significantly accelerated nongenetic neuronal conversion of mouse fibroblasts to iN cells. [74] A schematic representation of an artificial device used for providing such ES is shown in Figure 4B. [75] Highly conductive carbon nanomaterials, such as graphene or reduced graphene oxide (rGO), have gained significant interest in recent years, [76] due to their remarkable properties such as high surface area, high mechanical strength, and ease of functionalization. Modified 0.5% rGO has been incorporated into poly(ester amide) and found to reduce film resistance by at least 10 4 Ω sq À1 . [76b] rGO has been reported to have low dispersibility in different polymer matrices. To overcome this problem, Gou et al. increased the dispersibility of rGO polymer matrix by grafting trimethylene carbonate (TMC) oligomers onto rGO. [77] With electrical pulses, rGO has been reported to accelerate nerve growth and increase expression of βIII-tubulin, MAP2, and nestin. [72b,75] This significant increase in neural marker expression was due to the influx of Ca 2þ induced by a depolarizing current, which consequently was found to activate the calmodulin kinases to elicit neurites outgrowth and development. Calcium influx can be assessed by the relative fluorescence intensity change during a pulse period when preincubated with Ca 2þ -dependent dye ( Figure 4C). [75] Even conductive materials without ES may be used to influence the cell behavior by enhancing transmission of electrical signals between cells. For example, graphene/polyelectrolyte 3D architecture enhanced neuron cell adhesion and neurite outgrowth. [72a] Polyurethane(PU)/silkfunctionalized multiwalled carbon nanotube scaffolds [78] likewise significantly improved expression of neural markers in vitro and increased axonal growth without any ES. Another approach to obtain artificial electrical active neural network systems involved coating metal such as gold onto the surface of electrospun fibers. [79] Besides inorganic nanomaterials, promising conductive polymers have been under investigation. Loosely packed 3D conductive PAN/PPy electrospun nanofibers increased differentiation and migration of cortical cells after ES. [80] Adding PPy to the polymer blend was found to enhance the growth of rat pheochromocytoma 12 (PC12) neural-like cells by 59% by increasing the electroactivity of nanofibrous scaffold. [69] Electrically conductive PANI/PCL microfibers hold a conductivity above that of biological fluids (7.7 Â 10 À2 S cm À1 vs 1.0 Â 10 À2 S cm À1 ), making these ideal candidates for in vitro neural differentiation studies under ES. [81] An iron-containing porphyrin, hemin, has likewise been doped to confer conductivity in serum albumin-based scaffolds (%2 mS cm À1 ). [82] Not only direct ES is useful in neural tissue regeneration, magnetic stimulation and light stimulation have likewise proven their potential. Magnetic fields generated from magnetostrictive filler graphene oxide (GO)/CoFe 2 O 4 nanoparticles together with piezoelectric polymer polyvinylidene difluoride (PVDF) induced mesenchymal stem cells (MSCs) growth and differentiation into neural cells without using chemical induction differentiation media. [83] Light stimulation of inorganic/organic semiconductive materials, such as g-C 3 N 4 / GO [84] and poly(3-hexylthiophene), [85] has been shown to trigger optoelectronic conversion, thus providing a novel means of ES. External electrical stimuli of 3D conductive scaffolds open the possibility of influencing cellular behavior, which may potentially benefit the therapeutic applications.
Equally important, neural cells in the native tissues tend to align in certain directions and a tissue's function is tied to its structure. Oriented electrospun fibers with aligned topographies can support growth and migration along specific directions ( Figure 5A,B). [86] Aligned scaffolds reportedly provide more contact guidance for neural differentiation, resulting in enhanced nestin and βIII-tubulin expression compared with random fibers (RFs). [87] Likewise, PEO/PCL/poly(norepinephrine) microfibers with aligned nanogrooved channels (GF) have been found to significantly promote neurite extension in 3D ( Figure 5C). [38b] Besides fiber structure (random or aligned), fiber diameter and density also have an effect on cell growth and neurite extension. Daud et al. found that 8 μm fiber diameter promoted neurite outgrowth for neuronal cells alone, and 1 μm fibers were found to be superior in terms of neurite outgrowth in the tested coculturing system with Schwann cells. [88] Therefore, under different conditions, the diameter should be optimized. High fiber density may result in neurite path alteration and reduce neurite linearity. [89] Scaffolds that allow aligned growth of neurons could hold particular promise for the repair of injured nerve fibers by bridging the gap between the two stumps of a severed nerve. So-called nerve guidance conduits (NGCs) are tubular structures designed to guide the regeneration of nerve cells, as shown in Figure 5D. [90] The highly aligned nanofibers in the nerve conduits served as guidance for axon spreading and cell migration of Schwann cells in vitro ( Figure 5E). [91] PC12 cells have been directly encapsulated in hollow coaxial fibers to form living 3D-connected neuronal networks ( Figure 5F), where excellent alignment of the cells was observed along the directions of the microfibers. [92] In vivo, the oriented 3D fiber structure facilitates not only cell migration and guidance of the axonal extension, but also provides increased flexibility and resistance to deformation in a rat model of spinal cord injury. [93] In another work, aligned fibers within NGCs enhanced the bridging of the injured sciatic nerve in rats. [94] Interestingly, randomly oriented nanofibers could be used as a coating layer to strengthen the mechanical properties of the aligned fibers. [95] The strength of electrospinning is that it allows the creation of a wide diversity of scaffold morphologies. Zhang et al. [84b] developed three different anisotropic gridded PCL scaffolds with varying spacings between the grid patterns ( Figure 6A). Tailoring of the fiber spacing ratio between two arms of the grid patterns was found to affect the neurite extension of the seeded PC12 cells ( Figure 6A-d). The largest fiber spacing (600 μm from 1-3 scaffold) was found to induce increased biased growth along the long arm direction (Figure 6Ak). Intersection angles were also an important parameter to control, as anisotropic neurite guidance was further strengthened when the intersection angles were reduced from 90 to 30 ( Figure 6B).
Due to the significance of aligned fibrous topography and ES, scientists have tried to combine the two properties to investigate the potential additive effects. In vitro, the combination of aligned fiber morphology with electrical activation increased the expression of neuron-specific cytoskeletal proteins and microtubule assembly of S42 Schwann cells in PC12 cells. [96] The directional alignment of neurites in PCL/poly(acrylic acid) conductive scaffolds was likewise reported to enhance protein expression of βIII-tubulin and neurofilament 200. [90] Also, a rolled-up MoS 2 ÀPVDF nanofiber film with good electrical conductivity and large surface area significantly increased cell attachment and proliferation.
[73a] Through a combination of bioactive cues, aligned topographies, conductive coating, and ES, CS/PPy-coated poly(L-lactic acid) (PLLA)/PCL fiber films with 100 mV of ES were found to enhance neural cell compatibility and neurite growth. [97] In vivo, the implantation of aligned conductive electrospun scaffolds in rats after spinal cord injury improved functional recovery and electrical signals measured as motorevoked potentials. [98] Thus, multicue scaffolds may hold great potential for application in neural tissue regeneration. The mechanisms underlying the beneficial effects of ES, however, require further exploration. Table 2 shows the 3D electrospun scaffolds for neural tissue regeneration according to the functional elements, such as 3D architecture, bioactivity, aligned topography, conductivity, as well as stem cell sources used for validation. As the nerve cells are highly organized in the spinal cord, scaffold topography is likewise an important element in mimicking the native ECM. Electrical stimulation is a relatively new method in neural tissue regeneration and is proposed to mimic the natural cellÀcell electrical communication and thereby stimulate cell growth and differentiation.
Cardiac Tissue Regeneration
Cardiovascular diseases are the leading course of death compared with any other disease, affecting millions of people globally and significantly decreasing their quality of life. [99] The human heart has low regenerative properties, making this tissue very vulnerable to injuries such as myocardial infarction (MI). After MI, the injured tissue is replaced by scar tissue, leading to great loss of the contractile ability, which consequently may lead to heart failure. For these reasons, cardiac tissue regeneration may be the key to restoring the contractile ability of the heart after an injury caused by cardiovascular disease. [100] 3D electrospun structures can mimic the ECM of the myocardium, providing a nanofibrous microenvironment for cardiac cell adhesion, maturation, and function. Loosely packed 3D PPy scaffolds can enable stable electroactive cellÀfiber construct formation and promote cell proliferation compared with a traditional 2D electrospun PPy fiber mesh. [65a] Wet-electrospun 3D alginate/ gelatin hydrogel scaffolds were found to support the maturation of human iPS cell-derived ventricular cardiomyocytes. [42c] Likewise, 3D PCL nanofibrous scaffolds have been reported to directly promote cardiomyocyte differentiation through Wnt/ β-catenin signaling. [101] 3D electrospun fibers have been manufactured from different ECM proteins, to provide suitable substrates for cardiac tissue regeneration. A scaffold containing the abundant ECM protein collagen maintained cardiomyocyte contractile function for a period of 17 days with high levels of desmin expression. [102] ECM decellularized from the porcine ventricular tissue has been reported to provide natural cardiac ECM compositionally. [9d,103] Besides ECM proteins, the use of other naturally derived proteins, such as keratin [104] and SF [105] that are easy to obtain, have likewise been investigated. Fibrous scaffolds manufactured from SF have been reported to enhance cell commitment of adult rat cardiac progenitor cells. [105] Collectively, 3D electrospun scaffolds have been shown to exhibit improved performance for cardiomyocyte culturing compared with regular flat culture systems. A 3D cardiac patch-based system has emerged as a potential regenerative strategy, as shown in Figure 7A. In this study, SF-modified CA 3D nanofibrous patches were used to restrain pathologic ventricular remodeling post-MI by attenuating myocardial fibrosis. [106] The patch/adipose tissue-derived(AD)-MSC group showed increased viability of engrafted AD-MSCs ( Figure 7B,C), expression of cardiac paracrine factors ( Figure 7D), reduced fibrotic area ( Figure 7E), and improved density of neovascularization ( Figure 7F) compared with the intramyocardially injected AD-MSC group and untreated groups. These results indicated that the 3D electrospun patch could be a good carrier, which allows high retention and survival of the engrafted AD-MSCs.
As the human heart is a pulsating tissue, the mechanical properties are very important to consider when developing a cardiac patch. For this reason, cell-laden polycarbonateÀurethane (PCU) scaffolds have received much attention, due to their optimal Young's moduli (0.75 MPa, PCU electrospun on polyester fabric template), comparable with that of a human heart. The scaffold allowed prolonged spontaneous synchronous contractility of rat cardiac myoblasts on the entire engineered construct for 10 days in vitro at a near-physiological frequency of %120 bpm. [21] As a cardiomyocyte is a muscle cell, ES and directional arrangement are crucial. Therefore, conductive scaffolds have gained great interest in cardiac tissue regeneration in recent years. In one study, cardiac cells were seeded on nanogold-coated PCLÀgelatin scaffolds, which lead to cellular assembling of elongated and aligned tissues. [107] In another study, conductive PLLA/PANI nanofibrous sheets were found to promote cardiac differentiation measured in terms of maturation index and fusion index. [108] Others have focused on designing electrospun scaffolds to guide cell growth in precise directions. Cardiac cells cultured on aligned electrospun nanofibers were found to exhibit elongation and orientation of the α-actin filaments, [109] while simultaneously displaying high expression of genes encoding a number of sarcomere proteins, calcium-handling proteins, and ion channels. [110] Ultimately, multifunctional cardiac patches consisting of various tissue layers conducting different functions could be a promising solution for cardiac transplantation. A bottom-up approach was proposed to assemble an impressively complex modular tissue ( Figure 8A). [1c] Through analyzing collagen fiber orientation in adult rat hearts, Fleischer et al. [1c] found that the alignment from the epicardial side to the endocardial side was a 100 shift ( Figure 8B), which they used to guide the assembling of artificial multilayers by assembling several tissue layers on top of each other. Microholes (40 AE 0.8 μm) on the ridges of each layer were developed to ensure sufficient mass transfer (exchange of nutrients and oxygen) ( Figure 8C). The grooved scaffolds were stacked with a slight angle shift to mimic the collagen fiber orientation in adult rat hearts ( Figure 8D).
The cells in the grooved scaffolds were found to assemble into aligned cardiac cell bundles similar to that of the natural cardiac microenvironment with a higher expression of connexin 43 proteins (green color) due to the electrical coupling between adjacent cells ( Figure 8E). Furthermore, double-emulsion PLGA microparticles ( Figure 8F) with VEGF were deposited into the cages of "channel þ cages" layer ( Figure 8G), thus creating a controlled release system for continuous supply of vascularization signals. Microtunnels with dimensions of 450 μm were patterned between cage-like structures for endothelial cells to form large, closed lumens. In addition, Fleischer et al. designed another layer with cage-like structures, and PLGA microparticles containing dexamethasone (DEX), an anti-inflammatory agent, were scattered on top ( Figure 8H,I) to attenuate the activation of macrophages and thereby decrease the immune response after transplantation. The PLGA microparticles were found to enable the long-term release of VEGF and DEX ( Figure 8J). A macroscopic view of the patches in rats and a cross sectioning of the patches revealed that the layered structure was maintained 2 weeks posttransplantation ( Figure 8K). The layers of the developed scaffold were found to mimic both the stiffness and the mechanical anisotropy of the heart muscle, hence improving the potential for the scaffolds to be integrated properly to the heart muscle. Table 3 shows the characteristics of 3D electrospun scaffolds designed for cardiac tissue regeneration. Scaffold functionality in terms of bioactivity, aligned topography, and conductivity should still be improved to advance the potential of 3D electrospun scaffolds in the field of cardiac tissue regeneration. Based on the complexity of cardiac tissue, the ultimate goal of cardiac tissue regeneration is to achieve one scaffold with great diversity of functional elements to mimic the natural cardiac tissue.
Bone Tissue Regeneration
As osteoporosis can increase the risk of bone fracture, it is the leading cause of broken bones among the elderly. For large bone defects beyond the ability of self-regeneration, artificial scaffolds are necessary to bridge the gap, assist cell adhesion, and accelerate repair. This method is not only applicable for the elderly but may also be helpful to younger people by greatly decreasing the healing time. Importantly, advances in 3D electrospun scaffolds as bone substitutes are enhancing our ability to create ideal ECM mimicking structures for bone regeneration. [111] The 3D electrospinning technology is a well-established nano-/microstrategy used to manufacture biomimetic fibrous constructs for bone tissue regeneration. A special 3D honeycomb-like architecture has been proposed to support osteogenic differentiation by enhancing alkaline phosphatase (ALP) production, calcium deposition, and specific gene expressions. [65b] In another study, conducted by Sankar et al., a 3D-patterned electrospun scaffold was seeded with human AD stem cell spheroids. Osteodifferentiation was reportedly enhanced without the need for an osteoinductive culture medium. [112] The scaffold shape can also be designed to match specific bones, such as the lumbar vertebra, as described by Su and coworkers. [54] In vitro, 3D electrospun scaffolds support cell development. Human osteosarcoma cells cultured on piezoelectric hydrophilic electrospun PVDF scaffolds showed well-defined actin stress fibers crossing the cell ( Figure 9A), and the scaffold was found to generate a local electric field that activated the osteoblasts ( Figure 9B). [113] The surface potential of PVDF fibers, furthermore, was found to regulate the production of mineralized collagen, a key component of the bone matrix, thus promoting the processes of bone regeneration. [114] More complex electrospun scaffolds have allowed reversible dynamic mechanical stimulation for the study of cell response to mechanical forces. A thermocontrollable and stiffness-tunable 3D electrospun microfibrous poly(N-isopropylacrylamide) scaffold was used to submit cells to alternating cycles of mechanical stimulations by switching scaffold resistance between soft and stiff. The scaffold was reported to initiate cytoskeletal organization and cell shape deformation to activate the Yes-associated protein (YAP) as well as the preosteogenic runt-related transcription factor 2 (RUNX2), which is known to mediate mechanotransduction and differentiation ( Figure 9C). [115] Besides providing mechanical stimulation, reverse thermosensitive polymer fibers have also been used to support MSCs within 3D mechanically stable PCL. [116] Incorporating bioactive inorganic materials with electrospun polymers, mimicking the native bone ECM, has gained tremendous interest for bone tissue reconstruction throughout the years. Various modified and unmodified apatite minerals have been proven to resemble the physical properties of the natural bone tissue and promote osteogenic cell function. Beta-tricalcium phosphate (β-TCP), [117] silicate-containing hydroxyapatite, [118] and boron doped hydroxyapatite (B-HAp) [119] have been proposed as bone-like biomaterials mimicking the hierarchical architecture and chemical composition of the bone ECM. In one study, hydroxyapatite was incorporated in layer-by-layer electrospun honeycomb PCL fibers using electrospraying (Figure 10Aa,b), mimicking the natural environment for maxillofacial bone reconstruction. [33] Both reverse transcription-quantitative real-time PCR (Figure 10Ac) and ALP activity (Figure 10Ad) results clearly supported the positive effect of the honeycomb scaffolds on differentiation toward the bone lineage in the absence of any small-molecule osteoinductive agents. Furthermore, the honeycomb structure resulted in significantly higher migration of cells from explanted bone tissue onto the scaffold compared with random PCL fibers. (Figure 10Ae). A natural occurring material, diatom shell, was incorporated into a 3D electrospun scaffold and was found to improve bone tissue regeneration. [120] Another material that has been studied in regard to bone tissue regeneration is ZnO nanoparticles. Due to their osteoconductive and antibacterial properties, they were electrospun into PCL and demonstrated great potential for treatment of periodontal defects ( Figure 10B). [121] Another advantage of electrospun fibers is their capability to act as a great drug carrier or release system. In several studies, 3D electrospun scaffolds have been coated with bone morphogenetic protein-2 (BMP-2) on the surface by hydrogel or polydopamine (pDA)-assisted immobilization and physical adsorption. This approach was biocompatible and osteoinductive in vitro and in vivo in a pilot study in rabbits. [122] A low dose of recombinant human BMP-2 has likewise been incorporated into 3D PCL/PLA scaffolds and promoted osteogenic differentiation and facilitated new bone formation after 6 weeks of implantation in vivo. [123] In another study, rifampicin was incorporated in 3D electrospun scaffolds to successfully prevent bone infection, resulting from implant or orthopedic surgery. [124] Alendronate, a nitrogenous bisphosphonate widely used in the therapy of metabolic bone diseases, was incorporated into electrospun scaffolds and promoted osteogenesis-related gene expression in human fetal osteoblasts. [125] Finally, Xu et al. produced electrospun SF/poly(L-lactide-co-ε-caprolactone) (PLCL) coreÀshell fibers that c) Percentage of activated cells grown on different PVDF scaffolds. d) Schematic representation of a cell cultured on hydrophilic electrospun PVDF scaffold; ES, coming from the 3D fibrous scaffold bending due to the cell forces applied upon attachment, causes the opening of plasma membrane channels (VGCC, SACC), which lead to an increase in calcium ions in the cytoplasm. A,B) Reproduced with permission. [113] Copyright 2019, Royal Society of Chemistry. C) Temperature-dependent changes a) in swelling ratio of crosslinked microfibrous hydrogels in response to temperature alternations. b,c) Ability of RD-MS to alter hMSCs underlying YAP and RUNX2 signaling activation after 7 days. Reproduced with permission. [115] were used to dual-deliver the osteoinductive peptide H1 from the core and HAp from the shell for collectively enhancing osteogenesis both in vitro and in vivo. This was proven by increased expression of osteopontin ( Figure 10C) and induced bone mineralization ( Figure 10D) compared with control scaffolds. [126] Table 4 shows different 3D electrospun scaffolds used for bone tissue regeneration. In the field, the cues of bioinorganic, electromechanical, and mechanical stimulation have been the main research direction in the recent years. Scaffolds with drug-carrying or releasing properties have also gained tremendous attention with a large number of published reports. All the electrospun scaffolds implanted in animal models mentioned in this Review were uncellularized, designed to stimulate the animal's own cells to produce new bone. However, it would be ideal to stimulate cells in vitro to form artificial bone with the same properties as native bone. The shape of artificial bone can be controlled by the shape of electrospun scaffolds by combining it with either 3D printing, MEW, NFE, or field-assist electrospinning.
Vascular Tissue Regeneration
If tissue regeneration of tissues larger than 200 μm ever is to succeed, the field needs to overcome the challenge of creating fully functional blood vessels. Vascularization of tissues is essential for supplying the cells with nutrients and oxygen and to help remove waste products. [127] Su and coworkers developed an artificial multihelix electrospun PCL/PCL þ PEO scaffold. [128] Here, the seeded human umbilical vein endothelial cells (HUVECs) were reported to exhibit a preferential cell distribution of around 86% on the PCL side rather than the PCL þ PEO side, resulting in a distinctive 3D Janus cellular pattern with increased vinculin and phosphorylated-focal adhesion kinase (pFAK) expression. Kim et al. developed a vascularized 3D tissue by stacking HUVEC cell sheets with skeletal myoblasts and fibroblast cell sheets in a layer-by-layer construction. [59c] In another work, the thickness of a vascularized tissue construct was controlled by the thickness of the electrospun scaffold through the increasing layer number. [129] Besides solution electrospinning, MEW [33] Copyright 2018, American Chemical Society. B) Rat periodontal defect model. Reproduced with permission. [121] Copyright 2017, Wiley-VCH. C) Immunofluorescence images of osteogenic differentiation for hiPS-MSCs on different coreÀshell scaffolds in osteogenic medium at week 3 (blue: 4 0 ,6-diamidino-2-phenylindole, DAPI; red: osteopontin, OPN; green: F-actin). D) X-ray micro-computed tomography (μCT) analysis of the calvarial bone retrieved 8 weeks after implantation using various coreÀshell scaffolds in critical-sized bone defects. C,D) Reproduced with permission. [126] Copyright 2019, Elsevier. has been used for coculture of HUVECs and normal human dermal fibroblasts (NHDF) through cell accumulation technique, which allowed the formation of capillary-like network structures. [130] Regarding artificial blood vessel architecture, 3D electrospinning is a simple and ideal method to fabricate tubular-shaped biodegradable scaffolds. Such scaffolds have been fabricated based on either rotating mandrel collectors or post-treatments. In one study, engineered vascular grafts with diameters matching vascular vessels were prepared for vascular reconstruction by simply adjusting the diameter of the mandrel ( Figure 11A). [131] Another strategy applied for obtaining tubular constructs was an automated fabrication strategy, which rolled 2D matrices into 3D tubular constructs by continuously bonding different functional layers (cells, hydrogels, and scaffold biomaterials) with varying diameters. [132] In addition to scaffold shape, mechanical properties are key factors to consider when developing material for vascularized tissue regeneration. In one study, PU was electrospun onto an airbrushed PCL tube to mechanically reinforce the scaffold. The tensile strength and Young's modulus of the reinforced scaffolds were reported to be 67.5 AE 2.4 and 1039 AE 81.8 MPa, respectively, allowing a 2 kg dumbbell to hang from the scaffold without breaking it ( Figure 11B). [64b] Materials like polyamide-6 and GO have likewise been proven to reinforce the mechanical and physical properties of electrospun scaffolds. [131,133] By combining electrospun PCL with 3D-printed PCL, both surface morphology and mechanical properties were found to be suitable for vascular reconstruction ( Figure 11C). [134] Another approach to obtain mechanically reinforced scaffolds is using multiple electrospun layers with different architectures. Such a scaffold was designed using longitudinally aligned nanofibers (inner and outer layer) and radially aligned nanofibers (middle layer) and successfully mimicked native artery structure ( Figure 11D). [135] Similarly, the NFE technique combined with electrospraying and electrospinning produced a mechanically reinforced threelayered scaffold with highly aligned strong fibers, which provided appropriate mechanical support for HUVECs in vitro and allowed infiltration of host cells after implantation into the abdominal aorta in vivo. [136] Ju and coworkers [137] manufactured a bilayered electrospun and cellularized vascular scaffold and assessed its preclinical feasibility in sheep. The electrospun scaffold was composed of randomly orientated fibers in the inner layer and aligned fibers in the outer layer ( Figure 12A). The scaffold was seeded with smooth muscle cells (SMCs) and endothelial cells and preconditioned using a pulsatile bioreactor system ( Figure 12B) prior to transplantation. The cellularized vascular constructs were found to maintain a high degree of graft patency with a constant luminal diameter ( Figure 12C), structural integrity with compliance ( Figure 12D), and contractile properties without eliciting an inflammatory response after a 6-month implantation period. Consequently, this scaffold was reported as a clinically applicable alternative to traditional prosthetic vascular graft substitutes.
Ahn et al. improved the cell seeding efficiency on vascular-like electrospun PCL and collagen type I fibers, by prefabricating an SMC sheet. The cell sheet was wrapped around the electrospun vascular scaffolds and found to significantly enhance not only the Human-induced pluripotent stem cellderived MSCs (hiPS-MSCs) [126] www.advancedsciencenews.com www.small-science-journal.com Small Sci. 2021, 2100003 Figure 12. A) SEM images of entire layer, outer layer, cross-sectional interface and inner layer of the bilayered vascular scaffold. B) Bioreactor setup consisting of a computer-programmed gear pump, flow reservoir, and the bioreactor housing unit. C) Inner diameter measurement of the transplanted engineered blood vessels revealed a stable lumen caliber for the duration of the 6-month follow-up. D) A representative CT scan shows the absence of an aneurysm along the entire length of the transplanted engineered blood vessel (arrows). A-D) Reproduced with permission. [137] Copyright 2017, Elsevier. Copyright 2018, Elsevier. C) Schematic illustration of the process used to fabricate 3D tubular artificial vascular scaffolds combining electrospinning with 3D-printed coating. Reproduced with permission. [134] Copyright 2015, Royal Society of Chemistry. D) Longitudinal view of photographic and scanning electron microscopic images of electrospun trilayered tubular scaffolds as well as the fast FFT analysis. Reproduced with permission. [135] Copyright 2018, Elsevier. E) Prefabricated cell sheets wrapped around an electrospun scaffold. Reproduced with permission. [138] Copyright 2015, Elsevier. F) Schematic of multilayer graft fabricated by electrospun tubular scaffold and hydrogel. Reproduced with permission. [140] Copyright 2018, Elsevier. G) The photo of an electrospinning collector with copper wire wound around it and the interior copper wire being easily pulled out. Grooves and cross-sectional morphology of the scaffold. Reproduced with permission.
www.advancedsciencenews.com www.small-science-journal.com Small Sci. 2021, 2100003 cell seeding efficiency, but also the maturation process and the cell-to-cell junctions compared with cells seeded directly on the electrospun fibers, yielding an implantable vascular graft ( Figure 11E). [138] Wang et al. [139] used the molecule-releasing properties of electrospun fibers. Here, resveratrol, a natural polyphenol, was incorporated into electrospun PCL fibers, allowing sustained and controlled release that was found to enhance the vascular regeneration process by promoting migration of endothelial cells and tube formation. Another study conducted by Post et al. [140] showed that designing a layered cell-free hydrogel system likewise led to rapid endothelialization ( Figure 11F). Hydrogels have also been used to coat electrospun scaffolds with copper wire-induced grooves ( Figure 11G) using a so-called dip-coating method. This technique promoted the attachment of HUVECs.
[63b] The hydrogels can increase hydrophilicity, biocompatibility, and mechanical strength and therefore improve affinity between cells and the electrospun nanofibers.
To overcome the challenge of anatomic differences between individual patients, Fukunishi et al. developed a patient-specific vascular graft. A 3D-printed patient-specific stainless steel graft was used as an electrospinning collector, allowing both SMC engraftment and endothelialization; hence, this method was found to be a promising technology for future vascular tissue regeneration. (Figure 11H). [20] Table 5 shows the 3D electrospun scaffolds for vascularized tissue regeneration highlighted in this Review. There are relatively few studies published on biological activity, cellular interaction, and drug release compared with other fields of tissue regeneration. This might, however, be explained by the intense focus on optimizing scaffold shape and mechanical properties for vascularized tissue regeneration. For this field to reach its full potential, future studies should closely examine both vascular cell response and scaffold architecture.
Skin Tissue Regeneration
3D electrospun scaffolds with high porosity and surface-tovolume ratio have been shown to exhibit tremendous potential for wound healing and injured skin tissue regeneration. One application of electrospun materials is to create antibacterial wound dressings. As bacterial infections may result in increased exudate at the wound site, a wound dressing carrying antibacterial drugs is a direct method to overcome this challenge. In one study, poly(3-hydroxybutyric acid) and gelatin loaded with curcumin were electrospun onto a keratinÀfibrinÀgelatin hydrogel substrate. This dual system was found to facilitate tissue regeneration, while preventing infection at the transplant site. [141] Similarly, thymoquinone and antibiotic tetracycline hydrochloride [142] were incorporated into 3D electrospun scaffolds and prevented common clinical infections and promoted healing. [143] Several other antibacterial materials, such as the peptide OH-CATH30 (OH-30), [144] CS, [145] medicinal plant extracts, [146] and silver nanoparticles, [147] have likewise been investigated for this purpose, with promising results. As zwitterionic polymers have excellent antibiofouling abilities, they have also been used for wound-dressing purposes. [148] As an example, electrospun PCL membranes were functionalized with a zwitterionic polymer. To this system, halloysite nanotubes were HUVECs [188] like sustained drug carriers ( Figure 13A) of the broad-spectrum antibiotic tetracycline hydrochloride. The scaffolds were found to possess great selective biocompatibility, promoting platelet and mouse fibroblasts L929 cell adhesion and decreasing accumulation of both plasma proteins and bacteria. The scaffold increased skin regeneration compared with the commercial 3M Tegaderm TM film ( Figure 13B). [149] He et al. [150] developed a scaffold composed of quaternized chitosan graft-polyaniline (QCSP) in PCL and found it to significantly accelerate the wound-healing process in mice compared with the commercial Tegaderm TM film. Finally, the analgesic drug, lidocaine hydrochloride, was incorporated in an electrospun scaffold together with the anti-inflammatory agent curcumin. The developed scaffold showed great antibacterial performance in vitro and has great potential in wound care applications, though it has not yet been tested in vivo. [151] Other types of wound dressings focus on accelerating cell proliferation, rather than preventing infection, to induce repair of the damaged skin. Without any anti-inflammatory drugs, electrospun PCL reportedly provides a suitable 3D environment for differentiation of melanocytes and can be a promising candidate for treatment of skin disorders. [152] Combining PCL with other elements could further enhance skin repair. For instance, heparin and fibroblast growth factor-2-modified PCL have been found to promote the formation of distinct fibroblast and keratinocyte layers. [153] Collagen-coated PCL scaffolds have likewise been found to increase attachment and proliferation rates of human endometrial stem cells compared with pure PCL. [154] Other types of 3D electrospun scaffolds composed of either GelMA [155] or CA/PUL [156] have shown promising results in skin tissue regeneration applications. Compared with PLCL and gelatin scaffolds, the GelMA scaffolds were found to promote cell proliferation ( Figure 14A), reduce remaining wound area ( Figure 14B), and increase expression of the ECM protein collagen I ( Figure 14C). [155a] SF is another biomaterial that has been widely investigated for its promising properties for skin tissue regeneration. Electrospun SF scaffolds have been fabricated using either cold-plate electrospinning or by dropping NaCl crystals, which have resulted in scaffolds with high porosity and controllable thickness, indicating that these scaffold types are potential candidates for artificial skin reconstruction. [29,157] SF has, furthermore, been used in combination with other polymers or nanoparticles. Incorporation of gold nanoparticles (AuNPs) into tissue scaffolds, has been shown to enhance mechanical stability. SF/PEO/AuNP 3D matrices is one such example and was successfully tested in a rat model of full-thickness skin wound. [158] In another study, 3D PCL/SF scaffolds have been found to increase the wound healing rate and collagen deposition in rats compared with PCL alone. [28b] Finally, SF combined with decellularized human amniotic membrane (AM) has been found to be an efficient antiscarring wound-dressing material for thirddegree burn wounds. [159] Table 6 shows some of the 3D electrospun scaffolds developed for skin tissue regeneration. The main goals are to achieve both bacteriostasis and cell proliferation and migration by functionalizing the wound-healing scaffolds with antibacterial components, drug-releasing carriers, and bioactive materials. The electrospun mat was also designed with human skin patterns to mimic the actual human skin. [160] The development of portable electrospinning devices with a hand-held spinneret could allow the scaffold to be directly applied to the damaged skin, therefore simplifying the fabrication process. [161] 3.6. Other Tissue Regenerations Though neural, cardiac, bone, vascularization, and skin regeneration have been intensely investigated, the unique properties of electrospinning have also been put to the test for repair of other tissues such as liver, kidney, and esophageal tissue. [162] Liver diseases affect millions of people every year and due to the limited treatment options, donor livers are the golden standard of treating such diseases. [163] However, lack of sufficient donors leaves space for alternative treatment solutions such as Furthermore, hepatocytes/fibroblasts coculture systems have been developed using fibronectin-coated CS nanofiber scaffolds to culture and maintain long-term liver function. [162b] Likewise, drug-induced hybrid PCLÀECM scaffolds were found to maintain both hepatocyte growth and function for 5 days in vitro, [162c] where the hybrid scaffolds were conducted by culturing and decellularizing 5637 human urinary bladder epithelials on PCL scaffolds. Finally, heparan sulfate-coated polyethersulfone (PES) scaffolds have been found to improve human endometrial stem cell differentiation into functional hepatocyte-like cells. [162d] Chronic kidney disease like liver diseases also affects millions of people worldwide, making it a major health problem. [164] Besides organ transplantation, dialysis is the current treatment for people suffering from chronic kidney disease. However, this Human foreskin fibroblast and keratinocyte cells [190] PCL/QCSP Random nanofibers Increasing spinning time Accelerated wound-healing processes compared with commercial dressing (Tegaderm TM ); allowed higher collagen deposition, granulation tissue thickness, and angiogenesis.
Red blood cells (RBC) and L929 [150] Alginate/ZnO Random nanofibers Increasing spinning time Mechanical and water-related properties are similar to those of human skin.
L929 fibroblasts and human keratinocyte HaCaT cell line [191] www.advancedsciencenews.com www.small-science-journal.com is a costly method and affects the quality of life of patients. A solution to this challenge may be found using electrospinning for tissue regeneration. Electrospun PLA scaffolds have been fabricated for kidney tissue engineering and found to sustain a multipopulation of kidney cells including aquaporin-1-positive proximal tubule cells, aquaporin-2-positive collecting duct cells, synaptopodin-positive glomerular epithelial cells, and von Willebrand factor-positive glomerular endothelial cells. [162g] PCL scaffolds have also been fabricated for kidney proximal tubules with sufficient mechanical stability, rapid diffusibility, tight cellular monolayer formation, and prolonged construct viability, exhibiting superior properties over existing proximal tubule models with regard to implantation purposes and continuous blood clearance. [165] Finally, electrospun PCL/poly(3hydroxybutyrate-co-3-hydroxyvalerate) bioresorbable scaffolds have been developed and can be promising for tissue-engineered urinary bladder augmentation. [162i] Esophageal tissue regeneration has started to get attention in the world of tissue engineering, as several medical conditions, such as esophageal cancer, require surgical procedures resulting in esophageal defects. As the current methods of reconstructing the damaged esophageal tissue are highly invasive and potentially fatal, tissue engineering using electrospinning techniques is a potential way of improving esophageal regeneration. [166] Zhuravleva et al. [162h] developed an electrospun polyamide-6-based scaffold for esophageal tissue regeneration. Papio hamadryas esophagus was decellularized to determine the morphology and physical properties of its ECM. This inspired the design of a scaffold with excellent mechanical properties, 90% porosity that allowed cell adherence, and elongation of AD-MSCs and bone marrow-derived mesenchymal stromal cells (BMD-MSCs). Alternatively, Wu et al. used electrohydrodynamic jetting (e-jetting) to manufacture aligned PCL/pluronic F127 fibers with controlled pore sizes. Here they found that scaffolds containing 8% pluronic F127 exhibited a similar ultimate tensile strength as that of the native esophageal tissue. In addition, the PCL/pluronic F127 scaffolds were found to improve primary human esophageal fibroblast proliferation and expression of VEGF compared with pure PCL scaffolds, thus providing a promising approach for esophageal tissue regeneration. [166a] Finally, Park and coworkers developed electrospun polyurethane nanofibers for esophageal reconstruction in rats. After 4 weeks of transplantation, kreatin13 immunostaining revealed that the nanofibrous scaffolds with and without preseeding of hAD-MSCs were found to significantly increase the thickness of the regenerated esophageal epithelium compared with 3D-printed PCL scaffolds.
[166b] Table 7 shows the studies highlighted in this section. Although there are comparatively few studies focusing on these tissue regeneration fields, electrospinning has demonstrated strong potential in these still underexplored applications. Future studies will hopefully take them one step closer to clinic.
Conclusion
Electrospinning is a versatile technology that allows innovative advancement with great promise to fabricate 3D fibrous scaffolds. Electrospinning possesses the ease of combining other additive technologies to create scaffolds with optimal mechanical, biological, and physical properties. The electrospinning technology is rapidly advancing with organ-shaped tailored collectors and 3D printing features.
Compared with the traditional, densely packed electrospun submicrometer fibers, loosely packed 3D electrospun scaffolds hold larger surface areas, tunable pore diameters, adjustable densities, and controllable shapes, which can promote cell infiltration and nutrients exchange. Such 3D electrospun scaffolds have been proven as superior biomaterials in various tissue regeneration fields, as summarized in our Review. However, the industrial application of the final 3D electrospun products is still in its initial stages. Although a wide range of biomaterials and bioactive components have been used in electrospinning with promising results and significant advances, the industrial scale production for the 3D electrospun scaffolds is still in its infancy, especially when the precise control of the physical architecture and biochemical functionalization at submicrometer scale is desired.
The 3D electrospun scaffolds are believed to hold great potential in many different areas of regenerative medicine; however, it is possible to further expand the research areas and accelerate their clinical applications. The future direction should address the new possibilities of large-scale production with precise control over physical structure and biochemical composition. | v3-fos-license |
2017-10-11T00:43:44.565Z | 2008-11-23T00:00:00.000 | 105853494 | {
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} | pes2o/s2orc | Asymmetric Synthesis of ( + )-Coniine , ( − )-Coniceine , ( + )-β-Conhydrine , ( + )-Sedamine and ( + )-Allosedamine by a Strategic Combination of RCM with Nucleophilic 1 , 2-Addition on SAMP-Hydrazones
A tactically new approach to the asymmetric synthesis of the piperidine alkaloids (+)-coniine, (−)-coniceine, (+)-β-conhydrine, (+)-sedamine and (+)-allosedamine has been developed. The key step is the elaboration of the piperidine template equipped with suitably functionalized appendages at the stereodefined C-2 position. Subsequent manipulation of the appropriate functionalities gave rise to the targeted compounds in high yields and high level of enantioselectivity. Introduction The piperidine ring is a ubiquitous structural feature of numerous naturally occurring alkaloids and can be frequently recognized in the structure of drug candidates [1]. The interest in piperidine alkaloids is well displayed by the wealth of published material detailing their sources and biological activities and their structural diversity makes them interesting proving grounds for organic chemists. As a consequence numerous methods have been developed for the synthesis of substituted piperidines in a stereoand enantioselective manner [2] and interest in their chemistry continues unabated. Piperidine alkaloids incorporating a stereogenic center at C-2, as exemplified by (+)coniine (1), (−)-coniceine (2), (+)-β-conhydrine (3), (+)-sedamine (4) and (+)-allosedamine (5) (Figure 1), fall into this category and consequently have attracted considerable attention from the synthetic community. Objectives and Synthetic Strategy We were then interested in developing a feasible and highly stereoselective route giving rise to the specific embodiment of these piperidine alkaloids presented in Figure 1. The salient features of the synthetic strategy are (i) the early creation of the C-2 stereogenic center of the piperidine unit on reliance with the highly diastereoselective 1,2-addition on chiral SAMP-hydrazones [3] and (ii) the creation of the piperidine template by ring closing metathesis which ranks highly in the hierarchy of synthetic tactics for the elaboration of nitrogen containing ring systems [4]. N Pr H (+)-Coniine (1) N (-)-Coniceine (2) N H OH H (+)-β-Conhydrine (3) N Ph H Me OH N Ph H Me OH (+)-Sedamine (4) (+)-Allosedamine (5) Figure 1. Enantioselective Synthesis of (S)-(+)-Coniine (1). Extension to the Synthesis of (−)-Coniceine (2). To test the viability of our conceptually new synthetic approach the synthesis of the structurally simple piperidine alkaloid (+)-coniine (1) was envisaged since it was anticipated that its elaboration could serve as a testing ground and provide foundation for extension to more complex and functionalized systems. 1. (S)-(+)-Coniine (1) The first facet of the synthesis was the assembling of the requisite dienehydrazide 8a, which was readily obtained by the three-step sequence depicted in Scheme 1. This olefinic precursor was obtained essentially as single diastereoisomer detectable by NMR making evident the high selectivity of the initial diastereofacial 1,2-addition process allowing introduction of the absolute stereochemistry early in the sequence. RCM reaction with Grubb's second generation ruthenium catalyst proceeded uneventfully to afford diastereopure enehydrazide (S,S)-9a. Catalytic hydrogenation afforded hydrazide (S,S)-10a and subsequent treatment of (S,S)-4a with BH3 . THF triggered off the formation of the NH free model with the concomitant release of the chiral appendage, thereby providing high yield of the targeted alkaloid 1 with excellent enantioselectivity.
Introduction
The piperidine ring is a ubiquitous structural feature of numerous naturally occurring alkaloids and can be frequently recognized in the structure of drug candidates [1].The interest in piperidine alkaloids is well displayed by the wealth of published material detailing their sources and biological activities and their structural diversity makes them interesting proving grounds for organic chemists.As a consequence numerous methods have been developed for the synthesis of substituted piperidines in a stereo-and enantioselective manner [2] and interest in their chemistry continues unabated.
Objectives and Synthetic Strategy
We were then interested in developing a feasible and highly stereoselective route giving rise to the specific embodiment of these piperidine alkaloids presented in Figure 1.The salient features of the synthetic strategy are (i) the early creation of the C-2 stereogenic center of the piperidine unit on reliance with the highly diastereoselective 1,2-addition on chiral SAMP-hydrazones [3] and (ii) the creation of the piperidine template by ring closing metathesis which ranks highly in the hierarchy of synthetic tactics for the elaboration of nitrogen containing ring systems [4].
Enantioselective Synthesis of (S)-(+)-Coniine (1). Extension to the Synthesis of (− − − −)-Coniceine (2).
To test the viability of our conceptually new synthetic approach the synthesis of the structurally simple piperidine alkaloid (+)-coniine (1) was envisaged since it was anticipated that its elaboration could serve as a testing ground and provide foundation for extension to more complex and functionalized systems.
(S)-(+)-Coniine (1)
The first facet of the synthesis was the assembling of the requisite dienehydrazide 8a, which was readily obtained by the three-step sequence depicted in Scheme 1.This olefinic precursor was obtained essentially as single diastereoisomer detectable by NMR making evident the high selectivity of the initial diastereofacial 1,2-addition process allowing introduction of the absolute stereochemistry early in the sequence.RCM reaction with Grubb's second generation ruthenium catalyst proceeded uneventfully to afford diastereopure enehydrazide (S,S)-9a.Catalytic hydrogenation afforded hydrazide (S,S)-10a and subsequent treatment of (S,S)-4a with BH 3 .THF triggered off the formation of the NH free model with the concomitant release of the chiral appendage, thereby providing high yield of the targeted alkaloid 1 with excellent enantioselectivity.
(−)-Coniceine (2)
This bicyclic alkaloid is a representative member of the biologically active indolizidine alkaloids that have been isolated from the skin secretions of neotropical amphibians [5].Its elaboration was readily achieved as depicted in Scheme 2 starting from the diastereopure (S,S)-9b.This diastereochemically enriched enehydrazide equipped with a protected hydroxypropyl appendage was accessed via the synthetic route depicted in Scheme 1 starting from the appropriate aliphatic aldehyde 6b.
Enantioselective Synthesis of (+)-β β β β-Conhydrine (3).
This piperidine alkaloid is representative of the hemlock alkaloids isolated from the seeds and leaves of the poisonous plant Conium maculatum L. whose extracts were used in the ancient Greece to get rid of criminals and undesirable intellectuals, for example Socrates [6].
Initially we planned to synthesize the carboxaldehyde derivative 13 and we surmised that treatment of diastereopure 13 with EtMgBr would provide the potential for a direct access to the piperidine template tailed with the required hydroxypropyl appendage.The highly functionalized compound (R,S)-13 was obtained in high yield and high enantioselectivity as outlined in Scheme 3. Disappointingly, when diastereopure (R,S)-13 was allowed to react with EtMgBr a 2:3 mixture of (R,R,S)-14 and (R,S,S)-14 diastereoisomers was obtained.We then set out to achieve the alternative strategy depicted in Scheme 4 which secured the stereochemistry of the hydroxyalkyl appendage on the piperidine template at an early stage of the synthesis.Interestingly, when bis-olefin 15, readily assembled as portrayed in Scheme 4, was subjected to RCM a 55:45 mixture of diastereomerically pure 16 along with the NH free piperidine 17 was obtained.This unprecedented phenomenon had no impact on the outcome of the process since the targeted (+)-β-conhydrine (3) could be readily accessed from 16 and 17 as well.Enantioselective Synthesis of (+)-Sedamine (4) and (+)-Allosedamine (5).
The 1,3-aminoalcohol moiety is found in many synthetic and natural products possessing physiological activities and is the integral part of a variety of potent drugs [7].It is also the most distinguishing structural feature of 2-(2-hydroxyalkylsubstituted)piperidine based alkaloids such as sedamine and allosedamine.This type of alkaloid has been shown to display memory enhancing properties and may be effective for the treatment of cognitive disorders [8].
A new synthetic approach to these compounds has been developed and is outlined in Scheme 5. Noteworthy, for the reduction of the carbonyl functionality of diastereopure 18 a variety of reducing agents were screened.By making use of K-selectride a 1:3 mixture of the diastereoisomeric forms 19 and 20 was obtained.
Conclusion
In summary we have devised a new, general and flexible method for the highly enantioselective synthesis of chiral piperidines.The method tolerates the presence of diverse functionalized appendages at C-2, namely 1-and 2-hydroxyalkylated moieties.
The main advantages of this synthetic method lie in the readily availability of the aliphatic and aromatic aldehyde SAMP-hydrazone precursors.In addition, with the present synthetic approach, optically active antipodes of the titled compounds should be readily accessed by simple choice of the RAMP chiral auxiliary. | v3-fos-license |
2014-10-01T00:00:00.000Z | 2011-02-09T00:00:00.000 | 45623625 | {
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} | pes2o/s2orc | 4,4′-Bipyridine–5-fluoroisophthalic acid (1/1)
Co-crystallization of 5-fluoroisophthalic acid (H2fip) with 4,4′-bipyridine (bipy) leads to the formation of the title compound [(H2fip)(bipy)], C8H5FO4·C10H8N2, with an acid–base molar ratio of 1:1. The acid and base subunits are arrange alternately in the crystal structure, displaying a wave-like tape motif via intermolecular O—H⋯N and C—H⋯O hydrogen bonds [carboxyl–pyridine synthon of R 2 2(7) hydrogen-bond notation], which are further combined into a two-dimensional architecture through C—H⋯F interactions involving the bipy and H2fip molecules.
Comment
In light of the importance of hydrogen bonds in crystal engineering, the supramolecular synthon approach has been widely applied to adapt desired supramolecules by using identified robust intermolecular interactions (Desiraju, 1995;Nangia & Desiraju, 1998). Co-crystallization is a current theme in several research groups to study hydrogen bonding through X-ray diffraction technique (Aakeröy & Salmon, 2005), for the synthesis of interpenetrated networks (Sharma & Zaworotko, 1996), and especially in pharmaceutical developments (Schultheiss & Newman, 2009). At this stage, strong hydrogen bonds, such as O-H···N or charge-assisted N-H···O, are always essential in the co-crystallization of carboxylic acids with pyridyl bases, usually combining the auxiliary weak C-H···O interactions, lead to the familiar carboxyl/pyridyl heterosynthon [R 2 2 (7)] (Shan et al., 2002). Although aromatic dicarboxylic acids have been verified to be excellent building blocks in binary co-crystal assemblies with bipyridine-type components (Du et al., 2005), halogen substituented dicarboxylic acids have been seldom studied in this aspect (He, et al., 2009). Doubtless, substituents will profoundly influence the structural assemblies by demonstrating distinct hydrogen-bonding capability and potential steric/electronic effect. To further investigate the hydrogen-bonding networks involving halogen substituents, 5-fluoroisophthalic acid (H 2 fip) was chosen to construct binary cocrystal with familiar 4,4'-bipyridine (bipy) component as a hydrogen-bonding participant for the first time.
In this work, the reaction of 5-fluoroisophthalic acid (H 2 fip) with 4,4'-bipyridine (bipy) under ambient conditions and evaporation from the mixed CH 3 OH/H 2 O (2:1) solution of the reactants yields the crystalline binary adduct [(H 2 fip)(bipy)] (I). Single crystal X-ray diffraction reveals that compound (I) contains one-dimensional supramolecular tape via the connection of predictable carboxylate-bipyridine O-H···N/C-H···O interactions of R 2 2 (7) heterosynthon. Then, further C-H···F interactions extend the adjacent tape moieties into a two-dimensional (2-D) corrugated layer. The molecular structure contains one H 2 fip and one bipy molecule (Fig. 1). The two pyridyl rings within the basic unit form a dihedral angle of 30.9 (2)°.
The heterosynthon R 2 2 (7) ring pattern of O-H···N/C-H···O bonds (synthon I in Fig. 2, Table 1), connecting the base and acid moieties, is responsible for the formation of a 1-D wavelike tape structure. Analysis of the crystal packing of (I) suggests that a further C-H···F interaction (Table 1) 2). Within the 2-D layer, a new hydrogen-bonding pattern denoted as R 2 4 (14) (synthon II in Fig. 2, Etter, 1990) is found to link two pairs of centrosymmetry related carboxyl-bipyridine motifs from adjacent tape structures. By comparison, a closely related 1:1 binary cocrystal of isophthalic acid and bipy exhibits similar tapes of acid:base components formed via R 2 2 (7) synthons. But these tapes extend to form supramolecular sheets via additional C-H···O interactions (Shan et al., 2002).
In conclusion, this work demonstrates the first example for H 2 fip as a good participant in co-crystallization with basic modules. When co-crystallizing with rod-like 4,4'-bipyridine building block, the H 2 fip subunits fulfill the reliable carboxylic-pyridine synthon R 2 2 (7). Although the associated C-H···O bonds are not present between adjoining tape motifs, the introduction of fluorine substituents leads to a new hydrogen-bonding synthon R 2 4 (14). This result presents a new challenge in the exploration of crystalline products based on such halogen substituted benzene dicarboxylic acids.
Refinement
One restraint was applied to bonded N1 and C5 atoms to equalize each anisotropic vector component parallel to the bond (DELU command). H atoms bonded to C atoms were positioned geometrically (C-H = 0.93 Å for pyridyl and phenyl H atoms) and included in the refinement in the riding-model approximation, with U iso (H) = 1.2 U eq (C). O-bound H atoms were refined as rigid groups, allowed to rotate but not tip. Isotropic displacement parameters were derived from the parent atoms with U iso (H) = 1.5 U eq (O) and O-H distance of 0.82 Å. Fig. 1. The molecular structure of compound (I) drawn with 30% probability ellipsoids.
Special details
Geometry. All esds (except the esd in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esds is used for estimating esds involving l.s. planes.
Refinement. Refinement of F 2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F 2 , conventional R-factors R are based on F, with F set to zero for negative F 2 . The threshold expression of F 2 > 2sigma(F 2 ) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F 2 are statistically about twice as large as those based on F, and R-factors based on ALL data will be even larger.
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å 2 )
x y z U iso */U eq C1 0.2245 ( | v3-fos-license |
2020-06-25T09:08:42.816Z | 2020-06-21T00:00:00.000 | 225723454 | {
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} | pes2o/s2orc | Synthesis of C60 Nanotube from Pyrolysis of Plastic Waste (Polypropylene) with Catalyst
Fullerene nanotube was synthesized in this research by pyrolysis of plastic waste Polypropylene (PP) at 1000 ° C for two hours in a closed reactor made from stainless steel using molybdenum oxide (MoO3) as a catalyst and nitrogen gas. The resultant carbon was purified and characterized by energy dispersive X-ray spectroscopy (EDX), X-ray powder diffraction (XRD). The surface characteristics of C60 nanotubes were observed with the Field emission scanning electron microscopy (FESEM). The carbon is evenly spread and has the highest concentration from SEM-EDX characterization. The result of XRD and FESEM shows that C60 nanotubes are present in Nano figures, synthesized at 1000 ° C and with pyrolysis temperature 400° C. The synthesis operation doing in one reactor and limited time.
Introduction:
Plastic materials are characterized by many properties that make them desirable in practical applications such as low cost, lightness and durability and as a result are necessary for our daily lives (1). Municipal solid waste is non-degradable and is not implemented in nature. It is disposed of by the way known as landfill, which accumulates multiple types of plastic waste. In these tombs there are many microorganisms that accelerate the degradation of organic matter associated with plastic waste (2). In many developing countries, the amount of plastic consumption is much higher than the average global consumption. The large production of plastic poses a major challenge to deal with these huge quantities of plastic waste after use. Plastic materials in solid waste release harmful chemicals in the soil that can then flow into the groundwater or other surrounding rivers and lakes so it can pose a significant risk to the organisms that drink contaminated water (3). Polypropylene is an attractive candidate for packaging applications and has a wide popularity in automobile and electronics field due to its excellent advantages of good thermal stability, chemical resistance, easy handling, good mechanical characteristics and inexpensiveness ) 4).
Department of Chemistry, College of science, University of Anbar, AL-Anbar, Iraq. * Corresponding author: [email protected] 8102 -8244 -0002 -https://orcid.org/0000 ID: ORCID * Waste materials from domestic wastes to industrial remains, rise harmful effects on environmental and human health regarded as a source of air, soil, water and marine pollution. However, wastes can be used as tools to produce useful goods. A significant technique to obtain this goal is pyrolysis. Pyrolysis relates to thermal decomposition that is operated in an air-free condition (5). Pyrolysis is a probable alternative to landfill for processing plastic waste, resulting decomposition products which can be used as" fuels instead of gas, diesel or fuel oils" (6). Additionally, pyrolysis of plastics has also been utilized to manufacture various types of Nano Carbon such as nanotubes, nanofiber, Nano rods, nanowires, etc., C 60 nanotube which have high value and exceptional physical and chemical properties because of their impressive characteristic like high surface area, porous-rich structure, high conductivity and excellent chemical stability, by blending plastics and catalyst in one reactor (7). The properties of carbon nanotubes and the percentage of the product depend mainly on raw materials. For instance, various methods have been developed to produce CNTs such as arc discharge, pyrolysis, laser ablation of carbon, plasma assisted deposition and chemical vapor deposition (CVD) (8)(9)(10). Fullerene is any molecule in the form of an ellipsoid, tubular or a hollow sphere structure composed entirely of carbon. They are generally referred to as "Buckyballs" (11). There are many types of fullerene such as C 60 rods and C 60 tubes (12). The research objectives are to increase the economic value to benefit from plastics waste and assist in addressing environmental problems associated with this waste and produce new nanomaterials that inter into the technological industry.
Preparation of system gas
The nitrogen gas bottle was connected to three traps, the first trap contained concentrated sulfuric acid (H 2 SO 4 ), which absorbed water from the gas, then the gas was passed to the second trap which contained a saturated solution of pyrogallol to absorb oxygen from the gas, finally the gas was passed to the magnesium sulphate (MgSO 4 ) to absorb the rest of the acid. Then the gas was passed to the reactor system through a trap.
Methods
The samples were washed, air-dried and shredded into small pieces of an area that's around 1mm². 25 g of shredded PP was placed inside a stainless-steel reactor that is filled with some inert gas (nitrogen) at low pressure (between 50 and 70 mbar). 0.5 g of (MoO 3 ) catalyst was placed in tube nozzle connected with reactor. The reactor was tightly closed and put in an electric furnace to be heated as shown in Fig.1. This reactor is connected to condenser and then to three neck round-bottom flask for products collection. Nitrogen gas was pumped at 25 ° C until the temperature reached 500 ° C. The temperature of the furnace was gradually raised. When the temperature of 400 ° C was reached and the wastes began to decompose, the catalyst was added from the tube nozzle. At this level the distillation process began and at the end of distillation the temperature was raised to required temperature. We used 1000 ° C for two hours, at a heating ramp rate of 13 °C/min, then allowed to cool to room temperature naturally. It was found that the final product in the reactor included carbon powder.
Carbon Nanotube Identification
The following equipments were used to identify C 60 nanotubes properties:
Field Emission Scanning Electron Microscopy (FE-SEM)
The morphology and size of samples were studied by scanning electron microscopy (SEM; FEG-SEM MIRA3 TESCAN, Czech Republic), which is configured to operate at (15.0 kV) various magnification level.
X-Ray Diffraction (XRD)
The X-ray diffraction (XRD, X'PERT PRO from Philips, Netherlands) was evaluated to determine the crystal structure and phase the samples, with Cu-Kα radiation (λ=1.54178 Å), operated at 40 kV and 40 mA, was measured in 2θ range from 10 o to 80 o , performed on a University of Kashan (Iran).
Energy Dispersive X-Ray Analysis (EDS)
The elemental composition of samples was studied by (EDS, MIRA3 TESCAN, Czech Republic) Figure 2A shows the XRD patterns of the PP pyrolysis at 1000ºC without catalyst and having the diffraction peaks at the value of 23º, 28.5º and 43º were ascribed to the (002), (100) and (101) reflections. Figure 2B shows the XRD patterns of the C 60 nanotubes from waste PP with MoO 3 , the diffraction peaks at the value of 23º, 28.5º and 43º were ascribed to the (002), (100) The other peaks notices refer to the additives of polymer and the substrate used in the measurement (17)(18) Average crystal size in the product that can be found using X-ray diffraction profile. Calculating the crystal size (D) can be done by using the Debye Scherrer equation: D =
Results and Discussion:
Where is the Scherrer constant, λ is the wavelength of light used for the diffraction, β is the full width at half maximum of the sharp peaks and θ is the angle measured. The Scherrer constant ( ) in the above formula accounts for the shape of the particle and is generally taken to have the value 0.9 (19). From Table1, we could calculate the average crystal size of C 60 NTs as shown below: Average crystal size = 84.17 nm.
The morphology of the sample was revealed by FESEM. Figure 3-A shows a typical FESEM image of the sample. It is found that large quantities of nanostructures (C 60 NTs) were obtained (12). These nanotubes are carbon (34.5-90.6) nm in diameter, and a few micrometers in length, as shown in Fig. 3 Figure 4 shows the high concentration of the carbon content which indicates high purity, and shows the amount of catalyst used in pyrolysis (20). The other elements noticed (Au, Si, Al and Cl) refer to the elements in standard in analysis device (21)(22), and the other components (K, Ca and Na) are additives to improve properties of polymer.
Conclusions:
CNT is successfully synthesized via a new experimental method by using one pyrolysis reactor of polypropylene at 400°C for about 30 minutes and decomposed the polymer chains at 1000°C for two hours with nitrogen ambiance. Resulting of XRD and FESEM shows there is carbon nanostructure at this temperature and marked by a peak intensity at 2θ = 23º, 28.5º and 43º. Moreover, the result of EDX shows that carbon is highest spread when compared with the others. | v3-fos-license |
2020-01-05T14:27:05.637Z | 2020-01-03T00:00:00.000 | 209747079 | {
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} | pes2o/s2orc | Role of offset and gradient architectures of 3-D melt electrowritten scaffold on differentiation and mineralization of osteoblasts
Background Cell-scaffold based therapies have the potential to offer an efficient osseous regenerative treatment and PCL has been commonly used as a scaffold, however its effectiveness is limited by poor cellular retention properties. This may be improved through a porous scaffold structure with efficient pore arrangement to increase cell entrapment. To facilitate this, melt electrowriting (MEW) has been developed as a technique able to fabricate cell-supporting scaffolds with precise micro pore sizes via predictable fibre deposition. The effect of the scaffold’s architecture on cellular gene expression however has not been fully elucidated. Methods The design and fabrication of three different uniform pore structures (250, 500 and 750 μm), as well as two offset scaffolds with different layout of fibres (30 and 50%) and one complex scaffold with three gradient pore sizes of 250–500 - 750 μm, was performed by using MEW. Calcium phosphate modification was applied to enhance the PCL scaffold hydrophilicity and bone inductivity prior to seeding with osteoblasts which were then maintained in culture for up to 30 days. Over this time, osteoblast cell morphology, matrix mineralisation, osteogenic gene expression and collagen production were assessed. Results The in vitro findings revealed that the gradient scaffold significantly increased alkaline phosphatase activity in the attached osteoblasts while matrix mineralization was higher in the 50% offset scaffolds. The expression of osteocalcin and osteopontin genes were also upregulated compared to other osteogenic genes following 30 days culture, particularly in offset and gradient scaffold structures. Immunostaining showed significant expression of osteocalcin in offset and gradient scaffold structures. Conclusions This study demonstrated that the heterogenous pore sizes in gradient and fibre offset PCL scaffolds prepared using MEW significantly improved the osteogenic potential of osteoblasts and hence may provide superior outcomes in bone regeneration applications.
Introduction
Bone lesions that results from fracture, infections and tumors require targeted strategies for effective clinical treatment [1]. The combination of a three-dimensional scaffold combined with growth factors and/or cells has significant potential in bone tissue engineering as an ideal bone substitute option. However such scaffolds are not widely applied in clinical medicine, with the inability to seed a large enough cell population into scaffolds being one key impediment [2]. A desirable porous interconnected scaffold is required to allow uniform cell diffusion and distribution throughout the entire scaffold structure [3]. Many studies have attempted to solve this problem with various approaches, such as using bioreactors for enhanced cell proliferation, chemical and biological surface modification and varying the pore size of the scaffolds in a coordinated fashion to geometrically represent a cross section of a bone defect for an individual patient as a result of trauma or cancer metastasis [4][5][6][7]. Notably, previous studies showed that cell differentiation is influenced by the porosity, elasticity and stability of the scaffold [8].
Melt electrowriting scaffold fabrication enables precise control of the porosity, pore morphology and pore size of the printed scaffold architecture [9]. This allows predictable strain and stress distribution within the scaffold, which can be further optimized to produce an environment favouring higher cell infiltration through appropriate interconnectivity within the scaffold and subsequent vascularization and angiogenesis [10].
Previous studies have shown rapid bone formation was associated with pore sizes between 290 and 310 μm. While smaller pore sizes enhance the mechanical properties of the scaffold, the optimal size for vascularization was noted to be~400 μm [11,12] . Therefore, fabrication of a 'gradient' porous scaffold, whereby the pore size gradually increases from one layer to the next, may overcome some of the individual limitations of both small and large homogeneous pore size scaffolds.
Gradient structures provide macropores suitable for vascularization, efficient gas/waste diffusion and nutrient supply at the expense of reduced mechanical stability. However, the denser structure of a heterogeneous gradient architecture improves ion signalling and protein adsorption as well as mechanical properties of the scaffolds, such as compressive strength. Additionally, they facilitate greater protein adsorption and better cell adhesion which decreases in the larger pore size of a homogeneous scaffold [13]. Interestingly, the gradient scaffold architecture is similar to the natural structure of native bone tissue that represents different mineral density from cancellous bone to cortical bone [14,15].
Furthermore, higher pore size in gradient scaffolds has been shown to enhance permeability, cell migration, as well as sufficient nutrients and oxygen tension in larger pores and up-regulate osteopontin and collagen type I expression, thus generating more bone mass, vascularization and blood vessel ingrowth while inhibiting the formation of cartilaginous tissue in the regenerating sites [16,17]. On the other hand, the smaller size of gradient pores promote cell seeding and growth by providing higher surface area [18].
The complex heterogeneous and hierarchical structure of bone tissue creates significant variation in the compressive and tensile strength of different regions within bone. It's been shown that the morphology of the pores influences the mechanical properties and the structure of the scaffolds i.e. with more complex morphological architectures, the compressive strength will be increased [19], however, the scaffolds with greater Young's modulus and smaller pore size are preferred in applications required to withstand greater loads [20]. According to Sobral et al., the simple architecture of homogeneous scaffolds is prone to collapse under high stress applied to the scaffolds, while the complexity of non-uniform porous scaffolds enables them to recover after deformation and maintain their elastic state which is critical for biomaterials implanted for bone applications [21].
Our previous studies have shown that, when compared to simpler homogeneous structures, scaffolds with offset and gradient architectures resulted in significantly higher cell proliferation following seeding (Additional file 1) [22]. As a logical extension of this work, this study will investigate the potential effects of heterogeneous porous scaffold architectures, particularly offset and gradient structures on osteogenic gene expression by osteoblasts seeded into these scaffolds and the rate of mineralization throughout the construct.
Preparation of microfibrous PCL membrane
A melt electrowriter (MEW) system was used to produce a fibrous scaffold with the fibre diameter in a range of 6-10 μm. The parameters for the components nozzle diameter, voltage, temperature and feeding rate were described in as previously reported [22]. Two different scaffold structures; homogeneous pore size (250 μm, 500 μm, 750 μm) and heterogeneous architecture including a tri-layer scaffolds with different pore sizes from 250 μm on top, 500 μm in the middle, and 750 μm at the bottom of the scaffold and offset scaffolds in which layers were printed with various offset values of 30 and 50% compared to the previous layer (Fig. 1).
Subsequent calcium phosphate (CaP) coating was perform using simulated body fluid (SBF). The surface modification was achieved in three steps; 1) pre-treatment of the samples in 1 M NaOH aqueous solution for surface activation for 0.5 h 2) immersion of the samples in 10X SBF at 37°C for 1 h 3) soaking the samples in 0.5 M NaOH for 0.5 h to obtain an uniform coating then rinsing with distilled water and allowing to dry.
Cell culture
Human osteoblast cells (hOB) obtaining from alveolar bone of a healthy female were seeded onto the scaffolds (2 × 10 4 in 15 μl) and cultured in either osteogenic or basal medium for 3, 14 and 30 days, according to previously reported protocols [22].
Scanning electron microscopy (SEM)
Cell morphology after 30 days culture on the electrowritten scaffolds was captured by scanning electron microscopy (SEM). The substrates from cell culture were fixed in 3% glutaraldehyde, then dehydrated by treating in 0.1 M Cacodylate buffer three times, each 10 min and postfixed with 1% Osmium Tetroxide for 1 h. After that, the specimens were washed in Milli-Q water two times, each 10 min. The samples were then rinsed in a concentration gradient of ethanol (40-100%) 10 min for each. To visualise cell penetration into the scaffolds, cross-sections of the samples were fragmented in liquid nitrogen in the middle stages of the dehydration process. Finally, hexamethyldisilazane (HMDS, Sigma Aldrich, UK) was used to replace the critical point drying step in biological samples for two times, each 30 min. The samples were mounted on the Al stubs using adhesive carbon tapes and were Au-coated for observation by the SEM (Zeiss Sigma FESEM).
Alkaline phosphatase (ALP) activity
The concentration of alkaline phosphatase (ALP) in cells was measured after 3, 14 and 30 days of culture using pnitrophenyl phosphate as a phosphatase substrate in a commercial Alkaline Phosphatase colorimetric assay Kit (Sigma Chemical, St. Louis, MO, USA) at a wavelength 405 nm according to the manufacturer's instructions. The ALP concentration was subsequently normalised to the total protein content (Quick Start Bradford protein assay, Bio-rad, Australia) of each sample.
Alizarin red staining (ARS)
To assess the osteogenic potential of the osteoblasts cultured in the melt electrowritten scaffolds with different pore sizes, alizarin red S staining of calcium deposited in the extracellular matrix of osteoblasts cultured on the scaffolds after 14 and 30 days was carried out. Briefly, the culture media was removed and the scaffolds washed using PBS. The cells within the scaffolds were fixed in 4% paraformaldehyde then placed in 500 μL of Alizarin Red S solution (40 mM) for 20 min at room temperature. After washing, the stained cells and scaffolds were imaged using an inverted phase contrast tissue culture microscope (Olympus, CKX 41, NY, USA). Quantification of these results was achieved by dissolving the samples in 1 mL 10% Cetylpyridinium Chloride for 1 h before transferring to 96 well plates and measuring the absorbance at 540 nm in a microplate reader (POLARstar Omega, BMG LABTECH, Germany). The average value of the negative controls (scaffold without cells) [22] was subtracted from the values of the corresponding experimental groups.
Micro-computed tomography (μ-CT) of cell-scaffold construct
To further evaluate mineralisation within the cell-seeded scaffolds, μ-CT analysis of constructs cultured in osteogenic differentiation medium was compared to the control group (basal medium) after 30 days of culture. The volume of mineralisation was measured by the μ-CT software (μCT40, SCANCO Medical AG, Brüttisellen, Switzerland). The average value of the negative controls (cell seeded scaffolds in expansion media) was subtracted from the values of the corresponding experimental groups. The sample was put inside the tube containing deionised water so that, it was located at the bottom of tube and cotton wool was used to stop moving of sample during scanning process. The tube was sealed to prevent solution evaporation. For scanning, the high-resolution mode was selected and the X-ray tube was applied at 45 kVp and 177 lA. Integration time was set to 300 m sec and a three-fold frame averaging was applied using the same μ-CT hardware, acquisition, and reconstruction parameters as above. Three-dimensional images of the scaffolds were reconstructed by the software package.
Real-time PCR analysis (q-PCR)
RNA was extracted from the osteoblast cells 14 and 30 days after seeding onto the scaffolds using a TRIzol extraction kit (Ambion, USA). Following cDNA synthesis from 100 ng of total RNA (Taqman cDNA synthesis kit, Life Technologies, USA), the expression of osteopontin (opn), osteocalcin (ocn), bone morphogenetic protein 2 (bmp-2), alkaline phosphatase (alp), collagen type Ia (col Ia), wingless-related integration site (wnt) family member 3a (wnt3a), and wnt family member 5a (wnt5a) was achieved with an ABI 7900HT real-time PCR system (Life Technologies, USA) using SYBR Green Real-Time PCR Master Mix (Life Technologies, USA) and the following protocol; 3 min at 95°C for polymerase activation followed by 40 cycles of 10 s denaturation at 95°C, 20 s annealing at 58°C, and 1 s extension at 72°C. β-actin expression was used as a house keeping gene and for normalization of the data.
Immunofluorescence staining
For immunocytochemical analysis of the osteoblast cells cultured on different pore size PCL scaffolds (tissue culture plate acted as the control group) for 14
Statistical analysis
Statistical analysis of any differences between means was performed using a two-way ANOVA with correction for multiple comparisons. The experiments were run in triplicate. A p-value of < 0.05 was considered as statistically significant.
Results
Cell-scaffold morphology Figure 2 SEM images show the morphology and pore arrangement of the MEW PCL scaffolds prior to cell seeding. Thirty days following seeding with osteoblasts, SEM images showed good attachment and growth of the osteoblasts onto the PCL scaffolds ( Fig. 3a, b, c). Optimum cell attachment and proliferation was identified on the 250 μm pore size homogeneous scaffold and the offset architecture scaffolds where the majority of cells appeared to be entrapped in the space between two displaced fibres in the offset groups. The 500 μm and 750 μm pore sizes appeared to be too large for the cells to interact with the scaffold fibers whereas in the gradient group the cells passed through the largest pore size at the top of scaffold then settled down into the smaller pores at the bottom of the construct (Fig. 3c) Under lower magnification, information on the degree of scaffold mineralization and the position of the cells within this mineralized matrix after 30 days culture can be observed (Fig. 3a, b). Reflecting the cell attachment results, calcified matrix production occurred predominately on the offset and 250 μm surface scaffolds. At higher magnification, globular shaped deposits could be distinguished in the vicinity of the more dense regions of cell-fibre junctions and in the smaller areas between the pores (Fig. 3c). In the gradient group, the cells appeared more concentrated at the bottom of scaffold structure having passed through the large pore size at the top of scaffold and settled down into the smaller pores at the bottom of scaffold (Fig. 3d).
Impact of porous scaffolds on ALP activity of osteoblast cells
Alkaline phosphatase (ALP) activity was very low at day 3 in all groups (Fig. 4a, b) in the similar ranges between 0.7 to 3.1 that the maximum amount was displayed for gradient, 250 μm and offset.30.70 scaffolds respectively. By day 14 ALP activity had increased in all groups with the highest levels of activity in osteoblasts cultured on the offset.50.50 and gradient scaffolds. However, it was peaked significantly for the gradient structure culturing in osteogenic media compare to the other groups. By 30 days of culture, a reduction of ALP activity was seen for all groups except the homogeneous 750 μm pore size scaffolds.
Influence of porous scaffolds on calcium deposition by osteoblast cells
To assess mineralized matrix formation on the scaffold, analysis of calcium deposition was performed after 14 and 30 days of culture in osteogenic and basal medium (Fig. 5a, b). The analysis of alizarin red staining showed a significant difference in calcium accumulation in the scaffolds cultured in osteogenic differentiation medium compared to basal medium as the control group. The data indicated that all scaffolds were able to significantly induce osteogenic differentiation through the augmentation of calcium in the extracellular matrix. The calcium deposition started to appear after 14 days and gradually increased up to 30 days in osteogenic medium. Figures 5c, d showed the quantitative analysis of mineralization in all of the groups where the average calcium deposition was higher on offset.50.50, offset. 30.70 and 250 μm PCL scaffolds respectively. The 750 μm showed the lowest amount after 30 days. Figure 6 compares extracellular matrix mineralisation of osteoblasts seeded onto the scaffolds and cultured in osteogenic media with those cultured in basal media. As expected from the corresponding alizarin red staining data, mineralization was distributed within the pores of the scaffold (Fig. 6a). Consistent with the alizarin red staining, pixel quantification of the μ-CT images (Fig. 6b) also confirmed that the offset.50.50 scaffold had the highest amount of mineralisation while the 750 μm pore size scaffold showed the lowest level of mineralization.
Effect of porous scaffolds on osteoblast gene expression of osteogenic markers
Expression of the osteogenic markers col Ia, alp, ocn and opn, as well as the osteogenesis associated signalling molecules bmp-2, wnt3a and wnt5a, in osteoblasts cultured on the scaffolds were analysed by quantitative 30.70 and gradient scaffold groups, but this was not statistically significant compared to the other groups. Following 30 days of cell culture, the mineralization-related markers ocn and opn were up-regulated. In all groups, the expression of alp was less than ocn and opn after 30 days. Therefore, all of the scaffold groups stimulated the upregulation of alp and col I expression at early stages of osteogenic differentiation after 14 days, while, the gradient and offset.30.70 scaffolds were able to express ocn and opn. Increase of bmp2 gene expression was observed in 250 μm, offset. 30.70 and gradient scaffolds, and at the highest quantity in the 750 μm group, compared to other groups. The assessed data demonstrated the high expression of wnt5 in the gradient and 50.50 offset scaffolds.
Osteoblast-related protein expression
To evaluate the osteoconductivity of microporous melt electrospun PCL scaffolds, osteoblast cells were seeded on these scaffolds and cultured in osteogenic medium for 30 days in vitro. Immunocytochemistry was subsequently used to visualise collagen type I, collagen type III and osteocalcin deposition within the cellular matrix (Figs. 9, 10). Confocal microscopy demonstrated similar biocompatibility of the scaffolds as shown by the uniform spatial distribution of the cells on all six scaffold types. As expected, the images confirmed that collagen type I, collagen type III and osteocalcin were all expressed on all the scaffolds cultured in osteogenic media for 14 days and 30 days (Figs. 9, 10). These data indicated that all of the scaffolds showed markedly better expression of osteocalcin compare to the other two proteins. It was obvious that the number of differentiated cells on the scaffolds increased with time. The pore spaces were filled and covered completely by the cells after 30 days, particularly in gradient and offset scaffolds. However, the expressions of collagen type I, collagen type III and osteocalcin on the scaffolds cultured in osteogenic media were much higher than those on the scaffolds cultured in basal medium (Figs. 9, 10).
Discussion
Several strategies can be utilized to attain porous scaffolds. As melt-electrowriting fabrication delivers a stable electrostatically drawn jet without whipping or random fibre deposition (as in solution electrospinning) it can be used for the formation of gradient structures mimicking native bone by precisely controlling fiber placement [23]. The proliferation and differentiation of cells within 3D scaffolds are affected by both the size and geometry of the scaffold's pores [24] but it's not clear however whether scaffolds with a uniform pore distribution of homogenous size, or constructs with a varying pore size distribution, are more suitable for bone regeneration applications. Therefore, the present study evaluated the effect of complex offset and graded porous structures on bone differentiation in contrast to the simpler homogeneous MEW PCL scaffolds.
According to the SEM observations, the presence of more anchorage points in offset and 250 μm structures allowed more cells to populate and grow compared to the other scaffolds, suggesting that the smaller pore volume induced more cell aggregation. Porous scaffolds with an offset designs creates a higher surface area that could result in more cell distribution and proliferation. Since the fibres positioning affected the flow path of the media during cell seeding, the cell attachment efficiency increased. This is due to the presence of the 'obstacles' in the heterogeneous (offset/gradient) pore design, which provides cells with additional anchorage points during the seeding process [25]. The enhanced cell growth in the smaller pore size scaffolds may also be attributed to the higher concave surfaces and curvature of the small pore size than the large pore size which results in minimizing the surface area and surface tension [26,27].
Surface tension or surface energy comes from the tendency of cells to approach each other to achieve a balanced energy i.e. neighboring cells have the least energy and the most stable state. This is the natural tendency of molecules to minimize their energy [28], therefore, to reduce their surface energy, the cells will select the corners of the pore space to enhance contact with the other cells. Because of the lower angle between the fibers of small pore sizes, it can provide a more suitable environment for the cells to interact with each other, thus minimizing their residual energy and becoming more stable [29,30].. This could be the reason for higher cell proliferation and faster growth in the smaller pores of 250 μm and offset.50.50 scaffolds with the higher curvature, compared to larger pore sizes of 750 μm.
While there are no experimental findings to support the hypothesis that minimal surface areas advance bone regeneration, concave surfaces compared to convex and planar surfaces have been shown to promote bone tissue regeneration, a process that increases with higher curvature [20,31,32]. This agrees with our study, which showed more mineralization in offset.50.50 and 250 μm scaffolds compared to the higher pore size of scaffolds. Cells at the centre of the pores have the highest energy levels, and larger pore sizes generate more perimeter and thus less curvature [33]. This may be the reason for low cell density with larger pore sizes despite more penetration of the cells into the 500 μm and 750 μm pore size scaffolds after 24 h, compared to the 250 μm pore size scaffold. The restriction on infiltration though promotes differentiation over proliferation since the inner space is filled faster due to more interactions between cells in the smaller area, while larger pore sizes have more empty spaces promoting proliferation instead.
Our results are in agreement with the study of Di Luca et al., that also showed an increase in cell number and mineralization by reducing the pore size to 500 μm as well as in the area with the smallest pores (500 μm) of a complex four zone gradient (500/700/900/1100 μm zones) using PEOT/PBT (poly (ethylene oxide therephtalate)/poly (butylene therephtalate) and PCL scaffolds fabricated by rapid prototyping [34]. They also showed that the variety in cell density in the different regions of gradient scaffolds could be attributed to the increase of hypoxia inducible factor (HIF) in the higher hypoxic regions of small pore size where the oxygen levels could drop [35]. Previous studies already suggested that rising hypoxia leads to osteochondrogenic differentiation, mediated via HIF-1 and triggered through the elevation of ALP and OCN levels and mineralization [36][37][38][39]. Interestingly, we also found more calcium mineralization in smaller pore sizes of 250 μm and offset.50.50 structures.
Other models have shown that the behavior of cells can be affected by pore size. For example, chondrocytes produce large amounts of glycosaminoglycan (GAG) and collagen II in 400 μm sized pores, while they proliferate in 200 μm sized pores [40]. On the other hand, human mesenchymal stromal cells (hMSCs) seeded within PEOT/PBT scaffolds had significantly higher amounts of GAG in 500 μm pore size (non-gradient) and gradient (500-1100 μm) scaffolds, compared to largest pore sizes [41].
In the present study alkaline phosphatase activity, an early indicator of matrix mineralization, increased significantly after 14 days in the gradient and offset.50.50 scaffolds. After 30 days ALP activity decreased (compared to day 14) in all scaffolds, except for the 750 μm pore size scaffold. This suggests that the structures with the greater porosity of 750 μm and gradient architecture stimulate ALP expression. This finding is also in accordance with the in vitro study by Di Luca et.al (2016) which demonstrated that an increase in pore size of poly (ethylene oxide therephtalate)/poly (butylene therephtalate) (PEOT/PBT) and poly(ε-caprolactone) (PCL) scaffolds enhances ALP levels during human mesenchymal stromal cell differentiation [34].
Our study also showed that ALP levels were greater in 750 μm scaffold after 30 days compared to the other scaffolds. This finding was in accordance with the study of Hutmacher et.al (2000) that indicated the structures with greater porosity could stimulate ALP expression, since countering hypoxic conditions in the smaller pore size and unavailability of O 2 and nutrient supply for the cells [42]. Kasten et.al (2008) demonstrated that the higher porosity of 65 and 75% β-tricalcium phosphate (TCP) ceramic scaffolds also enhanced ALP activity when compared to lower porosity (25%) scaffolds due to better nutrient and O 2 transportation [43]. However, it also needs to be acknowledged that the higher levels of ALP activity after 30 days in the present study might also be the consequence of less cells initially attaching to the larger pore structure of the 750 μm scaffold.
Although higher porosity increased ALP activity, our results showed the offset scaffolds demonstrated superior matrix mineralization. This may be related to the surface wettability which increases surface free energy resulting in better cell attachment due to greater protein adsorption of ECM-products [44], and the rougher surface of the offset structures after calcium phosphate (CaP) coating modification, which was shown in our previous study by alizarin-red staining and surface area (BET) analysis [22]. It has been displayed the CaP coating on the surface of the fibres within the cells, that the higher level of mineralization was identified on the offset and then 250 μm scaffold structures through alizarin-red staining and micro-CT analysis. This is in agreement with the study of Hammerl et al. (2019) that showed that CaP/PCL scaffolds formed a mineralized matrix regardless of the cell type cultured on these scaffolds [45], suggesting that released ions and wide contact area between the cells and fluids can evoke higher mineralization in short term cell culture, although evidence for this phenomenon is not yet provided. Although some studies have used the commercial materials to create a biologically active surface of the implant [46,47]. The study of Yeo et al. (2012) also confirmed increased mineralization in 50 and 100% offset polycaprolactone (PCL) and β-tricalcium phosphate (β-TCP) scaffolds compared to no-offset structures [25]. Furthermore, higher hydrophilicity creates more surface free energy that results in better cell attachment due to the greater protein adsorption as more ECM-products are achieved by higher cell number [44]. Our results indicated a high level of col Ia gene expression in offset scaffolds over 14 days, which were expressed at the middle stage of differentiation. Type I Collagen gene expression, an early marker of osteoblast differentiation which results in increased bone mass in combination with OCN and ß-catenin functions [49], as well as increased up-regulation of ocn gene expression was indeed seen in offset.30.70 scaffolds after 30 days. Increased bmp-2 gene expression was also observed in 250 μm, offset.30.70 and gradient scaffolds with the highest expression in 750 μm scaffolds. Activation of bmp signalling leads to osteocalcin and alkaline phosphatase expression [50] and after 30 days culture the activity of ALP increased in the 750 μm scaffolds compared to the other groups, while bmp expression was not upregulated. The gradient scaffolds also had a high level of wnt5 gene expression. The wnt family of secreted glycoproteins plays a critical role in bone formation, mediated through the expression of osteoblast-specific genes. Activation of this pathway also results in the expression of alkaline phosphatase, an early osteoblast marker associated with the Wnt pathway [51]. The gradient scaffold was associated with the highest expression levels of wnt5 which correlated with increased alp expression in gradient scaffolds after 30 days of culture.
A previous study showed that up-regulation of bmp-2 supressed wnt3a signalling in MSCs and induced osteoblast differentiation [52]. Similarly, our results also showed higher expression of wnt3a in homogeneous 500 μm scaffold compared to bmp2, which may mean that the cells in this scaffold were stimulated towards proliferation instead of differentiation, in contrast to gradient and offset scaffolds which had lower wnt3a expression.
High expression of ocn and opn is associated with the late stages of differentiation and mineralization [53,54]. Offset and gradient scaffolds had greater expression of ocn and opn, suggesting that these architectures enhanced osteoblast differentiation to exhibit the mature markers following 30 days. Sicchieri evaluated the effect of pore size on the osetogenic gene expression and showed that larger pore size of PLA-CaP scaffolds increased the expression of alp, type I col and ocn, similar to the higher expression of these markers in the gradient structure of this study, which may be influenced by the large pore size section in the gradient scaffold [55]. In the present study the increase in osteocalcin (OCN) expression especially in offset scaffolds suggests that this architecture could promote osteogenesis as osteocalcin modulates the matrix mineralization which is a later phase of osteogenic differentiation [56]. The findings of this in vitro study need to be confirmed in an appropriate in vivo model, to fully elucidate the effect of heterogeneous and homogeneous pore size of melt electrospun (MEW) scaffolds on bone regeneration.
Conclusion
This study has shown that heterogenous offset and gradient porous scaffolds are favourable structures for bone differentiation compared with uniform pore size scaffolds. The gradient architecture with three different pore sizes (250-500-750 μm) favoured ALP activity, while scaffolds with an offset 50% value allowed more matrix mineralization and the expression of late osteogenic markers, such as osteocalcin, which promoted maturation of differentiated osteoblasts. Also, both the gradient and offset scaffolds appeared to support ECM deposition in contrast to the homogeneous porous scaffolds. Taken together, the finding of this research demonstrated the gradient pore size and the offset architecture of MEW scaffolds are able to overcome the limitation of small and large uniform pore sizes associating with cell adhesion and mineralization during the in vitro bone differentiation process. | v3-fos-license |
2016-05-12T22:15:10.714Z | 2015-11-16T00:00:00.000 | 17078781 | {
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} | pes2o/s2orc | Tumor Repression of VCaP Xenografts by a Pyrrole-Imidazole Polyamide
Pyrrole-imidazole (Py-Im) polyamides are high affinity DNA-binding small molecules that can inhibit protein-DNA interactions. In VCaP cells, a human prostate cancer cell line overexpressing both AR and the TMPRSS2-ERG gene fusion, an androgen response element (ARE)-targeted Py-Im polyamide significantly downregulates AR driven gene expression. Polyamide exposure to VCaP cells reduced proliferation without causing DNA damage. Py-Im polyamide treatment also reduced tumor growth in a VCaP mouse xenograft model. In addition to the effects on AR regulated transcription, RNA-seq analysis revealed inhibition of topoisomerase-DNA binding as a potential mechanism that contributes to the antitumor effects of polyamides in cell culture and in xenografts. These studies support the therapeutic potential of Py-Im polyamides to target multiple aspects of transcriptional regulation in prostate cancers without genotoxic stress.
Introduction
Pyrrole imidazole (Py-Im) polyamides are non-covalent, sequence specific DNA binders that can alter DNA architecture [1,2]. Upon high affinity binding to the DNA minor groove, the molecules cause a 4 angstrom widening of the minor groove walls and a corresponding compression of the opposing major groove [3,4]. Despite the relatively large molecular weight of Py-Im polyamides, these molecules are cell permeable and localize to the cell nucleus to affect endogenous gene expression [5][6][7][8][9][10]. Due to their modular sequence specificity, Py-Im polyamides can be synthesized to target DNA sequences of similar size to a protein-DNA interaction site and therefore used to antagonize gene expression driven by specific transcription factors [7,[9][10][11][12][13]. One such transcription factor that has been studied previously is the androgen receptor (AR) [9].
The AR is a dihydrotestosterone (DHT) inducible nuclear hormone receptor whose transcriptional program has been implicated in the progression of prostate cancer [14][15][16]. Upon ligand induction, AR will homodimerize, translocate to the nucleus and bind to conserved sequences known as the androgen response element (ARE) to regulate transcription [17]. Each monomeric unit binds to a half site of the sequence 5'-TGTTCT-3' [18]. Polyamide 1 (Fig 1) was designed to target the sequence 5'-WGWWCW-3' (W = A/T), found in a subset of ARE half-sites, and has been shown to prevent AR binding at select AREs and attenuate AR signaling [9].
In addition to antagonizing AR signaling, polyamide 1 is also cytotoxic towards prostate cancer cells [19]. Experiments in mice have shown that polyamide 1 is bioavailable via several routes of administration, with a serum half-life of 5.2 hours [20,21]. In xenograft experiments, polyamide 1 has been shown to be active towards LNCaP xenografts at doses of 1 mg/kg [19]. LNCaP, however, expresses a mutated androgen receptor, and as a result, may not be representative of the majority of human disease [22]. It would therefore be useful to evaluate the efficacy of 1 against other forms of prostate cancer.
The VCaP human prostate cancer cell line expresses wild type AR and contains the TMPRSS2-ERG fusion [23]. Gene fusions between the TMPRSS2 5'-untranslated region and the ERG oncogene are found in approximately half of prostate cancer cases [24]. The fusion allows the AR regulated TMPRSS2 promoter to drive the expression of ERG, and overexpression of ERG in patients has been linked with higher incidences of metastasis and poor disease prognosis [25]. In cell culture, ERG overexpression in immortalized prostate RPWE epithelial cells and in primary prostate epithelial cells (PrEC) has been shown to increase cellular invasiveness [26]. Due to these characteristics, the VCaP cell line presents an ideal model for the study of Py-Im polyamide activity towards this common subtype of prostate cancer. In this study, we evaluated the activity of the ARE targeted polyamide 1 in VCaP cells.
Materials and Methods
Synthesis and quantitation of Py-Im polyamide 1 Chemicals were obtained from Sigma Aldrich or Fisher Scientific unless otherwise noted. Synthesis was performed using previously reported procedures as indicated [7,27]. Briefly, polyamides were synthesized by microwave-assisted solid phase synthesis on Kaiser oxime resin (Nova Biochem) [27] and then cleaved from the resin with neat 3,3'-diamino-N-methyldipropylamine. The triamine-conjugated polyamides were purified by reverse phase HPLC and subsequently modified at the C-terminus with isophthalic acid (IPA) or fluorescein-5-isothiocyanate (FITC isomer I, Invitrogen) [7]. The amine substituents of the γ-aminobutyric acid (GABA) turn units of the polyamides were deprotected using neat trifluoroacetic acid [28,29]. The final polyamide was purified by reverse phase HPLC, lyophilized to dryness, and stored at -20°C. The identity and purity of the final compounds were confirmed by matrixassisted laser desorption/ionization time-of-flight (MALDI-TOF) spectrometry and analytical HPLC. Chemical structures are represented in Fig 1 and S1 Fig Mass spectrometry Py-Im polyamides were dissolved in sterile dimethylsulfoxide (DMSO, ATCC) and quantitated by UV spectroscopy in either 4:1 0.1% TFA (aqueous):acetonitrile (ε(310nm) = 69,500 M -1 cm -1 ) or 9:1 water:DMSO (ε(310nm) = 107,100 M -1 cm -1 ) as dictated by solubility. Polyamides were added to cell culture solutions at 1000x concentration to give 0.1% DMSO solutions.
Cell culture
The VCaP cell line was obtained from the laboratories of Dr. Kenneth J. Pienta and Dr. Arul M. Chinnaiyan at the University of Michigan Department of Pathology, where the cell line was derived [30]. VCaP cells were received at passage 19 and cultured in Dulbecco's modified eagle medium (DMEM, Gibco 10313-039) with 4 mM glutamine (Invitrogen) and fetal bovine serum (FBS, Omega Scientific) on Corning CellBind flasks. All experiments were performed below passage 30.
Cellular uptake studies
For visualization of uptake using FITC-analog polyamides, VCaP cells were plated in 35-mm optical dishes (MatTek) at 7.5×10 4 cells per dish and allowed to adhere for 48 h. Media was then changed and cells were treated with 0.1% DMSO with polyamide for 24 or 48 h. Cells were imaged at the Caltech Beckman Imaging Center using a Zeiss LSM 5 Exciter inverted laser scanning microscope equipped with a 63x oil immersion lens as previously described [5].
WST-1 proliferation assay
VCaP cells were plated at 2x10 4 per well in 96-well plates coated with poly-L-lysine (BD Bio-Coat). After 24 h, an additional volume of medium containing vehicle or polyamide was added to each well. All medium was removed following polyamide incubation at the indicated time points and replaced with one volume of WST-1 reagent (Roche) in medium according to manufacturer protocol. After 4 h of incubation at 37°C, the absorbance was measured on a FlexSta-tion3 plate reader (Molecule Devices). The value of A(450 nm)-A(630 nm) of treated cells was referenced to vehicle treated cells. Non-linear regression analysis (Prism software, Graphpad) was performed to determine IC 50 values.
Gene expression analysis by quantitative RT-PCR (qPCR)
For DHT induction experiments, VCaP cells were plated in 6-well plates coated with poly-Llysine (BD BioCoat) in charcoal-treated FBS containing media at a density of 31k/cm 2 (3x10 5 cells per well). The cells were allowed to adhere for 24 h and then dosed with 0.1% DMSO with or without polyamide 1 for 72 h followed by the addition of 0.01% ethanol in PBS with or without DHT (1 nM final concentration). Cells were harvested after additional 24 h incubation. Cells treated with etoposide and camptothecin (Sigma) were co-treated with DHT (1 nM) and harvested after a 16 h incubation. For native expression experiments, VCaP cells were plated as above but using standard FBS media and harvested after 72 h of treatment. For all experiments, the mRNA was extracted using the QIAGEN1 RNeasy mini kit following the standard purification protocol. Samples were submitted to DNAse treatment using the TURBO DNA-free™ Kit (Ambion), and the mRNA was reverse-transcribed by using the Transcriptor First Strand cDNA Synthesis Kit (Roche). Quantitative PCR was performed by using the FastStart Universal SYBR Green Master (Rox) (Roche) on an ABI 7300 Real Time PCR System. Gene expression was normalized against GUSB. Primers used are referenced in S3 Fig.
Immunoblot of ERG protein levels
For assessment of ERG and beta-actin protein levels, 3x10 6 VCaP cells were plated in 10 cm diameter dishes with charcoal-treated FBS containing media for 24 h before treatment with 0.1% DMSO vehicle with or without polyamide 1 for an additional 72 h. Ethanol (0.01%) in PBS with or without DHT (1 nM final concentration) was then added. After 24 h incubation, cells were lysed in TBS-Tx buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X100) containing fresh 1 mM phenylmethanesulfonylfluoride (PMSF) and protease inhibitors (Roche). The samples were quantified by Bradford assay, denatured by boiling in Laemmli buffer, and total protein was separated by SDS-PAGE. After transfer to the polyvinyl difluoride (PVDF) membrane (Bio-Rad) and blocking with Odyssey Blocking Buffer (LI-COR), primary antibodies were incubated overnight at 4°C. Rabbit monoclonal anti-ERG antibody (Epitomics 2805-1) and rabbit polyclonal anti-actin antibody (Sigma A2066) were used. Goat anti-rabbit near-IR conjugated secondary antibody (LI-COR) was added and the bands were visualized on an Odyssey infrared imager (LI-COR). The experiment was conducted in duplicate and the data are representative of both trials.
Single cell electrophoresis (COMET) assay
VCaP cells (3x10 6 cells) were plated in 10 cm cell culture dishes and allowed to adhere for 24 h before addition of DMSO vehicle or polyamide stock in DMSO. After 72 h incubation, cells were washed with warm PBS (37°C), gently scraped, and counted. Samples were centrifuged, resuspended at 1x10 5 cells/mL, and treated according to manufacturer protocol (Trevigen) for neutral electrophoresis. Slides were stained with SybrGreen (Trevigen) and imaged at the Caltech Beckman Imaging Center using a Zeiss LSM 5 Pascal inverted laser scanning microscope equipped with a 5x air objective lens. Overlayed fluorescence and bright field images were obtained using standard filter sets for fluorescein. Images were analyzed using Comet IV software (Perceptive Instruments Ltd) with 200-600 comets measured per sample. A random sampling of 200 comets per condition was used for two-way analysis of variance (ANOVA) analysis (Prism software, GraphPad) of three biological replicates.
Xenograft assays
Male severe combined immunodeficiency (SCID) mice (4-6 weeks old) were obtained from a breeding colony maintained by the University of Michigan. Tumors were induced by subcutaneous injection of 1x10 6 VCaP cells (10 mice per dose group) in 200 μL of Matrigel (BD Biosciences, Inc., San Jose, CA) above the right flank. Tumor growth was monitored by caliper measurement until the tumor size reached 100 mm 3 using the formula 0.56 x L x W 2 . Groups were randomized and all mice were treated subcutaneously with control (DMSO) or with polyamide 1 as reported (3 times per week, 10 total injections). Tumor growth was followed weekly by caliper measurements. Animal husbandry and daily care and medical supervision was provided by the staff of the Unit for Laboratory Animal Medicine (ULAM) under the guidance of supervisors who are certified as Animal Technologists by the American Association for Laboratory Animal Science (AALAS) at the University of Michigan. Animals were monitored twice daily by both the research team and the veterinary staff. Health was monitored by weight (twice weekly), food and water intake, and general assessment of animal activity, panting, and fur condition. The experiments were performed in accordance with the guidelines on the care and use of animals set by the University Committee for the Use and Care of Animals (UCUCA) of the University of Michigan, and all procedures in this study were specifically approved by the UCUCA (Protocol Number 3848). In all cases, appropriate measures were taken to minimize discomfort to animals. All injections or surgical procedures were performed using sterile technique with efforts made to minimize trauma to the animals. When necessary, animals were anesthetized with a mixture of 1.75% isofluorane/air. Following injections animals were closely monitored and any that appeared moribund were immediately euthanized by administration of anesthesia, followed by inhalation of carbon dioxide until breathing ceased. Death was then ensured through cervical dislocation.
RNA-seq analysis
VCaP cells (1x10 6 cells) were plated in 20 cm cell culture dishes and allowed to adhere for 72 h in DMEM containing 10% FBS and 4 mM glutamine. Polyamide 1 or 0.1% DMSO vehicle were then added in fresh media and allowed to incubate for 96 h. Total RNA was collected by trizol extraction. Library building and sequencing were performed at the Caltech Millard and Muriel Jacobs Genetics and Genomics Laboratory. Sequenced reads were mapped against the human genome (hg19) with Tophat2 using Ensembl GRCh37 gene annotations [31]. Exon alignment was performed with htseq-count and differential expression was determined with DESeq2 [32,33]. Genes with padj < 0.05 and |log 2 (fold change)| ! 1 were submitted for connectivity map analysis online at http://lincscloud.org.
Topoisomerase inhibition assay
Topoisomerase inhibition kits were purchased from Topogen (Port Orange, FL). For Top2 relaxation assays, 540 ng Top2α-p170 fragment (16 units) was added to 250 ng supercoiled pHOT1 DNA in assay buffer (0.05 M Tris-HCl (pH 8), 0.15 M NaCl, 10 mM MgCl2, 0.5 mM dithiothreitol) plus 2 mM ATP with or without test compounds in a total volume of 20 μL. The DMSO concentration was standardized to 1% for all samples except the no-DMSO solvent controls. Reactions were incubated at 37°C for 30 min and then quenched with 2 μL 10% sodium dodecyl sulfate solution. Samples were then extracted with chloroform: isoamyl alcohol 24:1, mixed with 2 μL 10x glycerol loading buffer and loaded onto 1% agarose gels in tris-acetic acid-EDTA (TAE) buffer with or without 0.5 μg/mL ethidium bromide (EtBr). Gels run without EtBr were post-stained with SYBR-Gold (Invitrogen).
Nuclear uptake and cytotoxicity of Py-Im polyamide
To test the nuclear uptake potential of polyamide 1, a FITC-labeled derivative was prepared (1-FITC) and incubated with VCaP cells prior to imaging by confocal microscopy (S1 Fig). Polyamide 1-FITC signal was observed in the nucleus and also showed significant membrane binding. The overall level of uptake in VCaP cells was found to be qualitatively less than that in LNCaP cells [21]. Next, polyamide 1 was evaluated for antiproliferation effects in VCaP cells using the WST-1 assay under conditions similar to the gene expression experiment. After a 96 h incubation with polyamide, an IC 50 value of 6.5 ± 0.3 μM was determined for polyamide 1 (Fig 2A). At 72 h, the IC 50 value for polyamide 1 in VCaP cells was found to be over 30 μM (data not shown). For comparison, polyamide 1 has been found to have an IC 50 of 7 ± 3 μM after 72 h incubation in LNCaP cells [19].
Reduction of DNA damage in VCaP cells upon treatment with Py-Im polyamide
The effect of polyamide 1 on the high level of extant DNA damage in VCaP cells was also investigated. After incubation with polyamide, VCaP cells were submitted to the neutral Comet assay, which allows visualization of double-strand breaks through single cell electrophoresis (Fig 2B). The percentage of DNA in the "tail" of the comets was then compared using two-way ANOVA statistical analysis (S4 Fig). A significant reduction in DNA damage (p < 0.001) was observed with polyamide 1 over the vehicle control.
ARE-targeted Py-Im polyamide downregulates AR-driven TMPRSS2-ERG expression
Next the effect of polyamide 1 on AR signaling in ERG-positive cells was examined. Dosage concentrations were chosen based on previous reports of polyamide gene expression effects in LNCaP [28,34]. In VCaP cells, polyamide 1 was found to reduce the DHT-induced expression of the TMPRSS2-ERG fusion as well as other AR target genes, including PSA and FKBP5 ( Fig 2C). Corresponding decreased expression of ERG protein was confirmed by Western blot (Fig 2D). In the non-induced state, polyamide 1 was also found to reduce expression of several ERG influenced genes, including PLAT and MYC (S5 Fig).
Diminished growth in VCaP xenografts upon polyamide treatment
We next moved from cell culture studies to investigations of polyamide 1 in a VCaP mouse xenograft tumor model. Xenograft experiments were conducted in male SCID mice bearing subcutaneous VCaP cell xenografts. Treatments were started after tumor sizes in each group of mice reached~100 mm 3 and were administered three times per week through subcutaneous injection in DMSO vehicle for three weeks for a total of 10 injections. Dose-dependent retardation of tumor growth was observed in mice treated with polyamide 1 (Fig 3). After 5 weeks of monitoring, tumors treated with vehicle grew to approximately 6-fold the initial volume of that group while tumors treated with polyamide 1 at 5.0 mg/kg grew to approximately 1.6-fold the initial volume of that cohort.
Genome wide expression analysis
RNA-seq analysis was performed after 96 hours of polyamide treatment in order to assess gene expression changes after prolonged exposure and to identify potential mechanisms of polyamide induced toxicity. Differential expression analysis using DESeq2 showed that of the genes Tumor Repression of VCaP Xenografts by a Pyrrole-Imidazole Polyamide with padj < 0.05 and |log 2 (fold change)| ! 1, 342 were upregulated and 399 were downregulated upon polyamide treatment (Fig 4A). Connectivity map analysis of these genes returned several compounds known to be topoisomerase inhibitors (Fig 4B), suggesting that the polyamide may also be interfering with topoisomerase activity. Analysis of a previously published genome wide data set from LNCaP cells treated with polyamide 1 shows similar results (S6 Fig)[9].
Inhibition of topoisomerases 1 and 2
Topoisomerase inhibitors have been shown to attenuate AR signaling in multiple cell lines [35,36]. Similar results are also seen in VCaP cells, where treatment with etoposide and camptothecin is able to reduce DHT induced expression of select AR regulated genes (S7 Fig). Based on the Connectivity map results, we examined the inhibitory effects of polyamide 1 against topoisomerase 1 and 2 in vitro. Topoisomerase 1 (Topo1) functions by relieving DNA supercoils generated by transcription and replication and is a therapeutic target in cancer [37]. To determine if polyamide 1 inhibits Topo1 mediated DNA cleavage, we titrated polyamide 1 with supercoiled pHOT1 plasmid and measured conversion to open circular plasmid or relaxation upon addition of purified Topo1. A reduction in DNA relaxation indicates polyamide 1 was able to attenuate Topo1 mediated cleavage of DNA (Fig 5A). To differentiate between open circular and relaxed DNA, samples were also run on an EtBr gel. Unlike camptothecin (CMT), which traps the Topo1 cleavage complex and generates nicked open circular DNA, treatment with polyamide 1 did not prevent DNA re-ligation. Topoisomerase II cleaves double stranded DNA in an ATP dependent manner and is essential for strand separation of tangled daughter chromosomes during replication. Like Topo1, Topo2 is targeted in cancer therapy [38]. Similar to results seen for Topo1, polyamide 1 was able to inhibit Topo2 cleavage of supercoiled pHOT1 plasmid in a concentration dependent manner ( Fig 5B). Furthermore, samples were run with EtBr to allow unambiguous identification of linearized DNA, which allowed the identification of Topo2 cleavage complex (Topo2cc) formation (Fig 5B, lanes 5 and 6). The lack of Topo2cc formation in polyamide 1 treated samples as compared to linearized DNA and etoposide-treated samples is consistent with disruption of Topo2 binding.
Discussion
In this study, we evaluated the activity of an ARE targeted polyamide in VCaP human prostate cancer cells. Polyamide 1 has been previously shown to exhibit antitumor activity in cell culture and in xenografts of the androgen sensitive LNCaP cell line [19], but there are several important genotypic differences between these two cell lines. First, VCaP cells possess an amplified AR region, leading to higher levels of AR protein than LNCaP cells [30,39,40]. Additionally, VCaP cells belong to a subtype of prostate cancer that possesses the TMPRSS2:ERG fusion, resulting in the AR driven expression of ERG [23]. ERG, an oncogenic transcription factor, has been reported to increase double stranded DNA break formation in PrEC cells, while knockdown of ERG by siRNA in VCaP cells have been shown to decrease DNA breaks [41]. Studies have also shown that ERG overexpression increases cancer invasiveness and has been correlated to increased metastasis in the clinic [25,26].
In VCaP cell culture experiments, polyamide 1 exhibited antiproliferative activity and attenuated the DHT induced expression of select AR driven genes including TMPRSS2:ERG. Furthermore, in this cell line with high genomic instability due to ERG overexpression, treatment with polyamide 1 repressed the high level of DNA fragmentation found in the basal state, which may be attributed to diminished ERG protein. In vivo, VCaP xenografts treated with polyamide 1 exhibited reduced growth in a dosage dependent manner, demonstrating its potential as an anticancer therapeutic.
To further examine the mechanism of action for polyamide 1, we conducted gene expression analysis of VCaP cells after exposure to polyamide 1 in the same time frame as the cytotoxic experiment. Connectivity map analysis of gene expression signatures from treated VCaP cells indicated overlap with expression profiles of several topoisomerase inhibitors. In vitro assays for inhibition of both Topo1 and Topo2 confirmed that polyamide 1 is able to attenuate enzymatic activity of both enzymes. Similar results have been reported for other minor groove binders [42][43][44][45][46][47]. Furthermore, the lack of topoisomerase 2 cleavage complex formation in the inhibition assays suggests polyamide 1 functions by preventing protein-DNA interactions. This mechanism is in contrast to most drugs that target topoisomerases, which poison the enzymes. Drugs such as etoposide, doxorubicin, and camptothecin work by causing covalent adducts, which results in genotoxicity [48].
In addition to inhibition of Topo1 and Topo2, polyamide 1 has been reported to antagonize AR signaling, block RNA polymerase II elongation, and affect DNA replication by impeding Tumor Repression of VCaP Xenografts by a Pyrrole-Imidazole Polyamide helicase processivity [19,21,49]. These effects may be related, as inhibition of Topo1 has been shown to lead to RNA polymerase II and DNA polymerase stalling [50], and treatment of prostate cancer cells with topoisomerase inhibitors has been shown to attenuate AR signaling [35,36,51,52]. Taken together, these data suggest that by virtue of targeting DNA and DNA:protein interactions, polyamide 1 may exhibit antiproliferative effects on cancer cells through polypharmacological mechanisms without inducing genotoxic stress. Table of mRNA expression levels for AR-driven genes under DHT-induced conditions in response to 10 μM polyamide 1. VCaP cells were plated at 31k/cm 2 , treated with medium containing 0.1% DMSO (with or without polyamide) and charcoal-treated FBS (CT-FBS) for 72 h followed by induction with 1 nM dihydrotestosterone (DHT) or vehicle for an additional 24 h. Ã -mRNA expression levels below threshold. (B) mRNA expression data for polyamides at 1 and 10 μM concentration. VCaP cells plated at 31k/cm 2 were treated with medium containing 0.1% DMSO vehicle (with or without polyamide) for 72h. mRNA levels were measured by qPCR, referenced to GUSB, and the polyamide effects compared to vehicle treated samples. Data shown are average of the fold changes (treated/ untreated) for three or more biological replicates +/-standard error. | v3-fos-license |
2019-01-29T12:36:14.817Z | 2018-03-08T00:00:00.000 | 102345223 | {
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} | pes2o/s2orc | A Case Study on Security Recommendations for a Global Organization
In today’s world, computer networks form an essential part of any organization. They are used not only to communicate information amongst the various parties involved but also to process data and store critical information which is accessible to approved subscribers. Protecting critical data, ensuring confidentiality, and thwarting illegal access are primary concerns for such organizations. This case study presents security recommendations for any such organization, to assist them in defining security policies at various levels of the network infrastructure.
Introduction
In order to present the security considerations required in a corporate setup, we created a fictitious company by the name of MediSecure Corporation and tried to give it a real profile.The profile of MediSecure Corporation is as follows.
MediSecure Corporation (referred to as our company or the company hereon)
Portfolio of the Company
The various divisions vis-à-vis the computer networks and their functions in our company are described in Figure 1.The profile of each division, mentioned in Figure 1 is described below:
Research Center
Most pharmaceutical companies invest heavily in research, so they have advanced research facilities, which contain sensitive information that needs to be protected from data breach.Confidential data may include intellectual property pertaining to their on-going research, information about proprietary drugs, procedures regarding testing and trials and other procedures, all of which needs to be protected.
Manufacturing Sites
Our company specializes in mass production of drugs/healthcare products for millions of customers.The orders can be placed online via public website and also offline via phone or paper contracts.Shipments are delivered to customers, from all locations.As manufacturing is automated, the sites consist of many digitally controlled production devices, operated locally or remotely, as well as a large number of monitoring and controlling stations for ensuring quality.process should be such that the workstations get updated within a deadline of one day and employees are informed through pop-ups if reboot is required.Besides this, the growing trend is to support laptops as workstations due to their ease of mobility.Hence these devices must also be protected from getting stolen [6].Laptops may be a soft target for stealing confidential data.Hence data encryption should be enabled for all the data residing in the hard-disks of laptops.
When the laptop boots it should ask for a PIN which only the owner employee is aware of.This way even if the laptop is stolen, confidential data can be protected.
Code Server
The code server would be holding the data for the research (like chemical reaction simulations) being undertaken at various facilities.First level of security is to restrict access only to employees of research units who work on this data.
Second level of security is to back up the data in code servers (discussed in detail later) regularly.Third level of security is to station the servers separately in a secure and monitored network.There should be a network firewall and IDS monitoring the traffic to the servers.The fourth level of security is to physically station the servers in a restricted area which is well monitored by security cameras and only the administrators should have access to that area.
Mobile Devices
Mobile devices aid employee productivity by allowing them to connect to the internal network through Wi-Fi from any location in the company.They can keep track of their simulations etc. at their convenience from anywhere within the facility.Since mobile devices access multiple wireless networks inside and outside the company, some of which are unsecured, they are more prone to getting infected compared to other devices.More and more rootkits are surfacing which target mobile devices specifically [7].Hence there should be a well-defined security policy on what is allowed on the wireless networks.Highly critical data like code access should not be allowed through these devices.Also, the wireless network should be configured in such a way that it only allows the devices which have the VPN software (discussed later) installed on the phones.The phones should also have the company recommended anti-virus software installed on them.The VPN network server residing behind the access points should check the presence of these softwares on the devices before granting access to the network.If the mobile device doesn't meet the requirements it should be denied access to the network.To allow access a registration policy should be adopted.
This means that only devices registered with the network may be allowed to access it.This could be done by one-time registration of the IMEI number and then reading and matching the number from the database, every time a device tries to connect to the Wi-Fi.This wouldn't cause any inconvenience to the employees since they don't change the equipment very often and would probably be using the same phone most of the time.
Email & IM Server (Spam, Malware)
Emails and IMs bound to be one of the primary forms of communication as offices span over different geographical locations.Also, since emails are a documentary evidence of the communication, they might be preferred over other forms in certain contexts.Emails reside on the email server, and any security breach of the server can leak confidential information.A link clicked in malicious emails by an unnoticing employee, may result in a drive by download malware getting downloaded on their machines.This may then spread across machines in the network and breach the security to carry out attacks.An employee could receive malicious emails which may look genuine as an internal information broadcast asking them to change their password.By acting on it they may leak their username/password which may then be used to gain access to the internal network by the hackers.Hence it is important to have a good spam filter and an anti-virus on the email server which scans all attachments before downloading them on the client machine to save employees from getting phished.Suspected emails should be blocked and investigated before passing on to an employee, especially when they are from an outside network.Advanced file inspections as part of antivirus are necessary so that attachments are scanned from its contents.Recommendations on anti-virus and spam scanner are discussed later.
Network/IP Phones
Telephone network helps in managing communication across the various centers distributed globally.It helps in conducting conference calls for interacting with other teams.These conference calls may be used to communicate confidential information and thus it is necessary that they be secured like any other digital communication.Since our company uses IP phones for all its phone communication, it is important that the software on the phones be protected.Phones are forms of embedded devices with limited memory, processing power and software.They don't have any anti-virus or monitoring software installed in them.Hence, they can be soft targets.Upgrade of firmware on the phones is not a regular activity and may happen rarely.Hence, we recommend that the phones have a provision to hard lock the firmware upgrade, and the unlocking/upgrade be done only when an upgrade is absolutely essential.This will protect the phones from any malicious code getting installed on them.Also, the upgrades should be carried out through a secure network by the administrators, which should require additional login credentials to prevent employees from unlocking the phone and triggering upgrades (insider threat).
Printers
Our company's premises have a large network of printers with each facility hav- ing multiple printers stationed on every floor of the premises.The investment on the printers has been done because the employees would need to take print-outs of various documents to assist in their daily work.The documents may contain company confidential information and thus need to be secured.Besides this, most of the printers are multi-functional as they can carry out printing, copying and scanning of documents.Since the scanner needs to save the digital copy of the data, the printers have a small hard disk and support emailing.If not well protected they could be used to carry out attacks like sending spam through the email facility of printers, similar to the refrigerator attacks [8] which was carried out recently.Again, similar to phones, the software upgrades on the printers should be hard-locked.There should be a software installed on the printers to purge the data from the hard-disk once it has been retrieved by the user/employee so that there are no traces of it.Before decommissioning a printer, all the information must be deleted, hard-disk removed and cleaned.There should be well defined processes for all these activities so that any new administrator also knows the process to follow.
Lab Equipment
There may be various lab equipment used to carry out testing of drugs and in carrying out research.Some examples include equipment like centrifuges, chromatography testing equipment, analyzers and software which may be used to analyze the results from the equipment.To secure them, they should not be connected to the network.Many equipment may be digitally controlled but need not be remotely controlled.We recommend that for security reasons they should not be network operated/controlled or monitored.Individual workstations which are offline should be connected to them if needed.Also, the lab premises should be well secured with restricted access and monitored via cameras.
Internal network equipment can be categorized into the following:
Access Points
Wireless networks may be a part of the entire facility, so that the network is easily accessible from any location within the company.They can also be an easy medium to gain access to the internal network, especially if the wireless network is not well protected.To address this, it should be ensured that all access points should be password protected, and default password must be changed to something secure.They should have access control lists that allow only registered devices.
Routers
Routers form the backbone of wired network.There would be plenty of them deployed within the company to build a large internal network connecting all the departments at a particular facility.ments to adjacent ones, in an attempt to route traffic towards the malicious routers.To make routers secure, the first thing to keep in mind is that any default passwords must be changed to something strong.The routers are often advanced enough to support fine-grained control or even firewalling, which should be properly utilized to disable any form of traffic from an unlikely source.In order to address the OSPF and BGP vulnerabilities, it is a good idea to have a PKI system for routers so that every route advertisement can be signed, and the source of every route advertisement is known and authenticated.This scheme has limitations to the effect that it involves a complex certificate management system which can scale to a large number for a big firm.Also, it provides no security against falsely configured routers, and hence requires additional monitoring stations to verify that the routes are topologically correct.
Internal Websites
Companies usually don't care about internal website security compared to external ones.We suggest that same level of security should be applied to internal websites too since they can be hosting confidential data, and/or the servers may be used to gain access to other network resources.
Physical Security can be categorized into the following:
IDs
Access to the facilities is usually managed by RFID cards.Since the access is digital, it needs to be well protected.Unauthorized access would cause a security breach which may lead to damage to the company.Security access should be of different levels i.e.only required personnel have access to any of the restricted areas.In order to avoid spoofing the ID cards, the employees can each have a key-pair registered with the company, and the data on the ID card can be signed using their private key for authentication.This also ensures that once registered, the same card will work at any office within the company.
Biometrics
Biometrics could be used to further enhance the level of security provided by ID card access.This additional level of security would be useful in protecting the restricted areas within the company premises.But biometrics come with their own weaknesses, e.g.facial recognition can be fooled, or it may read incorrect data if face is altered naturally, or voice recognition can fail in case of an illness.
Similarly, fingerprints can be spoofed using gels, etc.The solution is to use biometrics under supervision so that spoofing using physical means can be avoided.
Also, biometrics should be used in conjunction with another identifier like names so that matching time is reduced, instead of using the digitized biometric itself for a lookup.
Security Cameras
Security cameras stationed at all the critical places can act as deterrent to people trying to sniff.Also, they can provide critical evidence in case there is a break-in and hence the camera feeds should be well secured.There should be an additional security staff managing the security at the facilities.Security cameras should be installed in tandem so that there are no blind spots.Access to camera feed should be restricted, and the videos should be backed up in addition to being saved.Access to those tapes can be restricted based on the biometrics for the security personnel.
Digitally Controlled Equipment
There would be mechanical equipment controlled digitally to manufacture and package medicines.An attack on such equipment could potentially turn the company bankrupt.This is because if for example the manufacturing equipment are compromised to print wrong label on the medicines, it could affect people's lives, the company could be sued and may have to close down.There can be other similar attacks like contaminating a particular medicine with chemicals being used to manufacture other medicines and so on.Hence very strict security policy measures are required to protect such equipment.These measures are discussed in detail while discussing impact of Stuxnet and how to prevent it from disrupting manufacturing sites and their digitally controlled equipment.
Workstations
Being an advanced facility, each manufacturing site may have multiple workstations for employees and personnel, as well as multiple monitoring / controlling stations to regulate operations.It is important for the company to ensure that these workstations are not compromised.All of the measures for workstations discussed in workstations at research centers apply here as well.However, there are some additional measures that need to be taken into account which are discussed while discussing impact of Stuxnet.
Public Facing Network
Since public website is accessible from anywhere, it's also a highly sought-after target for attackers.There is no need to be part of the internal network to attack the servers.The attacks on web-servers are both in form of traditional (generic) attacks as well as advanced ones targeted towards specific web-servers [9].Some of the known forms are explained below: • SQL injection attacks: Insertion of malicious SQL statements into an entry field for execution.This is mostly used against data driven applications, which may be relevant to our company as it has various databases to maintain for customer-data, credentials, company-data, etc.If the user-input is not strongly validated, unexpected SQL code may be embedded into it, and may get executed in this manner.• URL interpretation attacks: This attack is possible in situations where an attacker can adjust the parameters of a request.The syntax of the URL is maintained, but its semantic meaning is altered.For example, changing the email address parameter in the GET request of a password-reset page.• input validation and buffer overflow attacks: This is a very common type of attack against web servers, which is made possible when the scripts/programs handling the data entered by the user are not written securely, and don't perform sanitization or bounds checking, allowing execution of malicious code.
• cross site scripting: This particular vulnerability allows attackers to inject client-side scripts into web pages.It may also be used to bypass access controls.It typically uses known-vulnerabilities in web applications, their servers, or plugins.
• attacks on the medical professionals' portal: Communication on this portal requires login credentials, which make it a good target for attackers.Leakage of this data can enable attackers to listen in on confidential discussions or to masquerade as another user.
• attacks on the customer transaction site: Since this operation has a financial facet, it is also an attractive target for attackers.Needless to say, a successful compromise of this subsystem can lead to theft of financial details of users, which has also been seen in many recent attacks like the one on Sony PlayStation server which lead to breach of data as well as loss of credit card information putting customers at risk [10].
The attacks related to inputs and code injection can be handled by proper input validation and sanitization before passing on the input to the backend scripts or databases.Server application(s) should start as non-privileged user, so it can't compromise protected files.Scripts should be allowed only in certain directories which can be maintained easily, and suEXEC (Apache Web Server) or similar feature should be used to switch ID's before running scripts.Proper patches must be applied to the server software so that it remains bug-free.Access to server must be over TLS, with the key stored in encrypted form, readable only by the administrator.
For the professionals' portal, login must be done using credentials stored securely in a database.This particular portal can preferably be hosted on a separate server.Firewalls should be in place to ensure that there is no way to contact the server bypassing security filtering layers.Thus, the company should have a DMZ-minded layer of firewalls to ensure network isolation.
For the customer's financial transactions, the traffic should be over TLS and authenticated.It is best to keep the server hosted on a separate machine so that the compromise of any other service should not weaken the security of this part.
Help of a 3 rd -party vendor can also be taken for this.A good example is OAUTH.
Backup and Disaster-Recovery Management
In the unfortunate event of a complete or partial data loss, it's important to have a backup from where the last known good state can be re-instated.
Taking the Backup
Following parameters need to be taken care of while backing up date: • Content to be backed up.
• Source to get data from.
• Frequency of the backup operation.
• Location of storage (backup server).
• Duration for which the backup has to be kept.
Since backing up involves sending data over the network to a backup server, the operation is vulnerable to network attacks and vulnerability in backup mechanism may make it a soft target for attacks.As a counter-measure, the company can use certificate-based encryption for securing the backup data during transit.Validation of the backup server ensures that the data is being backed up to an authentic server, and not leaked to some other location.Validation of the client certificate ensures that the source of the data is genuine and from within the company and doesn't contain anything malicious.
Backup data is likely to have a large size, and the longer it stays in transit, the more vulnerable it gets.In order to minimize the transfer duration, it might be a good idea to compress the backup before sending.Less sensitive/important data may be backed up at lower frequencies to reduce transfers.To further minimize the transit operation, each location can have its own backup server, backing up independently.
It makes more sense to have backup servers in the same location where R & D takes place, instead of routing it over to the central headquarters (which may be done at a much lower frequency).Blind backup which involves backing up everything indiscriminately should be avoided.Priority should be given to more sensitive data and data which changes frequently like codebases and research data.Some data changes are in-frequent like databases for employees and customer details, etc.In order to maintain consistency, the data may be updated at the back-up node along-with the primary node whenever there is an update to the database.This would ensure consistency on the back-up node and also avoid the need to back-up such data.Incremental back-ups could be adopted as a general measure.However, backups should be taken frequently enough, either daily or semi-weekly, to be minimize data loss in case of an eventuality.Moreover, there should be policy which rotates the validity of the backup, before the expiry of which, a new backup must be taken to replace the old one.Backed up data has an advantage over live data.Since backups are stored more frequently than being retrieved, a back-up versioning system could be used, with older backups getting removed after a certain period of time.Even if reading from backups (which is rare and only in case of damage to live data) is extremely slow due to highest levels of encryption, it can be adopted as an acceptable trade-off.
Securing the Backup
When a backup server gets decommissioned, there must be a policy in place which mandates the erasure of all data on the server before disposing off the machine.
External Access via VPN
Access to VPN allows employees access to company's secure network through a VPN tunnel.It is used to connect to the research center and the manufacturing sites.Being run over an unsecure public network, it makes VPN communication an attractive target for attackers.Issues compromising VPN tunnel: • VPN fingerprinting: while not an attack in itself, it does give the attacker useful information by identifying the type, version, model, etc. of the device.• Insecure storage of credentials by clients: storing the user credentials in unencrypted form, or using weak forms, whether in the memory or the registry.
• Username Enumeration: usage of pre-shared keys enables this kind of attack.
• Offline Password Cracking: This type of attack is made possible when a valid username is obtained using the previous vulnerability, and the hash of the username can be obtained from the VPN server to launch an offline password cracking attack.
The issue of username enumeration is quite new and can be addressed from the vendor's side.Addressing this will take care of the password-cracking attack too.In order to address the remaining threats, it's imperative to use the strongest possible encryption methods, like EAP-TLS and IPsec.Usage of two-factor authentication products like RSA SecurID is also recommended.VPN access should be made available to personnel with a valid business reason.A strong password policy should be implemented and enforced, which is different from the one used in internal networks, so that compromised credentials only allow access to what is available through VPN.The limitation with this security is that the remote user may himself be compromised in the first place, which can then render some of these protections less effective.A safe approach would be to ensure that the remote users themselves have strong antivirus, antispam and personal firewall, and they must be required to use it.One method is to allow only company issued devices for VPN access.The devices can be validated through the serial keys of the hardware.Also, the system could be checked for meeting the security requirements every time a user logs in, before granting access to the network.
Additional Class of Attacks
There are two more classes of attacks which apply to almost all the divisions described above.We chose to address them separately, as they apply to multiple scenarios, and are quite important from that perspective.They can be listed as follows:
Insider Attacks
We mention insider attacks [11] separately, as they apply to almost all aspects of the company in terms of security.The following observations were notable: • It is important to identify the several most important entities that need to be protected within the company and secure them with the highest priority.This doesn't mean just encryption, but also restriction on access, and rigorous monitoring of who interacts with it, and under what circumstances.The circumstances can be used as a basis for a set of policies that clearly outline when the access is allowed.Physical security of assets is also one aspect which should not be ignored at any cost.
• The company should be extra careful in times of someone's resignation or employment termination, especially if they're from a high post.Suspicious behavior must be brought to notice.
• Centralized logging tools can be used to track all data exfiltration.Proper auditing should be enforced so that no operation on sensitive data goes unmonitored.
• Access to sensitive data should be allowed on a need-to-know basis only, and privileges must be rescinded once the project/operation is done.An employee must be asked to submit a valid, signed (digitally or otherwise) reason for accessing sensitive data.
• It should be possible to remotely erase a disk on a smartphone or laptop in case of theft.
• There should be a proper training course based on general security guidelines and best practices which trains users about the importance of strong password policies, proper emailing protocol for different situations and browsing recommendations, sharing of information with outsiders or colleagues, etc.
Peripherals and Removable Devices
Devices like USB sticks, flash drives, external hard disks, smart cards, and mobile phones are examples of set of entities which are very effective as tools for at-tackers.In many cases, they turn out to be either the starting or ending point of a successful attacks, involving mostly theft of data by carrying it off on one of these.Since they can be used with a variety of devices, in different environments and without need of prior installation of any software, they prove to be an effective out-of-band channel for attacks.They are also harder to track because of their removable nature and the human factor involved.We chose to mention them separately, as they apply to all of the divisions of the company as presented above.
We recommend the following guidelines for dealing with them: • In general, it's best to avoid the use of removable devices as much as possible.
• If the use of removable devices cannot be avoided, it's best to have them registered so that they can be tracked.Un-registered or ad-hoc devices must be rejected.
• Some removable devices like USB sticks can have native password protection.
It's recommended to use those, to protect data against theft.
• Workstations, and any other devices which have USB ports or CD trays, must have the auto play option permanently disabled.
• The antivirus software on the host device must be configured to scan the removable device as soon as it's attached.
• The HIDS must monitor ALL data travelling to/from a removable device, even if it's registered.
• It may be possible to encrypt data if it's copied out to one of these removable devices.
• For mobile phones, registration should be recommended, and the user must be required to have sufficient protective measures like antivirus, GPS locators, etc. on the phone.We also discussed mobile phone registration earlier while discussing threats on the research center devices.
The limitation of the above strategies is that they might turn out to be too cumbersome to implement and carry out in a large company.The tradeoff of security vs usability states that if the users tend to face a lot of delays or difficulties in working with these restrictions, they may tend to bypass them.Hence, employees should be trained against the harmful effects of such negligence.
Measurement of Security Posture
Measurement of security posture refers to the steps the company can take to measure how secure they are at any given point in time.It can be interpreted in a systematic way if we look at different things the company can look at, in order to determine the state of their security.
These "things" can include the security alarms generated by their: • host-monitoring systems
• physical security mechanisms
The best way to look at these alarms is to use some kind of an IDS visualiza-tion tool.Since the company has a distributed architecture containing many locations, each having many machines, all connected through LAN's and WAN's, a good example for visualization can be IDSradar [12] tool.The tool has provisions to visually depict each node from each location and their network connections within a single representation, which gives a holistic idea of what is going on with the company in terms of security threats.
The following properties of IDSradar can prove very helpful in obtaining this representation: • servers and workstations-nodes are shown as arranged in circles indicating a common corporate network.The bigger nodes are those with high priority.
Locations are ordered by IP address.This can be used to take a look at any of the nodes in the whole network, and to see any security-relevant statistics.
• alert types-each alert type is shown in a separate color, and the width of each arc is corresponding to its percentage.This gives a kind of visually proportionate size to the importance of an attack.• interactive design-interactive filtering is provided in the form of clicks on hosts, servers, alerts types, etc. as well the ability to zoom-in/out, play, stop, etc. Detailed information about any entity can be seen by mouse-hover.This provides information in various ways, and being visual, it's easy to be interacted with by personnel who may not know about the setup or commands or similar technicalities of a particular system.
Apart from visualization, the company can consolidate the security alarms and reports from different locations and try to use behavior-based learning methods to glean patterns or statistics out of the reports.The statistics can be collected based on location, time, type of attack, or any relevant metric which can help in detecting any kind of trend in the attacks.If so, the company can take steps to address that particular concern proactively.This methodology of pro-active threat assessment will ensure that the companies resources are secure, and breaches can be avoided before they take place.elements on which products from different vendors could be compared.This can be taken as a baseline.There is multiple criterion which must be considered before selecting a security product to be installed in the company.Each criterion should be matched by the network requirements of the company and whether any of those criteria would act as bottleneck in case the network resources are expanded.Also, the projected network growth and pricing of security products must be considered before purchasing/deploying a product to ensure least security cost to company.Other parameters include:
Selecting a Security Product
• Network speed supported by the device when the security features are enabled.• Number of concurrent sessions supported.
• Inter-operability with other vendor security appliances already installed in the network or the ones which would be installed.
• Inter-operability with other TYPES of appliances.For example, inter-operability of vendor X network IDS with vendor Y Host IDS.
• Support of required features.
A list of security features (not exhaustive but informative) provided by various security devices in the market: (They can be either at the host level or the network level or both) In order to arrive at this list, we referred to Juniper Data Sheet [14], Cisco Data Sheet [15], McAfee Data Sheet [16], Palo Alto Networks Data Sheet [17] and some other articles [13] [18] [19] [20] [21] on product comparisons.[22] is an informative resource on Intrusion Detection Technologies and [23] can be referred to learn more about anomaly-based Intrusion Detection Systems.
As attackers get more and more sophisticated, even anti-viruses (specialized to thwart viruses/malware/spyware) are becoming ineffective in dealing with the latest threats [24].Hence, a package of products functioning and collaborating at different levels must be used.
There are some organizations like NSS Labs which evaluate IDS and other security products from different vendors and publish their report annually.Such third-party reports may be more reliable in reporting actual figures than the company data sheets which usually report the best-case figures to beat the competition.These reports are available for a subscription [25].We recommend relying on at-least one such report before finalizing on the product.
Similar Threats Faced by Others
We looked at some threats faced by some other companies/organizations.There were quite a few of them related to healthcare industries, which we enumerate first, followed by some other relevant examples: • Utah Department of Health, March 2012-a breach caused by weak password policy (default password wasn't changed) on a network server exposed protected information of 780,000 individuals, which included their Social Security numbers.This is a classic case of negligence/misconfiguration brought about due to ignorance of user training.Enforcement of proper regulations (like password format restrictions in the company) would have avoided this easily.
• Emory Healthcare, Georgia, April 2012-lost 10 backup disks containing information more than 300,000 patient records, two-thirds of which contained Social Security numbers.This strengthens our claim made earlier in the document stressing the security of the backups.In fact, backups need more security as they may contain data This is a good example of insider threats, resulting when a firm makes the mistake of trusting any or all entities within its physical/network boundaries, and fails to monitor their activities.To avoid/detect these kind of attacks, we think it's imperative to track all kinds of operations performed on sensitive documents or machines.This may impose a heavy burden on the monitoring tool as the number of such entities (which are potentially sensitive and should be monitored) can be very large in a big company.The company can then try to compartmentalize documents using security levels and monitor the ones at the highest levels only.
• Stuxnet, Iran-Stuxnet was propagated to a high security facility with only PLCs through a USB drive inserted in devices/workstations outside the high security facility.Stuxnet works in the following way: After infecting a less secure workstation (through a USB drive), it propagates to other networked computers and then scans for specific software manufactured by Siemens for controlling a PLC.If the computer is found with the software AND it is controlling the PLC, it introduces the rootkit by infecting both the software and the PL.Otherwise it becomes dormant on the system.Once both the software and PLC are infected, it can send unexpected commands to the PLC while the software still reports normal operation.The design of networks which were infected by Stuxnet are similar in nature to the manufacturing facility of our company.Hence it is a good idea to learn from the way Stuxnet propagates and operates, while designing the security systems at our company.
First of all, it is important to highlight that traditional security methods including anti-virus and IDP would not have helped prevent Stuxnet since it exploited 0-day vulnerabilities and there were no signatures available for this attack before it was discovered.
The above examples cover a good variety of threats like weak protection policies, insufficient protection of backups, insider attacks which are all witnesses to the fact that healthcare industry requires proper security no less than any other industry.It also covers highly targeted attacks using malware like Stuxnet, which are relevant for healthcare firms which are involved in mass production of drugs using specialized devices and sophisticated machinery.
Impact of Snowden's Disclosures about NSA
The Snowden's disclosures on NSA [26] reveal a nexus of international agencies having an elaborate network of global surveillance.Many intelligence agencies like NSA are spying on the citizens of their country by spying on the digital footprints of people through emails/phone conversations and so on.The agencies are able to spy on individuals with relatively simple methods using various kinds of tools.This is alarming because this highlights that other agencies could use similar tools to spy on their targets.For example, a particular pharmaceutical company could develop means of spying on our company to carry out an espionage and steal critical information from the company's network.With Advanced Persistent Threats, it is possible to carry out such an espionage for a long period of time before it even gets detected.So, such threats are not a one-time affair and can last over long periods of time from months to years.Snowden's disclosures also highlight some of the tools which can be used to carry out targeted attacks.Some of the tools are listed below:
Computer Implants
1) Sparrow II: A small device that could be implanted at a strategic location to spy on the wireless networks and collect data.
2) Firewalk: It is capable of filtering and regressing (outputting) network traffic over a custom RF link and can inject traffic into the network as commanded.
3) SWAP: Can be used to exploit motherboard BIOS before the operating system loads 4) CottonMouth I, II, III: USB Implant that provides a wireless bridge into a target network and also the capability to load exploit software on target PCs in the target network.
Likewise, there are about 18 hardware/software tools listed as part of the disclosure which can be used as computer implants to spy on the network/data.It is possible that other organizations may be able to develop such tools.Hence it is important to be aware of their presence and take action to prevent their use in spying on the network.Since all the above equipment is a computer implant, it would need to be physically implanted at a strategic location within the company premises.It cannot be done remotely and thus needs an insider to plant the devices.Refer the section on insider threats on strategies to mitigate them.
Many such software implants can be used to install backdoors in servers and firewalls, which can be used to leak data from the networks.This highlights the importance of protecting servers and firewalls from such tools.There should be a policy to protect the firewalls themselves, especially when updating their operating system, software, changing the firewall rules, or in general carrying out any management tasks on the devices.
Covert Listening Devices
Another set of devices listed in the disclosures are the covert listening devices like LoudAuto, NightWatch, CTX4000, PhotoAnglo, Tawdryyard.These devices can be used to listen to wireless data.There may be many similar devices which could be used to pry on the wireless networks within our company.Hence it is important to use wireless routers whose range is limited to the company premises and don't have very high ranges beyond the premises which cannot be monitored.It is better to use multiple wireless routers with small ranges rather than using high range routers which may have sufficient range making it possible to listen to their traffic beyond the company premises.
Mobile Phone Implants
Mobile Phone implants like Picasso, Genesis, Crossbeam, Candygram, Dro-poutJeep, GopherSet highlight that mobile phones can be compromised either during manufacturing phase or during their operation.It is also possible that certain employees might bring in modified hardware devices to help in spying on the networks.Otherwise, a mobile phone user could be a target of an attack which may lead to software like DropoutJeep getting installed in their phone.This will compromise the privacy of the user and may also leak company information exchanged through conference calls, emails, SMS messages through the phone.It is worth noting that such devices are used at multiple networks (public hot-spot) and thus more susceptible to attacks unless the user is careful in using them.Hence it is important for the company to highlight best-practices to employees regularly, so that they are careful in using their devices, and opening links in emails etc.Also, it is important to restrict the data access which is available through these wireless devices on the company's wireless network.
Measures to Avoid Sophisticated Attacks like Stuxnet
Our recommendations are as follows: • Isolate the network of the manufacturing units from the internal LAN of the company.This would not have prevented Stuxnet from spreading (it used USB to inject itself initially).However, it is still an important practice to follow • The computer systems connected to the manufacturing units and machines should have very limited connectivity to the outside world.Security measures should be built in to provide additional credentials for connecting USB drives/CD-ROM etc. to the systems.This will prevent anyone who has operational access to the systems from trying to modify it.
• Modifying the firmware on the manufacturing units should not be easy.It should have multiple levels of security.Even after downloading the firmware on the computer systems from (say) USB drive, there should be additional privileges required to upgrade the firmware.This should be through a specific set of computers placed in a high security zone which are NOT used for the operational activities of the units.• There should be mandatory integrity checks carried out on the new version of firmware before it is passed for installation.If the integrity checks fail, the systems used for upgrading should lock out.The integrity checks should be carried out on a separate system/network than the one used for upgrading.Some experts recommend using cloud services to carry out the integrity checks which can be considered too.
• To ensure tightest security, firmware upgrades for mechanical units should be locked in hardware through jumpers.Only when upgrades are mandated, they should be unlocked and upgraded.Although this is difficult to implement on large scale manufacturing facilities with 1000 s of units for manufacturing, it must be noted that firmware upgrades are not carried out frequently and the equipment run for many months or years without the need for any change in operational logic.The risks of damage that can be caused are way higher than the lack of convenience in upgrading the firmware.
Hence, we strongly recommend this method.
• Any change in software of the systems (besides firmware) should use the same level of security.Consider the extent of damage that can be caused by printing wrong labels on the medicines and sending them out in the hospitals/ stores.
Proactive Threat Protection
Besides a reactive strategy for security threats i.e. taking action once an attack occurs or is attempted, as most organizational measures deploy, we recommend pro-active threat prevention strategies along-with the strategies discussed till now.In this section we see what kind of innovative measures can be used to not only prevent attacks but also to catch the attackers and identify their intentions by monitoring their activities on the company network.Although at first glance, the recommendations might appear to be costly to implement, the cost is certainly less than the risks involved in security breaches.Our company, as a pharmaceutical company has the functions of manufacturing and delivering medicines promptly which help in saving lives.Any delays in these procedures or errors in these procedures could affect human lives and lawsuits could potentially lead to closure of the company.Considering this aspect, our recommendations in this section must be seriously considered by our company.
Our first recommendation is to deploy multiple HoneyPots at significant locations at all the facilities of our company.A honeypot is a system that's put on a network, so it can be probed and attacked.Because the honeypot has no production value, there is no "legitimate" use for it.This means that any interaction with the honeypot, such as a probe or a scan, is by definition suspicious [27].
Computer systems which may appear to contain confidential data like research information, payroll information, financial bills etc. which are fake could be placed at strategic locations within the company.They could be setup to monitor any network activity on them.A legitimate employee would not access these systems as they don't need to.However, an insider trying to steal data may find these systems online while scanning the network and could get caught easily by the monitoring of the system.This would counter insider threats.A masquerader or an outsider accessing the network through an insider's credential or in general posing as an insider may try to access the resources on honeypots too and get caught by their activities on them.Honeypots may be used to fingerprint the activity of attackers and identify what they are looking for, why they are attacking the network, and also perhaps who they are.Recognizing the identity of attackers would help our company in catching them and thus help in reducing the threats they pose to the company.
Honeypots can help prevent attacks in several ways.The first is against automated attacks, such as worms or auto-rooters.These attacks are based on tools that randomly scan entire networks looking for vulnerable systems.If vulnerable systems are found, these automated tools will then attack and take over the system (with worms self-replicating, copying themselves to the victim).One way that honeypots can help defend against such attacks is slowing their scanning down, potentially even stopping them.Called sticky honeypots, these solutions monitor unused IP space.When probed by such scanning activity, these honeypots interact with and slow the attacker down.They do this using a variety of TCP tricks, such as a Windows size of zero, putting the attacker into a holding pattern.This is excellent for slowing down or preventing the spread of a worm that has penetrated your internal organization [28].
Our recommendation is to deploy multiple HoneyPots throughout all the facilities of our company and at strategic locations in the network.This network of HoneyPots is usually termed as HoneyNet.A honeynet is a type of honeypot.Specifically, it is a high-interaction honeypot designed to capture extensive information on threats.High-interaction means a honeynet provides real systems, applications, and services for attackers to interact with, as opposed to low-interaction honeypots which provide emulated services and operating systems.It is through this extensive interaction we gain information on threats, both external and internal to an organization.What makes a honeynet different from most honeypots is that it is a network of real computers for attackers to interact with.These victim systems (honeypots within the honeynet) can be any type of system, service, or information the company wants to provide [29].
The second recommendation is to deploy decoy documents.These decoy documents are usually also referred to as HoneyFiles.These are documents that look enticing i.e. appear to contain important information but actually contain bogus information.They won't be accessed by regular employees either because of their permissions and/or because they are not useful for them.However, an insider or a masquerader trying to steal information would try to look at them.These documents can be embedded with a beacon code through the Decoy Document Distributor system so that whenever they are accessed they trigger (say) an email alert, alerting the administrators of a suspected malicious activity [30] [31].This way they can be caught.
Since the deployment of HoneyFiles, HoneyPots and HoneyNets may require some level of advanced security knowledge, our company may consider hiring security experts full-time to manage their security or they could consult them for recommendations on a regular basis.
Counter Measures for Future Threats
We looked at several aspects of security which are based on the current state of technology, both software & hardware, and which may change in the near future.These are certain areas which may need to be addressed separately in the future.Here's our view of it and a few examples: • Legacy systems, hardware and code: There are many systems which use legacy code or software, e.g.Windows XP, or software written in languages like C/C++, which may be outdated in the future, or lose its support.Care should be taken to keep them to their latest patched versions, or to replace them with a secure version.This process may be difficult because in order to keep the network homogenous, the same changes may have to be done on a large number of machines, which may cause delay or downtimes, or may not be feasible immediately in cases like email server.
• IPv6: As the IPv4 address space is slowly expiring [32], the networking world is switching over to IPv6.Many contemporary security features may not work with IPv6 without some form of modification.The company should look into making itself IPv6 compliant in terms of security software as soon as possible.With the advent of IPv6, there is a possibility of using stronger security features like encryption, key-passing and signatures.
• Cryptographic capabilities: A lot of protection depends on the security capabilities provided by the present state of security algorithms like RSA.Day by day, attackers are coming up with newer attacks and also newer hardware is coming closer to crossing the computational barrier on which many secure algorithms rely.In the future, the security experts should look at methods which are based on multiple factors rather than computational difficulty.Also, older, insecure protocols should be replaced by newer, more secure ones as they arrive.The difficulty obviously lies in the scale of the transition and the fact that all machines may not be able to support all kinds of newer cryptographic operations and may require replacement as well.
• Bypassing of learning methods by "wrong training" of IDS systems: This is another trick used by attackers and takes place over a really long time, so it is not immediately noticed by monitoring systems.This is in the form of a particular attack which is consistent, and over a long period of time it makes the behavior engine in the IDS system raise its score much higher in comparison to other attacks.Later, the attackers launch an attack which is not much different in nature and is classified by the IDS as low severity.The company's IDS should have features that watch out against this type of "wrong" learn- Most of the latest laptops and mobile phones have a front-camera.The company could mandate using ONLY devices with a front camera.The VPN software could ask for permissions to the front camera, and if permissions were not granted, the software could be disabled.The VPN software while logging into the official network could take a picture of the user trying to log-in and use it as an additional validation.Further, the camera could be accessed randomly during the user's session, ESPECIALLY when dubious activity is detected.This would help catch insiders attacking the system, remotely, which would otherwise get evaded by traditional systems.A similar approach may be adopted for fingerprint scanners.With the (remote) use of biometric hardware on the user's device, the security of access from remote locations would be similar to access on-site.
Conclusions
As part of this case study, we have tried to cover all aspects of computer networks that would typically be found in a large scale global organization with offices spread in different geographies.We defined a fictitious company and chose a network profile for the organization.The network profile was carefully chosen so that it is relevant, generic and nearest to the actual profile of most global organizations.However, any real organization may have some deviations from profile we have chosen.Hence, we advice that while taking the suggested recommendations, they should also be tweaked based on the actual profile of the organization being considered.We have tried to address each aspect of the network independently, with suggestions to ensure security of that part of the network.We have studied and discussed some of the recent threats that have been in the news relevant to the profile of our fictitious organization, and how those threats can be dealt with.We also tried to suggest parameters that should be used while selecting a security product from the available vendors, which would best meet the needs of the organization.By listing the different features that most security products offer and the types of appliances that are available, working at different levels of the network (ranging from host devices to network devices), we have tried to help any organization make an informed decision, in order to ensure the security of their systems, in the best possible manner.
It must be noted that just following best practices and ensuring compliance with the security certifications are not sufficient for an organization.The personnel involved in defining the security measures must understand the organization's computer systems and how to best provide security in the current threat scenario.Hence, as part of this document, we have not looked at solutions from compliance perspective.Instead our focus has been based on the design of the networks.
. Tandon, P. Parimal DOI: 10.4236/jcc.2018.63010129 Journal of Computer and Communications satellite locations.Each location has an internal LAN, and the LANs from different satellite locations are connected by a private WAN.
Figure 1 .
Figure 1.Network model of the company.
D
. Tandon, P. Parimal DOI: 10.4236/jcc.2018.63010136 Journal of Computer and Communications From the D. Tandon, P. Parimal DOI: 10.4236/jcc.2018.63010137 Journal of Computer and Communications point of view of security, it mainly involves looking at the following two processes: It is just as important to secure the backup, as securing the live servers.Stealing the backup from central back-up servers gives attackers more advantage as then there's no need for stealing the same data individually from their respective sources (which is more difficult).Security of the backup is essentially a case of D. Tandon, P. Parimal DOI: 10.4236/jcc.2018.63010138 Journal of Computer and Communications host-based security and involves mostly the same parameters.Apart from beingcompressed, the backup data must be encrypted even in stored form.The individual subparts of the backup may be encrypted using separate keys (for each source) so that the compromise of a particular key doesn't put the security of the whole backup at risk.The backup server must have a good host-based IDS which can monitor any operation being performed on the backup files within the server.Physical security of backup server should be ensured too and any physical access to the servers must be allowed only when absolutely necessary and restricted using biometrics.
• timeline & histogram-time is depicted using animation, and the histograms below each alert type are drawn clockwise along the alert-type arc in real time.They're updated every few minutes.This can help the company keep track of attacks/alerts over time, and can be used to figure out a pattern, if any exists.• attack correlation-triangles are used to connect source IP, destination IP and the top of the bar of histogram below the alert type, in the current time span.The nodes in an alert are highlighted.This can be especially helpful in case the hosts/networks in different physical locations are being targeted systematically.The correlation can help filter out random attacks from deliberately targeted ones.
ing.Periodic evaluation/audit of threat scores of various types can help the D. Tandon, P. Parimal DOI: 10.4236/jcc.2018.63010150 Journal of Computer and Communications company in finding these types of anomalies.• Biometrics: The state of biometrics has evolved in the past few years leading to technologies like facial recognition, retina scans and fingerprint readers starting to get used in devices like smartphones for authentication.They are accurate in reading the biometric input, however, the technology being fairlynew is prone to spoofing.As biometrics starts getting used at a wide scale, attackers would try to come up with new ways to spoof the input.The technology may work well for a single user-based phone, but altogether replacing password authentication and certificate signing on large networks may take a while.Nevertheless, the company should be aware of this technology and prepared to adopt it in its systems where evaluations prove it to be better than conventional methods.
Table 1
gives an example of a set of generic security features and a list of evaluation | v3-fos-license |
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} | pes2o/s2orc | Validation and Characterization of Persistent Organic Pollutant Emissions from Stack Flue Gases of an Electric Arc Furnace by Using a Long-Term Sampling System ( AMESA ® )
A long-term sampling system, called the Adsorption Method for Sampling of Dioxins and Furans (AMESA), was used for long-term sampling (up to 168 hours) of an electric arc furnace (EAF) to obtain the representative flue gas samples. In order to have a comprehensive view of the emissions of persistent organic pollutants (POPs) from EAFs, six POPs, including polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), polychlorinated biphenyls (PCBs), polychlorinated diphenyl ethers (PCDEs), polybrominated dibenzo-p-dioxins and dibenzofurans (PBDD/Fs), polybrominated biphenyls (PBBs) and polybrominated diphenyl ethers (PBDEs), were investigated. Tests showed the breakthroughs of PBDD/Fs and PBDEs are much larger (8.9%–43%), while the others are less than 3%. A significant increase in breakthrough with the increase in halogen numbers is observed, because highly halogen-substituted POPs tend to partition to the particulate-phase. Except for PCBs and PCDEs, whose percentages of the POPs in the rinses to the total POPs collected from the long-term samples (cartridges + rinses) are less than 7%, those of the other POPs are all greater than 30%. Therefore, the solvents from the rinses of the sampling probe and other components need to be combined with an XAD-2 cartridge for analyses. The PCBs and PBDEs are the most abundant pollutants in the stack flue gases of the EAF, and their mass concentrations are one to three orders higher than those of the other POPs. With regard to POPs with dioxin-like toxicity, the percentages of the contributed toxicities by PCDD/Fs, PCBs and PBBD/Fs are 87.1%, 11.7% and 1.2%. The close association that PBDEs and PBDD/Fs have with the concentrations and congener profiles is not present for PCDEs and PCDD/Fs, suggesting the need for further studies of the formation mechanisms of these analogues.
INTRODUCTION
Electric arc furnaces (EAFs) are used to produce carbon and steel alloys, primarily from melting iron and steel scraps, and play an important role in iron/steel making.A typical EAF includes the stages of feeding, smelting, oxidation, reduction and steel discharge.The melting of scrap ferrous material contaminated with varying amounts of chlorinated compounds (i.e., PVC plastics, cutting oils, coatings and paints) provides conditions favorable to the formation of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) (Wang et al., 2003b;Lee et al., 2005).EAFs have been reported as one of the major PCDD/F emission sources (European Commission, 2000;Lee et al., 2004).Together with sinter plants (Wang et al., 2003c), they contribute 99% of the aggregate PCDD/F health risk to residents in densely populated areas of a city in southern Taiwan (Kao et al., 2007).
In addition to PCDD/Fs, EAFs have also been recognized as an important emitter of polybrominated diphenyl ethers (PBDEs) (Odabasi et al., 2009;Wang et al., 2010bWang et al., , 2011a, c) , c) and polybrominated dibenzo-p-dioxins and dibenzofurans (PBDD/Fs) (Wang et al., 2008(Wang et al., , 2010c)).PBDEs and PBDD/Fs are emitted from EAFs when they are not completely destroyed in the feeding scraps, and are also formed during the combustion process.This suggests that it is necessary to further characterize the other analogues that are contained in the emissions of EAFs, such as polychlorinated biphenyls (PCBs), polybrominated biphenyls (PBBs) and polychlorinated diphenyl ethers (PCDEs).Except for PCDEs, the various pollutants noted above, such as PCDD/Fs, PBDD/Fs, PCBs, PBBs and PBDEs, all appear on the list of persistent organic pollutants (POPs) in the Stockholm Convention.These POPs are bio-accumulative, toxic, and susceptible to long-range transport (LRT) (Wania, 2003;Wang et al., 2011b).For example, PBDEs can have various harmful effects on humans, such as developmental neurotoxicity, hepatotoxicity, embryotoxicity, and decreased reproductive success (Chao et al., 2010(Chao et al., , 2011;;Shy et al., 2012).
PCDEs are a group of halogenated aromatic compounds, which are structurally similar to PCBs and PCDFs.The difference in the chemical structures of PCDEs and PBDEs, which have been extensively used as brominated flame retardants (BFRs) in a large variety of consumer products, is that bromine substitutions are all replaced by chlorine.Similar to their analogues, PCDEs with lipophilic and persistent properties tend to bioaccumulate and biomagnify in food webs (Domingo, 2006).Some PCDEs may have biochemical and toxic effects similar to those of PCBs and PCDD/Fs (Becker et al., 1991).Furthermore, PCDEs may be converted to or form toxic PCDD/Fs by photolysis or pyrolysis (Norström et al., 1976;Lindahl et al., 1980;Liu et al., 2010), and have been observed in the flue gas and fly ash of waste incinerators (Kurz and Ballschmiter, 1995;Nakao et al., 2006).Nevertheless, in terms of being a product of incomplete combustion, PCDEs have attracted little attention compared to related combustion-originated POPs, such as PCDD/Fs (Wang et al., 2007;Lin et al., 2008;Li et al., 2011), PCBs (Chang et al., 2014), PBDD/Fs and PBDEs (Wang et al., 2010a, c).
The regulated methods for sampling PCDD/Fs in stack flue gases, such as US EPA modified Method 23 and EN 1948-1,2,3, are used for manual short-term sampling, with the time for one stack flue gas sample usually ranging from three to six hours.Therefore, the PCDD/F emissions that occur during the sampling time are of little relevance to the overall emission level, especially for EAFs that have significant variations in the properties of the raw feeding scraps (Lee et al., 2005).Another issue is that some POPs, such as PBDD/Fs and PBBs, have very low concentrations in the stack flue gases of combustion sources, and several stack flue gas samples are thus usually combined into one for the PBDD/F measurement in order to meet their detection limits (Wang and Chang-Chien, 2007;Wang et al., 2010c).
To solve the representative issue resulting from spot PCDD/F measurements using manual short-term sampling, which occur for only a few hours each year, three longterm sampling systems and two semi-real-time continuous monitoring systems have been developed (Mayer et al., 2000;Lee et al., 2008;Vicaretti et al., 2012).One of these is the Adsorption Method for Sampling of Dioxins and Furans (AMESA), which is a fully automatic long-term sampling system for industrial processes based on isokinetic flue gas sampling and the PCDD/F adsorption that occurs on an exchangeable resin-filled cartridge (Lee et al., 2008).This system has been tested, undergoing certification procedures (Mayer et al., 2000;Idczak et al., 2003;Lee et al., 2008;Vicaretti et al., 2012), and is obligatory for incinerator plants in Belgium, France and the Lombardy region of Italy (Rivera-Austrui et al., 2012).The European Committee for Standardization (CEN) is currently developing a standard for long-term sampling of PCDD/Fs and dioxin-like PCBs (pr EN 1948-5).While AMESA has been widely installed in incinerators, especially in Europe, and some related studies with regard to PCDD/Fs and PCBs have been reported (Mayer et al., 2000;Idczak et al., 2003;Lee et al., 2008;Vicaretti et al., 2012), few works have discussed its application with regard to EAFs or for sampling other POPs, such as PCDEs, PBDD/Fs, PBBs and PBDEs.
In this study, AMESA was used for long-term sampling (up to 168 hours) of an EAF to obtain the representative POP flue gas samples.In order to have a comprehensive view of the POP emissions from EAFs, six POPs, including three chlorinated ones (PCDD/Fs, PCBs and PCDEs) and three brominated analogues (PBDD/Fs, PBBs and PBDEs) were investigated.Breakthrough tests of XAD-2 cartridges and rinses of the sampling probe were conducted to evaluate the feasibility of the long-term POP sampling of EAF by AMESA.Furthermore, the concentrations, emission factors and congener profiles of the six POPs were compared with each other to clarify which one is the most influential atmospheric pollutant, as well as the similarities or differences among their formation mechanisms.
Basic Details of the Investigated EAF and AMESA
The EAF investigated in this study is operated intermittently, with scrap (104 tonnes/hr), alloying agents (1.1 tonnes/hr), flux (2.1 tonnes/hr) and coke (1.8 tonnes/hr) used as its raw feeding materials, and bag filters as its air pollution control devices (APCDs).It can produce carbon steel as a rate of 100 tonnes/hr.
The operation of AMESA complies with the cooled probe method of EN-1948.The flue gas is isokinetically sampled by using a titanium probe to cool the temperature of flue gas down to less than 50°C, before it is introduced into the XAD-2 cartridge.Fifty grams of XAD-2 resin were filled into the cartridge, which is greater than that used in the manual short-term sampling method.Instead of using filters to collect particles, quartz wool is placed in the front of XAD-2 cartridge.AMESA can thus collect flue gases for up to four weeks at a time.In this study, an additional XAD-2 cartridge is mounted in series to check the breakthrough of the POPs.
Sampling Procedures
A total of six stack flue gas samples were collected from the EAF by using AMESA, and were performed by an accredited laboratory in Taiwan.Prior to sampling, XAD-2 resin was spiked with isotopically labeled PCDD/F surrogate standards.The AMESA system was manually started and stopped to be consistent with the batch time of processes.Each stack gas sample accumulated ~168 hour sampling time (about a week and half of sampling).The sampled flue gas volumes were normalized to the dry conditions of 760 mmHg and 273 K, and denoted as Nm 3 .
The sampling probe and other components of the sample train were rinsed after each sampling.The nozzle, probe and probe lines were brushed while rinsing three times with acetone, three times with methylene chloride and then three times with toluene.The rinsate were collected and analyzed for POPs.To ensure that the collected samples were free of contamination, one field blank were also taken during field sampling.
Analytical Procedures
In contrast to other POPs, there is little data associated with PCDEs emitted from combustion sources and in the environment, because native and mass-labeled PCDEs have not been commercially available until recently (Domingo, 2006).In this study, each sample was analyzed for seventeen 2,3,7,8-substituted PCDD/F, twelve dioxin-like PCB, six PCDE, twelve 2,3,7,8-substituted PBDD/F, five PBBs and fourteen PBDE congeners.Internal standards were spiked into the samples before Soxhlet extraction with toluene, and were used to monitor the extraction and cleanup procedures.After the sample was extracted in a Soxhlet extractor with toluene for 24 hours, the extracts were concentrated, and then treated with concentrated sulfuric acid, and this was followed by a series of sample cleanup and fractionation procedures, including a multi-layered silica column, alumina column and an activated carbon column.During the alumina column cleanup, non-planar PCBs and PBBs were eluted with 15 mL hexane, and were then further eluted with 25 mL DCM/hexane (1/24, v/v) for activated carbon column use.The activated carbon column was sequentially eluted with 5 mL toluene/methanol/ethyl acetate/hexane (1/1/2/16, v/v) for PCDE, PBDEs, planar PCBs and PBBs, which was further followed by 40 mL of toluene for PCDD/Fs and PBDD/Fs.Before instrument analyses, the planar and nonplanar PCBs/PBBs eluates were mixed together, representing the PCB and PBB samples.The eluate was concentrated to approximately 1 mL and transferred to a vial.The concentrate was further concentrated to near dryness, using a stream of nitrogen.10 μL of the standard solution for recovery checking was added to the sample extract immediately prior to injection to minimize the possibility of loss.The detailed analytical procedures are given in our previous works (Wang et al., 2010a, b, c;Chang et al., 2013;Chang et al., 2014).
Instrumental Analysis
A high-resolution gas chromatograph/high-resolution mass spectrometer (HRGC/HRMS) was used for the POP analyses.The HRGC (Hewlett-Packard 6970 Series gas, CA) was equipped with a silica capillary column (J&W Scientific, CA) and a splitless injector, while the HRMS (Micromass Autospec Ultima, Manchester, UK) was equipped with a positive electron impact (EI+) source.The selected ion monitoring (SIM) mode was used with a resolving power of 10,000.The electron energy and source temperature were specified at 35 eV and 250°C, respectively.The detailed instrumental analysis parameters of PCDD/Fs, PCBs, PBDD/Fs, PBBs and PBDEs are given in our previous works (Wang et al., 2010a, b, c;Chang et al., 2014).The instrumental parameters of the PCDEs analyses are the same as those of the PCDD/Fs.
Quality Assurance and Quality Control (QA/QC)
Prior to sampling, XAD-2 resin was spiked with PCDD/F surrogate standards pre-labeled with isotopes, including 37 Cl 4 -2,3,7,8-TCDD, 13 C 12 -1,2,3,4,7,8-HxCDD, 13 C 12 -2,3,4,7,8-PeCDF, 13 C 12 -1,2,3,4,7,8-HxCDF and 13 C 12 -1,2,3,4,7,8,9-HpCDF.The recoveries of precision and recovery (PAR), surrogate, and internal labeled standards of POPs all met the relevant standards.Field and laboratory blanks were carried for each batch of sampling and analyses.The total amounts of PCDD/Fs and PBDEs in the field and laboratory blanks were all < 0.1% and 0.3% of the real stack flue gas samples.As for the other POPs, the blanks were all below the detection limits.Furthermore, breakthrough tests of XAD-2 cartridges and determination of the accumulated POP levels onto the surface of the sampling probe were conducted.The detailed results are discussed in the following section.
Breakthrough Tests
Two XAD-2 cartridges are mounted in series and analyzed individually to evaluate the POP breakthrough for the long-term sampling of the EAF by AMESA.The breakthrough of POPs is calculated as follows.
where A is the mass (toxicity) adsorbed by the first XAD-2 cartridge, and B is the mass (toxicity) adsorbed by the second XAD-2 cartridge.The results of chlorinated and brominated POPs breakthroughs are listed in Tables 1 and 2, respectively.Less than 3% breakthroughs are found for the mass and toxicity of PCDD/Fs, PCBs, PCDEs and PBBs.The breakthroughs are lowest for PCBs (only 0.0533% for mass and 0.236% for toxicity).However, for PBDD/Fs and PBDEs, their breakthroughs are much larger, and are 43.4% and 14.9% for PBDD/Fs mass and toxicity, and 8.91% for PBDEs mass.As for the individual congeners of these POPs, there was a significant increase in breakthrough with the increase in halogen numbers for the PCDD/Fs, PCBs, PBDD/Fs and PBDEs.
We know that as the halogen numbers of POPs increase, their boiling points rise and vapor pressures drop (Gajewicz et al., 2010).In order to clarify the relation between the breakthrough and halogen numbers, the breakthrough and the subcooled liquid vapor pressure (P L ) of these POPs, which is an important property affecting the gas-particle partition of POPs in the environment, are analyzed by Pearson correlation analyses.We find that the logarithms of the breakthroughs for these POPs are significantly and negatively correlated with the logarithm of P L (r = -0.904,p < 0.0001) (see Fig. 1).This strong negative correlation implies that the highly halogen-substituted POPs with lower P L tend to partition to the particulate-phase, resulting in their greater breakthrough in the AMESA.This is because AMESA uses quartz wool instead of filters to collect particles, and a higher percentage of the smaller particles emitted from the stack flue gases pass through the first cartridge to the second one.
Rinse of the Sampling Probe
The operation of AMESA complies with the cooled probe method of EN-1948, and thus the particulate-and gaseous- phase POPs in the flue gas will adhere and condense onto the surface of the sampling probe and other components of the sample train.To evaluate their accumulated POP levels, four rinses of the sampling probe and other components of the sample train were performed after each sampling, and then individually analyzed for POPs.
The percentages of the POPs in each rinse to the corresponding total POPs collected from long-term sampling (cartridges + rinses) are listed in Table S1 of the supporting information.The amount of POPs in the first rinse of the sampling probe and other components of the sample train are one to three orders higher than those in the second one.Except for the first rinse, the POPs in the subsequent rinse of the sampling probe and other components contributed less than 3% of the total POPs.The results show that the rinse procedures adopted in this study are effective with regard to the removal of POPs from the surface of the sampling probe and other components after long long-term sampling by AMESA.
Table 3 lists the percentages of the POPs in the whole four rinses to the total POPs collected from long-term sampling (cartridges + rinses).The percentages of the rinses based on total mass are 58.0%for PCDD/Fs, 2.6% for PCBs, 2.9% for PCDEs, 55.1% for PBDD/Fs, 31.6% for PBBs and 32.1% for PBDEs, while those based on total toxicity are 40.8% for PCDD/Fs, 7.0% for PCBs and 49.8% for PBDD/Fs.Except for PCBs and PCDEs whose total percentages are less than 7%, those of the other POPs are all greater than 30%.
A significant trend seen in these figures is that the percentages increased along with the increase in halogen numbers for these six POPs.That is attributed to the fact that the highly halogen-substituted POPs in the gaseous phase more easily condense onto the surface of the sampling probe than the lower halogen-substituted ones.Furthermore, the particles that adhere onto the surface of the sampling probe contain more highly halogen-substituted POPs.Therefore, it is crucial to carefully perform the cleaning procedures, and the solvents from the rinses of the sampling probe and other components need to be combined with XAD-2 cartridges for analyses to obtain the real POP concentrations in the stack flue gases.Although performing rinses could remove most POPs which are adherent to the surface of the sampling probe and components, residues might cause memory effects if the next sampling is a short-term one, because the difference between the sampling volumes of the flue gases could reach about 200-fold.
Concentrations and Congener Profiles of POPs in the Stack Flue Gases
The POP concentrations in the stack flue gases of the EAFs are listed in Table 4.The mass concentrations are 0.439 ng/Nm 3 for PCDD/Fs, 7.53 ng/Nm 3 for PCBs, 0.0115 ng/Nm 3 for PCDEs, 0.163 ng/Nm 3 for PBDD/Fs, 0.145 ng/Nm 3 for PBBs and 8.03 ng/Nm 3 for PBDEs.The PCBs and PBDEs are the most abundant pollutants, and their mass concentrations are one to three orders higher than those of the other POPs.Regarding POPs with dioxin-like toxicity, the toxicity concentrations are 0.0601 ng I-TEQ/Nm 3 for PCDD/Fs, 0.00804 ng WHO-TEQ/Nm 3 for PCBs and 0.000869 ng TEQ/Nm 3 for PBBD/Fs, and the sum of the dioxin-like toxicity concentrations of these three POPs is 0.0690 ng TEQ/Nm 3 .The toxicities contributed by PCDD/Fs, PCBs and PBBD/Fs are 87.1%,11.7% and 1.2%, respectively.
Table 5 lists the POP concentrations in the stack flue gases/ exhausts of various emission sources, such as incinerators (Kim et al., 2004;Wang et al., 2010a), sinter plants (Kuo et al., 2012;Wang et al., 2003c), EAFs (Lee et al., 2005;Wang et al., 2010c;), power plants (Dyke et al., 2003;Hutson et al., 2009;) and diesel engines (Wang et al., 2010b).As yet, no data regarding PCDEs and PBBs in flue gases emitted from combustion facilities have been reported in the literature.Compared to the results in previous studies of EAFs (Wang et al., 2010b, c;Lee et al., 2005), the concentrations of PCDD/Fs, PBBD/Fs and PBDEs obtained in this work are lower, but still within one order.Generally, the PCDD/F and PCB concentrations in the flue gases of stationary sources are higher than those in the exhausts of diesel engines or vehicles, although diesel engines seem to emit higher or at least similar levels of PBDD/F and PBDE concentrations (Wang et al., 2010b) than stationary sources.
The congener profiles of these POPs in the stack flue gases of the EAF are shown in Fig. 2. The congener profiles of PCDD/Fs, PBDD/Fs and PBDEs obtained from this study are consistent with the results of previous works (Hofstadler et al., 2000;Wang et al., 2003c;Lee et al., 2004Lee et al., , 2005;;Wang et al., 2010c).The PBDE congeners in the stack flue gases leaned more to the low to medium brominated congeners, namely BDE-28, -47, -100 and -99.These abundant low to medium brominated congeners may be attributed to the thermal desorption of the commercial penta-BDE mixtures, which were impurities in the feeding scrap (Wang et al., 2010c).Among the highly brominated-substituted congeners, BDE-209 was the most abundant.BDE-209 could be formed through combustion processes, which is similar to that of sinter plants (Wang et al., 2010c), and from the undestroyed BDE-209 in the feeding scrap.
For the PCBs, the dominant congeners in the stack flue gases were in the order of PCB-118, -105, and -77, showing that tetra-and penta-CBs were the major PCB homologues.This assembled to the results found for the PCDF homologue.Although PBBs, like PCBs, were also dominant in lower halogenated-substituted congeners, the bromination pattern was different from that seen for PBDD/Fs.
The PCDE congener profile is dominated by CDEs-28 and -99, the lower chlorinated-substituted congeners.The homologue profile clearly shows that the formation of PCDEs is favorable to the lower chlorinated ones, while the highly chlorinated ones remain minor components.Similar phenomena occurred with regard to PCDFs of the EAF, which had higher fractions of the lower chlorinated congeners compared to other combustion sources, such as vehicles (Chang et al., 2014), incinerators (Wang and Chang-Chien, 2007) and crematories (Wang et al., 2003a).We speculate that Fe 2 O 3 , which should be abundant in fly ashes of EAFs, may shift the formation of PCDEs towards the lower chlorinated congeners (Liu et al., 2013).The close association between PBDEs and PBDD/Fs does not exist for PCDEs and PCDD/Fs.Furthermore, the formation mechanisms between PBDEs and PCDEs, as well as those between PBDD/Fs and PCDD/Fs, are not very similar, suggesting the need for further studies on the relationships among the formation mechanisms of these analogues.
The congener profiles of PBDD/Fs and PBDEs, especially for PBDD/Fs, which were more dominated by the highly halogen-substituted congeners than other POPs, resulted in their breakthroughs much larger than those of other POPs.
Emission Factors of the POPs
Table 6 lists the POP emission factors of EAFs obtained from this study and from elsewhere.The emission factors are calculated based on the total weights of steel production or feedstock, including scraps, alloying agents, flux and coke.For the same measurement, the obtained emission factors based on the product will be 10%-15% higher than those based on the feedstock, because the transformation rate of feedstock to produced steel is commonly around 90% or lower.
The emission factors of the investigated EAF in this study are 0.177 µg I-TEQ/tonne-feedstock for PCDD/Fs, 0.0232 µg WHO-TEQ/tonne-feedstock for PCBs, 0.0343 µg/tonnefeedstock for PCDEs, 0.00247 µg TEQ/tonne-feedstock for PBDD/Fs, 0.416 µg/tonne-feedstock for PBBs and 24.0 µg/tonne-feedstock for PBDEs, which are all one order lower than those reported in other studies.The much higher sampling time of AMESA should include more high emission events, which will then cause higher emission factors.Therefore, we think that the lower emission factors obtained in this study should be attributed to the influence of feeding materials and the operating condition of EAFs (Lee et al., 2005;Wang et al., 2010a, c), not the samplings by AMESA.
CONCLUSIONS
The PBDD/F congener profile was dominated by 1,2,3,4,6,7,8-HpBDF and OBDF (highly brominated PBDF congeners), while the others were all quite minor components or below the detection limits.Among the highly brominated congeners, the congener profiles of PBDEs showed a similar pattern to those of the PBDFs.The PCDD/F congener profile, which was dominated by lower chlorinated-substituted PCDFs, was very different to the corresponding PBDD/F congener profiles.De novo synthesis of PCDD/Fs is known to dominate in the post-combustion zone of EAFs, and therefore the formation of PBDD/Fs from PBDE precursors is much more significant than that from de novo synthesis.The logarithms of the breakthroughs of these POPs are significantly and negatively correlated with the logarithm of P L (r = -0.904,p < 0.0001).This strong negative correlation implies that the highly halogen-substituted POPs with lower PL tend to partition to the particulate-phase, resulting in their greater breakthrough in the AMESA.Therefore, PBDD/Fs had much higher breakthroughs than the other POPs.
The emission factors of the investigated EAF in this study are one order lower than those from other studies.This should be attributed to the influence of feeding materials and the operating condition of the EAFs, not samplings by AMESA, because the much higher sampling time of AMESA should include more high emission events.
Fig. 1 .
Fig. 1.Relation between the logarithms of the breakthroughs and the logarithm of P L among the investigated POPs.
Fig. 2 .
Fig. 2. Congener profiles of these POPs in the stack flue gases of the EAF.
Table 3 .
Mean percentages (%) of the POPs in the whole four rinses to the total POPs collected from long-term samplings (cartridges + rinses).
Table S1 .
Mean percentage (%) of the POPs in each rinse to the corresponding total POPs collected from long-term samplings (cartridges + rinses). | v3-fos-license |
2019-12-08T21:14:15.803Z | 2019-12-01T00:00:00.000 | 208878369 | {
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} | pes2o/s2orc | Lemon Peel Polyphenol Extract Reduces Interleukin-6-Induced Cell Migration, Invasiveness, and Matrix Metalloproteinase-9/2 Expression in Human Gastric Adenocarcinoma MKN-28 and AGS Cell Lines
Among plant polyphenols, lemon peels extract (LPE) from the residues of the industrial processing of lemon (Citrus limon) shows anti-proliferative properties in cancer cells and anticholinesterase activity. In this study, we analyze the anti-cancer properties of LPE on migration and invasiveness in MKN-28 and AGS human gastric cancer cell lines either in the absence or presence of the pro-inflammatory cytokine IL-6. We find that the pretreatment with non-cytotoxic concentrations (0.5–1 μg/ml of gallic acid equivalent) of LPE inhibits interleukin-6 (IL-6)-induced cell migration and invasiveness in MKN-28 and AGS cells, as analyzed by wound and matrigel assays. Pretreatment with LPE is able to prevent either IL-6-induced matrix metalloproteinases (MMP)-9/2 activity, as assessed by gel zymography, or mRNA and protein MMP-9/2 expression, as evaluated by qPCR and Western blotting analysis, respectively. These LPE effects are associated with an IL-6-dependent STAT3 signaling pathway in MKN-28 and AGS cells. Furthermore, LPE shows acetylcholinesterase inhibitory activity when assayed by the Ellman method. In conclusion, our results demonstrate that LPE reduces the invasiveness of gastric MKN-28 and AGS cancer cells through the reduction of IL-6-induced MMP-9/2 up-regulation. Therefore, these data suggest that LPE exerts a protective role against the metastatic process in gastric cancer.
Introduction
Gastric cancer (GC) is one of the most common malignant tumors in the world, with relatively high mortality [1]. Due to the high metastatic ability of gastric cancer cells, the GC prognosis is complex to determine. A key step in the complex processes of invasion and migration of cancer cells is caused by the degradation of the extracellular matrix (ECM). Among different enzymes acting in Polyphenols were recovered by maceration as follows: 1 g of powdered waste was suspended into 25 ml of 80% EtOH and left under stirring at room temperature for 2 h. Partial purification was performed by discarding the resting solid material by centrifugation at 10,000× g for 40 min (Centrifuge Avanti TM J-25, rotor JA14 Beckman Coulter TM ). The liquid phase was filtered on paper-filter (Whatman®cellulose Grade 1) and successively reduced to 10-15 ml in a rotary evaporator (Buchi Rotavapor R-210) at 40 • C under vacuum. The total phenolic content in the extract was determined according to the adapted Folin-Ciocalteu colorimetric method [21] and the results were expressed as mg Gallic acid equivalent (GAE) per g of dry sample. The raw extracts were concentrated at 40 • C under vacuum by a rotary evaporator and dissolved in the suitable solvent for the biological assays.
Cell Cultures and Treatments
The human gastric adenocarcinoma cell lines MKN-28 [17] and AGS (American Type Culture Collection, ATTC CCL-107) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen, Life Technologies, Italy), 1.5 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin under humidified atmosphere of 5% CO 2 at 37 • C. The treatments of sub-confluent cells with increasing concentration (10-100 ng/ml) of rIL-6 [19,20] or lemon peel extract (LPE) (0.5-50 µg/ml) [18] were performed for 24 h in serum-free DMEM to determine the IC 50 value. The evaluation of LPE protective effects was performed by the pretreatments with two concentrations of LPE (0.5 or 1 µg/ml) for 6 h that was followed by exposure to rIL-6 (50 ng/ml) for further 24 h. The treatments were performed under serum-free conditions in the presence of 0.5% (v/v) final concentration EtOH used as a vehicle for LPE.
Cell Proliferation Assay
Cell viability was evaluated as mitochondrial metabolic activity using the PrestoBlue TM (PB) cell viability reagent (Cat. N. A13262, Invitrogen, Life Technologies, Italy) [22], according to the manufacturer's protocol. Briefly, the cells were plated onto 96-well plates (1 × 10 4 cells/well), treated and then incubated with (PB) reagent at 10% final concentration for 1 h. The absorbance was read at 570 nm with a reference wavelength set at 600 nm using a microplate reader (Labsystems Multiskan, MS). Control wells without cells, containing DMEM in the absence or the presence of the different LPE concentration, were used for background subtraction. The cell viability was expressed as a percentage relative to the untreated cells, cultured in serum-free medium, set as 100%.
RNA Extraction, Reverse Transcription (RT), and Quantitative Real-Time Polymerase Chain Reaction (qPCR)
Total RNA was purified by the ultrapure TRizol reagent (GibcoBRL) according to the manufacturer's instructions. The concentration and purity of RNA were determined spectrophotometrically by reading the absorbance at 260 and 280 nm. Aliquots (1 µg) of total RNA were subjected to DNase I digestion (Thermo Fisher Scientific) and reverse transcribed using Sensi FAST TM cDNA synthesis kit (Cat. N. BIO-65054, Bioline, Italy) according to the manufacturer's protocol. Real-time PCR was carried out using the PowerUP Sybr green master mix (Thermo Fisher Scientific), the Quant Studio 7 Flex instrument, and the fast gene-expression method with the following conditions: a first denaturation step a 95 • C for 20 s followed by 40 cycles at 95 • C for 1 s, 60 • C for 30 s and then melting curve analysis was performed raising temperature from 60 • C until 95 • C with a 0.5 • C/s increase. Reactions were carried out in triplicate, and the 18S gene was used as an internal control to normalize the variability in expression levels. The 2 −∆∆CT (cycle threshold) method [20] was used to calculate the results and mRNA expression levels were determined as fold-induction relative to Ctrl cells, set as 1. PCR primers for MMP-2, MMP-9, and 18S were designed using Primer Blast software based on sequence deposited in the GeneBank Database (NCBI: NM_004530.5, NM_004994.2, and NR_145820.1, respectively). Reactions were carried out in triplicate, and the 18S gene was used to normalize the variability in expression levels. The mRNA expression levels were determined as fold-induction relative to control cells, set as 1. The primers used for the qPCR reactions are the following:
Gelatin Zymography
Gelatinolytic activity in the cell-conditioned medium was assayed by SDS-PAGE zymography, as described previously [7,17]. Samples were analyzed under non-reducing conditions without boiling, through a 12% SDS-polyacrylamide gel co-polymerized in the presence of gelatin (1 mg/ml, cat. n. G1890, Sigma-Aldrich, Italy). Electrophoresis was conducted at 35 mA for 90-120 min at 4 • C. After the run, the proteins in the gels were renatured in a 2.5% Triton X-100 solution for 1 h. The gels were then incubated with 50 mM Tris-HCl pH 7.5, 200 mM NaCl, 5 mM CaCl 2, and 5 µM ZnCl 2 at 37 • C for 48 h, which allows substrate degradation. Finally, the gels were fixed in 30% methanol, 10% acetic acid for 30 min, and stained with 0.5% Coomassie Brillant Blue R-250. Proteolytic bands were visualized by destaining with 50% methanol and 5% acetic acid.
Wound Assay
The cell migration was assessed using a wound assay [25]. Cells (2 × 10 5 cells/well) were seeded into each well of a 6-well plate and incubated with complete medium at 37 • C and 5% CO 2 . After 24 h of incubation, the cells were scrapped horizontally and vertically with a sterilized P10 pipette tip, subjected to different treatments in medium with 0.5% FBS and two views on the cross were photographed on each well at 0, 12, and 24 h, using a Zeiss Axiovert 40 CFL inverted microscope (Carl Zeiss, Milan, Italy) 10 × objective. The microscope was equipped with a 12.1-megapixel CCD digital video camera (Canon, PowerShot G9, Italy) with a digital image software (Remote Capture DC, Canon). Quantitative analysis of the scratch assay was performed by measuring the gap area using the free image-processing software ImageJ version 1.47.
Matrigel Invasion Assay
Cell invasion assay was performed by BD Falcon BioCoat Matrigel invasion chambers (cat. n. 354480, BD Bioscience, Bedford MA; 8 µm). The cell culture inserts were rehydrated and prepared as previously described [7,17]. Briefly, 2 × 10 4 cells in 0.5 ml of DMEM with 0.5% FBS were seeded in the upper chamber, and 750 µl medium with 5% FBS was placed in the lower chamber. After 24 h, cells in the upper chamber were removed with the cotton swab, and the cells at the bottom of the filters were fixed and stained with a Diff-Quick kit (cat. n. B4132-1A, Becton-Dickinson). After two washes with water, the inserts were allowed to air dry, and phase-contrast images were captured as described above using an LD A-Plan 20×/0.30 Ph 1 objective. To assess cell invasion ability, the total number of cells was determined by cell counting in five fields randomly selected per membrane.
Acetylcholinesterase Inhibition Assay
Cholinesterase activity was assayed by the Ellman method [26] using acetylthiocholine as substrate. The reduction of dithiobisnitrobenzoate by the thiocholine, produced by the enzymatic hydrolysis of thiolated substrates, was followed colorimetrically (412 nm) at room temperature (22-27 • C). Briefly, an 1 ml aliquot of LPE was dried under a liquid nitrogen stream, and then the pellet was resuspended in the same volume of PBS. The reaction mixture (500 µL) contained 330 µM 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB), 500 µM acetylthiocholine as substrate, and different amounts of LPE (7-60 µg/ml final concentration) in 0.1 M sodium phosphate buffer pH 7.4. The reaction was started by the addition of 100 mU/ml AChE, and the initial rate of the reaction was derived from the linear portion of the kinetics. The concentration of LPE required to reduce the enzymatic activity to 50% (IC 50 ) was derived from semi-logarithmic plots. Linear curve fits were obtained with the least-squares method, and the significance of the correlation was estimated from the squared correlation coefficient r 2 .
Statistical Analysis
The data were expressed as mean ± SD of at least three independent experiments performed in triplicate. Statistical significance of treated samples against control cells (cultured in serum-free medium) was determined by a one-way analysis of variance (ANOVA), followed by Bonferroni's test (p-values of * p < 0.05; § p < 0.01; # p < 0.001).
Effect of LPE Treatment on Cell Proliferation
In a first approach, we analyzed the ability of the LPE (prepared as reported in Section 2.2) containing a total polyphenol content of 0.46 mg/ml GAE to affect proliferation of MKN-28 and AGS cells. To this aim, the cell lines were exposed to increasing amounts of LPE, ranging from 0.5 to 50.0 µg/ml [18] in serum-free conditions for 6 and 24 h and then subjected to a viability assay. As shown in Figure 1, cell proliferation was inhibited by LPE in a time and concentration-dependent manner in a range from 1.0 to 50.0 µg/ml GAE. The IC 50 values expressed as GAE were determined by non-linear regression analysis in a hyperbolic binding equation ( Figure A1) at 6 (12.14 and 17.06 µg/ml for MKN-28 and AGS, respectively), and 24 h stimulation (7.46 and 5.08 µg/ml for MKN-28 and AGS, respectively). In the light of these results, the two concentrations of 0.5 µg/ml, that did not affect cell viability and 1.0 µg/ml that exhibited little effect on the cell viability (~90%), were selected and used in the subsequent experiments to evaluate the protective effect of LPE on IL-6 exposed MKN-28 and AGS cells.
The LPE Treatment Reduces the rIL-6-Stimulated Migration and Invasiveness of MKN-28 and AGS Cells
Since IL-6 causes an increase of migration and invasiveness in gastric cancer cells [1,14,15,27,28], we investigated the effect of LPE treatment on rIL-6-induced migration and invasive ability by wound healing and matrigel invasion assays, respectively, in MKN-28 and AGS cells. The wound-healing assay ( Figure 2) shows that either serum deprivation (Panel A,B images a,c,i) or the treatment with LPE alone (Figure 2, Panel A,B images f,j) do not affect the wound area compared to that of untreated cells (Figure 2, Panel A,B images a,e,i) in both cell lines. Conversely, in both cell lines, the exposure to rIL-6 alone led to a significant reduction in the wound area (Figure 2, Panel A,B images g,k) compared to that of untreated cells. Quantitative analysis of wound area (Figure 2, Panel C) shows that the effect induced by rIL-6 is more prominent in MKN-28 cells that show a major percentage of wound area reduction (~70% or 97% at 12 and 24 h, respectively) compared to AGS cells (~50% or 90%, at 12 and 24 h, respectively). Interestingly, the pre-treatment with LPE prevents the rIL-6 induced reduction of wound area, either in MKN-28 (Figure 2, Panel A images h,l) (~5% or 40% at 12 and 24 h, respectively) or in AGS (Figure 2, Panel B images h,l) (~10% or 30% at 12 and 24 h, respectively) as compared to the cells stimulated with rIL-6 alone.
The matrigel invasion assay ( Figure 3) shows that, in both cell lines, rIL-6 induces a 2-fold increase in number of cells that had invaded through the membrane (Figure 3, Panel A images c,g) compared to untreated (Figure 3, Panel A images a,e) or LPE exposed cells (Figure 3, Panel A images b,f). Cells seeded in trans-well chambers for the matrigel invasion assay were pretreated with LPE (0.5 or 1 µg/ml GAE) for 6 h and then exposed to rIL-6 (50 ng/ml) for further 24 h. Thereafter, the cells at the bottom of the filters were fixed, stained, and observed by a phase-contrast-microscope (10 × objective). (A) Representative photomicrographs of random fields of MKN-28 and AGS cells keep in serum-free medium (a,e); exposed to LPE alone (b,f); treated with rIL-6 alone (c,g); pretreated with LPE and then exposed to rIL-6 (d,h). (B) The invasive capability was determined by cell counting in five fields, randomly selected, per membrane. Quantification was relative to untreated cells, cultured in serum-free DMEM, set as 100%. Results are presented as mean ± SD (n = 3). ( § p < 0.01, statistically significant vs. untreated cells or treated with rIL-6).
Interestingly, the pre-treatment with LPE leads to an invasive ability similar to that observed in the absence of the inflammatory stimulus caused by rIL-6 ( Figure 3, Panel B).
These results clearly indicate that non-cytotoxic LPE concentrations prevent the rIL-6 exerted pro-metastatic effect in MKN-28 and AGS gastric cancer cells.
Effect of Exposure to rIL-6 on MMP-9 and MMP-2 Gelatinolytic Activity and Expression Levels in MKN-28 and AGS Cells
As cell invasiveness is strictly related to IL-6-induced upregulation of MMP-9 and MMP-2 expression levels in human gastric cancer cells [1,10,15], we have explored the effect of LPE in rIL-6 exposed MKN-28 and AGS cells. To this aim, we first analysed the effect of increasing amounts of rIL-6 (10-100 ng/ml) on MMP-9/2 gelatinolytic activity and expression levels. Gel zymography performed on cell-conditioned media from MKN-28 and AGS cells ( Figure A2, Panel A) revealed that rIL-6 was able to induce MMP-9 and MMP-2 gelatinolytic activity in a concentration-dependent manner, with a maximum increase of 3.1-and 4.9-fold for MMP-9 and 2.7-and 3.1-fold for MMP-2, respectively, in MKN-28 and AGS cells compared to that of unstimulated cells. The increase of gelatinolytic activity was detected in both cell lines, although to a major extent in AGS cell-conditioned media.
We also evaluated whether this increase of MMP-9/2 gelatinolytic activity was due to an rIL-6-dependent increase of MMP-9/2 mRNA expression levels. The result of qPCR analysis ( Figure A2, Panel B) shows that both MMP-9 and MMP-2 mRNA expression levels, which were very low in untreated cells, greatly increased both in MKN-28 and AGS cells exposed to rIL-6. The up-regulation of MMP-9/2 expression level was concentration-dependent in both cell lines; however, the maximal induction of MMP-9/2 mRNA expression levels was reached at 50 ng/ml rIL-6, in agreement with previous result [27]. Therefore, this concentration was chosen for further studies.
The above data indicate that rIL-6-induces an up-regulation of MMP-9 and MMP-2 gelatinolytic activity, which is correlated to an increase of their mRNA expression level in MKN-28 and AGS cells.
Effect of LPE on rIL-6 Induced Up-Regulation of MMP-9/2 Expression Levels in MKN-28 and AGS Cells
To analyze the effect of LPE on rIL-6-induced MMP-9/2 expression levels, the conditioned medium of cells pretreated with LPE and then exposed to rIL-6 were subjected to gelatin zymography and qPCR analysis (Figure 4). The pre-treatment with LPE reduces the rIL-6-induced MMP-9 and MMP-2 gelatinolytic activity in the culture medium of both cell lines (Figure 4, Panel A). This effect was LPE concentration-dependent, and the highest concentration tested (1 µg/ml GAE) completely suppressed the rIL-6 induced MMP-9/2 enzyme activity up-regulation, which was similar to that observed in untreated cells.
Similar behavior was observed when the effect of the LPE pre-treatment was evaluated on rIL-6 dependent induction of MMP-9/2 mRNA expression level (Figure 4, Panel B). The pre-treatment with 1 µg/ml LPE for 6 h induces a decrease of IL-6 induced MMP-9/2 mRNA expression levels in both cell lines. However, as can be observed (Figure 4, Panel B), LPE greatly reduces the effect on the MMP-2 mRNA levels that are stimulated by rIL-6 in AGS cells. In addition, the exposure to LPE is able to counteract the rIL-6 induced MMPs mRNA levels up-regulation although to a different extent in MKN-28 or AGS cells. In particular, the highest reduction in mRNA expression has been observed for MMP-2 levels in AGS cells. In the latter case, the pre-exposure to 1 µg/ml LPE leads a MMP-2 expression level that is similar to that on untreated cells. Figure 4. Effect of LPE exposure on rIL-6-induced MMP-9 and MMP-2 gelatinolytic activity and expression level in MKN-28 and AGS cells. Cells, pretreated with LPE (0.5 or 1 µg/ml GAE) for 6 h, were then exposed to rIL-6 (50 ng/ml) for further 24 h. After the treatments, the cells were harvested for preparation of total RNA and protein extracts. Cell conditioned media were collected, concentrated by ultrafiltration, and volumes corresponding to 40 µg of cellular proteins were analyzed under non-reducing conditions through a 12% SDS-polyacrylamide gel co-polymerized in the presence of gelatin (1 mg/ml) for (A) gelatin zymography of MKN-28 and AGS cells. (B) Quantitative qPCR analysis of MMP-2 and MMP-9 mRNA expression levels of MKN-28 and AGS cells. 18S was used as the internal control. Results are presented as mean ± SD (n = 3). (* p < 0.05; # p < 0.001, statistically significant from untreated cells or treated cells with rIL-6).
Effects of LPE on IL-6-Dependent STAT3 Phosphorylation Levels in MKN-28 and AGS Cells
As the IL-6-induced transcriptional stimulation of MMP-9/2 results from the activation of STAT3 via the JAK2/STAT3 pathway in gastric cancer and AGS cells [27,28], we tested the STAT3 phosphorylation level [29] in MKN-28 and AGS cells exposed to rIL-6 for different time points. Panel A in Figure 5 shows that in untreated cells, kept in serum-free medium, a negligible amount of pSTAT3 protein level is detectable whereas, remarkably, the treatment with IL-6 strongly stimulated STAT3 phosphorylation at Serine 727 residue (Ser 727 STAT3) in a time-dependent manner.
The pSTAT3 protein level progressively increased until 24 h in MKN-28 cells (~4.5-fold); conversely, in AGS cells, a~1.9-fold increase was observed at 12 h stimulation. In both cell lines, no difference in STAT3 total protein level was observed in comparison to pSTAT3 levels. The pretreatment with LPE ( Figure 5, Panel B) was able to reduce the rIL-6 stimulated pSTAT3 level compared to that of cells exposed to rIL-6 alone, although with a different efficacy between the two cell lines. The major inhibitory effect was observed at the higher LPE concentration (1 µg/ml GAE) that yield~2-fold decrease of pSTAT3/STAT3 level compared with that observed in cells treated with rIL-6 alone, in both cell lines. A low pSTAT3 signal was immuno-detected in cells treated with LPE alone that was similar to that of unstimulated cells, and no changes in the abundance of the total STAT3 protein level were found in the cells exposed to the different treatments. These findings indicate that LPE can affect the IL-6-dependent STAT3 phosphorylation in MKN-28 and AGS cells.
Effect of LPE on Acetylcholinesterase Activity
Up to today, acetylcholinesterase inhibitors represent the major approved drugs to treat Alzheimer's disease, and therefore, many efforts have been devoted to the identification of novel compounds able to inhibit AChE as potential drugs for the treatment of this neurodegenerative disease [30,31]. Since a previous study [18] demonstrated that LPE shows an acetylcholinesterase inhibitory activity when assayed by TLC bioautographic assay [32], we performed a further characterization of this activity. We used an in vitro AChE enzyme assay [26] that allows an accurate determination of the inhibition values. The assay was performed in the absence or the presence of different LPE concentrations, and the result ( Figure 6) indicates that LPE exhibits AChE inhibitory activity ( Figure 6).
The analysis of the data by a semilogarithmic plot ( Figure 6, Panel B) allowed the determination of the inhibitor concentration required to get inhibition of half of the activity (IC 50 ) corresponding to 36 µg/ml GAE, a result in agreement with those previously obtained [18].
Discussion
Several studies on dietary polyphenols have demonstrated their chemopreventive activities [17,[33][34][35]. Regarding lemon polyphenols, the exploitation of their different beneficial effects on human health, such as the reduction of blood glucose levels, the regulation of lipid metabolism, and the reduction of inflammation, has already been reported [36][37][38]. In previous studies, the lemon peel industry's wastes have been reported to represent a sustainable source of such valuable phenolic compounds [18,39,40]. HPLC analysis of these residues' extracts showed the presence of main phenolic compounds such as benzoic and p-coumaric acids, hesperetin, naringenin, naringin, and quercetin [18]. This polyphenolic mixture exhibited promising properties as antioxidant and anti-proliferative agents against some cancer cells [18]. These results prompted us to further investigate the biological potential of lemon wastes' polyphenols as anticancer agents.
The results here reported demonstrate novel biological properties of LPE that are able to prevent IL-6-induced invasiveness, to reduced IL-6-stimulated MMP-9/2 up-regulation in gastric MKN-28 and AGS cancer cell lines, and to inhibit in vitro AChE activity. First, we demonstrated that LPE affects cell proliferation in a concentration-dependent manner in MKN-28 and AGS cells in agreement with the anti-proliferative effect observed in neuroblastoma SH-SY5Y, colorectal adenocarcinoma Caco-2, and hepato-carcinoma HepG2 human cells (1 µg/ml GAE) [18].
As the inflammatory cytokine IL-6 also promotes migration and invasiveness of gastric cancer cells [15], we analyzed the effect of LPE in the IL-6 stimulated human gastric AGS and MKN-28 cell lines. We observed that the pre-treatment with LPE is able to counteract the rIL-6-induced increase in the migration and invasive abilities of MKN-28 and AGS cells, in agreement with the scientific literature [1,14,15,27,28].
Here we also demonstrated that LPE is able to prevent the rIL-6-induced up-regulation of MMP-9 and MMP-2 gelatinolytic activity in the conditioned medium of MKN-28 and AGS cells. Further, we demonstrated that LPE chemo-preventive effects are correlated with MMP-9 and MMP-2 down-regulation both at mRNA and protein expression levels.
Following the IL-6 binding to its transmembrane receptor gp130, the activation signal transduction requires the activation of Janus kinase (JAK), which is followed by phosphorylation of STAT (STAT1 and STAT3) [20], thus, suggesting that JAK-STAT pathways play an important role in the gastric cancer process. We found that rIL-6 increased the STAT3 serine phosphorylation both in MKN-28 and AGS and this effect was inhibited by LPE pre-exposure before rIL-6 stimulation. This data strongly demonstrates that LPE is able to prevent rIL-6 induced effects by impairing the JAK2/STAT3 activation pathway in MKN-28 and AGS cells.
Conclusions
In conclusion, our results demonstrate that LPE appears to be a protective agent against the insurgence of human gastric adenocarcinoma since it can reduce the upregulation of key enzymes such as MMP-9 and MMP-2 that occur during the inflammation process. Moreover, LPE also shows a neuroprotective effect for its ability to inhibit AChE activity, an enzyme involved in Alzheimer's disease. Although further studies will be necessary to clarify the molecular and cellular mechanisms underlying LPE anticancer effects, our data highlight the beneficial effects of LPE in the prevention and/or therapy of human gastric cancer metastasis and Alzheimer's disease. Moreover, the biological properties of LPE could be exploited not only in the pharmaceutical field but also in food industries. In fact, LPE preparation represents a useful strategy for the recycling of vegetable waste that allows the production of novel additives or functional compounds with new biological properties. | v3-fos-license |
2019-04-09T13:09:07.357Z | 2018-01-01T00:00:00.000 | 102634929 | {
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} | pes2o/s2orc | Design, automation and remote management of a water purification plant
This study presents a water purification plant that uses the waste cake from the process of oil extraction of Moringa oleifera seeds. The particularity of this purification plant is that it should be autonomous to work in isolated areas. To do so, the design counts on solar panels and batteries controlled by a Supervisory Control And Data Acquisition (SCADA) system. The main objective of this study is the design and automation of the purification power plant so it can be used either manually or remotely by means of a web server and a micro controller in charge of data collection and to proceed orders from and to the web platform. In pursue of a cost reduction, caused by the development and implementation of hardware and software, this project is conceived using open source systems. Additionally, the plant counts on an Energy Management System that should optimize the energy consumption of the control system and actuators. This system is designed in such a way that it can be used independently in isolated mode or connected to the grid in regions where local authority regulations allows the connection of energy storage systems to the grid.
Introduction
The access to drinkable water is considered a Human Right since 2010 by the United Nations General Assembly.In spite of the efforts and cooperation projects related to drinkable water and sanitation access, there are still 663 million people without access to drinkable water according to the World Health Organization [1].Therefore, the differences in quantity and quality of the water supply, especially in impoverished countries, supposes the main cause of diseases due to intestinal parasites, having a significant effect on infant population [2].
Additionally, due to regulations regarding the environmental impact in farming and stock breeding zones, agro industry should introduce water treatment systems to treat water before returning it to rivers or water basins to eliminate nitrates and fertilizers that are very pollutant [3].It is in these scenarios where the necessity to implement com-pact, portable and autonomous water treatment plants is stated.Due to the difficulties to reach these isolated water treatment plants in person, it is appreciated to install the capability to control and supervise remotely its functioning and working state.
There are multiple studies related to water treatment processes, such as the ones presented in the recent comparison done by Lopez-Grimau et al. [4] that shows how some studies consider the development of pilot plants to treat urban wastewater while other focus the attention on industrial wastewater.The novelty of the system presented in this study in comparison to other pilot water treatment plants is the possibility to be used in isolated locations without access to electricity.In fact, this study presents the working details of the pilot water treatment plant that presented better environmental results in a previous work [5].
Considering that the plant should work in isolated places, where human intervention is more difficult, the automation of the plant is justified.However, automation needs control.In this case, automation is done using a Supervisory Control And Data Acquisition (SCADA) sys-
M.M Rafique et al. / Desalination and Water Treatment
(2017) 1-8 2 tem that allows equally the control of the plant in a manual or a remote mode.
Nowadays, software to optimize water and energetic resources is used in a wide range of productive sectors [4].Particularly, in the sector of water purification automated plants, the control of water and energy consumption together with the monitoring and activation of actuators is done by SCADA [6].The SCADA plays an important role in the industrial communication in real time.These systems combine hardware and software such as the Main Terminal Unit (MTU), Remote Terminal Units (RTU), actuators, sensors and Human Machine Interfaces (HMI) [7].Moreover, SCADA systems can be designed and implemented in such a way that they can work either with expensive but function oriented private software or with low cost open source programming software [8].Generally, the automation takes advantage of Programmable Logic Controllers (PLC) [9] to run the supervision and control of sensors and actuators connected through an OCP server to communicate between them [10].
Being possibly installed in isolated locations, the presented prototype should be capable to work powered by distributed electricity generation systems for the cases where no electricity grid is in reach, increasing the cost of the solution.This paper also presents an interesting alternative of batteries to power the water treatment plant in such cases.
In summary, the proposed system stands out because it is compact, economic, energetically sustainable and reliability, so it allows the implementation of the water purification plant almost anywhere.
Methodology
The design of the plant has several requirements to follow: First, the plant should be compact; second, the plant should be inexpensive, reason why it uses free software; and third, the plant should be self-sufficient, so the energy consumption of all the control, sensors and actuators has to be measured to correctly dimension the amount of solar panels and the number of battery packs to store energy.
The presented water purification plant follows the basics of the Patent P201430600 that takes advantage of gravity to operate.This base plant is totally mechanical, using the difference in height and in volume of three containers (water to treat, dissolution and treatment or agitation containers) to dose the coagulants/flocculants by means of a Venturi tube and to proceed with the fast and slow agitation phases that are necessary to properly eliminate the solids in suspension and to disinfect [2] water.
This study uses a similar compact concept adding an automation system, which entails the need of electricity power.Electricity could be obtained by connecting the water treatment plant to the electricity grid, if available, or using electricity generation systems such as solar panels and lithium ion batteries, which is the case of the designed prototype presented in this study.Notice that the batteries used in this prototype come from a waste product, as they are taken from dismantled electric vehicles (EV).In fact, EV batteries are considered no longer useful for traction purposes when they have lost a 20% of its capacity [11].When this happens, these batteries are taken out from the car and they are normally recycled.In this case, though, it is considered that an 80% capacity is good enough to fulfill the power plant needs.By incorporating re-used EV batteries, the plant incorporates compact batteries as Li-ion batteries have higher energy density than all other battery types at the moment [12] and reduces the acquisition costs [13].
The resulting prototype is shown in Fig. 1, which has a tarpaulin to protect it from the rain (upper-right image).
Roughly, the system works as follows: In container number 1 (Fig. 1) the system stores the water to treat.In a preliminary stage, the tests were generally done mixing kaolin to drinkable water, but several synthetic waters where also satisfactorily tested.In the following container (2) there is a beater that shreds the M. oleifera waste cake to prepare the coagulant/flocculants mixing the M. oleifera with water that is dosed through a Venturi tube to the water to treat.The residual cake used comes from a process that presses M. oleifera seeds to obtain oil.The process to obtain oil is simple, using standard presses having holes in the lower part that allow the oil to flow while capturing the rests, although other possible continuous presses could be used [14].This residual cake may have big parts of seeds, in consequence, the prototype incorporates the aforementioned beater.
As it is visible in Fig. 1, this container counts with a mixer to prepare the coagulant based on the Moringa oleifera residual cake but it also has a 0.45 µm glass fiber filter.This is done in the central tanks, circled in orange in Fig. 1 with a dosage of coagulant of 100 mg/L for turbidities between 30 and 150 NTU.For better results, the system may incorporate sodium chloride (NaCl) 0.25 M to the M. oleifera coagulant in tank number 3. If another coagulant should be used, the system should inject the predefined dose of this other coagulant in this container.This mix is pumped (manually or electrically) into the elevated tank (number 5 in Fig. 1) where water follows the processes of fast (150 rpm) and slow (20 rpm) agitation for 10 and 30 min respectively.After that, there is 1 h resting period to facilitate the natural sedimentation.Containers 4 and 5 are dimensioned so the slow agitation is done naturally in the manual configuration thanks to the entrance at 90º of the tube that has a length of 1 m and a diameter of 20 cm.Treated water is stored in container 6.When purged, all containers send the residual mud to container 7. The purge operation is defined to be done once every 3 treatment batches.The different size and
M.M Rafique et al. / Desalination and Water Treatment (2017) 1-8 3
positioning of the containers respond to the possibility to use the plant in a manual or electrically powered mode.In this prototype plant there are two optional tanks, the first one contains water to treat and the last one (the one more to the left in Fig. 1) will accumulate slot.This process is better explained in the results section.
With the presented configuration, the prototype is able to work in totally isolated areas using the manual configuration (having no instant control of the purification quality).In electrically isolated areas the system could be powered by solar panels and a battery stack using wireless systems to remotely communicate with the system controller (such as the ones used in mobile phones).Finally, in locations where it can be connected to the electricity grid there is no need to install the solar panels and batteries.
The study continues with the implementation and programming of the SCADA that manages and controls the water purification plant.A representation of the piping and instrumentation diagram (P&ID) is visible in Fig. 2.This diagram shows how water is pumped and how it is transferred from one container to another by pipes and valves.Notice that the coagulant/flocculants, consisting in the residual cake of the process of oil extraction from M. oleifera seeds, is grinded and then mixed with water.To dose and program the agitation time and pauses so the purification and disinfection processes are done correctly, the system uses a table relating these parameters according to the degree of turbidity of the water to treat.
As shown in Fig. 2, the system counts on sixteen valves, three electric motors (one for the beater that shreds the residual cake, another to prepare the coagulant and the last one for the fast and slow agitation), five ultrasonic sensors to control the water level in the containers and one turbidity sensor (although another one is expected to be incorporated in the nearby future).
All these elements are also visible in Table 1 together with a description of their use and the PIN used in the Arduino Mega board (which is presented in the results section).All these variables are essential for the correct use of the purification plant under the remote mode.
The control of the water treatment plant counts on the low cost micro controller Atmega 2560, which is powerful enough and it is easy to integrate to SCADA systems following industrial communication protocols [15].Additionally, Atmega 2560 is an open source hardware installed in an Arduino Mega board.
For communications between the automated water treatment plant and user, the network counts on a master (web platform), in charge of the reception and sending of orders from and to the screen controller.As depicted in Fig. 3, the communication between these two devices can be done by LAN or WAN network using the Web sockets internet communication protocol detailed in the document RFC 6455 [16].
The control interface (HMI) and the data storage follow a LAMP (Linux Apache Mysql) architecture due to the collection of open source elements that it can combine to create all type of Web applications [17].In our case, the web server is Apache, the database is Mysql managed by Phpmyadmin and for the rest of control applications the system uses Java, due to a higher robustness, stability [18] and security in comparison to PHP [19].
The web platform is configured in a server on the TR5 building in the Terrassa Campus of the Universtitat Politècnica de Catalunya.All the aforementioned structure is implemented between this server and the water purification pilot plant installed in the rooftop of this same building.
Finally, the HMI interface design was done in the integrated development environment of Netbeans that is compatible with Java.Netbeans is a robust and user friendly
M.M Rafique et al. / Desalination and Water Treatment (2017) 1-8 5
environment that supports several program languages and web applications.
To dimension the electricity generation system, the study measured the consumption and power of the SCADA, sensors, motors and actuators of the water purification plant.For the energy storage, the study takes into account that the systems should be able to work for 2 d even though there is no sun in the cases where the plant is located in isolated places.However, being the actual implementation of the water treatment plant on the rooftop of the R5 building of the Terrassa Campus of the UPC, there is no need to install that amount of solar panels or storage capacity, as the treatment plant may take the energy from the electricity grid if necessary.In consequence, an energy management system (EMS) was specially designed to take the maximum profit of the use of energy.The fact that the prototype re-uses EV battery modules permitted a reduction in the costs and to provide an additional value to a waste from the automotive sector that still has an 80% of its initial capacity [20] The last part of the study presents a comparison of the raw and treated water characteristics to evaluate the efficiency of the proposed process based on previous works that studied the life cycle assessment of this methodology in Burkina Faso using two coagulants: the traditional aluminum sulfate (Al 2 (SO 4 ) 3 ) and M. oleifera.
Results
A representative scheme of the water treatment process including all the control and automation elements is visible in Fig. 4. As it can be appreciated, the minimized system counts on four tanks.Tank 1 and 2 are needed to shred the M. oleifera and to prepare the coagulant respectively.Then, by means of a Venturi tube, the coagulant/ flocculants is dosed naturally when water to treat passes through a transversal tube.This mix enters into the third tank, where the processes of fast and slow agitation (15 min for the fast agitation and 1 h in slow agitation mode) takes place.During this process, the coagulant/ flocculants catch the suspended solids that fall to the bottom of the tank by decantation.The fourth tank is conceived to store all treated and drinkable water.It can be also appreciated that there is a purgative system in all tanks to clean them.
The SCADA counts on an Arduino mega 2560 together with an Ethernet Wiznet W5100 board, which characteristics are presented in Table 2.
M.M Rafique et al. / Desalination and Water Treatment (2017) 1-8 6
Both boards were assembled to build the controller.These boards and the developed algorithm permit the control of all sensors and actuators of the system.The Ethernet board assigns to the Arduino card an IP direction and configures it with the protocol TCP-IP to allow remote access through an intranet or by internet.
All the other sensors in the treatment plant are described below: • Ultrasonic module HC-SR04: Precision distance 2-450 cm, measurement angle <15º and working voltage of 5 V • Turbidity meter: Analogic 0-5 V exit signal • End of movement: incorporated to the electro-valves.
Regarding the actuators, the system counts with: • Water pump Calpeda MMM 1/AE: Nominal power of 0.37 kW (0.5 HP) and a flow of 1-4.2 m 3 /h and a range of height of 16.3-22 m. • Electric beater Novital: Nominal power 0.72 kW, diameter 2.5, 4, 6 and 8 mm.• Mixer Rubimix9: Nominal power of 1.2 kW, two velocities: 620 rpm y 820 rpm.• Electro valves: Voltage 12 V DC and 1.8 A current.Some laboratory tests were performed to evaluate the automation system.In the first place the system was built substituting sensors by power regulators, LEDs and pushers as Fig. 5 (left) shows.This process was useful to validate the electric schema (Fig. 5
right).
Then, to validate and verify the program, all sensors and actuators where added one by one independently until it was clear that the system was able to control all of them correctly.More details of the plant are accessible in [21].Finally, tests where done in the definitive circuit and the whole device was installed to the prototype water treatment plant.
To design the HMI, a client, server and controller were defined.The controller receives the state values from sensors and actuators through the messages from the client (the purification plant in this case).The server sends HTML code to present results graphically and stores the operations in a database.
Communications through web socket between the server and the controller of the plant were verified prior to its final implementation.The system refreshes the parameters that rule the plant every one second.In fact, shorter refreshing periods entailed problems in the reception and sending of messages.Thus, as the water treatment process does not require fast responses, this delay in time response is acceptable.
In the actual implementation on the rooftop of the R5 building at the university, when the water treatment plant works on automatic mode, it is able to take the energy from the different power sources.These sources are: solar panels, 2 nd life EV batteries and the electricity grid that powers the entire building.The EMS choses the power source by calculating the best economic option taking into account the building and plant consumptions together with the electricity tariff, as graphically depicted in Fig. 6.
The water treatment plant is capable to treat 1000 l of water in about 2 h.Thus, the energy consumption of the plant, taking into account all the elements described and that it should work 16 h to produce 8000 l of drinkable water, is close to 11,178 kWh per day.
In such circumstances, considering the characteristics of the installed solar panels (210 W/m 2 ) and that it should be able to work for almost 2 days without any additional power supply in the case of being installed in isolated locations, the plant needs an extension of 40 m 2 of solar panels and a capacity of the battery to store near 22 kWh, which is almost the average of the actual battery capacity of commercial EV.
Finally, a summary of the raw and treated water characteristics presented in further detail in [23] is described to verify the goodness of the process.
M.M Rafique et al. / Desalination and Water Treatment (2017) 1-8 7
The dosage of coagulant applied was based on the studies carried out by Pritchard et al. [24] and by Bhuptawat et al. [25] multipurpose tree whose seeds contain a high quality edible oil (up to 40% by weight, which is comprised between 25 mg/L, 50 mg/L and 100 mg/L for turbidities within the range of 30 and 150 NTU.These studies demonstrated that concentrations of 100 mg/L reached a 95% of turbidity removal, which was the initial concentration selected to carry out the tests (Table 3).
As it is appreciable in Table 3, the lower concentrations of M. oleifera and alum did not comply with the international Standards.In consequence, to increase the effectiveness of M. oleifera as a natural coagulant, the addition of 0.25 M of sodium chloride (NaCl) increased the turbidity removal dramatically (going from 40 NTU to 3.3 NTU).In addition, it should be mentioned that the pH of water was not altered by M. oleifera, and its impact on conductivity is minimal, being still into the international standards [8].
Moreover, Fig. 7 compares the visual aspect of the raw water used in the tests against the treated water.As it can be observed, the process clearly improves the turbidity of the sample.The chemical analysis done afterwards confirmed the good response of the process.
As mentioned above, the capacity of M. oleifera as coagulant/flocculants is clearly boosted when mixing it with 0.025 M of sodium chloride (NaCl).
From the experience in developing this prototype, there are some aspects that should be considered for its future replicability.For example, the addition of an UPS (uninterrupted power supply) is recommended to avoid the loss of data or control of the plant in case of shutdown.Secondly, communication messages through web socket are limited to 120 characters, which is too low for some applications that might need auxiliary systems to avoid collapse and connection loses.
Conclusions
This study presents a remote control water purification plant that can work either in automated or manual mode.The plant uses the residual cake from the oil extraction process from M. oleifera seeds.This method provides and additional value to the waste from oil production process to treat water.
This study presents the SCADA structure, the elements of the plant and the HMI that allows the remote control of the water purification plant.Additionally, the system was designed to be autonomous, sustainable and compact.To do so, the project took advantage of solar panels and lithium ion batteries.In fact, these batteries come from old electric vehicles, when they are not useful for traction purposes.Therefore, this is another step of re-valorization of waste, in this case, from the automotive sector.
Thus, the final design, exploitation and maintenance costs are low.In the first place, the investment costs are low because most of the elements used where conceived to be inexpensive (containers, software, re-used batteries…).Secondly, because the system uses a waste product as the principal input material, not needing additional expenses for the plant to work.
The compact water treatment plant is able to treat 1000 L of water every 2 h, and it proved to give satisfactory results water treatment.
Fig. 1 .
Fig. 1.Image of the water purification prototype plant.
Fig. 2 .
Fig. 2. Piping and instrumentation diagram of the water purification plant.
Fig. 3 .
Fig. 3. Scheme of the network used for the communications between the user and the plant controller.
Fig. 4 .
Fig. 4. Screen shot of the web representation of the water treatment plant.
Table 1
Detail of the variables in the web platform, Pin used in the Arduino board and identification in the P&ID | v3-fos-license |
2019-05-17T13:08:36.165Z | 2019-05-01T00:00:00.000 | 155101241 | {
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} | pes2o/s2orc | Bioassay-Guided Isolation of Triterpenoids as α-Glucosidase Inhibitors from Cirsium setosum
Cirsium setosum (C. setosum) has a potential antihyperglycemic effect, but it is unclear what bioactive components play a key role. According to the α-glucosidase inhibition activity, three new taraxastane-type triterpenoids of 3β-hydroxy-30-hydroperoxy-20-taraxastene (1), 3β-hydroxy-22α-methoxy-20-taraxastene (2), and 30-nor-3β,22α-dihydroxy-20-taraxastene (3), as well as five known taraxastane triterpenoids of 3β,22-dihydroxy-20-taraxastene (4), 20-taraxastene-3,22-dione (5), 3β-acetoxy-20-taraxasten-22-one (6), 3β-hydroxy-20-taraxasten-22-one (7), and 30-nor-3β-hydroxy-20-taraxastene (8) were obtained from the petroleum ether-soluble portion of the ethanol extract from C. setosum. All chemical structures of the compounds were elucidated by spectroscopic data analysis and compared with literature data. Compounds 4–8 were identified for the first time from this plant, and compounds 1, 2, 4, and 7 exhibited more potent α-glucosidase inhibitory activity—with IC50 values of 18.34 ± 1.27, 26.98 ± 0.89, 17.49 ± 1.42, and 22.67 ± 0.25 μM, respectively—than acarbose did (positive control, IC50 42.52 ± 0.32 μM).
The purpose of this study is to explore new α-glucosidase inhibitors (AGIs) from C. setosum. In a bioassay-guided fractionation of an EtOH extract of C. setosum, we found that the petroleum ether-soluble fraction showed potent α-glucosidase inhibitory activity. Further separation from the above inhibitory activities component against α-glucosidase resulted in the isolation of eight triterpenoids inhibitors. Among these eight compounds, three are new structures and two are found to be more active than the acarbose that is available clinically. This work elucidates the relationship between triterpenoids constituents and hypoglycemic functions of C. setosum. Findings of this study contain important empirical implications in terms of developing future hypoglycemic functional food and improving its quality standards.
Results and Discussions
The crude extract of stems of C. setosum was suspended in H 2 O and then partitioned with petroleum ether and EtOAc. Our random bioassay revealed that the petroleum ether-soluble portion had the highest activity against α-glucosidase, with an inhibitory rate of 87.6 ± 1.23% (300 µg/mL). Bioassay-guided isolation yielded eleven fractions (Sh1-Sh11) via silica gel column chromatography, eluting with a gradient of acetone (0-100%) in petroleum ether (60-90 • C). Fraction Sh8 showed significant activity against α-glucosidase, with an inhibitory rate of 99.2 ± 2.19% (300 µg/mL). Fraction Sh8 was further isolated by the combination of silica gel column chromatography, low pressure liquid chromatography, Sephadex LH-20 chromatography, and high-performance liquid chromatography (HPLC), generating three new (1-3) and five known (4-8) compounds ( Figure 1).
Molecules 2019, , x FOR PEER REVIEW 2 of 10 The purpose of this study is to explore new α-glucosidase inhibitors (AGIs) from C. setosum. In a bioassay-guided fractionation of an EtOH extract of C. setosum, we found that the petroleum ethersoluble fraction showed potent α-glucosidase inhibitory activity. Further separation from the above inhibitory activities component against α-glucosidase resulted in the isolation of eight triterpenoids inhibitors. Among these eight compounds, three are new structures and two are found to be more active than the acarbose that is available clinically. This work elucidates the relationship between triterpenoids constituents and hypoglycemic functions of C. setosum. Findings of this study contain important empirical implications in terms of developing future hypoglycemic functional food and improving its quality standards.
Structural Elucidation of the Three New Compounds
Compound 1 was obtained as a white amorphous powder. The IR spectrum of 1 suggested that it contained hydroxyl groups (3417 and 3165 cm −1 . The 13 C-NMR spectrum displayed 30 carbon signals, which were classified as seven methyls, ten methylenes (one oxygenated), seven methines (one oxygenated and one olefinic), and six quaternary carbons (one olefinic carbon) on the basis of DEPT and HSQC spectra. These data suggested that 1 was very similar, with one known 30-hydroperoxy-ψ-taraxasteryl acetate [18], except for lacking acetate group located at C-3, which was confirmed by the comprehensive analysis of the 2D NMR spectra of 1, especially 1 H-1 H COSY and HMBC ( Figure 2). carbons, the other functional groups and the above five structural fragments was mainly achieved by the analysis of the HMBC spectrum ( Figure 2). HMBC correlations from 3H-23 (H 1.25) to C-5, C-3 and C-24, and from 3H-24 (H 1.06) to C-4, C-3, C-5, and C-23 indicated that Me-23 and Me-24 were attached to C-4. The HMBC correlations of 3H-25 (H 0.91) to C-5, C-1, C-10, and C-9; 3H-26 (H 1.00) to C-9, C-7, and C-8; 3H-27 (H 0.97) to C-13, C-15, and C-8; 3H-28 (H 0.96) to C-16, C18, and C-22; and 2H-30 (H 4.66 and 4.91) to C-19, C-20, and C-21 not only confirmed the presence of A/B/C/D/Ering systems but also located the Me-25, Me-26, Me-27, Me-28, and -CH2OOH-30 at C-10, C-8, C-14, C-17, and C-20 respectively. The structure of 1 was, therefore, determined as 3β-hydroxy-30hydroperoxy-20-taraxastene. Compound 2 was obtained as a white amorphous powder. The presence of hydroxyl groups (3362 cm −1 ) and a double bond (1673 cm −1 ) functionalities were evident in its IR spectrum. Its molecular formula was deduced as C31H52O2, from the negative HRESIMS at m/z 455.3890 [M − H] − (calcd for C31H51O2 455.3895) and 13 C-NMR spectrum. This indicated six degrees of unsaturation. The 1 H and 13 C-NMR spectra of 2 were very similar to those of compound 4, a known 3β,22α-dihydroxy-20-taraxastene that was also isolated from this plant [19], with the only difference being the replacement of the hydroxyl group by a methoxy moiety at C-22 (Table 1). This inference was confirmed by the HMBC correlation of 3H-OMe/C-22. The configuration of H-22 was assigned as βequatorial on the basis of the coupling constant (5.8 Hz) with the vicinal olefinic proton H-21 and the NOESY correlation with Me-28. Thus, compound 2 was deduced to be 3β-hydroxy-22α-methoxy-20taraxastene.
Compound 3, a white amorphous powder, had the formula of C29H48O2 on the basis of the negative HRESIMS at m/z 427.3585 [M − H] − (calcd for C29H47O2 427.3582) and the 13 C-NMR spectrum. The IR spectrum showed absorption bands at 3656, 3405, 1657, and 1607 cm -1 due to the hydroxyl groups and double bond. The NMR spectra of 3 and a known 3β,22α-dihydroxy-20-taraxastene (compound 4, which was also isolated from this plant) were closely comparable [20], with the only difference being the lack of a methyl group at C-20. The structure of 3 was confirmed by the 2D NMR HSQC, COSY, HMBC, and NOESY data. The NOESY correlation of Me-28 with H-22, and the coupling constant (6.0 Hz) of H-22 with the vicinal olefinic proton H-21 indicated that H-22 was βoriented. The structure for 3 was thus assigned as 30-nor-3β,22α-dihydroxy-20-taraxastene.
Compound 2 was obtained as a white amorphous powder. The presence of hydroxyl groups (3362 cm −1 ) and a double bond (1673 cm −1 ) functionalities were evident in its IR spectrum. Its molecular formula was deduced as C 31 H 52 O 2 , from the negative HRESIMS at m/z 455.3890 [M − H] − (calcd for C 31 H 51 O 2 455.3895) and 13 C-NMR spectrum. This indicated six degrees of unsaturation. The 1 H and 13 C-NMR spectra of 2 were very similar to those of compound 4, a known 3β,22α-dihydroxy-20-taraxastene that was also isolated from this plant [19], with the only difference being the replacement of the hydroxyl group by a methoxy moiety at C-22 (Table 1) The IR spectrum showed absorption bands at 3656, 3405, 1657, and 1607 cm -1 due to the hydroxyl groups and double bond. The NMR spectra of 3 and a known 3β,22α-dihydroxy-20-taraxastene (compound 4, which was also isolated from this plant) were closely comparable [20], with the only difference being the lack of a methyl group at C-20. The structure of 3 was confirmed by the 2D NMR HSQC, COSY, HMBC, and NOESY data. The NOESY correlation of Me-28 with H-22, and the coupling constant (6.0 Hz) of H-22 with the vicinal olefinic proton H-21 indicated that H-22 was β-oriented. The structure for 3 was thus assigned as 30-nor-3β,22α-dihydroxy-20-taraxastene.
α-Glucosidase Inhibitory Activity of the Isolates
All the isolates were evaluated for their α-glucosidase inhibitory activities using p-nitrophenyl-α-dglucopyranoside (p-NPG) as the substrate and acarbose as the positive control ( Table 2). All of the eight compounds that showed inhibitory rates higher than 50% at the concentration of 100 µM, were further evaluated for their IC 50 values. As shown in Figure 3, Figure 4, and Table 2, IC 50 values of the eight compounds were in the range of 18.34 to 80.07 µM. were further evaluated for their IC50 values. As shown in Figure 3, Figure 4, and Table 2, IC50 values of the eight compounds were in the range of 18.34 to 80.07 μM. The tested concentration of all samples was 100 μM, IC50 values represent the concentrations that caused 50% activity loss. The value of each activity is expressed as mean SD (n = 3). were further evaluated for their IC50 values. As shown in Figure 3, Figure 4, and Table 2, IC50 values of the eight compounds were in the range of 18.34 to 80.07 μM. The tested concentration of all samples was 100 μM, IC50 values represent the concentrations that caused 50% activity loss. The value of each activity is expressed as mean SD (n = 3).
Plant Material
The
General Experimental Procedures
The HRESIMS data were generated on a Thermo QE UPLC-Orbitrap MS spectrometer (Thermo Scientific Inc., Waltham, MA, USA). The specific rotations data were obtained with a Rudolph Research Autopol III automatic polarimeter (Rudolph Research Analytica, Hackettstown, NJ, USA). The UV data and circular dichroism spectra were recorded on a JASCO J-810 circular dichroism spectrometer (JASCO Corporation, Tokyo, Japan). IR spectra were acquired on a Nicolet Impact 400 FT-IR spectrophotometer (Nicolet Instrument Inc., Madison, WI, USA). 1D-and 2D-NMR spectra were acquired in C 5
Extraction and Isolation
The air-dried stems of Cirsium setosum (Willd.) (10 kg) were ground into powder and extracted with 90%, 80%, and 70% aqueous EtOH sequentially at room temperature for 120 min under sonication. The extract was evaporated under reduced pressure to yield a dark brown residue, which was suspended in H 2 O and then partitioned with petroleum ether and EtOAc. The petroleum ether-soluble portion (468.5 g) was fractionated via silica gel column chromatography, eluting with a gradient of acetone (0-100%) in petroleum ether (60-90 • C), to give eleven fractions (Sh1-Sh11).
α-Glucosidase Inhibitory Effect Assay
The α-glucosidase inhibitory assay was carried out spectrophotometrically, according to the previously described method, with slight modifications, in which acarbose was used as the positive control [23].
A total of 200 µL of reaction mixture, containing 70 µL of 0.1 M phosphate buffer (pH 6.8), 10 µL of 1.0 mg/mL reduced glutathione solution, and 10 µL of the sample solution (test concentration at 0.1 mg/mL), was added to each well of a 96-well plate, followed by the addition of 20 µL of 0.5 U/mL α-glucosidase solution. The plate was incubated at 37 • C for 15 min, and then 20 µL of p-Nitrophenyl α-d-glucopyranoside substrate was added to the mixture to start the reaction. The reaction mixture was incubated at 37 • C for 30 min, and then 70 µL of 0.1 M Na 2 CO 3 solution was added to the mixture to terminate the reaction. All samples were analyzed in triplicate with three different concentrations near the IC 50 values. The absorbance (A) was immediately recorded at 400 nm, using a spectrophotometrical method to estimate the enzymatic activity. The inhibition percentage was calculated by the following equation: Inhibitory rate (%) = [1 − (A test − A blank )/(control A test − control A blank )] × 100%.
Here, A test represents the absorbance value of the experimental sample, A blank represents the absorbance value of sample blank, control A test represents the absorbance value of the control, and control A blank represents the absorbance value of the blank.
The results suggest that triterpenoids from C. setosum could be the key and potential functional food ingredients for a new antidiabetic agent. Due to the relatively high contents and potent α-glucosidase inhibitory activity of compounds 1, 2, 4, and 7 in C. setosum, we speculated that those four compounds could be the main bioactive components responsible for the α-glucosidase inhibitory effect of C. setosum. This work provides a scientific basis for the development of C. setosum as a hypoglycemic functional food, and also a theoretical basis for the establishment of a quality test method for the bioactivity factor of C. setosum as a dietary supplement for hypoglycemic products.
Supplementary Materials: The following are available online, IR, UV, HRMS and NMR spectra of compounds 1-8 as well as other supporting data. | v3-fos-license |
2018-04-03T01:08:23.318Z | 2017-01-23T00:00:00.000 | 18564439 | {
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} | pes2o/s2orc | The Biomineralization of a Bioactive Glass-Incorporated Light-Curable Pulp Capping Material Using Human Dental Pulp Stem Cells
The aim of this study was to investigate the biomineralization of a newly introduced bioactive glass-incorporated light-curable pulp capping material using human dental pulp stem cells (hDPSCs). The product (Bioactive® [BA]) was compared with a conventional calcium hydroxide-incorporated (Dycal [DC]) and a light-curable (Theracal® [TC]) counterpart. Eluates from set specimens were used for investigating the cytotoxicity and biomineralization ability, determined by alkaline phosphatase (ALP) activity and alizarin red staining (ARS). Cations and hydroxide ions in the extracts were measured. An hDPSC viability of less than 70% was observed with 50% diluted extract in all groups and with 25% diluted extract in the DC. Culturing with 12.5% diluted BA extract statistically lowered ALP activity and biomineralization compared to DC (p < 0.05), but TC did not (p > 0.05). Ca (~110 ppm) and hydroxide ions (pH 11) were only detected in DC and TC. Ionic supplement-added BA, which contained similar ion concentrations as TC, showed similar ARS mineralization compared to TC. In conclusion, the BA was similar to, yet more cytotoxic to hDPSCs than, its DC and TC. The BA was considered to stimulate biomineralization similar to DC and TC only when it released a similar amount of Ca and hydroxide ions.
Introduction
Bioactive glass-based materials have been introduced as promising hard/pulp tissue regenerative materials in dental fields, not only because of their apatite-forming ability, but also for their biomineralization ability for hard tissue (i.e., bone, enamel, dentin, and cementum) formation [1,2]. To date, these materials have been applied to various types of dental materials, such as dental sealants/composite resins, bone or dental tissue scaffold matrices, and regenerative endodontic materials [3][4][5][6][7][8]. Recently, a bioactive glass-incorporated light-curable pulp capping material (pulp regenerative material, Activa6 Bioactive, Pulpdent, Watertown, MA, USA) has been introduced as the first bioactive dental product approved by the FDA in terms of bioactivity for indirect pulp capping materials; it provides quick setting times of less than 20 s for clinical convenience and biomineralization ability for hard tissue regeneration, and it has excellent biocompatibility [9].
Several in vitro and in vivo studies have evaluated the cytotoxicity, biocompatibility, or biomineralization ability of bioactive dental materials [10,11]. Many studies have determined whether the mineral trioxide aggregate-(MTA-) like light-curable bioactive pulp capping materials can promote biomineralization to the same degree as conventional calcium hydroxide-incorporated chemically curable pulp capping materials, which have been considered the "gold standard" for approximately 20 years [12][13][14]. However, there is still a need for confirmatory testing with ionic supplement solutions that have the same amount of ions from extracts to confirm the role of biomineralization from released ions. In addition, there are newly developed bioactive glass-incorporated lightcurable pulp capping materials that are still being investigated for their cytotoxicity and biomineralization compared with calcium hydroxide-incorporated chemically curable pulp capping materials [9].
Biomineralization, including alkaline phosphatase activity and mineral deposition from dental pulp stem cells Blend of diurethane and other methacrylates with modified polyacrylic acid (∼53.2%), silica (∼3.0%), sodium fluoride (∼0.9%), and so on * Composition was filled with the manufacturers' available information including material safety data sheets.
(DPSCs), is an essential biofunctional characteristic of pulp capping materials [15]. When the dentin-pulp complex is damaged by dental caries or traumatic injuries, clinicians' efforts should allow the regenerated pulp tissue to be functionally competent, capable of forming mineralized tissue quickly to repair lost structure and to seal the clean pulp environment from the potentially contaminated external oral environment [16].
Reports have shown that isolated human DPSCs (hDP-SCs) from extracted human teeth can be induced to differentiate into odontoblast-like cells in odontoblastic differentiating media and produce dentin-like mineral structures in vitro [17]. Therefore, they are widely used in investigations of dentin-pulp regeneration due to their clinical mimicking of pulp origin and their easy accessibility, which allows them to be readily isolated from extracted teeth [18][19][20]. However, to the best of our knowledge, no studies have been carried out to determine whether the newly developed bioactive glass-incorporated light-curable pulp capping material can promote biomineralization of hDPSCs or to determine the key mediators (possibly ions) required to induce biomineralization.
Therefore, the aim of this study was to perform a comparison of the cytotoxicity and biomineralization capability of the newly developed bioactive glass-incorporated lightcurable pulp capping material with the calcium hydroxideincorporated chemically curable product as the calcium releasing gold standard control and the MTA-like incorporated light-curable pulp capping material as the light-curable counterpart. In addition, the key players (ions) necessary for biomineralization induction were studied to investigate the underlying mechanism. Table 1. Each pulp capping material was applied in a Teflon mold, which was 10 mm in diameter and 2 mm in height. TC was dispensed from a syringe and applied in two layers of 1 mm in depth, and each layer was light cured with an LED light curing instrument (Litex 695, Dentamerica Inc., Industry, CA, USA) for 20 s according to the manufacturer's instructions (curing 1 mm depth per 20 s of light curing). BA was mixed using the dispensing syringe, added to the mold, and cured for 20 s using a curing light (Litex 695) according to the manufacturer's instructions (light curing setting time of 20 s for 4 mm depth of cure). Chemically self-cured calcium hydroxide liner (DC) was mixed according to the manufacturer's protocol, applied to the mold, and incubated at room temperature (23 ∘ C) with 50% relative humidity for 3.5 minutes (thermo-hygrometer, Daihan, Wonju, Korea). All procedures were performed on a sterilized clean bench to prevent any possible contamination. Each specimen from the mold was sterilized by ethylene oxide gas before further experiments.
Collection of Extract.
Each pulp capping specimen was extracted at a ratio of 3 cm 2 /mL following the recommendations of ISO 10993-12 using distilled water (DW) [21]. Because the total surface area of the sample was 2.2 cm 2 , the samples were incubated in 0.73 mL of DW. To simulate the clinically adjustable environment, the eluates of all specimens were collected at 37 ∘ C for 24 hours in a shaking incubator (120 rpm).
Primary
Culture of hDPSCs. The hDPSCs were from extracted third molars and were used after less than 10 passages throughout the experiments. Approval for their use was received from the institutional review board of Dankook University Dental Hospital (IRB number H-1407/009/004). Pulp tissues were antiseptically collected and added to phosphate-buffered solution (PBS) (Gibco, Grand Island, NY, USA) containing 1% penicillin/streptomycin (Gibco). After the addition of 0.08% collagenase type I (Worthington Biochemical, Lakewood, NJ, USA), the solution was incubated for 30 minutes with tapping every 10 minutes. Following enzymatic digestion, the hDPSCs were recovered by centrifugation at 1,500 rpm for 3 minutes. The cells were cultured in a humidified atmosphere of 5% CO 2 at 37 ∘ C with supplemented media consisting of -MEM with 10% fetal bovine serum (Gibco), 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA), 2 mM GlutaMAX (Gibco), and 0.1 mM L-ascorbic acid (Sigma Aldrich, St. Louis, MO, USA). All culture systems adhered to the above conditions. The stem cell functionality of hDPSCs was confirmed by positive expression (over 90%) of CD73, CD105, and CD106, and negative expression (less than 1%) of CD34 and CD45 by flow cytometry (data not shown). To promote biomineralization of the hDPSCs, we cultured the cells in odontogenic supplemental medium (OM) that included 50 g/mL ascorbic acid, 100 nM dexamethasone, and 10 mM -glycerophosphate, as described previously [22].
Cell Viability.
The cell viability test was performed according to ISO 10093-5 [23]. Briefly, 100 L of 1 × 10 5 cells/mL were cultured in each well of a 96-well plate (SPL Life Sciences, Pocheon, Gyeonggi-do, Korea) with supplemented media (described in Section 2.3) in a humidified atmosphere of 5% CO 2 at 37 ∘ C for 24 hours. After being washed with PBS (200 L), the cells were cocultured with 50 L of supplemented media and 50 L of extract or serially diluted extract by DW for another 24 hours. The percentages of the final concentrations of extract in the culture media were 50%, 25%, 12.5%, 6.25%, and 3.125%, and a mixture of 50 L of DW with 50 L supplemented media was used as the control (0%).
Cell viability was assessed with the MTS assay (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega, Madison, WI, USA) according to the manufacturer's protocol, and the results are expressed as the optical density percentage of each test group ( = 6) compared with each control group ( = 6). The optical absorbance was measured by a microplate reader (SpectraMax M2e, Molecular Devices, Sunnyvale, CA, USA) at a wavelength of 490 nm. To determine the toxicity of the tested pulp capping materials, a live-dead assay was performed. After the culture medium was removed, the cells were rinsed with PBS and incubated for 30 minutes with calcein AM (0.5 M) and ethidium homodimer-1 (4 M, Molecular Probes, Eugene, OR, USA). Images of live and dead cells were acquired using confocal laser scanning microscopy (CLSM, LSM700, Carl Zeiss, Thornwood, NY, USA) to confirm the above cell viability data. Live cells were identified by the presence of intracellular esterase activity, determined by the enzymatic conversion of the nonfluorescent cell-permeable calcein AM to green fluorescent calcein, which is retained within live cells. Dead cells were identified as stained with red fluorescent ethidium homodimer-1, indicating the loss of cellular mem-brane integrity. All analyses were independently performed in triplicate, and the representative means ± standard deviations (SD) or images are shown.
Alkaline Phosphatase Assay.
Biomineralization was firstly estimated using an alkaline phosphatase (ALP) activity assay. ALP is an early biochemical marker for biomineralization. To measure ALP activity, we incubated noncytotoxic diluted extract (12.5%) with 1.2 mL of 10 5 /mL of hDPSCs in each well of a 12-well plate containing supplemented media or OM. Every 3 days, the media was replaced with fresh diluted extract (12.5%) conditioned OM, with the addition of DW (12.5%) for the positive control and the addition of DW (12.5%) to the supplemented media for the negative control. At days 14 and 21, the medium was removed, and 1 mL of 0.2% Triton X-100 (Sigma Aldrich, St. Louis, MO, USA) was added to each well. After the cell lysates were placed in a 1.5-mL centrifuge tube, the samples were processed through three freeze-thaw cycles (−70 ∘ C and room temperature, 45 minutes each) to rupture the cell membranes and extract the proteins and DNA from the cells, which were then centrifuged for 20 minutes at 13,000g at 4 ∘ C. Using the supernatant, alkaline phosphatase (ALP) activity ( = 6) was measured using pnitrophenyl phosphate (at a final concentration of 20 mM, Sigma Aldrich) as the substrate in 1.5 M 2-aminomethyl-1-propanol (pH 10.3, Sigma Aldrich) and 1.0 mM MgCl 2 (Sigma Aldrich). The absorbance at 405 nm was measured with a plate reader (SpectraMax M2e) and was normalized to the dsDNA quantity measured by a PicoGreen assay ( = 6); dsDNA from the supernatant was quantified using the Quant-iT PicoGreen Kit (Invitrogen) following standard protocols. Briefly, 100 L of each cell lysate solution was added to 100 L of PicoGreen reagent and incubated in the dark at room temperature for 5 minutes. The absorbance was read at an excitation/emission of 480/520 nm on a plate reader (SpectraMax M2e). All analyses were independently performed in triplicate, and the representative means ± SD are shown.
2.6. Alizarin Red Staining. Alizarin red staining (ARS) staining was performed to determine the mineralization ability of the noncytotoxic diluted extract (12.5%) using 1.2 mL of 10 5 /mL hDPSCs in each well of a 12-well plate. Every 3 days, the media was replaced with fresh diluted extract (12.5%) conditioned OM; then, DW (12.5%) was added to the OM as a positive control, and DW (12.5%) was added to the supplemented media as a negative control. After 21 days of incubation with supplemented media or OM, with the media being changed every 3 days, the cells were rinsed, fixed with 10% formaldehyde for 30 minutes, and stained with 40 mM alizarin red S (pH 4.2) for 30 minutes. After staining, the morphology was observed using light microscopy (Olympus lX71, Shinjuku, Tokyo, Japan). Quantitative analysis was carried out after the addition of 10% cetylpyridinium chloride (Sigma Aldrich) in 10 mM sodium phosphate (pH 7.0) for destaining. The concentration of ARS was determined by an absorbance measurement at 562 nm on a microplate reader (SpectraMax M2e). All analyses were independently performed in triplicate, and representative means ( = 5) ± SD or images are shown.
Detection of Calcium and Silicon
Ions. The release of calcium (Ca), silicon (Si), or other possibly detected cationic ions (Zn, Ba, Na, Ti, and Fe) according to the composition of the products was measured from the extract with inductively coupled plasma atomic emission spectrometry (ICP-AES) (Optima 4300DV, Perkin-Elmer, Waltham, MA, USA), according to a previous protocol [24]. The detection limit was determined to be 0.1 ppm for all cations. To measure the released OH ions, the pH of the original extract and diluted extract in 12.5% with -MEM were measured using a digital pH meter (Orion 4 Star, Thermo Scientific Pierce, IL, USA). Three measurements ( = 3) in each sample were performed, and all analyses were independently performed in triplicate to confirm reproducibility. The representative means ( = 3) ± SD were recorded.
Biomineralization from Released Ions.
To prepare the ionic supplement added to BA, the ions that were differentially released between the TC and BA extract were added to the BA extract using CaCl 2 (Sigma Aldrich) and NaOH (Sigma Aldrich) to match the ionic conditions of TC, which had 110 ppm Ca ions and pH 11. After the ionic supplement was added to BA, both BA and TC were diluted to 12.5% with OM to obtain a noncytotoxic extract, and they were cultured in OM for 21 d. DW diluted to 12.5% with OM was added to the culture with or without OM as negative and positive controls, respectively. The ionic supplement added to DW under the OM culture condition was used for investigating the effect of the ions released ions in the extract on the biomineralization of hDPSCs. Calcium deposition was measured using alizarin red staining (ARS) according to the above ARS section. After ARS was observed under the microscope, 10% w/v cetylpyridinium chloride monohydrate was used for quantitative analysis. The measurements were independently performed in triplicate, and representative averages ( = 5) with SD or images are presented.
Statistical Analysis.
All experiments were independently carried out in triplicate to confirm reproducibility. The data were analyzed using one-way analysis of variance (ANOVA), followed by Tukey's post hoc test. The level of significance was < 0.05. Figure 2: The effects of extracts from light-curable pulp capping materials (TC and BA) as well as the gold standard pulp capping material (DC) on alkaline phosphatase (ALP) activity (at 14 and 21 days) (a) and on calcium deposition using alizarin red staining (ARS) (at 21 days) (b and c). After ARS was observed under the microscope (c), 10% w/v cetylpyridinium chloride monohydrate was used for quantitative analysis (b). The cells were incubated for 7 to 21 days with 12.5% diluted extract in odontogenic supplemental medium (OM) for the differentiation of hDPSCs or supplemented media only for the negative control. Different letters indicate significant differences among the groups at < 0.05. The measurements were performed in triplicate, and representative averages ( = 5) with SD or images are presented.
Cell
showed less than 70% cell viability, which was significantly less than the control (0%). In particular, DC and BA had the lowest cytotoxic effect (almost 10% cell viability) among the groups (Figure 1(a), < 0.05). With incubation in the 25% extract, all groups showed significantly less cell viability than the control (0%, Figure 1(a), < 0.05), and DC had less than 70% cell viability. The 12.5%, 6.25%, and 3.125% groups were not significantly different compared to the control (Figure 1(a), > 0.05). The CLSM images obtained after incubation in 50% extract in the supplemented media with different pulp capping materials confirmed these cell viability results. The results are shown in Figure 1(b). A significant number of dead cells (red) and few live cells (green) appeared in the DC, TC, and BA groups, while the control groups showed only live cells.
3.2.
Biomineralization. ALP activity from the "pulp capping material extract"-treated cells was minimal on day 14 and increased on day 21 (Figure 2). At 14 and 21 days of culture, statistically significant increases in ALP activity were noted in the DC and TC extract (12.5%) treated cells compared with OM-treated control cells (Figure 2(a), < 0.05), but this difference was only observed at 21 days of culture in the BA extract (12.5%). At 21 days of culture, the DC and TC groups had significant ALP activity compared to the BA group (Figure 2(a), < 0.05). Moreover, there was a clear (c and d) After an ionic supplement including the ions that were differentially released between the TC and BA extract was added to the BA extract to match the ionic conditions, the ionic supplement-added 12.5% BA extract was cultured with odontogenic supplemental medium (OM) for 21 days to compare the biological effects of 12.5% TC, BA, and ionic supplement. Calcium deposition was measured using alizarin red staining (ARS). After ARS was observed under the microscope (c), 10% w/v cetylpyridinium chloride monohydrate was used for quantitative analysis (d). The measurements were performed in triplicate, and representative averages ( = 5) with SD or images are presented. Different letters indicate significant differences among the groups at < 0.05. difference in the ARS-stained images from the DC and TC groups compared to the BA-or OM-treated control cells, and quantification of the eluted products revealed a statistically significant difference in the DC and TC groups compared to the other groups (Figures 2(b) and 2(c), < 0.05).
Biomineralization of the Released Ions.
The release of Ca and OH ions was observed to be significantly higher in the DC and TC groups compared to the BA group (Figures 3(a) and 3(b), < 0.05). Other ions, such as Zn, Ba, and Ti, were detected at less than 1 ppm or were not detected. Si and Na ions were detected in all groups but at levels less than 1.5 and 6 ppm, respectively. Ionic supplement-conditioned BA had statistically similar mineralization ability compared to the DC and ionic supplement only groups under OM culture (Figures 3(c) and 3(d), > 0.05).
Discussion
Pulp capping materials have been shown to play a vital role as restorative materials in the successful regeneration of the dentin-pulp complex [3,25,26]. Pulp capping materials have not only pulp sealing effects but also biological properties such as biomineralization, which leads to dentin-pulp complex regeneration [25]. Recently, a bioactive glass-incorporated light-curable pulp capping material was released to the market, but investigations of the biological activities in mammalian cells that result from its use are limited. After evaluating the cytotoxicity compared with calcium hydroxide-incorporated and MTA-like light-curable pulp capping materials, we investigated the biomineralization of a commercially available bioactive glass-incorporated light-curable pulp capping material to determine its potential as a dentin-pulp regenerative material.
Cytotoxicity.
First, the elutes were serially diluted with DW and added to the culture media for 24 hr. The method by which pulp capping materials meet the hDPSCs is via the extract through dentinal tubules, not by direct contact. Therefore (diluted) extract was added to hDPSCs, and cytotoxicity was evaluated. Cytotoxicity over 30% (cell viability less than 70%) was revealed in the 50% culture conditions in all experimental groups and in the 25% culture conditions for DC, most likely due to the alkaline pH (>10) and high concentrations of ions. The calcium hydroxide-incorporated (DC) and MTA-like light-curable (TC) pulp capping materials showed excellent biocompatibility according to in vivo and clinical studies, but they also showed in vitro cytotoxicity in these results. Therefore, we concluded that the newly developed bioactive glass-incorporated light-curable pulp capping material (BA), which revealed similar or greater in vitro cytotoxicity compared to both DC and TC, has relatively biocompatible characteristics. Most of light curing materials including BA are polymerized from monomers with the help of a photoinitiator such as camphorquinone, and when these components are released into the applied tissue, they can be toxic to the surrounding cells. According to the components in BA listed in the MSDS and the polymerizing mechanism, unpolymerized monomers, silica, fluoride ions, and camphorquinone could be among the reasons for the induction of cytotoxicity. Further studies investigating the releasable cytotoxic inducers are needed to comprehend the cytotoxicity of BA. To exclude the potential adverse effect on biomineralization from the cytotoxicity-induced biological cascades, which negatively affect the therapeutic characteristics, a 12.5% culture condition was chosen, which showed less than 30% cytotoxicity in all tested groups at the first time point.
Biomineralization of hDPSCs.
ALP activity and ARS staining, which are well-known biomolecule assays for investigating biomineralization [27], were detected at significant levels in the TC and DC groups compared with the OMtreated control group ( < 0.05) but were not significantly expressed in the BA group ( > 0.05). ALP activity and ARS are correlated with calcium-phosphate matrix formation in dentin prior to the initiation of mineralization [28]. High ALP activity provides high concentrations of phosphate at the site of mineral deposition. ARS is used to quantitatively determine calcific deposition by cells, which is a crucial step toward the formation of the calcified extracellular matrix in a later stage. Differentiated odontoblasts from hDPSCs produce dentin, which consists of calcium and phosphate. Therefore, the above two assays were promising for the evaluation of biomineralization [3]. We concluded that the bioactive glass-incorporated light-curable bioactive pulp capping material has less potential for similar therapeutic effects in terms of biomineralization from hDPSCs compared to calcium hydroxide-incorporated and MTA-like incorporated light-curable pulp capping materials.
Direct comparisons among the above-evaluated products in terms of cytotoxicity and consequent biomineralization is beyond the scope of this discussion; however, compared with the calcium hydroxide-incorporated pulp capping material (DC), the MTA-like light-curable pulp capping material (TC) has shown comparable biocompatibility and biomineralization owing to released Ca and OH ions [29]. However, the bioactive glass-incorporated pulp capping material (BA) did not show comparable biomineralization, possibly due to a lack of released Ca and OH ions. In general, compared with the conventional calcium hydroxide-incorporated pulp capping material, light-curable bioactive pulp capping materials have quick setting times in light-activated systems, which are more clinically beneficial for the restoration of dentin and enamel on pulp capping materials. Of course, light-curable Dycal (Prisma VLC Dycal) could be used as control, but its biomineralization ability was considered less than that of the conventional chemical curing Dycal (DC) because released calcium ions were not detected [30,31]. For use in base materials, bioactive light-curable pulp capping materials have advantages such as greater stability and mechanical properties [12,31]. The major component of light-curable bioactive pulp capping materials is composite resin, which, when adjusted in dentin, has higher mechanical and chemical stability against internal and external stimuli, such as dentin fluid and biting force [32,33]. Future in vitro and in vivo studies are needed to directly compare the in vivo biomineralization and sealing effects of calcium hydroxide-incorporated pulp capping materials and the two other tested types of pulp capping materials in dentinal tubules to use bioactive glassincorporated light-curable pulp capping materials as a base and liner material in clinical settings.
Biomineralization from Released Ions.
When extracts from pulp capping materials affect hDPSCs, the ions released in the extract are key players in the induction of biomineralization. It has already been revealed that extracellular Ca 2+ , Si 4+ , and OH − combine to regulate the biomineralization of hDPSCs [21,34]. In previous experiments, osteogenic or odontogenic biomineralization from stem cells was encouraged by the dissolution of ions (Si 4+ , Ca 2+ , and OH − ) [35,36]. Concentration of released Ca 2+ and OH − was significantly increased in the TC and DC treated groups compared to that from BA treated group. Even though Na + and Si 4+ were detected at significant levels of approximately 5 ppm and 1 ppm, respectively, in the BA group, these ions were not present at effective dosages for stem cells, including hDPSCs [37]. Therefore, to confirm the role of released ions in terms of biomineralization, an ionic supplement matching the ionic environment of TC with Ca 2+ (110 ppm) and OH − (pH 11) was added to the BA-treated group, and 12.5% diluted TC and BA, and ionic-supplemented BA were compared in terms of mineralization. The ionic supplement-conditioned BA and DW group under OM culture media had similar mineralization abilities compared to the TC group ( > 0.05), which suggested that the released ions played key roles in inducing biological properties in hDPSCs. By combining the above results with the data on the released ions, within the limitations of this study, it can be postulated that lightcurable pulp capping materials stimulate biomineralization at the same level as that from the calcium hydroxideincorporated gold standard materials when they release the same amount of Ca and OH ions. Of course, other in vitro assays, such as ALP activity and mRNA/DNA expression assays, are needed to confirm the essential role of Ca and OH ions for biomineralization. In addition, further in vivo studies of the biocompatibility and biomineralization, as well as mechanical/physical properties, such as compressive strength, microleakage, and biodegradability, are needed to clarify the suitability or performance ability of bioactive glassincorporated light-curable pulp capping materials for clinical applications compared to calcium hydroxide-incorporated and MTA-like light-curable pulp capping materials.
Conclusions
Extracts from a bioactive glass-incorporated light-curable pulp capping material were evaluated for cytotoxicity and biomineralization of hDPSCs. The eluates from the bioactive glass-incorporated light-curable pulp capping material (BA) were similar to and more cytotoxic to hDPSCs than those from calcium hydroxide-incorporated (DC) and MTA-like incorporated (TC) pulp capping materials. In addition, the BA extract promoted biomineralization along with ALP activity and ARS staining compared to the OM-treated control. However, BA only showed similar mineralization after matching the ionic conditions to those of the other tested pulp capping materials. Therefore, within the limitations of this in vitro study, BA exhibited the potential to stimulate biomineralization at the same level as other pulp capping materials it released the same amount of Ca and OH ions. Further in vitro studies (odontogenic differentiation markers at the gene or/and protein level) and in vivo or clinical studies are necessary to compare the regenerative potential of BA compared to other pulp capping materials on pulp tissue. | v3-fos-license |
2019-04-03T23:36:56.508Z | 2007-01-01T00:00:00.000 | 97718750 | {
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} | pes2o/s2orc | Synthesis and Characterization of 9-Phenyl-9 H-purin-6-amines from 5-Amino-1-phenyl-1 H-imidazole-4-carbonitriles
9-Phenyl-9H-purin-6-amine derivatives have been synthesized in high yields by reaction between 5-amino-1-phenyl-1H-imidazole-4carbonitrile with HC(OEt)3 and Ac2O followed by reaction with ammonia.
Introduction
Purines are a class of heterocyclic compounds which play an important role in many biological processes 1-5.The most important natural occurrence of purines is in the nucleotides and nucleic acids; compounds which perform some of the most crucial functions in fundamental metabolism.The chemotherapeutic uses of purines and purine analogues have prompted tremendous efforts towards their synthesis, both in academia and in the pharmaceutical industry [6][7][8][9] .
As the purine ring system is a fusion of two aromatic heterocyclces, pyrimidine and imidazole, a logical starting point for ring synthesis is an appropriately substituted pyrimidine or imidazole from which the second ring can be constructed by a cyclization process 10,11 .
Experimental
All solvents purified and dried using established procedures.The 1 H NMR spectra were recorded on Hitachi-Perkin-Elmer R24B (60 MHz) or Bruker XL 300 (500 MHz) instruments (with J-values given in Hz), 13 C NMR spectra either on a Bruker WP 80 or XL300 instrument, and IR spectra on a Shimadzu IR-435 spectrophotometer.Mass spectra were recorded on a Kratos Concept instrument.The melting points were measured on an Electrothermal digital melting point apparatus and are uncorrected.
General procedure for the preparation of 1-aryl-4-cyano-5-[(ethoxymethylene)amino]imidazoles (4a-b)
A mixture of 5-amino-1-aryl-4-cyanoimidazole (0.5 g), triethyl orthoformate (12.0 M equivalent), and acetic anhydride (6.0 M equivalent) was heated gently at 70-80 0 C under an argon atmosphere for several hours.After TLC showed that no starting material remained the resulting yellow-brown solution was evaporated under vacuum to give a residue which, upon treatment with a mixture of dry diethyl ether / hexane (1:1) afforded a precipitate which was filtered off, washed with the same mixture and dried under vacuum to give the products 4a-b.
Once TLC confirmed complete consumption of the starting material, the result in solution were evaporated under vacuum to give a residue that upon treatment with a mixture of dry diethyl ether and hexane (1:1), gave the required imidates 4a and 4b as a solid in 81-87% respectively yield.The products were recrystallised from a mixture of dry diethyl ether and hexane (1:1).In the infrared spectra the presence of the cyano and C=N stretching vibrations were observed in the range of 2210-2220 and 1630-1650 cm -1 respectively.
The 1 H NMR spectra of the isolated ethoxyimidates (4a-b) showed the presence of the H-2 proton of the imidazole ring in the range of δ7.26-8.28ppm.The H-7 proton appeared in the range δ8.28-8.66ppm.The CH 2 and CH 3 of the ethoxy group had clear quartet and triplet patterns as expected in the regions of δ1.32-1.55 and δ4.31-4.5 ppm respectively.The other bands were in agreement with expected structures.The 13 C NMR spectra of the imidazoles had the expected number of bands with the C-2 carbon of the imidazole ring in the region of δ135.4-140.9, the C-7 carbon at δ159.9-165.5 and the C-4 carbon within the region of δ98.9-103.0ppm.
The imidates (4a-b) were converted to the corresponding 9-phenyl-9H-purin-6amines (5a-b) by treatment with ammonia in the minimum amount of methanol.The reaction was carried out under an argon atmosphere at room temperature.During the first 20 minutes a white precipitate started to form.After 2-3 h TLC showed no staring material, and filtration of the reaction mixture gave the purines as a powder in 67-83% yield.The purines (5a-b) were fully characterized by microanalysis and spectroscopic methods.The elemental analysis and mass spectra of isolated 9-phenyl-9H-purin-6-amines (5a-b) were satisfactory.In the infrared spectra, the NH stretching vibrations were observed as 2-3 bands in the range of 3300-3150 and C=N absorption band in the range 1650-1660 cm -1 .The NH 2 protons were observed in the range δ5.70-5.93ppm, the proton at position H-2 of the purines system appeared in the regions of δ8.12-8.26ppm and the proton at position H-8 were seen as a singlet in the range of δ8.08-8.22ppm.The 13 C NMR spectra of the compounds (5a-b) had the expected number of peaks.The C-8 carbon of the imidazoles ring appeared in the region of 143.5-144.0ppm.The carbon at positions C-2 and C-6 of the purines system appeared at δ152.0-152.6 and 158.2-158.4ppm respectively. | v3-fos-license |
2014-10-01T00:00:00.000Z | 2007-08-22T00:00:00.000 | 34898404 | {
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} | pes2o/s2orc | An Improved Synthesis of 4-Chlorocoumarin-3-sulfonyl Chloride and Its Reactions with Different Bidentate
An improved synthetic method affording 4-chlorocoumarin-3-sulfonyl chloride (4) in very good yield (ca. 85%) is reported. This compound was reacted with various bidentate nucleophiles such as 2-aminopyridines and 2-aminothiazoles in order to obtain substituted pyrido- and thiazino-1,2,4-thiadiazino-benzopyranone dioxides (potential anticancer and anti-HIV agents). These reactions occurred rapidly at room temperature giving yellowish precipitates, which are insoluble in common organic solvents, making the purification process challenging. Further investigation has shown that these fused heterocycles are not stable and decompose with opening of the 1,2,4-thiadiazine ring.
Introduction
Heterocyclic chemistry is one of the largest areas of research in organic chemistry and it is growing rapidly.Of all published organic chemistry literature, papers on heterocyclic synthesis accounted for around 60 % in 1998 [1], but nowadays the fraction is much larger considering that novel heterocyclic compounds are published in different fields such as biochemistry, pharmaceuticals, materials and others.A similar trend is seen for coumarin, a heterocyclic system with a very large number of different derivatives.Coumarin is a compound with varied biological activities and in 1954 it was classified as a carcinogenic substance [2,3].Main representatives of the class are its hydroxy derivatives 4-hydroxycoumarin (1) and 7-hydroxycoumarin (umbeliferone), also biologically active and very important for synthesis of other coumarin derivatives.Until now, an enormous number of compounds with coumarin systems in their structure have been synthesized.Those derivatives have shown a remarkably broad spectrum of pharmacological and physiological activities and they are used as anticoagulant [4][5][6], antibacterial [7,8], antiviral [9,10], antitumor [11][12][13][14], bactericidal [15], fungicidal [16], and anti-inflammatory agents [17].Also, in recent times there are references to derivatives with anti-HIV activity [18][19][20][21].
On the other hand, the nitrogen and sulfur heterocyclic system families are very interesting because of their physicochemical properties with relevance to the design of new drugs and new materials, especially those relating to molecular conductors and magnets [22].1,2,4-Thiadizines are also used for treatment of HIV infection [23,24], as cardiovascular agents [25], for their antimicrobial effects [26], and as diuretics and antihypertensive drugs [27,28].Based on these facts we wanted to combine the coumarinic system with 1,2,4-thiadiazines in the hope that the resulting novel heterocycles would be biologically active, especially as anticancer and anti-HIV agents.Also, we designated positions 3 and 4 of coumarin for annellation, because these positions are mainly attacked by electrophiles and nucleophiles, respectively [29].
Results and Discussion
Checchi et al. had previously reported one of the target compounds, pyrido[1',2':2,3]-1,2,4thiadiazino [6,5-c]benzopyran-6-one 7,7-dioxide (7a), as one of two possible structures [30].It was obtained by reaction of 4-chlorocoumarin-3-sulfonyl chloride (4) with 2-aminopyridine (5a) in dry benzene.In our attempts to obtain the same compound 7a and its derivatives, unsatisfactory results were obtained and difficulties followed the synthesis of 4 as the key substrate.These problems were avoided by modification of the reaction route used, simplifying the preparation of the sodium salt 3 of 4-hydroxy-coumarin-3-sulfonic acid (2).Thus, considering that 2 is very soluble and stable in aqueous solution, 3 was obtained by simple mixing aqueous solutions of 2 and sodium chloride.Contrary to the abundant literature mentioning the good solubility of alkali-metal salts of sulfonic acids [31], the salts we prepared are not soluble in water and can be easily isolated in nearly quantitative yields from aqueous solutions by simple filtration (Scheme 1).Compound 3 was reported once by Huebner and Link [32], but the product was only characterized by its elemental analysis for sodium.This paper presents the full characterization of 3. In the next step 3 was chlorinated by refluxing with POCl 3 for approximately three hours.Afterwards, instead of removing POCl 3 by vacuum distillation, the suspension was poured onto a mixture of crushed ice and water to afford a nice precipitate of 4 in 85 % yield (lit. [30]: ca. 25 %) (Scheme 2).Modification of this step was based on the report that the chlorine atom at position 4 cannot be attacked by water [29].Also, it was found that in general the sulfonyl chlorides are insoluble and stable in water [31].Structural assignment of 4 was based on 1 H-and 13 C-NMR, IR and MS spectral analysis.The signals of four aromatic protons at 7.34-7.85ppm could be observed in the 1 H-NMR spectrum, as well as nine signals in the expected regions of the 13 C-NMR spectrum.In the mass spectrum (ESI, +ve mode) there was a peak at m/z 302 corresponding to the [M+Na] + species.These data confirmed that the product isolated from water is identical with that isolated by vacuum distillation.Substrate 4 is very soluble in organic solvents and it was used without further purification.
% crude yield
Reactions of 4 with 2-aminopyridines 5a-h and 2-aminothiazoles 6a,b were performed in acetonitrile as a polar and aprotic solvent.Reactions were very fast at room temperature and after a few minutes crude yellowish precipitates of 7a-f and 8a,b were formed, respectively (Scheme 3).
Using single crystal X-ray diffraction the structure of 7a has been determined and the dilemma about its configuration was eliminated (Figure 1).The compound crystallizes in monoclinic space group P21/n with cell dimensions a=16.3541(5)Å, b=9.2246(2)Å, c=16.7064(5)Å, α=90.00°,β=110.802(4)°,γ=90.00°,V=2356.04(11)Å 3 , Z=8 [33].All atoms in the molecule are in one plane, except for the two oxygen atoms of the SO 2 group, which are arranged symmetrically out of plane.All products 7a-f and 8a,b are very insoluble in common organic solvents, with the exception of DMSO, which caused difficulties when purifying them.Purifications were performed on chromatographic columns with slow gradient elution using dry solvents.Further investigation confirmed that compounds 7a-f and 8a,b are not stable.Analysis carried out on 7d proved that those systems annulated on coumarin decompose with opening of the 1,2,4-thiadiazine ring.Decomposition in the solid state was slow, but when crystals were dissolved in DMSO, where complete elimination of traces of water is not possible, decomposition was rapid (within a few hours).Therefore, it was assumed that these heterocycles are sensitive to moisture.The reaction with water releases sulfuric acid.Since the decomposition is slow in the beginning it is possible that the reaction is autocatalytic, so when the amount of sulfuric acid increases decomposition is more rapid (Scheme 4).4-(5-Chloropyridin-2-yl-amino)-benzopyran-2-one ( 9) was isolated in pure form as white crystals after decomposition of 7d.In the IR spectrum one sharp band at 3328 cm -1 , corresponding to an NH vibrational mode and a C=O band at 1672 cm -1 , which is evidently lower than the band of the annulated heterocycles at 1710 cm -1 , were noticed.The 1 H-NMR spectrum showed a singlet at 9.5 ppm corresponding to the NH proton, seven aromatic protons ranging from 7.4 to 7.9 ppm and one singlet at 7.3 ppm, corresponding to the proton at the 3-position of the coumarin ring.In the 13 It was also observed that the substituent in the 2-aminopyridine moiety is very important because of its influence on the reaction, rate and the stability of derivatives.Substrate 4 did not react with any 2-aminopyridines possessing a nitro group in positions 3, 4 or 5 (10a-c, Scheme 5).This is understandable considering that the nitro group has a strongly negative inductive effect (-I), as well as a negative resonance effect (-M).As a result of both effects, the electron density on the nitrogen atoms is much lower, which consequently weakens the nucleophilicity [34].In view of these facts it was concluded that weak bidentate nucleophiles cannot substitute the chlorine atoms in 4, to form a 1,2,4thiadiazine ring, and that only strong nucleophiles are able to do so.To prove this, comparative reactions of 4 were performed with urea as a weaker and thiourea as a stronger bidentate nuclophile.A yellowish precipitate of 8H,10H-1,2,4-thiadiazino[6,5-c]benzopyran-9-thioxo-6-one 7,7-dioxide (11, Scheme 5) was formed within a few minutes after mixing an acetonitrile solution of 4 and thiourea crystals at room temperature, while all attempts to obtain a product from the reaction with urea failed.
General
Melting points were determined on a Reichert heating plate and are uncorrected.C, H elemental analysis was carried out on a Coleman Model 33 carbon-hydrogen analyzer.N elemental analysis was carried out by the Dümas method.NMR spectra were recorded on a Bruker 400 MHz instrument using DMSO-d 6 as solvent and tetramethylsilane as internal standard.Infrared spectra (KBr pellets) were measured on a Perkin-Elmer System 2000 FT IR.ESI-TOF mass spectra were measured using an LCT mass spectrometer (Waters) equipped with a lockspray dual-electrospray ion source combined with Waters Alliance 2695 HPLC unit.The X-ray structure of 7a was measured on a CCD Xcalibur S diffractometer.All non-hydrogen atoms were refined anisotropically and all hydrogen atoms were placed in ideal positions.All the reagents and solvents were obtained from commercial sources and were used without further purification.4-Hydroxycoumarin-3-sulfonic acid (2) was synthesized following a literature method [30,35], as described below in more detail. | v3-fos-license |
2021-05-16T00:03:38.898Z | 2020-12-01T00:00:00.000 | 234577814 | {
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} | pes2o/s2orc | Ciprofloxacin Release from Polymeric Films. Modeling and Pharmaceutical Parameters Determination †
: Ciprofloxacin (Cipro) is a broad-spectrum antibiotic used against both Gram (+) and Gram ( − ) bacteria. Its biological half-life is very short (4–5 h) and its conventional administration forms present a limited absorption efficiency. For this reason, the aim of this work was to study other administration strategies based on topical films. Sodium alginate (SA), a naturally occurring polymer, and a recombinant elastin-like polymer (rELP) produced by advanced genetic engineering techniques were evaluated as potential carrier systems. The films were obtained by the casting tech-nique, adding the Cipro by direct dispersion in the polymer solution using 16.6% w / w rELP or 1.5% w / w SA. The in vitro release assays were performed at 37 °C in physiological solution and with orbital shaking at 90 rpm. Cipro concentration was determined by ultraviolet (UV) spectrophotometry at 276 nm. The release profiles were analyzed and adjusted using the Lumped model developed and validated by our research group. Pharmaceutical interest parameters were calculated and com-pared for both polymer-Cipro systems: the time required to reach 80% of the drug dissolved ( t 80% ), the dissolution efficiency ( DE ) and the mean dissolution time ( MDT ). The SA-Cipro platform released the 80% of the drug in 35 min, while this parameter was 209 min for the rELP-Cipro system. The MDT 80 % was 8.9 and 53 min for the SA-Cipro and rELP-Cipro, respectively, while the DE , evaluated at 200 min, was 66.6 and 58.8 for each platform, respectively. These parameter values demon-strate that the rELP films were able to modulate the drug release rate and for the SA ones, release can be considered immediate. Therefore, both systems are promising strategies for the topical application of Cipro.
Introduction
Ciprofloxacin (Cipro) is a pale yellow crystalline powder. It responds to the chemical structure of 1-cyclopropyl-6-fluoro-1,4-dihydro 4-oxo-7-(1-piperazinyl)-quinoline-3-carboxylic acid ( Figure 1) which belongs to the family of quinolones. The central structural unit is a quinolone ring with a fluorine atom in position 6, a piperazine group in position 7, a cyclopropyl ring in position 1 and a carboxyl group in position 3 [1]. The Cipro molecule has amphoteric characteristics due to the presence of the carboxyl and amino groups ( Figure 2). It is well known that Cipro can form complexes with certain multivalent cations, according to the degree of ionization and the prevalence of certain chemical variants present in the aqueous solution [2]. The antibacterial effects of Cipro are due to the inhibition of bacterial topoisomerase IV and DNA gyrase, preventing the replication and transcription of bacterial DNA [3]. Cipro belongs to the group of synthetic fluoroquinolone antibiotics with broad antimicrobial activity. It is commonly used for infections of the urinary tract, intestinal, among others, but it has a very short biological half-life of approximately 4 to 5 h [4]. On the other hand, the limited absorption efficiency of the drug in conventional form prompted the development of new delivery systems. Among them, we can mention the transdermal systems which have numerous advantages, such as their application in a specific site, painless application, less frequent replacement, and greater dosage flexibility.
Among the materials used to prepare these systems, polymers stand out, particularly those that are biodegradable, biocompatible and from natural sources such as chitosan, sodium alginate (SA) and cellulose, among others. SA is an anionic polysaccharide, derived mainly from brown algae and bacteria. This polymer represents an outstanding class of materials for its biocompatibility, biodegradability, low toxicity and low relative cost [5]. On the other hand, rELPs are self-gelling, biodegradable, and biocompatible polymers, tailored designed for different applications in human medicine. This material was synthesized from recombinant elastin-type protein polymers (rELPs) [6]. rELPs are a class of protein polymers that have promising applications in the fields of biomedicine and nano-biotechnology. These are synthesized by recombinant DNA technology produced by fermentation of Escherichia coli and purified by thermo-dependent reversible segregation cycles. This polymer is capable of self-assembling and forming nanoparticles at low concentrations and hydrogels at high concentrations with a very strong and irreversible stability. The molecular mass of this polymer is 101.12 kDa [7].
The aim of this work was to evaluate the feasibility of developing films based on rELP and SA for the controlled release of Cipro. With this framework, films loaded with Cipro were prepared and characterized by evaluating drug release profiles. The data obtained were analyzed using the "Lumped" model, which allowed determining the initial release rate and parameters of pharmaceutical relevance, such as the mean dissolution time (MDT), the time to release 80% of the drug (t80%) and dissolution efficiency (DE) [8,9] to compare the different formulations developed.
Materials
The rELP was designed and synthesized by the BIOFORGE group-University of Valladolid (Spain), SA was purchased from Tododroga (Córdoba, Argentina) and the Cipro hydrochloride from Parafarm ® (Buenos Aires, Argentina).
Cuantification of Ciprofloxacin
The Cipro concentration was determined by UV spectrophotometry (JENWAY) at 276 nm. These tests were performed in triplicate.
rELP Films Preparation
To obtain the films, 10 g of aqueous solutions were prepared with concentrations of 16.6% w/w of rELP, to which 10 mg of Cipro was added. They were subsequently subjected to 0 °C for 10 min to ensure complete dissolution of the polymer. These solutions were placed in glass plates covered with non-stick material and placed in an oven at 37 °C for 24 h.
Sodium Alginate Films Preparation
For these films, 0.45 g of SA and 100 mg of Cipro were weighed, and 30 mL of distilled water was added. This solution was homogenized under magnetic stirring (150 rpm) at room temperature and then poured into Petri dishes and oven-dried at 37 °C for 24 h. Finally, crosslinking was carried out for 7 min in a 0.2 M calcium chloride solution.
In Vitro Drug Release Tests
To carry out the release tests, 1 × 1 cm 2 samples were cut from the different films and their weights and thicknesses were determined ( Table 1). The samples were carefully placed in test tubes containing 3 mL of physiological solution, used as a release medium, at 37 °C and with orbital shaking at 90 rpm. At pre-established time intervals, samples were taken by complete removal of the release medium, replacing the same volume with fresh medium to re-establish the maximum driving force at each sampling point. The
Data Analysis
The data obtained from the release tests were analyzed using a second-order kinetic model, called the Lumped model, developed and validated by our research group [8,9]. To compare the release profiles, the experimental data were adjusted using the Polymath 6.0 program and parameters of pharmaceutical relevance were calculated: the MDT, the time to release 80% of the drug (t80%) and the DE. Furthermore, the model makes it possible to determine the initial release rate (a).
In Vitro Drug Release Tests
In vitro drug release tests are used to characterize the release of a drug from a certain pharmaceutical form, the dissolution test being the most relevant. According to the United States Pharmacopeia (USP), drug dissolution and release tests are required for dosage forms in which absorption of the drug is necessary for the product to exert the desired therapeutic effect [10].
A central goal in the modified release of the drugs is to establish the desired release kinetics of a given drug for a specific application.
The experimental data obtained from the Cipro release tests of the 2 systems are shown in Figure 3. In the platforms developed, the drug is distributed homogeneously in a continuous matrix formed by the polymeric network that controls the release rate, forming what is called a monolithic system. In this type of devices, release is usually controlled by diffusion through the monolith matrix material or through aqueous pores, and an initial burst of drug release from the surface is often observed. Over time, the rate of drug release decreases as the distance of drug diffusing to the surface increases. This can be seen in Figure 3, where a high initial rate of drug release from both polymer platforms is observed due to the transfer phenomenon that occurs as a consequence of the presence of drug on the surface of the films. Then, a moving front of solvent advances through the polymeric film, allowing the Cipro to diffuse to the surface face so that it is available for dissolution in the release medium. As the distance between the surface and the advance of the moving front increases with time, the release rate decreases. The object of any mathematical model is: (i) to be able to accurately represent the processes associated with drug release, (ii) to be able to describe/summarize experimental data with parametric equations or moments, and (iii) to predict processes under varying conditions. However, when describing the processes involved, some models developed often suffer from being too complex to be useful in practice. Under these premises, the mathematical model used to adjust the experimental values was the Lumped model developed by our research group in pharmaceutical technology. (Equation (1)). It considers the grouped effect of diffusion within the film and the transfer to the physiological solution.
where Mt(%) is the cumulative percentage amount of drug released at the moment t. Equation parameters a (%/min) and b (min −1 ) can be obtained graphically. However, the best procedure to fit the model with the experimental data is through a nonlinear regression analysis using the values a and b found graphically as a first approximation. Nonlinear regression analysis was performed using Polymath 6.0 program. The model adjusted well to the experimental values for the 2 polymers evaluated (Figure 3). The model also allows us to determine the initial dissolution rate, since the rate at any time will be: Therefore, when t = 0, the initial rate of dissolution will be: Table 2 shows the values of the parameters a and b of the equation, as well as other parameters of pharmaceutical relevance, such as t80%, DE and MDT and M∞, which are calculated from the following equations: It is observed that SA systems have an initial rate of about 6 times higher than that of rELP systems. Furthermore, the t80% parameter, which is the time required to reach the dissolution of 80% of the drug available for dissolution, is 35 min for the SA films. The pharmacopeia states that if this parameter is lower than 45 min, the release can be considered immediate [11], as in the case of SA platforms. In a system that modulates the dissolution of a drug, it is desirable a t80% high value, as is the case with the rELP films, which would indicate a delay or control in the release process. The DE is defined as the area under the dissolution profile up to a certain time (tF), expressed as the percentage of the area of the rectangle described by 100% dissolution at the same time. These DE values are very close for both systems.
Finally, the MDT value is a widely used pharmaceutical parameter to characterize the release rate of drugs from a specific dosage form that provides information about the ability to delay the release of the active ingredient from the polymer platform. A high MDT value indicates a greater ability to delay release. In this case, the behavior of MDT80% is correlated with that presented by t80%, showing a value about 6 times higher for rELP systems than for SA ones.
It is known that for topical products, understanding of safety and efficacy is based on the release of the active drug from its dosage form to the surface of the skin. Once the drug is in contact with the surface of the skin, it penetrates through the stratum corneum to achieve its pharmacological action. For this reason, the determination of the release rate through in vitro studies is a relevant parameter to control the quality of a topical product, in the same way that dissolution tests are important for solid dosage forms administered via the oral route.
Conclusions
Platforms based on rELP polymer have a greater ability to release the drug gradually, while the systems based on SA would be useful for topical applications where rapid delivery of the drug is needed, providing high concentrations for a short time. Therefore, it can be concluded that it is possible to modulate the release rate of Cipro through the films developed based on both polymers, with characteristics suitable for different applications. Consequently, both systems are promising strategies for the topical application of Cipro.
Finally, managing the ability to modulate drug release through various polymeric platforms can reduce the variability of the performance of pharmaceuticals. This last aspect is increasingly important given the current emphasis on "quality by design" by regulatory agencies such as the FDA.
Institutional Review Board Statement: Not applicable
Informed Consent Statement: Not applicable Data Availability Statement: The data presented in this study are available on request from the corresponding author. | v3-fos-license |
2019-04-08T13:05:39.273Z | 2016-07-02T00:00:00.000 | 55402924 | {
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} | pes2o/s2orc | A Pseudomonas aeruginosa Strain Ikw1 Produces an Unusual Polymeric Surface-Active Compound in Waste Frying Oil-Minimal Medium
A Gram-negative bacterium isolated from the sub-surface water of Ikang River, Niger Delta region, Nigeria produced an unusual biosurfactant in waste frying oil-minimal medium. Cultural and biochemical characterizations as well as 16S rRNA sequencing identified the bacterium as a strain of Pseudomonas aeruginosa with 100% sequence homology with Pseudomonas aeruginosa strain HNYM41. Biochemical characterizations, thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and Fourier transform-infrared (FTIR) spectrometry identified the active compound as a glycolipopeptide (peptidoglycolipid) composed of 40.36% carbohydrates, 20.16% proteins and 34.56% lipids. The biosurfactant reduced surface tension of water from 72.00 to 24.62 dynes/cm at a critical micelle concentration (CMC) of 20.80 mg/L indicating excellent effectiveness and efficiency properties. Commendable oil-washing property (79.92% oil recovery) with an elution rate of 0.68 mL/min at 70°C, foaming and foam stability, excellent emulsification activity in kerosene, crude oil and palm oil and a significant (P = 0.000; R = 0.9901) oil solubilization property indicate excellent oil recovery, detergency and remediation potentials of the biosurfactant. Oil displacement, emulsifying and antimicrobial activities of the compound were relatively stable at relevant temperatures, pH and NaCl levels suggesting suitability for applications in hydrophobic compound remediation, emulsion stabilization and preservation of formulations.
Introduction
Surface active compounds, otherwise called surfactants, are amphiphilic molecules with hydrophilic and hydrophobic domains which reduce the free surface enthalpy per unit area [1] at air/water interfaces and the interfacial tension at oil/water interfaces [2]. They are about the most sought-after process chemicals worldwide. Their applications range from agriculture and environment [3] [4] to industries including food, cosmetics and pharmaceuticals not to mention petroleum [5]. These applications derive from their nature and the physicochemical properties which determine their various types including glycolipids, lipopeptides, neutral lipids, phospholipids and the polymeric types [6]- [8].
Synthetic chemistry is at the forefront of production of these molecules derived predominantly from petrochemicals which make them cheap and commercially available [9]. Nevertheless, the bulk applications of these chemicals especially in environmental bioremediation [10] [11] and to a lesser extent, the industries, whose effluents still end up in the environment, have been a source of environmental concern owing to their toxicity and environmental incompatibility [7].
Green surfactants or biologically-derived surfaceactive molecules, especially those of microbial origin called biosurfactants, are suitable alternatives to their chemical counterparts by reason of their wider applications, biodegradability, low to non-toxicity and environmental compatibility [12]. Biosurfactantproducing microbial strains from different taxonomic groups especially bacteria [13] [14] and yeasts [15] [16] have been isolated and employed in production. In recent times, a new area, called green chemistry, has been developed where biosurfactants are applied in the production of nanoparticles [17].
In the industries, most of the biosurfactants are used as emulsifiers but extensive applications in this respect have been limited by relatively high production and recovery costs [1]. Production economics is frequently encountered as the major drawback of biotechnological processes including biosurfactant production and has greatly limited its commercial applications. However, production costs can be reduced by careful selection of producing strains with improved yield, improved product formation rates and use of cheap (often waste) substrates. Recommended protocol for isolation of biosurfactant-producing bacteria involves a combination of screening concepts [18]. However, multiple substrates are required for successful isolation and selection of a diversity of biosurfactantproducing bacteria with abilities to produce novel biosurfactant types [13]. The successful use of wastes like olive oil mill effluent, dairy waste and waste frying oil as substrates for biosurfactant production has been reported by few researchers and with encouraging results [19]- [21].
Here, we report the isolation of a strain of Pseudomonas aeruginosa from the mesotidal waters of Ikang River, Niger Delta area, Nigeria, with ability to elaborate an unusual surface-active compound when grown on waste frying sunflower oil. A waste frying oil disposal problem currently exists in Calabar, the capital city of Cross River State, Nigeria, where it is emptied into drainages resulting in unbearable stench in the capital city. Government has already shut down a number of fast foods in the city thereby worsening the unemployment problem of the state and country. Utilization of the waste frying oil for biosurfactant production will go a long way to solving waste oil disposal and subsequently unemployment problems. To the best of our knowledge, this is the third time a strain of Pseudomonas aeruginosa has been reported to produce a glycolipopeptide biosurfactant but the first report of that production on waste frying oil.
Medium formulation for biosurfactant production
Purified cultures of all morphologically-distinct bacteria were grown in minimal medium supplemented with different carbon sources including glucose, glycerol, rice processing effluent, crude oil and waste frying oil at 1% (v/v or w/v) as sole source of carbon and energy. The minimal medium contained (g/L) KH2PO4 1.0; K2HPO4 0.5; MgSO4.7H2O 0.2; NaCl 0.5; NH4Cl 1.0; FeSO4.7H2O 0.01 with pH adjusted to 7.0 using 1 M HCl/IM NaOH. Twenty milliliter volume of minimal medium was dispensed into 100 mL Erlenmeyer flasks. Flasks were sterilized by autoclaving at 121°C for 15 min and inoculated, upon cooling, with 2% (v/v) 18 h-old Luria broth culture of each bacterial isolate. Flasks were incubated at room temperature (28 ± 2°C) on a rotary shaker agitating at 150 rpm for 72 h.
Qualitative screening of isolates for biosurfactant production
Cell-free fermentation broth of each flask corresponding to each bacterium was obtained by centrifugation at 8,000 x g for 10 min followed by membrane filtration with 0.2 µM (Millipore) and subjected to qualitative screening for biosurfactant production by the combination of rapid drop collapse test [13], emulsifying activity test [22], salt aggregation test [23] and oil displacement test [6].
Quantitative biosurfactant screening
The initial quantification of biosurfactant in cell-free broth of bacteria which tested positive to at least 75% of the qualitative screen tests followed the oildisplacement assay. This assay is sensitive enough to detect the presence of 10 µg or 10 nmol of biosurfactant in sample solutions [6]. In this test, 15 µL of crude oil (Nigerian medium crude-Adaax) was added to the surface of 40 mL of distilled water in a Petri dish of diameter 15 cm to form a thin uniform oil layer. Plates were allowed to equilibrate for 1 h. Thereafter, 10 µL of each cell-free fermentation broth was gently placed on the center of the oil layer. The diameter of clear halo visualized under visible light was measured with a meter rule after 30 s and area of clear zone calculated using the formula A = πr 2 ; where A is the area of the oil film, r the radius and π a constant of value 3.14. The larger the diameter of clear halo, the larger the area and hence the greater the amount of biosurfactant. All determinations were made in triplicates. Biosurfactant hyper-producing strains were selected by consideration of mean oildisplaced areas of their cell-free fermentation broth.
Identification of biosurfactant -producing bacterium
The selected bacterium was identified morphologically and biochemically using the MICROGEN ID Kit (Microgen Bioproducts Limited, UK) in conjunction with Microgen identification system software as well as by 16S rRNA sequencing.
The sequencing protocol made use of primers 271 and 1492R and utilized a sequence mix composed of NZYTaq 2 x Green Master Mix (Nzytech). Amplification was done by denaturation at 94°C for 3 min, cooling to 5°C for 1 min and raising the temperature again to 72°C. This was repeated through 30 cycles and then held at 72°C for 10 min. The polymerase chain reaction (PCR) products were purified with the JETQUICK PCR purification Spin Kit and used as template for the sequencing reaction. Sequences were compared to GenBank sequences by using standard nucleotide basic local alignment search tool (BLAST). The bacterium was deposited at the University of Calabar collection of Microorganisms (UCCM) and had since been given the collection's code name.
2.4.
Determination of the effectiveness of biosurfactants by surface tension reduction measurement The surface tensions of sterile clear amber cell-free biosurfactant solutions of four isolates were determined by the ring method [20] at room temperature with a tensiometer (CSC Du-Nouy Tensiometer) fitted with a platinum ring. A volume of 70 mL of each cell-free biosurfactant broth was dispensed into 100 mL beakers and placed on the seat of the tensiometer. The seat was then raised by an adjustment knob until contact was made between the surface of biosurfactant liquid and the platinum ring. The liquid film produced beneath the ring was stretched as attempt was made to bring the ring out of the liquid by means of the adjustment knob. The force needed to break the ring free of the liquid to the surface was read off a scale calibrated in dynes/cm. This was repeated 5 times for each of the biosurfactant solutions and mean determinations obtained and presented as surface tension of biosurfactants from the bacterial strains tested.
2.5.
Recovery and characterization of biosurfactant 2.5.1 Biosurfactant recovery Most effective biosurfactant was recovered from the sterile clear amber liquid (obtained by centrifugation of fermentation broth at 8,000 x g for 10 min followed by sterilization by membrane filtration) by acidification of 5 mL sterile biosurfactant solution to pH 2.0 with 6 N HCl [24]. Thereafter, acidified biosurfactant treatments were allowed to stand for 10 h at 4ºC after which equal volumes of the acid-treated biosurfactant and chloroform-methanol mixture (2:1) were prepared in separatory funnels The preparations were allowed to stand for 30 min and the bottom layer (organic phase) separated and subjected to rotary evaporation at 35°C. The brown oily substance that ensued was weighed and expressed as g/L. [13] for the detection of carbohydrates followed by heating at 125°C. Some were steamed in iodine vapour for the detection of lipids (fatty acids) and some sprayed with ninhydrin reagent containing 0.5 g ninhydrin in 100 mL anhydrous acetone for the detection of peptides (proteins). Negative ninhydrin tests prompted digestion of biosurfactant with 6N HCl followed by heating to 105°C for 24 h for the detection of free amino acids [28] and the ninhydrin test repeated. Spot colour was noted. Surface active fractions were confirmed by oil displacement activity of needle-point scrapings from the plates.
Biosurfactant analysis by high performance liquid chromatography (HPLC)
The active fractions of crude biosurfactant obtained from analytical TLC were subjected to HPLC analysis using a Gemini C18 column (100 x 4.6 mm) with a particle diameter of 5 µM accompanied with a Varian 335 diode array detector to facilitate UV detection over a wavelength range of 200 and 220 nM. Sample analysis followed a linear gradient with 85% eluent A comprising of 0.1% ortho-phosphoric acid and 15% eluent B made of acetonitrile at 0 min, only to increase eluent B to 100% after 40 min at an eluent flow rate of 1.0 mL/min [29].
Biosurfactant analysis by Fourier transform-Infrared (FT-IR) spectrometry
The infrared spectra of the partially-purified biosurfactant were recorded on a Fourier transforminfrared system (Spectrum BX-Perkin Elmer) in the 4000-400 cm -1 spectral region at 2 cm -1 resolution. The sample was spread on 0.23 mm KBr cell and cell inserted into the IR transforming system. The spectra were displayed on a connecting computer monitor after Fourier-transformation.
Evaluation of biosurfactant effectiveness and efficiency
The effectiveness of the crude biosurfactant, given by surface tension reduction, was determined by the ring method [20] at room temperature with a tensiometer (CSC Du-Nouy Tensiometer) fitted with a platinum ring. The efficiency of the biosurfactant, given by its critical micelle concentration, CMC was determined by measuring surface tension values of increasing concentrations of the biosurfactant. The logtransformed biosurfactant concentrations were regressed on their respective surface tension values using a second-order polynomial function. The CMC was defined as the minimum biosurfactant concentration above which no further reduction in surface tension occurred.
2.7.
Activity characterization of biosurfactant 2.7.1. Biosurfactant-enhanced oil recovery (washing activity) by the sand pack method Biosurfactant enhanced recovery of Bonny light crude oil was conducted using the modified sand pack column technique [30]. One hundred and fifty grams (150 g) of 140 µM size sand particle was packed into a glass column of 20 mm x 25 mm x 85 mm dimensions with a 100 µM pore size sieve. The column was fitted with a tap outlet at the bottom to permit escape of recovered oil. A volume of 100 mL of Nigerian medium crude oil was poured into the column and allowed to stand for 3 h. Then 100 mL of sterile biosurfactant solution was added to the column and the column incubated at 30, 50 and 70°C to ascertain temperature influence on biosurfactantenhanced recovery. The experiment was set up in triplicates with 50 mL of Milli-Q water serving as control. Volume of oil released by biosurfactant enhancement was measured at 30 min interval for 3 h at the different temperatures and compared with that of the control. Oil recovery rate was determined and expressed as mL/min.
Biosurfactant-enhanced solubilization of Nigerian medium crude
Crude oil solubilization test was performed following a batch solubilization technique [31]. Twenty-five milliliters (25 mL) of Nigerian medium crude oil (ADAAX Oil Nigeria) and different concentrations of crude biosurfactant (0, 50, 100, 200, 300, 500, 700, 1000, 1300, 1650 and 2000 mg/L) solutions were mixed in equal volumes in glass-stoppered 250 mL Pyrex separatory funnel. The funnels were placed on a rotary shaker agitating at 200 rpm, at 30°C for 36 h. A 12 h settling period was allowed followed by withdrawal of the aqueous phase from the bottom of the funnel with minimal disturbance. Samples, now called water soluble fractions (WSF), were analyzed for total petroleum hydrocarbons (TPH) by the nhexane method. Absorbances of n-hexane extracts from the different treatments were taken at 450 nm with HACH DR 300 Spectrophotometer and amounts determined gravimetrically from a standard curve. Regression statistics (Excel 2007) was used to analyze data obtained to show relationship between biosurfactant concentration and solubilization of crude oil at 95% confidence limit.
Assessment of antimicrobial activity of biosurfactant
Assessments were conducted by the agar disc diffusion method [28] in Muller-Hinton agar for bacteria and Potato dextrose agar for fungi. Discs of 6 mm size were punched from filter paper (Whatman No 3; Millipore) and sterilized by autoclaving in capped bottles. Aliquots of 20 µL (50 µg/mL) of crude biosurfactant solution were absorbed by each disc and allowed to equilibrate for 24 h. Equilibrated discs were aseptically introduced onto the center of Muller-Hinton and Potato dextrose agar plates preseeded with appropriate dilutions of test cultures/spores. Cell/spore densities of test cultures were as follows (cfu/mL); 10 6
Measurement of emulsifying activity of biosurfactant
The emulsifying property was measured as described in Asitok and Antai [22] and results expressed as per cent E24 (% E24). Kerosene, Nigerian medium crude oil and palm oil served as hydrophobic compounds.
Assessment of foaming property of biosurfactant
The foam power and stability of the glycolipopeptide was evaluated according to the method of Chen et al.
[32] with slight modifications. A volume of 200 mL of sterile biosurfactant solution was allowed to flow through a burette from a height of 90 cm into a 500 mL measuring cylinder. The turbulence generated foam whose height was noted immediately and then after 5 min. Foam heights were also measured every 1 h, then every 24 h. The foam height at time 0 min was considered the foam power while the R5, defined as the ratio of the foam height at time 5 min to that at time 0 min, was considered an indication of foam stability.
Stability characterization of biosurfactant 2.8.1. Temperature stability
To determine the thermal stability of a 5 g/L crude biosurfactant solution, the test biosurfactant preparation was maintained at temperatures of 20, 40, 60, 80, 100, 120, 130 and 140°C for 15 min, cooled at room temperature and oil displacement, emulsification and antimicrobial activity tests of the glycolipopeptide performed as described by Abouseoud et al. [33].
pH stability
The effect of pH on biosurfactant activity was performed by adjusting the pH of 5 mL of 0.5% (w/v) crude biosurfactant solution with 1M HCl/1M NaOH through a range from 4 to 11 and held for 15 min. Oil displacement, emulsification and antimicrobial activity tests of the glycolipopeptide were conducted as described previously [33].
Salinity (NaCl) stability
Crude biosurfactant concentration of 0.5% (w/v) was prepared by dissolving 0.5 g of crude biosurfactant in 100 mL of different concentrations of NaCl solution. The concentrations used included 5, 10, 15, 20 and 25% at a pH of 7.0. The preparations were held at 30°C for 15 min to determine the effect of salt concentrations on activities of the glycolipopeptide. Oil displacement, emulsification and antimicrobial activity tests were performed as previously described [33].
3.1.
Substrate-specific isolation of biosurfactant-producing bacteria The results of isolation of biosurfactant-producing bacteria from the 20 samples analyzed in our study, as mediated by the different substrates, are presented in Table 1. The table reveals that a total of 858 morphologically-distinct bacteria were isolated from all 20 samples with Ifondo water (IFW) harboring the highest number of distinct bacteria of 69. The table also reveals that a total of 141 (16.43%) biosurfactant-producing bacteria were selected using different substrates with waste-frying oil contributing 50 (35.46%). Generally, miscible substrates selected fewer biosurfactant-producing bacteria than hydrophobic substrates. A two-way analysis of variance revealed significant (P < 0.05) influence of substrates on the isolation of biosurfactant-producing bacteria.
3.2.
Quantitative screening for industriallyrelevant biosurfactant-producing bacteria Table 2 presents the results of the quantitative screening of primarily positive biosurfactantproducing bacteria from the three major kinds of samples. The results show that only 28 (19.59%) of the total biosurfactant-producing bacteria were truly positive for biosurfactant production by the oil displacement assay method. Mean oil-displaced areas of 4 best isolates were IKW1 (66.5 cm 2 ) > IFW (55.3 cm 2 ) > OB6 (52.8 cm 2 ) > R15B (50.3 cm 2 ). Results also show very clearly that 3 (75%) of the best isolates were obtained from water samples.
3.3.
Identification of biosurfactant-producing bacteria A summary of the results of characterization tests leading to the identification of the best 4 biosurfactant-producing bacteria is presented in Table 3. The table reveals that isolate IKW1 was Pseudomonas aeruginosa with a 100% sequence homology with Pseudomonas aeruginosa strain HNYM41 with a GeneBank accession number of JN999891A. Isolate R15B, on the other hand, was identified as Bacillus cereus with a 100% sequence homology with Bacillus cereus strain F2 with an accession number of JQ579629A. The table also reveals that 3 (75%) of the 4 best biosurfactantproducing bacteria were Gram-negatives. The high performance liquid chromatography (HPLC) analysis of preparatory thin layer chromatographic fractions revealed just one peak indicating that the biosurfactant was purified to 95% purity level. indicating the presence of amino and carboxylic groups. These characteristics, in combination with biochemical and chromatograhic results strongly suggest carbohydrate (glyco), lipid (lipo) and protein (peptide) composition for this biosurfactant. The surface-active compound could therefore be safely referred to as a glycolipopeptide (or a peptidoglycolipid).
3.6.
Determination of glycolipopeptide efficiency with the critical micelle concentration (CMC) The efficiency of the glycolipopeptide biosurfactant, given by its critical micelle concentration (data not shown) revealed that the glycolipopeptide has a CMC of 20.80 mg/L. The second order polynomial used to fit the regression model revealed a significant goodness-of-fit (P = 0.000; R 2 = 0.9683) of the model.
Enhanced oil recovery (washing activity), solubilization, emulsification and foaming activities of glycolipopeptide
Results of the sand pack experiment demonstrating the oil recovery potential of the glycolipopeptide (data not shown) revealed that highest recovery rate of crude oil of 0.87 mL/min occurred at 50°C at 120 th min of incubation. Biosurfactant-enhanced recovery rate of crude oil at 30°C increased linearly with time until it peaked at 0.63 mL/min at 150 th min. A nearperfect parabolic curve with a peak of 0.68 mL/min at 90 min was attained when temperature was raised to 70°C with an oil recovery effectiveness of 79.92%. Only 21.37% of oil was recovered with Milli-Q water (control) with a constantly increasing recovery rate throughout the 3 h holding time.
The results of the solubilization potentials of the glycolipopeptide revealed significant (P < 0.05) positive linear relationship (R 2 = 0.9901); oil solubilization increasing as biosurfactant concentration increased.
Emulsifying activity of the glycolipopeptide was tested with kerosene, crude oil and palm oil as hydrophobic compounds. The results showed that emulsification indices of the surface-active compound were 79.71%, 84.87% and 87.54% in kerosene, crude oil and palm oil respectively.
Results of the foaming property experiment of the biosurfactant revealed that the active compound could produce small densely-packed foam with a height of 7.2 ± 0.3 cm at time 0 min. The foam was stable for > 48 < 72 h. R5 of the foam was 1.
3.8.
Antimicrobial potential of glycolipopeptide The results of the antimicrobial potentials of the glycolipopeptide showed that the biosurfactant could most inhibit Bacillus subtilis UCCM 0006 to a zone diameter of 34 mm. However, the surface-active compound showed no inhibitory activity against Serratia sp.UCCM 0003. Overall spectrum of antimicrobial coverage of the glycolipopeptide was narrow.
3.9.
Stability characterization of glycolipopeptide activities The results of the effect of temperature, pH and NaCl on the oil displacement activity of the glycolipopeptide are presented in Figure 2. Figure 2A reveals that oil displacement activity of the glycolipopeptide was stable up to 80°C after which it dropped gradually. The glycolipopeptide was observed, in Figure 2B, to be stable at alkaline pH levels with maximal stability of oil displacement activity at pH between 7 and 9. The oil displacement activity of the surface-active compound in Figure 2C was decreased gradually with increase in NaCl concentrations. NaCl concentration of up to 10% still demonstrated commendable displacement activity.
Results of the effect of temperature, pH and NaCl on the emulsifying activity of the glycolipopeptide are presented in Figure 3. Figure 3A shows that the emulsifying activity of the glycolipopeptide increased as temperature increased. An activity of 79.71% at 30°C in kerosene increased to 91.43% at 140°C while that of 82.87% in palm oil increased to 95.49% at 140°C. Figure 3B shows the influence of pH on the emulsifying activity of the biosurfactant and reveals that the activity increased as the pH increased from 4 to 7 and stabilized at the alkaline region. The influence of NaCl on the emulsifying activity of the biosurfactant is shown in Figure 3C and reveals that 77.21% of the emulsifying activity was retained at 10% NaCl. Figure 4 is a presentation of the results of the effect of temperature, pH and NaCl concentrations on the antimicrobial activity of the glycolipopeptide. Figure 4A shows that the antimicrobial activity was retained up to 60°C and reduced after that until 140°C. Figure 4B shows that the antimicrobial activity of the surface-active compound was high at acidic and extremely alkaline pH but was moderate at neutral and weakly alkaline pH (8 to 9). The influence of NaCl concentrations on the antimicrobial activity of the biosurfactant is presented in Figure 4C and reveals that the activity remained stable up to 10% NaCl concentration but gradually decreased at higher NaCl concentrations.
Discussion
The results of this study are very emphatic on the influence of carbon substrates on the isolation of biosurfactant-producing bacteria as opposed to its influence on microbial selection for an industrial process. Our results show significant (P < 0.05) influence of substrates on isolation or distribution of biosurfactant-producing bacteria from/in the samples but non-significant (P > 0.05) influence of nature of samples on the same property. This infers that whatever the nature of sample, a tendency exists to isolate a biotechnologically-relevant biosurfactantproducing organism if the substrate in the screening medium appropriate for the organism. These results ratify the suspicions of Bodour et al. [13] that nature of carbon substrates is a major reason for the difficulty encountered in the many attempts at isolating biosurfactant-producing bacteria from samples.
The substrate-dependence of biosurfactant elaboration by bacteria in particular and microbes in general is borne out of the different physiological roles to which the producing organism puts the surface-active agent post-synthesis. It would be strange for a bacterium to elaborate biosurfactants for solubilization when a highly soluble substrate like glucose is the carbon source in the screening medium. If the organism did, then the biosurfactant would be for a purpose other than solubilization. The dominant selection of 29 (54.72%) biosurfactantproducing strains when waste frying sunflower oil served as carbon substrate in the screening medium suggests its better nutrient level and/or mix which directs biosurfactant synthesis in most of the bacteria. Successful production of biosurfactants in waste frying oil has been covered in the review by Makkar and colleagues [34]. Only few reports exist in the use of waste frying oil for biosurfactant production and even fewer for isolation of biosurfactant-producing bacteria in nature.
True biosurfactant-producing bacteria were confirmed in our study from a combination of four qualitative tests in addition to the rapid drop collapse test. The ability of an organism to test positive for biosurfactant-production in one screening method but fail in another suggests diversity in physiological roles of biosurfactants and their very chemical nature. For instance, diffusion restraints in blood agar may not allow high molecular weight biosurfactants to be detected by blood cell hemolysis [20]. Mabrouk et al. [18] combined four different methods to be able to isolate the glycolipopeptide-producing Bacillus sp. E34. The need for multiple substrates for successful isolation of biosurfactant-producing bacteria underscores both the tedion involved in biosurfactant bio-prospecting and the unpredictability of appropriateness of selection protocols in industrial organism search.
The results of the quantitative screening assessed by the method of oil displacement present a rather interesting trend. The method was selected by reason of the significant (P = 0.000; R 2 = 0.9997) linear relationship between area of displaced oil and amount of surface-active compound demonstrated by sodium dodecyl sulphate (SDS). This strong relationship had earlier been reported by Satpute et al. [14]. Apart from ES2 isolated from Eketai sediment sample, the immediate 9 most efficient biosurfactant-producing bacteria were obtained from water or crude-oil samples suggesting the physiological need for bulk production of the surface-active agents in liquid media/systems which compensates naturally for the dilution effect of the liquid environment. Strain HNYM41 and Bacillus cereus Strain F2 respectively, suggests that these strains are the same but there has been no report on the biosurfactant-producing potentials of these bacteria.
The effectiveness of a biosurfactant is given by its measure of surface/or interfacial tension reduction. Pseudomonas aeruginosa Strain IKW1 surface-active compound reduced the free surface enthalpy per unit area of water from 72 dynes/cm to 24.62 dynes/cm thus capable of bringing 65.44% of hydrophobic substances into solution to form emulsion. The second best surface-active compound obtained from Bacillus cereus Strain R15B was able to reduce surface tension from 72 dynes/cm to 29.40 dynes/cm. The most powerful biosurfactant, surfactin, was reported to reduce the surface tension of water to 27.50 mN/m [7] thus, making the active compound from Pseudomonas aeruginosa Strain IKW1in this study a more effective biosurfactant.
The efficiency of a biosurfactant is, on the other hand, given by its critical micelle concentration; defined as the minimum concentration of the biosurfactant above which no further reduction in surface tension occurs and supramolecular structures like micelles and bi-layers form. In this study, the critical micelle concentration (cmc) of the surfaceactive compounds of Pseudomonas aeruginosa Strain IKW1 and Bacillus cereus Strain R15B were 21 and 12 mg/mL respectively. Different surface tension reduction and cmc values of biosurfactants have been reported by various authors and biosurfactants with surface tension reduction up to 35 dynes/cm and cmc between 1 and 150 mg/L are reported to be good biosurfactants [7]. This indicates that the best two bio-molecules in our study are effective and efficient and therefore suitable for their desired applications. Different solvents and/or their mixtures have been adopted for biosurfactant isolation depending on the chemical nature of the biosurfactant. In this study, acidification, followed by solvent extraction with chloroform:methanol recovered 4.39 g/L of crude biosurfactant.
The successful recovery of biosurfactants from cell-free fermentation broth by this method had earlier been reported by Onbasli and Aslim [36].
Biochemical characterizations, thin layer chromatography, high performance liquid chromatography and Fourier transform-infrared (FT-IR) spectrometry identified surface-active compound by Bacillus cereus strain R15B as a lipopeptide (data not shown) and that from Pseudomonas aeruginosa strain IKW1 as a polymeric compound composed of 40.36% carbohydrates, 20.16% proteins and 32.56% lipids. As far as we know, this is the third report on the ability of a strain of Pseudomonas aeruginosa to produce a glycolipopeptide (or peptidoglycolipid) biosurfactant. The first appearance of this report came from Koronelli et al.
[37] while the second came from Ilori and Amund [38]. This therefore contradicts the speculation that Pseudomonas aeruginosa only produces rhamnolipid (glycolipid) type of biosurfactant. Two decades before the brilliant review of Desai and Banat [7] on biosurfactants, a strain of Pseudomonas aeruginosa [39] was reported to produce a protein-like activator PA for n-alkane oxidation. Thavasi et al. [21] recently isolated a strain of Pseudomonas aeruginosa from salt-water that produces a lipopeptide biosurfactant. The implication of these is that different strains of Pseudomonas aeruginosa obtained from different samples at different climatic and ecological conditions would produce surface-active compounds of differing chemical nature and composition to facilitate their competitiveness in those environments.
The commercial applications of biosurfactants are intrinsically related to their activities and a number of such activities were investigated in our study. The washing activity of the glycolipopeptide investigated by the sand pack method at different temperatures was targeted at demonstrating the oil recovery potential of the biosurfactant. The dual influences of holding time and temperature on the recovery rate of Nigerian medium crude oil at a constant biosurfactant concentration were significant (P < 0.05). Our results of 79.92% recovery of the oil at 70°C with the highest elution rate of 0.68 mL/min occurring at 90 min suggests sublime efficiency of the glycolipopeptide to recover crude oil from producing and abandoned oil reservoirs.
The ability of hydrophobic organic compounds to be solubilized and transported into the immediate vicinity of bacterial cells capable of metabolizing (degrading) them is potentially the rate-limiting step in bioremediation [40]. The underpinning activities of microorganisms able to degrade hydrophobic organic compounds include solubilization; which makes the compound bio-available, and emulsification; which makes the compound bio-accessible, to the bioremediating microorganisms. The solubilization ability of our study glycolipopeptide was excellent increasing with glycolipopeptide concentration and suggesting significant (P = 0.000) influence of biosurfactant on the amount of oil solubilized. The linear regression model used to fit the data revealed that 99.01% of the solubilized oil was due to the biosurfactant and that 0.99% of the dissolved oil would be due to other factors especially water and temperature. The glycolipopeptide in this study was able to emulsify kerosene, crude oil and palm oil to emulsification indices of 79.71%, 84.87% and 87.54% respectively. Our results are in agreement with those of a glycolipopeptide produced by Bacillus sp.E34 [18] with similar emulsification index of 77% in kerosene and 84.5% in crude oil. Our results of soil washing experiment as well as those of solubilization and emulsification indicate the potentials for application of our glycolipopeptide in enhanced oil recovery, remediation of environments polluted with petroleum and petroleum products as well as treatment of palm oil effluents.
The usefulness of biosurfactants with antimicrobial properties in bioremediation of hydrocarbonimpacted ecosystems has been a research question in our labouratory for some time. Inhibitory activity of our glycolipopeptide against Bacillus subtilis UCCM 0006; a most potent crude oil degrader in the University of Calabar Collection of Microorganisms (UCCM) goes to suggest the non-suitability of this surface-active compound for bioremediation when the major remediating organism is Bacillus subtilis. Interestingly, the biosurfactant was metabolized by Serratia sp. Strain DW2 (100% sequence homology with Serratia sp. Strain ZJ-I-JQ954966A) as sole source of carbon and energy and so demonstrated no antimicrobial activity against it. This indicates the biodegradability potential of the biosurfactant and suggests its suitability for applications in environments dominated by this bacterium. The antimicrobial potentials of our biosurfactant suggest the need for microbiological characterization of hydrocarbon-polluted sites before biosurfactantenhanced remediation attempt.
A property that is immediately visible upon agitation of a biosurfactant solution is foaming and this is characteristic of all detergents. The glycolipopeptide under study was found to demonstrate excellent foaming power and therefore excellent detergency. The small densely-packed foam ensured strong intermolecular forces responsible for the attraction of the foam molecules together ensuring good activity and stability; a property that facilitates emulsion formation and stabilization of the surface-active compound. The foam power and its stability for more than 48 h suggest suitability for applications in the detergent and food industries.
The oil displacement activity of a biosurfactant is a property that ensues as the contact angle at the oilwater interface is changed by a surface-active compound and varies directly with the amount of biosurfactant in the sample. The more the active compound at the oil-water interface the more its pressure; the more the pressure the lower the contact angle and the greater the solubilization of the hydrophobic component in the solvent. This activity is therefore a rapid indirect assessment of the solubilization potential of a biosurfactant and its stability to the influences of temperature, pH and NaCl is essential for oil recovery and bioremediation exercises. The oil displacement activity of the glycolipopeptide in this study was significantly stable to temperatures up to 80°C and pH from 7 to 9 but decreased linearly with increase in NaCl concentrations. These, in consonance with its oilwashing activity, suggest the suitability of the surface-active compound for biosurfactant-enhanced recovery of crude oil.
On the other hand, the emulsification activity of the glycolipopeptide increased with increase in temperature indicating the thermal stability of the bonds involved in the formation of the polymer. The emulsions formed at high temperatures, with kerosene as the hydrophobic compound, were stable for more than 4 months at the ambient conditions of our labouratory. The emulsifying ability of the glycolipopeptide was stable over a wide range of pH values (6-10 for kerosene and 7-10 for palm oil) but decreased gradually with increasing NaCl concentrations. Only 23.47% of the emulsifying activity of the biosurfactant was lost at 10% NaCl concentration which underpins the suitability of the compound for tertiary recovery of oil.
The antimicrobial activity of the glycolipopeptide was stable up to 60°C and decreased as temperature increased. The loss of antimicrobial activity at this temperature and the stability or improvement of oil displacement and emulsifying activities suggests the probable loss of the peptide moiety of the glycolipopeptide and its possible non-requirement for emulsifying activity. This is at variance with the hypothesis that protein moiety is responsible for the emulsifying activity of biosurfactants [41]. If this was so, then rhamnolipids would have no emulsifying activities. pH and NaCl significantly (P < 0.05) affected the antimicrobial property of the glycolipopeptide, nevertheless, the activity was stable at alkaline pH and NaCl concentrations below 10%. Acidic pH values have been shown to influence the electrostatic interactions between polar head groups of biosurfactant molecules [42]. This suggests the applicability of the surface-active compound for antimicrobial coverage in preparations of food and pharmaceutical formulations where emulsion developments are desirable.
5.
Conclusion Pseudomonas aeruginosa Strain IKW1 was isolated from the mesotidal waters of Ikang River, Niger Delta area, Nigeria and demonstrated commendable ability to elaborate a rare but effective and efficient surface-active compound identified as a glycolipopeptide. The active compound demonstrated excellent properties of oil washing, solubilization, emulsification, foaming and antimicrobial action. These activities were stable at moderately high temperatures, alkaline pH and NaCl concentrations below 10%. The bacterium is recommended for large-scale production of the biosurfactant on waste frying oil as a restaurant waste management option and for applications in tertiary oil recovery, bioremediation of hydrocarbon-impacted environments and the development of detergent, food and pharmaceutical preparations where emulsion development, stabilization and preservation are desired. 11.9 ± 0.8 EW6 11.3 ± 1. | v3-fos-license |
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} | pes2o/s2orc | Fine Tuning of the Properties of Organically Modified
Oxopolimetalatos organicamente modificados do “tipo Keggin”de for mula [R]4[SiW11O40(SiR’)2], (R=Bu4N, R’ = -C2H5, -C10H21, -CH=CH2, -CH2CH=CH2, -OH, -C6H5, -C10H7, -C6H4NH2 (o, p), -C6H4NMe2 (p), -C6H4CH=CH2, -C6H4CF3, -C6H4CH=CHC6H5) foram sintetizados. O núcleo óxido apresentou propriedades re dox reversíveis que foram sintonizadas, escolhendo-se o modificador orgânico. As variações nos deslocamentos químicos na RMN de W, induzidas pelos modificadores, foram também medidas e ambos os efeitos correlacionados com as propriedades de doação-aceitação eletrônica do rad i cal orgânico. As mag ni tudes destes efeitos foram comparadas com aqueles causados pelo eletrólito.
In tro duc tion
Over the last de cade, there has been great in ter est in the study of polyoxometallates (POM) as mod els for tran si tion metal ox ides 1 .These com pact ox ide clus ters of small size (around 10 Å) pres ent unique re dox prop er ties and the abil ity to mimic tran si tion metal ox ide fea tures 2 .The redox prop er ties are sen si tive to the POM com po si tion and struc ture.Strong ef fects are also mea sured with changes of elec tro lyte 3 .Re cently, the graft ing of or ganic rad i cals to a Keggin-type ox ide core was de scribed 4,5,6 .Asa first ex am ple of the `mod i fier ef fect', we re ported pre vi ously the graft ing of polymerizable groups to POM structrures as a pow er ful way to de velop mixed or ganic-inorganic poly mers with ad just able prop er ties 7,8 .
In this Pa per, the in ter ac tions be tween the or ganic modi fier and the ox ide core are mea sured for or gan i cally mod ified POM (OMPOM) with var i ous sub stitu ents.Re dox po ten tials (cy clic voltametry) and NMR chem i cal shifts ( 29 Si and 183 W) are used as sen si tive probe for such in ter actions.were syn the sized fol low ing two pro ce dures pre vi ously described 5 .Trichlorosilane (Cl 3 SiR') in wa ter or trialkoxysilane ((OEt) 3SiR') in acid i fied wa ter were added to lacunary Keggin POM [SiW 11 O39 ] 8-(Ta ble 1).The charac ter iza tion of a few OMPOMs has al ready been de tailed by el e men tal anal y sis, IR, time of flight mass spec trom e try, 29 Si and 183 W NMRs.These anal y ses as sert the graft ing of two R' groups, sym met ri cally an chored to the edges of the hole in the lacunar [SiW 11 O39 ] 8-clus ter, as pro posed by Knoth 9 (Fig. 1).All com pounds were pu ri fied twice by recrystallization in DMF/wa ter or DMF/ac e tone mix tures and the pu rity of these com pounds was shown to be above 95% (main im pu rity is [Bu 4 N] 4 [SiW 12 O 40 ]).
NMR spec tros copy
1 H, 13 C-{ 1 H} and 29 Si-{ 1 H} NMR spec tra were recorded to check the pu rity of the com pounds (Bruker AC250 spec trom e ter). 183W NMR spec tra were re corded on a Bruker AM500 spec trom e ter fol low ing usual pro cedures 5 .OMPOM salts were dis solved in DMF/DMSO-d 6 Ta ble 1 .Prop erties of [Bu 4N] 4[SiW 11 O40 (SiR') 2] salts.Syn the sis pro ce dures for the organo-silicon pre cur sor : (a) (OEt) 3SiR' com mer cial, (b) Cl 3SiR' com mer cial, (c) (OEt) 3SiR' from Si(OEt) 4; R'Br; Mg/Ether, (d) 183 W : 1.0 M so lu tion of Na 2WO 4 (pH = 10).With re spect to the low sen si tiv i ties of these tech niques, mea sure ments could be only ob tained when suf fi cien t quan ti ties of com pounds were avail able.W1, ... W6 la bels are ar bi trary as signed in re gard to the POM struc ture.Elec tro chem is try: Re duc tion po ten tials ( vs. SCE) for the two first re dox stages in Bu 4NBF 4 (0.1M)/DMF elec tro lytes, scan rate 50 mV/s.
Elec tro chem is try
Elec tro chem is try mea sure ments were per formed in differ ent elec tro lytes, fol low ing usual pro ce dures 10 .In all exper i ments, the con cen tra tion of the elec tro lyte was 0.1 M in sup port ing salt, and the con cen tra tion of OMPOM was fixed to 10 -3 M. The work ing elec trode was a 0.07 cm 2 disk of glassy car bon, pol ished be fore each mea sure ment (1 µm grain size SiC paste).The ref er ence elec trode was a sat urated cal o mel elec trode (SCE), ex cept in acetonitrile where an Ag/Ag + elec trode had to be used.So lu tions were degazed with dried ar gon prior to mea sure ments.Cy clic voltammetry ex per i ments were per formed with scan rate from 1000 mV s -1 down to 1 mV s -1 and char ac ter is tic poten tials were re pro duc ible within 5 mV.Two suc ces sive monoelectronic re vers ible stages pro ceeded, as shown by the 60 mV mea sured be tween re duc tion and ox i da tion stages, coulometric mea sure ments and the in ten sity-scan rate re la tion ship (I = v 1/2 ).
Re sults and Dis cus sion
Ta ble 1 pres ents the prop er ties of 14 OMPOM as their tetrabutylammonium salts.Vari a tions in the chem i cal shifts of all nu clei can be seen with the na ture of the or ganic mod i fier group. 29Si sig nals are very sharp, and changes in the chem i cal shifts are noted for both the or ganic bonded sil i con atom and the cen tral oxometallate sil i con atom.The first ef fect is due to σ−π in ter ac tions be tween the or ganic group and the sil i con at oms and par al lels those ob served for trichloro or trialkoxy sil anes 11 .NMR Si-W cou plings were ob served on this first sig nal, and their sim i lar val ues re flect the iden ti cal struc ture of the dif fer ent OMPOMs.The mag ni tude of the ef fect on the sec ond sil i con is much lower and one should as sume a mod er ate vari a tion of the sil i con par tial charge 12 .Changes in chem i cal shifts are also ob served for all W nu clei.The mag ni tude of this ef fect varies from 0.8 ppm for W1 to up to 8.4 ppm for W6.Assuming also that this ef fect arises mainly from changes in the par tial charge on the dif fer ent tungstens, the stonger effect mea sured on W5 and W6 as serts the prox im ity of the or ganic groups to these nu clei 13 .
A sig nif i cant vari a tion in the re duc tion po ten tials, measured in DMF/Bu 4 NBF 4 was ob served for the two first redox steps of the dif fer ent OMPOMs, rang ing from 1000mV ( vs. SCE) up to -890 mV.Ta ble 2 re ports the data ob tained for some of these com pounds in dif fer ent solvents.The ferrocene/ferricinium (Fc/Fc + ) cou ple was used for stan dard iza tion of all mea sure ments in or der to compare the re sults on a com mon scale.Se vere vari a tion of the re dox po ten tials was ob served, de pend ing on both the solvent and sup port ing salt.Ob vi ously, the sol vent strongly af fects the re dox po ten tials (it could be parametrized by the ac cep tor num bers 14 ), while the na ture of the sup port ing salt af fects mainly the dif fer ence be tween the re dox stages.How ever, the rel a tive re dox po ten tials of the dif fer ent OMPOMs are not af fected by chang ing the elec tro lyte.This fun da men tal ob ser va tion dem on strates the in trin sic ef fect of the or ganic substituent on the re dox po ten tial, which ap pears to be re lated to the elec tronic ac cep tor/do nor be hav ior of the or ganic substituent.The abil ity to be reduced is in creased when the ox ide core is elec tron de pleted, i.e. , when the R' or ganic group is more electronegative 15 .Fig ure 2 cor re lates the data ob tained from elec tro chem is try and 183 W NMR mea sure ments on some of these compounds.A strong re la tion ship be tween these two sets of mea sure ments is ev i denced by the par al lel ef fects and both should be re lated to the π-ac cep tor char ac ter of lacunary polyoxometalate 16 and the elec tronic ef fect of the or ganic group.The strong π-con ju ga tion be tween both or ganic and in or ganic moi eties is ex alted in com par ing the re dox po tentials of para and ortho aminophenyl de riv a tives, re spectively -1000 and -890 mV ( vs. SCE).Only in the first of these com pounds, the lone pair may be con ju gated with the ox ide core, lead ing to a rather low re duc tion po ten tial.This work re ports the strong syn ergy be tween or ganic and in or ganic com po nents in a se ries of or gan i cally mod ified heteropolymetalates.Two tech niques were used, both mea sur ing the elec tronic charge on the tung sten core.Elec -tro chem is try av er ages the charge ef fect on the whole ox ide clus ter (EPR of 1e -re duced spe cies dem on strates the electron delocalization at room tem per a ture), while 183 W NMR spec tros copy could pro vide a se lec tive map ping of the charge on the dif fer ent tung sten at oms.Mod er ate con ju gation ef fects were mea sured and a fine tun ing of the re dox po ten tial was ob tained by the choice of the mod i fier group.How ever, for these dif fer ent com pounds, the mag ni tude of the intramolecular ef fect is low in com par i son with the solvent ef fect.This could be due to the siloxane bond which is cer tainly not the best elec tronic junc tion.Other mod i fi cation meth ods of polyoxometallates have al ready been described 2,4,16 .They could be a key to ob tain functionnalized mol e cules with un usual be hav ior for the fields of mo lec ular elec tron ics and non lin ear op tics.
Fig ure 2. Cor re la tion be tween elec tro chem i cal (re duc tion po ten tial in Bu 4NBF 4/DMF elec tro lyte ( vs. SCE)) and 183 W NMR chem i cal shifts (for W6 at oms) data.The dots rep re sent the decyl, ethyl, allyl, vi nyl and phenyl spe cies re spec tively, from left to right.
JSiR
(80-20) mix tures (0.1 to 0.4 M).Vari a tions of 183 W chem ical shifts for the dif fer ent sig nals were less than ±0.1 ppm with changes of OMPOM con cen tra tions and slight changes of sol vent com po si tion.The sig nal of the silicotungstate salt was used as an in ter nal ref er ence.The spec tra of the dif fer ent com pounds ex hib ited six sig nals. | v3-fos-license |
2018-04-03T05:23:55.905Z | 2011-08-19T00:00:00.000 | 16089741 | {
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} | pes2o/s2orc | Lime pretreatment of sugar beet pulp and evaluation of synergy between ArfA, ManA and XynA from Clostridium cellulovorans on the pretreated substrate
Sugar beet pulp (SBP) is a waste product from the sugar beet industry and could be used as a potential biomass feedstock for second generation biofuel technology. Pretreatment of SBP with ‘slake lime’ (calcium hydroxide) was investigated using a 23 factorial design and the factors examined included lime loading, temperature and time. The pretreatment was evaluated for its ability to enhance enzymatic degradation using a combination of three hemicellulases, namely ArfA (an arabinofuranosidase), ManA (an endo-mannanase) and XynA (an endo-xylanase) from C. cellulovorans to determine the conditions under which optimal activity was facilitated. Optimal pretreatment conditions were found to be 0.4 g lime/g SBP, with 36 h digestion at 40 °C. The synergistic interactions between ArfA, ManA and XynA from C. cellulovorans were subsequently investigated on the pretreated SBP. The highest degree of synergy was observed at a protein ratio of 75% ArfA to 25% ManA, with a specific activity of 2.9 U/g protein. However, the highest activity was observed at 4.2 U/g protein at 100% ArfA. This study demonstrated that lime treatment enhanced enzymatic hydrolysis of SBP. The ArfA was the most effective hemicellulase for release of sugars from pretreated SBP, but the synergy with the ManA indicated that low levels of mannan in SBP were probably masking the access of the ArfA to its substrate. XynA displayed no synergy with the other two hemicellulases, indicating that the xylan in the SBP was not hampering the access of ArfA or ManA to their substrates and was not closely associated with the mannan and arabinan in the SBP.
Introduction
Sugar beet (Beta vulgaris) is grown commercially for its high sucrose content and contributes to 35% of sucrose production worldwide (Foster et al. 2001). The residue remaining after the extraction of sucrose is known as sugar beet pulp (SBP). Current uses of SBP include use as an animal feed (Deaville et al. 1994;Serena and Knudsen 2007), as a pectin source for the food industry (Turquois et al. 1999) and in the production of paper (Vaccari et al. 2005). However, SBP could also be a potential source of renewable biomass for biofuel production (Foster et al. 2001).
The chemical composition of SBP, as reported in literature, reveals a large hemicellulose fraction (45-61% dry weight), with 20-24% cellulose, as well as 7-8% protein and 1-2% lignin (Foster et al. 2001). According to Micard et al. (1996), SBP has a high arabinose content of 20.9% of the dry weight of the SBP, compared to 1.1% mannose and 1.7% xylose. This probably points to the presence of an arabinose-based polysaccharide such as arabinan. The presence of lignin is considered one of the most important stumbling blocks for the effective enzymatic hydrolysis of lignocellulose substrates (Merino and Cherry 2007;Varnai et al. 2010). Therefore it has become a general practice to pretreat lignocellulose substrates through some chemical pretreatment step, such as acid or alkaline hydrolysis and steam explosion (See reviews by Hendricks and Zeeman 2009;Sun and Cheng 2002). Depending on the type of pretreatment, the lignin portion may be removed or simply disrupted and in some cases the hemicellulose fraction may also be degraded during pretreatment (Hendricks and Zeeman 2009). Enzymatic degradation of cellulose has been considered the most important as most strains of Saccharomyces cerevisiae are only able to ferment glucose (Himmel et al. 2007). However, enzymatic degradation of hemicellulose has become more important to improve the overall yield of sugars which can also be fermented into bioethanol using other microorganisms (Dien et al. 2003;Fortman et al. 2008;Merino and Cherry 2007). As the hemicellulose fraction of SBP is very significant, a pretreatment method was therefore chosen that would disrupt the lignin without degrading the hemicellulose, namely lime pretreatment (Kumar and Wyman 2009).
Lime pretreatment using slake lime (calcium hydroxide) is often employed as it is inexpensive and can be recovered through neutralising to insoluble calcium carbonate and regeneration through lime kiln technology (Kaar and Holtzapple 2000). The optimal lime pretreatment conditions for lignin removal in SBP has not been reported in literature; thus the conditions of lime load, temperature and time were investigated in this study. Pretreatment conditions were evaluated by using three hemicellulase enzymes to enzymatically act on the various pretreated SBP fractions. The resulting activity (as total reducing sugars produced) was considered to be a measure of the pretreatment conditions that caused the greatest enhancement of hydrolysis.
Synergy between enzymes exists when the combination of enzymes displays a higher activity than the sum of the individual activities on the same substrate. It is a ratio that measures the cooperative interaction between enzymes in the degradation of a substrate and the ability of an enzyme to enhance the activity of another enzyme. A study of synergistic interactions assists in determining the areas of recalcitrance in a substrate and the mechanism by which enzymes cooperate to overcome this.
ArfA is an a-L-arabinofuranosidase, a non-cellulosomal family 51 glycosyl hydrolase from C. cellulovorans (Kosugi et al. 2002a). Apart from arabinofuranosidase activity, ArfA also has high activity on arabinoxylan and arabinan. XynA is an endo-xylanase, a family 11 glycoside hydrolase from the cellulosome of C. cellulovorans (Kosugi et al. 2002b). XynA also contains an acetyl xylan esterase module that contributes to deacetylation of acetylated substrates and displays a synergy with the endo-xylanase catalytic domain (Kosugi et al. 2002b). ManA is an endomannanase, a cellulosomal enzyme from C. cellulovorans and is classified as a family 5 glycosyl hydrolase with narrow substrate specificity (Tamaru and Doi 2000).
Several synergy studies have previously been completed on ArfA and XynA (Kosugi et al. 2002a;Koukiekolo et al. 2005); however, the C. cellulovorans mannanase A (ManA) has not been studied in combination with ArfA and XynA. The role of mannanases in the degradation of lignocellulose substrates has only received limited attention. Studies have indicated that an endo-mannanase acted synergistically in the hydrolysis of a lignocellulose substrate, namely sugarcane bagasse (Beukes et al. 2008;Pletschke 2010, 2011). However, whether it may contribute in a similar manner to the degradation of other lignocellulose substrates has not been investigated. Studies have suggested that addition of an endo-xylanase in an enzyme cocktail greatly enhanced the ability of cellulases to hydrolyse a substrate, even where the xylan is only present in very low concentrations (Garcia-Aparicio et al. 2007). Therefore we wanted to investigate whether this may also be the case for a lignocellulose substrate containing low levels of mannan.
The aim of this work was therefore to optimise the lime pretreatment of SBP and then to assess the synergistic interaction (degree of synergy) of the three C. cellulovorans hemicellulases (ArfA, ManA and XynA), using SBP as substrate.
Materials and methods
Expression and purification of ArfA, ManA and XynA Cloned genes of arfA (AY128945) (Kosugi et al. 2002a), manA (AF132735) (Tamaru and Doi 2000) and xynA (AY604045) (Kosugi et al. 2002b) were kindly donated by Prof. Roy Doi (University of California Davis, CA). Purification of ArfA, ManA and XynA was carried out according to the method described in Beukes et al. (2008). The efficiency of the purification was determined by electrophoresis of purified protein samples on a 12% SDS-PAGE gel (Laemmli 1970). The activity of the purified protein samples was confirmed using commercial substrates as described below. Substrates used for each enzyme were birchwood xylan for XynA, locust bean gum for mannanase activity and p-nitrophenyl-a-L-arabinofuranoside for ArfA.
Protein analysis
The Bradford protocol (Bradford 1976) was utilised to determine the protein concentration using bovine serum albumin (BSA) as a suitable standard.
Enzymatic activity: reducing sugar analysis Hemicellulase activity was assayed in 50 mM sodium citrate buffer (pH 5.5) at 40°C using the DNS method described by Miller (1959). Assays were performed in triplicate and the released reducing sugars (as D-xylose equivalents) were reported in units (U) of activity, with 1 U defined as the amount of hemicellulase liberating 1 lmol of reducing sugar per minute.
Substrate preparation
The fresh sugar beet was stored at -20°C upon arrival. The size of the pulp was reduced by chopping it into small pieces and then homogenising with a Waring blender. Multiple wash and filter steps were performed to ensure no residual reducing sugars were present on the surface of the sugar beet. The filtrate was monitored until no reaction with the DNS assay was observed. At this stage the sugar beet was described as 'sugar beet pulp' (SBP). Air-drying of the SBP for 24 h was performed at room temperature and the SBP was stored in an air-tight container.
Optimisation of lime pretreatment
A 2 3 factorial design with central replicate points, with the factors lime loading, time and temperature, was utilised to determine the optimal conditions for a slake lime pretreatment. Individual runs were carried out on 4 g of dried SBP. Wash and filter steps were completed to remove residual lime. The filtrate was tested to ensure that no residual sugars remained present on the SBP after pretreatment. The SBP was air-dried for 24 h at room temperature after completion of pretreatment.
The extent to which different pretreatment conditions were able to enhance enzymatic hydrolysis was determined using an enzyme assay containing equal concentrations (5 lg/ml) of ArfA, ManA and XynA in 50 mM sodium citrate (pH 5.5) for 24 h at 37°C. The design matrix response was noted as the total reducing sugars (TRS), released as a result of hydrolysis using the three hemicellulases. The DNS assay was used to measure TRS with the highest levels indicating optimal pretreatment conditions. A one-way analysis of variance (ANOVA) with Design-Expert Ò 6.0.4 software was utilised to examine the pretreatment data.
Scanning electron microscopy (SEM)
Samples of the SBP before and after lime treatment were prepared by the Rhodes University Electron Microscopy Unit. The untreated and lime pretreated SBP samples were air dried at room temperature. Sample preparation for SEM included a sputter coating of gold (Balzers Union Sputtering Device) on the various SBP samples and subsequent examination on SEM.
Optimisation of hemicellulase synergy Synergy assays were set up with combinations of the three enzymes at different ratios with the total enzyme concentration always amounting to 40 lg/ml. Each enzyme was added at concentrations of 0.0, 12.5, 25.0, 37.5, 50.0, 62.5, 75.0, 87.5 and 100.0% of the total protein concentration. Triplicate reactions were conducted in 50 mM sodium citrate (pH 5.5) at 40°C for 5 days. Enzyme activity was measured using the DNS method described above. The degree of synergy that was obtained in the synergy studies was calculated by dividing the actual activities of the recombinant enzymes obtained with the enzyme assays with the sugar beet pulp, by the theoretical sum of the enzyme activities of individual enzymes, i.e., Degree of synergy ¼ actual observed activity of enzymes in combination theoretical sum of activities of individual enzymes
Enzyme purification
ArfA, ManA and XynA were purified to a single band on an SDS-PAGE gel and identity confirmed through activity assays on commercial substrates (data not shown). Specific activities for ArfA, ManA and XynA were calculated as 72.68, 2.4 and 11.07 U/mg, respectively.
Lime pretreatment
The statistical model was a 2 9 2 9 2 factorial design with the factors lime loading, time and temperature. The response of the design matrix, used to evaluate optimal pretreatment conditions, was the release of total reducing sugars (TRS, lmol reducing sugar per gram of SBP over 24 h) as a result of enzyme activity from the hydrolysis of the SBP using ArfA, ManA and XynA (Table 1). An equal concentration (5 lg/ml) of each of the hemicellulases was used for hydrolysis. The highest levels of TRS was found to be 42.4, which resulted from a lime loading of 0.4 g/g SBP and a pretreatment time of 36 h at a temperature of 40°C. With similar conditions for lime loading and pretreatment time but with the highest temperature (70°C), TRS was much lower at 6.3, but it is not clear why the increased temperature had this effect.
One-way analysis of variance (ANOVA) verified the model with a significant F-value of 8.61 (Table 2). A confidence level of 95% was chosen; thus the terms A (lime loading), B (time of pretreatment), AC (lime loading and temperature), BC (time and temperature) and ABC (lime loading, time and temperature) were all identified as significant as the probability values fell below 0.05. Term AC, representing lime loading and temperature, had the highest F-value at 23.44 indicating that these two conditions displayed the highest significance. However, temperature (C) had a high p value of 0.7255 thus confirming the discrepancy of the temperature effect. A curvature Fvalue of 7.79 showed that there was significant curvature within the model. The triplicate central replicate points of the design matrix were included to give curvature and there is only a 1.07% chance that a curvature F-value could be related to noise.
Three-dimensional perturbation plots were used to compare the three different parameters for pretreatment on resulting enzyme activity. Figure 1a represents the effect of the concentration of lime loading on the enzyme activity, while Fig. 1b represents the effect of duration of pretreatment and Fig. 1c the effect of temperature during pretreatment on enzyme activity.
In Fig. 1a, the x-axis (time) and y-axis (temperature) were consistent with the pretreatment conditions, the z-axis (reducing sugars) represents the response to the enzyme assays run subsequently to the pretreatment. A significant increase in activity (as TRS) with a increase in lime loading was observed (from 0.1 g lime/g SBP to 0.4 g lime/g SBP, see Fig. 1a). The highest activity observed for 0.1 g lime/g SBP, which resulted in TRS of 16.4, was compared to 42.4 for 0.4 g lime/g SBP. Figure 1a displayed a fairly flat gradient at a low concentration of lime (0.1 g lime/g SBP) and changes in temperature or time had little effect in increasing the TRS. Temperature had a relatively greater effect than time at this low concentration of lime, as at 40°C activity released 0.9 TRS (at 12 h) and 3.8 TRS (at 36 h) compared to 13.9 TRS (at 12 h) and 16.4 TRS (at 36 h) at 70°C. With a lime loading of 0.4 g lime/g SBP, a three-factor interaction was observed with an increase in activity and TRS from 8.7 to 42.4 (12 to 36 h) while the temperature was kept constant at 40°C.
The three-dimensional plots in Fig. 1b and c confirm the results from Fig. 1a. Figure 1b, investigating the effect of temperature, clearly demonstrates that the longer pretreatment time of 36 h resulted in the highest enzyme activity (42.4 TRS) compared to an enzyme activity of 15.1 TRS at 12 h. The effect of temperature (Fig. 1c) indicated that at low temperature, lime loading and time of pretreatment were the most important factors. At 70°C, these factors did not appear to have the same impact (Fig. 1c).
The factors lime loading and incubation time produced significant responses within the design model. The third factor, temperature, was not significant with a F-value of 0.13 (Prob [ F 0.7255). Industrially, a lower temperature is more favoured as there is a lower energy demand, resulting in lower costs, and with high temperatures a pressure vessel would be required (Wyman et al. 2005). Thus the effect of temperature actually posed an advantage. Therefore, the optimal conditions from this study of 40°C, 0.4 g lime/g SBP and 36 h were used as the optimal pretreatment conditions for all subsequent experiments. As the SBP contains quite low amounts of lignin (1-2%), optimal enhancement . 1 The effect of lime loading (a), duration (b) and temperature (c) during pretreatment of SBP on the TRS as a result of enzyme activity of a combination of ArfA, ManA and XynA (5 lg/ml each). The TRS recorded is displayed as released reducing sugar on the z-axis of hydrolysis by pretreatment appeared to require a much lower severity than found with other substrates.
Effect of lime pretreatment on structure of SBP using SEM The scanning electron micrographs in Fig. 2 display the impact of the lime pretreatment on the SBP. The untreated pulp fibres (Fig. 2a) had a structured architecture and the fibres were clearly visible. However, after pretreatment, the structure of the SBP appears to become more amorphous and the fibres were indistinguishable (Fig. 2b), indicating that fibre disruption had taken place.
Synergy studies with C. cellulovorans ArfA, ManA and XynA on pretreated SBP Various combinations of the three hemicellulases were tested on the optimised lime pretreated SBP. The results in Fig. 3 are depicted in a three-dimensional triangle that can be read according to the tri-directional arrowed key. Relative concentrations of the enzymes are depicted on the three axes, with the combinations which produced the highest activity indicated. Figure 4 depicts the same three enzyme combinations, but reflects the degree of synergy displayed by the different ratios of enzymes in the assay, rather than the actual activity. The highest activity was noted at 100% ArfA (4.2 U/g protein), indicating that the arabinofuranosidase contributed extensively to hydrolysis of this substrate. As ArfA also displays arabinase activity, it was able to act on the high concentration of arabinan in the SBP and convert it to increased levels of reducing sugars. The surrounding combinations had a slightly lower activity ranging between 2.3 and 3.2 U/g protein, indicating that the higher activity levels favoured higher ArfA concentrations. Achievement of high hydrolysis yields would also require the addition of cellulases within an optimised enzyme mixture, but the results clearly indicate that an arabinase/arabinofuranosidase would be the most important hemicellulase to use in combination with cellulases.
Synergistic associations, which revealed cooperative interaction between the three hemicellulases, were shown to be optimal (i.e. exhibit the highest degree of synergy) at 75% ArfA: 25% ManA (Fig. 4). The low mannan composition of the SBP explains the relatively low requirement for ManA activity. But the fact that synergy between these Fig. 3 Specific activities of ArfA, ManA and XynA with SBP. The relative specific activities are indicated using the code provided. The three enzymes were mixed in various ratios of protein concentration to a total of 40 lg/ml. Each enzyme is displayed on the axes as a percentage of total protein and the arrows indicate the directions of axes for each enzyme concentration Fig. 4 Degrees of synergy of ArfA, ManA and XynA with SBP. The relative degrees of synergy are indicated using the code provided. The three enzymes were mixed in various ratios as a percentage of the total protein of 40 lg/ml in every assay. Each enzyme is displayed on the axes as a molar percentage and the arrows indicate the directions of axes for each enzyme concentration. The degree of synergy that was obtained in the synergy studies was calculated by dividing the sum of the actual activities of the recombinant enzymes obtained with the enzyme assays with the sugar beet pulp, by the theoretical sum of the recombinant enzyme activities two enzymes was established, indicated that the presence of ManA enhanced the activity of the ArfA, possibly due to the mannan in the substrate hampering access of ArfA to the arabinan in the SBP. The presence of XynA did not increase activity at a synergistic level, despite a similar content of XynA to ManA in SBP. This may suggest that the xylan was not closely associated with mannan or arabinan in the SBP.
The results obtained for specific activity and degrees of synergy using the various combinations of hemicellulases therefore highlighted the high requirement for ArfA, although ManA enhanced activity of ArfA. The low mannan content in SBP as reported by Micard et al. (1996) did not suggest that an endo-mannanase would be required for the synergistic degradation of SBP. However, it is possible that the role of mannanases have thus far been understated in the degradation of complex substrates.
A similar observation was made in the study by Beukes et al. (2008) on the synergistic degradation of sugarcane bagasse by enzymes from C. cellulovorans, where it was observed that mannanase was required for high synergy on this substrate even though bagasse only contained low levels of mannan. It was therefore suggested by Beukes et al. (2008) that the mannan fibres were obstructing XynA and EngE from acting on the xylan and cellulose fibres.
Some indication of the importance of arabinases/arabinofuranosidases in SBP hydrolysis has been suggested in literature. Spagnuolo et al. (1999) reported that incubation of beet pulp with two arabinases (a-L-arabinofuranosidase and endo-arabinase), used singularly or in combination, led to the hydrolysis of arabinan, which produced arabinose in the hydrolysate. In addition, Van der Veen et al. (1991) found that sugar beet pulp was an inducer of arabinase activity in Aspergillus niger N400. Three arabinan degrading enzymes (a-L-arabinofuranosidase A, a-L-arabinofuranosidase B and endo-arabinase) were produced. Utilisation of SBP as a substrate for bioethanol production should therefore include arabinase/arabinofuranosidase, as well as mannanase, in an enzyme consortium for enzymatic degradation.
This study provides information on the structure of SBP and the manner in which the hemicellulose fibres are associated within the pulp. While ArfA proved to be the most important hemicellulase for degradation of the hemicellulose fraction of SBP, the unexpected result was the role of ManA in this process. Based on the very low mannan content in SBP, 1.1% according to Micard et al. (1996), mannanase would have appeared insignificant for the degradation of SBP hemicellulose. However, it was demonstrated that ManA had a clear synergistic relationship with the ArfA and therefore should be included in an enzyme consortium for degradation of SBP. SBP has the potential to be utilised as a second generation biofuel source and optimised lime treatment and exposure to arabinase/arabinofuranosidase activity hold the key to the efficient enzymatic hydrolysis of SBP. | v3-fos-license |
2019-01-22T22:30:21.078Z | 2018-12-09T00:00:00.000 | 57758514 | {
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} | pes2o/s2orc | Lipid Extraction from Spirulina sp. and Schizochytrium sp. Using Supercritical CO2 with Methanol
Microalgae are one of the most promising feedstocks for biodiesel production due to their high lipid content and easy farming. However, the extraction of lipids from microalgae is energy intensive and costly and involves the use of toxic organic solvents. Compared with organic solvent extraction, supercritical CO2 (SCCO2) has demonstrated advantages through lower toxicity and no solvent-liquid separation. Due to the nonpolar nature of SCCO2, polar organic solvents such as methanol may need to be added as a modifier in order to increase the extraction ability of SCCO2. In this paper, pilot scale lipid extraction using SCCO2 was studied on two microalgae species: Spirulina sp. and Schizochytrium sp. For each species, SCCO2 extraction was conducted on 200 g of biomass for 6 h. Methanol was added as a cosolvent in the extraction process based on a volume ratio of 4%. The results showed that adding methanol in SCCO2 increased the lipid extraction yield significantly for both species. Under an operating pressure of 4000 psi, the lipid extraction yields for Spirulina sp. and Schizochytrium sp. were increased by 80% and 72%, respectively. It was also found that a stepwise addition of methanol was more effective than a one-time addition. In comparison with Soxhlet extraction using methylene chloride/methanol (2:1, v/v), the methanol-SCCO2 extraction demonstrated its high effectiveness for lipid extraction. In addition, the methanol-SCCO2 system showed a high lipid extraction yield after increasing biomass loading fivefold, indicating good potential for scaling up this method. Finally, a kinetic study of the SCCO2 extraction process was conducted, and the results showed that methanol concentration in SCCO2 has the strongest influence on the lipid extraction yield.
Introduction
Fossil fuels, which include coal, petroleum, and natural gas, comprise 81% of the total energy consumed by mankind in 2014 [1]. Compared to other energy types, developing and producing fossil fuels is the most economical way of energy production. Also, the energy density of fossil fuels is above other alternative energy sources, which means fossil fuels are the most efficient energy sources. However, the use and consumption of fossil fuels has caused many problems. Because fossil fuels are considered to be a nonrenewable and nonsustainable form of energy, their production will decline and eventually be exhausted. Since the combustion of fossil fuels emits greenhouse gases (GHGs), the use of fossil fuels is a major contributor to global warming [2]. The extraction of fossil fuels also causes other forms of environmental degradation [3]. Because of these disadvantages, developing sustainable and cleaner energy forms is a global necessity and has become an urgent pursuit in scientific research.
Biodiesel is a renewable energy form that can be used as a substitute for fossil fuels. It is considered to be one of the best candidates to replace fossil diesel, because it can be directly used in any compression ignition engine without major modifications [4]. Biodiesel can be used in its pure form, but due to its high production cost and poor cold flow properties, it is often used as a diesel additive. Commercially used biodiesel is primarily composed of fatty acid methyl esters (FAMEs) that are produced via transesterification of triglycerides obtained from plants, animal fats, and microalgae [5].
BioMed Research International
Among all of the biodiesel production feedstocks, microalgae are considered to be the most promising feedstock due to their high rate and efficiency of photosynthesis. Microalgae can accumulate high lipid content in their biomass (up to 77% of total biomass), depending on the type of microalgae [6]. Compared to other available feedstocks for biodiesel production, microalgae have higher oil productivity and require less land area for cultivation. Most importantly, commercial-scale microalgae farms would not be in competition with food production [5].
Nevertheless, the main drawback of microalgae as a feedstock for biodiesel production is the energy intensive and costly production process, due mainly to the harvesting and the lipid extraction processes [7].
It is therefore necessary to develop effective methods to reduce the cost of these processes. For lipid extraction, two methods are often used: organic solvent extraction and supercritical CO 2 (SCCO 2 ) extraction. Organic solvent extraction requires a large volume of expensive and toxic organic solvents. It also requires an energy intensive lipidsolvent separation process after extraction, and not all of the solvent can be recycled. In addition, the lipid extraction rate is slow [8].
Compared to organic solvent extraction, SCCO 2 extraction has several advantages. SCCO 2 extraction is faster and has a higher selectivity toward biodiesel desirable lipid fractions, and the solvent-liquid separation process is not required, since SCCO 2 will become gas after pressure is reduced [8]. The main drawback of SCCO 2 extraction is its high equipment installation cost and its high energy requirement [9].
Previous researcher has demonstrated effective results for SCCO 2 lipid extraction from microalgae. Halim et al. [10] studied the effect of SCCO 2 on extracting lipid from the marine microalga Chlorococcum sp. They found that 80 min of SCCO 2 extraction at 4400 psi and 30 ∘ C resulted in a maximum lipid yield of 7.1 wt% of dry biomass, which was equivalent to 5.5 h of hexane extraction using a Soxhlet apparatus. Mendes et al. [11] studied the SCCO 2 lipid extraction of Arthrospira maxima with the addition of ethanol. They found that the addition of a polar modifier such as ethanol could greatly improve the results of SCCO 2 extraction. Under the optimal experiment conditions of 60 ∘ C and 5000 psi, the addition of ethanol could increase the -linolenic acid (GLA) extraction yield from 0.05 wt% of dry biomass to 0.44%. Other researchers also have reported high efficiency supercritical CO 2 extraction [12][13][14].
However, many of these experiments were conducted at the bench scale, and few of them used microalgae masses beyond 200 g. For a larger amount of microalgae sample, the effectiveness of SCCO 2 extraction is not well investigated. Moreover, most of the SCCO 2 extraction experiments were conducted at high pressures (at or above 5000 psi). Operating at such high pressures at commercial scale is more costly and could be potentially dangerous. Further, adding a polar modifier to the SCCO 2 extraction process has been demonstrated to improve extraction efficiency [11]. Lorenzen et al. [15] conducted an industrial scale lipid extraction on the microalga Scenedesmus sp. using SCCO 2 at low pressure (1740 psi) without any modifier or cosolvent in the extraction process, resulting in a long extraction time requirement (9 h) for a high lipid extraction yield.
The goal of this study is to investigate the scaling up potential of SCCO 2 extraction for microalgae. Our study focused on SCCO 2 extraction at a larger scale (from 200 g to 1 kg) at moderate pressures (below 5000 psi) with a modifier (methanol) added. A comparison between SCCO 2 extraction and Soxhlet extraction was also conducted. The fatty acid profile in the extracted lipids was analyzed in order to predict the quality of biodiesel produced. Finally, a kinetic model was established to analyze the effects of pressure, methanol concentration, and CO 2 flow rate on the lipid extraction yield.
Optimizing the Operating Pressure of SCCO 2 Extraction.
The SCCO 2 extraction unit used in this research is shown in Figure 1(a). Figure 1(b) is a schematic representation of the supercritical extraction process. 200 g of dry microalgae biomass is first placed in an extractor. An air-driven gas booster was used to pump CO 2 from a cylinder to the extractor and increased the pressure in the extractor. The operating pressure has a significant impact on the SCCO 2 extraction process, because the solubility of SCCO 2 is a function of the pressure.
A pressure regulator was installed at the exit of the extractor in order to reduce the pressure in the separators, where the pressure was kept at 350 psi. The CO 2 became a supercritical fluid and was able to extract lipids from the microalgae in the extractor, returned to a gas phase in the separators, and then was pumped back to the extractor by the air-driven gas booster. The extracted lipids were automatically collected in the separators. A valve was installed at the bottom of each separator to collect lipids after the operation. After the extraction process, the CO 2 in the system was pumped back to the CO 2 cylinder for recycling. Although two separators were used in the process, most of extracted lipids were collected in the first separator. The purpose of the second separator was to provide extra retention time to ensure pure CO 2 was pumped back to the CO 2 cylinder. The entire system was maintained at 50 ∘ C.
In order to investigate the effect of pressures, three different operating pressures were tested during the SCCO 2 extraction process: 2000 psi, 3000 psi, and 4000 psi. Since the CO 2 flow in the system was controlled by the air-driven gas booster, the pressure in the extractor also affected the CO 2 flow rate in the system. The corresponding CO 2 flow rates for the gas booster operated under 2000 psi, 3000 psi, and 4000 psi were 7.5, 7.1, and 6.8 standard cubic feet per minute (scfm), respectively, based on the performance curves of the gas booster. The residence time of CO 2 in the extractor can be roughly estimated using the ratio of CO 2 mass in the 5 L extractor to the CO 2 mass flow rate. Under 2000 psi, 3000 psi, and 4000 psi of pressure, the residence times of CO 2 were calculated as 0.13, 0.18, and 0.20 h, respectively.
Two different species of microalgae were tested in the SCCO 2 extraction. Spirulina sp., a blue green alga commonly used as a food supplement or animal nutrition [16] with a lipid content of less than 33.3% dry weight of biomass (as reported by the producer), was purchased from Ojio5 (India). The other species was Schizochytrium sp., which is a marine microalga characterized by its high content of polyunsaturated fatty acids [14], had a lipid content of 25% dry weight of biomass (as reported by the producer), and was purchased from Xiamen Huison Biotech Co. (China). The lipid content of this microalga was determined after docosahexaenoic acid (DHA) extraction conducted by the company. Both microalgae were obtained as dry powders and were used directly in the extraction without pre-treatment. CHNS analysis and TGA analysis were conducted for both species to determine the elemental composition in microalgae biomass. For Spirulina sp., the composition was 45.83 wt% C, 6.52 wt% H, 10.73 wt% N, 0.66 wt% S, 20.76 wt% O, 6.46 wt% moisture, and 9.04 wt% ash. For Schizochytrium sp., the composition was 62.57 wt% C, 9.55 wt% H, 0.82 wt% N, 0 wt% S, 22.89 wt% O, 0.48 wt% moisture, and 3.69 wt% ash. These results were generally consistent with published data [17,18]. Lower nitrogen and sulfur contents in biomass are preferred, since the biodiesel produced by that biomass will have lower exhaust emissions (such as NO x and SO 2 ) when used in a diesel engine [17]. Lower oxygen percentage in biomass is also desirable, since it can affect the stability of the produced biodiesel [17].
The SCCO 2 extraction experiments were operated for 2, 4, and 6 h, and the extraction yield was measured after each time period during operation. The lipid extraction yield was calculated using the mass loss of the dried biomass before and after extraction, using the following equation: where i is the initial weight of the dried biomass (g) and f is the final weight of the dried biomass (g). The equation could overestimate the lipid extraction yield, since nonlipid components such as chlorophyll could also be extracted; however the equation was considered to be appropriate to compare the effectiveness of the extraction method for the tested conditions. Due to the physical characteristics of the extraction system, extracted lipids remaining in the extractor could not be easily measured; therefore the total mass of lipids was not used in the evaluation.
For comparison, lipid extraction using the organic solvent method was also investigated. A Soxhlet apparatus was charged with 25 g dry biomass, and 150 mL of methylene chloride/methanol (2:1, v/v) was refluxed for 6 h [19]. The extraction yield was calculated according to (1). An independent samples t-test was conducted to determine whether the difference was significant for the two extraction methods.
Investigating the Addition of Methanol and Its
Effect on SCCO 2 Extraction. Because SCCO 2 can be treated as a nonpolar solvent, it will be difficult for SCCO 2 to extract lipid complexes that are linked to the proteins on the microalgae cell membrane. Therefore, mixing SCCO 2 with a polar solvent such as methanol might be a good way to increase the efficiency of SCCO 2 extraction [20]. Methanol was chosen instead of ethanol due to its relative inexpensiveness, lower boiling point, and good solvation potential for polar components.
To investigate the effect of adding methanol to the SCCO 2 extraction, 200 mL of methanol was added to the 5 L extractor before the extraction, so that the volume ratio of methanol/SCCO 2 was 1:25 v/v. Experiments were conducted on both species mentioned previously. A stepwise addition of methanol was also tested, where 200 mL of methanol was added at 0, 2, and 4 h, for a total addition of 600 mL. The operating pressure used was similar to the previous experiments. The lipid extraction yield was measured and compared with SCCO 2 extraction without the addition of methanol.
Investigating the Biomass Loading and Its Effect on SCCO 2
Extraction. As stated previously, many experiments in the literature focused on small quantities of microalgae biomass. To assess the potential of scaling up the extraction process, the mass of microalgae in the extractor was increased from 200 g to 1 kg, while maintaining the porosity and the surface area. SCCO 2 extraction was evaluated to see if similar lipid extraction yields could be achieved with larger biomass loading rates. The operating pressures and the amount of methanol added in the extractor were similar to those in the previous experiments. The residence time of CO 2 in the extractor was kept constant when the biomass increased (see Section 2.1). An independent samples t-test was conducted to determine whether the difference was significant for different biomass loadings.
Characterization of the Extracted Lipids.
The fatty acid profile of lipids in microalgae can affect the quality of biodiesel produced from microalgae lipids. Normally, cisunsaturated fatty acids are favored over saturated fatty acids because the FAMEs derived from cis-unsaturated fatty acids often have advantageous cold flow properties [21]. The fatty acid profiles of the lipids from the two species were reported previously in published data [22,23]. Therefore, matching the fatty acid profile in the extracted lipids and comparing to reported data in the literature can be a good indicator of the lipid extraction effectiveness of SCCO 2 .
The extracted lipids from the two species were characterized using gas chromatography (GC). Before the characterization, a derivatization step was conducted to convert the different compounds in the lipids to FAMEs. 1 mL of extracted lipid sample was collected in a test tube, and 1 mL of toluene and 2 mL of 1% sulfuric acid in methanol were added into the test tube. This test tube was then sealed and stored in a 50 ∘ C oven for 8 h. After cooling, 5 mL of 5% sodium chloride in water was added to the test tube to encourage phase separation. After phase separation, 1 mL of the top organic phase was collected in a GC vial for analysis. An Agilent 7890B GC containing a DB-23 column (60 m length and 0.15 m film) and a flame ionization detector was used with helium as the carrier gas. The FAME mixture (C8-C24) (Sigma Aldrich) was used as the calibration standard with methyl myristate as the internal standard. The operating conditions of the GC system were selected according to the literature [24].
Kinetic Study of the SCCO 2 Extraction.
The lipid extraction from microalgae using SCCO 2 can be expressed using first-order kinetic as follows [10]: where M e is the amount of extracted lipids at time t (g/g biomass), M i is the total lipid content in microalgae cell (g/g biomass), t is the extraction time (h), and k (h −1 ) is a time constant correlated to the lipid mass transfer from the microalgae cells to SCCO 2 . For a typical mass transfer problem, the time constant k is a function of Reynolds number (Re) and Schmitt number (Sc), as shown in the following equation: In SCCO 2 extraction, the values of the dimensionless numbers Re and Sc are dependent on pressure, temperature, concentration of methanol, and CO 2 flow rate. Therefore, the time constant k can be expressed as where P is pressure (psi), T is temperature ( ∘ C), [ME] is the concentration of methanol in SCCO 2 (v/v), and Q is the flow rate of CO 2 (scfm). To simplify the model, the effect of temperature was eliminated in (4) due to the constant temperature used in this research. The extracted lipids were treated as a single substance instead of a compound. It was assumed that the all of the extractable lipids could be extracted eventually; therefore all of curves under different operating conditions will have the same asymptotes. M i of the two microalgae species was assumed to be 0.25 g/g biomass. In addition, the methanol concentration in SCCO 2 was assumed to be constant (4%) during the extraction process. Also, the methanol concentration during the stepwise addition was also assumed to be constant (4%). To study the effects of P, [ME], and Q on the time constant k and lipid extraction yield, we assumed a first-order equation to express the relation: where a, b, c, d are unknown constants. These four constants can be estimated by fitting the kinetic model with the experimental data obtained from the previous sections. MATLAB programming platform was used to perform the model fitting, and these constants were determined by minimizing the sum of squared errors between experimental and calculated values of lipid extraction yield. The minimization was performed using the MATLAB toolbox fmincon. Since the lipid mass transfer coefficients may be different for different microalgae species, the estimation of the constants was conducted separately for the two species.
To validate the kinetic model, SCCO 2 extraction data obtained by a previous research conducted by Nobre et al. [25] was also modeled. The authors of the research investigated the lipid extraction yield from microalga Nannochloropsis sp. under different pressures, flow rates, and ethanol concentrations in a laboratory scale SCCO 2 extraction system (5 cm 3 extraction vessel). To perform the model fitting, the ethanol concentration was used in place of [ME] (methanol) in (5). The data used for parameter fitting included the lipid extraction yields at 40 ∘ C, pressures of 1800, 2900, or 4350 psi, CO 2 flow rates of 6.64×10 −3 or 1.18 ×10 −2 scfm, and ethanol concentrations of 5, 10, or 20 wt%. M i used for Nannochloropsis sp. was 0.45 g/g biomass, based on the reported results by the authors, and the biomass loading of Nannochloropsis sp. was 1.25 g.
After obtaining the constants, a sensitivity analysis was conducted to analyze the influence of P, [ME], and Q on the simulated results. Sensitivity analysis was carried out by calculating the lipid mass transfer coefficient (k) and lipid extraction yield at 4 h under the conditions where the value of one parameter was changed by 50% without changing other parameters. The experiment conditions used in the sensitivity analysis were 4000 psi of pressure, 200 mL of methanol with stepwise addition, and 6.8 scfm of CO 2 flow rate. For microalga Nannochloropsis sp., the experiment conditions used were 4350 psi of pressure, 1.18×10 −2 scfm of CO 2 flow rate, and 5 wt% of ethanol concentrations [25].
Lipid Extraction Yields of SCCO 2 under Different
Operating Pressures. Different operating pressures of SCCO 2 resulted in a significant difference in lipid extraction effectiveness from Spirulina sp. (Figure 2(a)). As operating pressures increased, the extraction yield increased, and 4000 psi SCCO 2 resulted in the highest lipid extraction yield. A similar trend was observed in SCCO 2 extraction from Schizochytrium sp. (Figure 2(b)). These results suggest that higher operating pressures should be selected when conducting SCCO 2 extraction; therefore 4000 psi was chosen as the operating pressure for subsequent experiments.
Lipid Extraction Yields of SCCO 2 with Addition of
Methanol. As shown in Figure 3, adding 200 mL of methanol as a cosolvent increased the lipid extraction yield of SCCO 2 for both species in the first 2 h. However, after the initial 2 h, the effectiveness of extraction due to the added methanol decreased and eventually became insignificant after 4 h of extraction. These results indicated that the methanol added to the SCCO 2 extractor might be washed away with the SCCO 2 flow during the extraction process resulting in no increase in lipid extraction yield observed after 6 h of extraction. These results suggested that adding methanol once may not be a good method. To examine this possibility, a stepwise addition of methanol was applied in subsequent experiments. SCCO 2 lipid extraction with a stepwise addition of methanol was also tested by adding 200 mL of methanol every 2 h into the extractor to keep the methanol concentration in SCCO 2 at 4% (v/v), as shown in Figure 4. The stepwise addition of methanol significantly increased the lipid extraction yield for both species compared with pure SCCO 2 extraction. An 80% increase in lipid extraction yield was found for Spirulina sp., while a 72% increase was found for Schizochytrium sp. These results suggest that the stepwise addition of methanol overcame the decreasing methanol concentration and was more effective than adding methanol once. Average yields of 0.164 ± 0.006 and 0.188 ± 0.003 were obtained after 6 h of SCCO 2 extraction with stepwise methanol addition for Spirulina sp. and Schizochytrium sp., respectively.
Soxhlet Lipid Extraction Using Organic Solvents. The
Soxhlet extraction of lipids was conducted for both microorganisms and compared with the highest extraction yield obtained from the SCCO 2 extraction, which was achieved at 4000 psi of SCCO 2 with methanol added sequentially ( Figure 5). The Soxhlet extraction resulted in higher lipid extraction yield for both species. For Schizochytrium sp., the methanol-SCCO 2 extraction produced 91.4% of the lipid extraction yield obtained from Soxhlet extraction after 6 h, but the difference was not statistically significant. For Spirulina sp., the methanol-SCCO 2 extraction produced 79.6% of the lipid extraction yield obtained from Soxhlet extraction, which was statistically significant. While these results indicate that methanol-SCCO 2 extraction is not superior to Soxhlet extraction, the methanol-SCCO 2 method demonstrated comparable extraction yield while avoiding the use of a toxic solvent such as methylene chloride.
Investigating the Scale-Up Potential.
To investigate the scale-up potential of methanol-SCCO 2 extraction, experiments were conducted with increased biomass loadings of up to 1 kg while maintaining other operating conditions (4000 psi, 200 mL methanol added sequentially every 2 h). As shown in Figure 6, increasing the biomass loading from 200 g to 1 kg resulted in a slight decrease in lipid extraction yields for both species. The lipid extraction yield for Schizochytrium sp. decreased by 7.4%, while lipid extraction yield for Spirulina sp. decreased by 7.8%, although neither difference was statistically different. These results demonstrated that the loading amount of microalgae biomass could be increased in SCCO 2 extraction without a significant loss of extraction yield.
Characterizing the Fatty Acid Profile in Extracted Lipids.
For Spirulina sp., the fatty acids extracted by the Soxhlet method differed significantly from those extracted by SCCO 2 , particularly in the composition of decanoic acid (C10), palmitic acid (C16), and total unsaturated fatty acids ( Table 1). The total unsaturated fatty acids were the sum of myristoleic acid (C14:1), palmitoleic acid (C16:1), oleic acid (cis C18:1), linoleic acid (cis C18:2), and linolenic acid (C18:3). On the other hand, the methanol-SCCO 2 extraction resulted in a similar fatty acid composition to the Soxhlet method. The Soxhlet method was presumed to be most representative of complete lipid extraction. The fatty acid compounds were mostly C16 and C18, while total of 47.25% of unsaturated fatty acids was found in the extracted lipids.
The SCCO 2 method extracted significantly lower amounts of unsaturated fatty acids compared to the Soxhlet method, while greatly increasing the content of C10 compounds. However, when combining SCCO 2 with methanol, the fatty acid profile of the extracted lipids was similar to that obtained by the Soxhlet method, with C16 and C18 as the main compounds and total unsaturated fatty acids of 45.11%. These results indicate the content of unsaturated fatty acids in extracted lipids was greatly affected by the polarity of the extraction fluid in that increasing the polarity of the extraction fluid increased the total unsaturated fatty acids extracted.
For Schizochytrium sp., it can be observed that the three extraction methods used in this research resulted in 8 BioMed Research International similar fatty acid compositions with no significant differences ( Table 2). Unlike Spirulina sp., no unsaturated fatty acids were found in Schizochytrium sp.; therefore the difference induced by the polarity of extraction fluid became insignificant, which resulted in the similar fatty acid profiles.
Kinetic Study of the SCCO 2 Extraction.
The estimated values of a, b, c, and d after model fitting with MATLAB are shown in Table 3. The parity plots of calculated lipid extraction yields versus experimental lipid extraction yields for all experiments in this research are presented in Figure 7. For Schizochytrium sp., an average error of 12.7% was found between the experimental data and calculated values while for Spirulina sp., the average error was 16.6%. Figure 8 shows the comparison of simulated and experimental lipid extraction yields at 2000 and 4000 psi, as well as 4000 psi with the addition of methanol. Generally good agreement was observed for both species. However, for Spirulina sp., the simulated results were more accurate under high operating pressures; therefore 4000 psi was chosen as the operating pressure for sensitivity analysis.
Applying the kinetic model to the results from Nobre et al. [25], the estimated values of a, b, c, and d are shown in Table 3.
The parity plot of calculated lipid extraction yields versus experimental lipid extraction yields (Figure 9) showed an average error of 35.6%, indicating the model was less accurate for the SCCO 2 extraction of Nannochloropsis sp. However, model agreement improved between simulated values and experimental data when the extraction was conducted at high operating pressures ( Figure 10).
Results of the sensitivity analysis suggest that the influences of pressure, methanol concentration, and CO 2 flow rate on lipid extraction yield were species dependent (Table 4). However, for all species the methanol concentration (or ethanol concentration) in SCCO 2 had the strongest influence on the lipid extraction yield, followed by the operating pressure. While the CO 2 flow rate showed some influence on the lipid extraction yield for Schizochytrium sp., it was insignificant for Spirulina sp. and Nannochloropsis sp.
Discussion
In our study, results showed that the effectiveness of SCCO 2 lipids extraction increased with an increasing pressure for both species. This is an expected result and matches the findings of the work published by other researchers [26]. It is generally believed that, at a constant temperature, increasing the pressure increases the density of SCCO 2 , which increases the solubility of lipids [8]. Thus, a higher operating pressure of SCCO 2 would produce a stronger lipid extraction power. Therefore, the highest operating pressure that the equipment could sustain, which was 4000 psi, was chosen as the optimal operating pressure for subsequent experiments.
The effect of methanol as a cosolvent in SCCO 2 extraction was shown to increase the lipid extraction yield at least for the first 2 h. The reason for the improved extraction yield can be explained by the polarity of CO 2 and methanol. The CO 2 molecule is a nonpolar molecule and can attach to nonpolar acylglycerols in microalgae cytoplasm by Van der Waals forces during the extraction process [27]. However, some of the acylglycerols in microalgae were associated with proteins on cell membrane via hydrogen bonds. The Van der Waals forces were not strong enough to disrupt the hydrogen bonds. On the other hand, methanol molecules are polar and can easily form hydrogen bonds with the proteins on the cell membrane and replace those lipids, leaving the lipids to be extracted by SCCO 2 [27] thus increasing lipid extraction yield when methanol was added.
Nevertheless, the effectiveness of the methanol addition decreased over time. It was assumed that the methanol added to the SCCO 2 extractor was washed away with the SCCO 2 flow during the extraction process, since methanol was easily soluble in SCCO 2 . Because the SCCO 2 would return to gas status in the separator, methanol would accumulate in the separator, which led to the reduction of methanol concentration in the extractor. In order to overcome this problem, a stepwise addition of methanol was applied for the SCCO 2 extraction, and the results indicated that adding methanol sequentially to the SCCO 2 extractor resulted in higher extraction efficiency compared to adding methanol at once.
Soxhlet extraction using methylene chloride/methanol (2:1 v/v) was reported to be a superior effective lipid extraction method and a good substitute for the Bligh and Dyer method for its lower toxicity. The lipid extraction yields after 6 h of Soxhlet extraction reached 0.21 g/g biomass and 0.22 g/g biomass for Spirulina sp. and Schizochytrium sp. These results were consistent with the reported lipid content on the label (below 33 wt% for Spirulina sp. and 25 wt% for Schizochytrium sp.). The methanol-SCCO 2 extraction method was not significantly different from, or only slightly less effective than, the Soxhlet extraction. Further, the methanol-SCCO 2 extraction excelled in its low toxicity and requirement of organic solvent, since only 600 mL of methanol was used for a 5 L extraction chamber, which can be loaded with 1 kg of biomass (0.6 mL/g). By comparison, the Soxhlet extraction required 100 mL of methylene chloride and 50 mL of methanol for every 25 g of biomass (6 mL/g). Methylene chloride usage, which is considered carcinogenic, was also avoided. Overall, the results indicated that the methanol-SCCO 2 extraction method can produce high lipid extraction yield and might be a good replacement for the organic solvent extraction. In addition, further improvements could be considered to increase the efficiency of the methanol-SCCO 2 extraction method, such as optimizing the methanol amount added at each stage of stepwise addition, or optimizing the temperature provided for the extraction system. Increasing pressure could also be an option, although it may also increase the capital and operating expenses.
The effect of biomass loading rate was also investigated, and it was found that a statistically insignificant decrease in lipid extraction yield occurred when the biomass loading rate was increased from 200 g to 1 kg. The decrease in yield may be explained by the fact that increasing the biomass loading increases the packing density of the microalgae. This might hinder overall extraction as the lipids may reabsorb on the microalgae surface or cause nonhomogeneous extraction via fluid channeling effects [28]. However, the decrease in yield was less than 8% for both species implying that scale-up can be successful and that the methanol-SCCO 2 Extraction yield (g / g biomass) Time (h) and experimental lipid extraction yields (symbols) from microalga Nannochloropsis sp. [25] under different conditions. Red square, 2900 psi of pressure, 6.64×10 −3 scfm of CO 2 flow rate, r 2 = 0.960; blue circle, 4350 psi of pressure, 6.64×10 −3 scfm of CO 2 flow rate, r 2 = 0.931; green triangle, 4350 psi of pressure, 1.18×10 −2 scfm of CO 2 flow rate, 5 wt% of ethanol concentration, r 2 = 0.802; black inverted triangle, 4350 psi of pressure, 1.18×10 −2 scfm of CO 2 flow rate, 20 wt% of ethanol concentration, r 2 = 0.982. extraction method is feasible for 1 kg biomass loadings with our apparatus.
For Spirulina sp., the fatty acid compositions obtained from the Soxhlet and methanol-SCCO 2 method supported published data [22,29], while the SCCO 2 extraction without methanol showed a significant decrease in the extraction of palmitic acid and total unsaturated fatty acids. These results indicate that using SCCO 2 alone was insufficient in obtaining the ideal fatty acid composition from Spirulina sp. due to its ineffectiveness in targeting the lipids associated with cell membrane proteins. Adding methanol to SCCO 2 increased the extracting power of SCCO 2 by extracting all types of fatty acids in the cells. For the Schizochytrium sp., the three extraction methods did not show significant differences. These results also supported published data except for the composition of DHA [23,30]. Since the microalgae used in these experiments were obtained after a DHA extraction process, the absence of DHA was expected. In general, methanol-SCCO 2 extraction could recover lipids with fatty acid composition similar to that extracted by the Soxhlet extraction, proving the effectiveness of this method.
In the kinetic study of the SCCO 2 extraction, the estimated values of a, b, c, and d varied greatly as a function of species and operating parameters. However, the sensitivity analysis of the kinetic modeling showed similar results for all species, indicating that both the operating pressure and concentration of methanol (or ethanol) in SCCO 2 greatly affected the lipid extraction yield. Therefore, to increase lipid extraction yield, the addition of a polar modifier (such as methanol) in the SCCO 2 process might be preferred to increasing pressure, since the increase in pressure would also increase the operating cost. On the other hand, it is not recommended to increase the CO 2 flow rate since it had little or no influence on the lipid extraction yield and the mass transfer coefficient. Similar results were also found by Nobre et al. [25] where the authors noted that the flow rate of CO 2 had no influence on lipid extraction yield.
Conclusions
SCCO 2 combined with methanol demonstrated a high efficiency in lipid extraction from microalgae, as well as a high potential for process scale-up. The high lipid extraction yield and the fatty acid composition in the extracted lipids indicated that the methanol-SCCO 2 extraction method can be used as a good substitute for the Soxhlet extraction method, while the requirement of organic solvents was significantly reduced. At same time, the lack of usage of toxic organic solvents such as methyl chloride showed that the methanol-SCCO 2 extraction was safer than organic solvent extraction, suggesting it might be a preferable method in commercialscale extraction. It should be noted that dry biomass was used in this research. Since the drying process is typically energy intensive, it would be preferable if wet biomass was used in the extraction process. Future research involving the extraction of wet biomass is needed. In addition, further studies regarding the optimization of the rate of methanol addition during the extraction process, as well as temperature effects, should be investigated in order to optimize both the extraction efficiency and economic feasibility of supercritical CO 2 lipid extraction from microalgae.
Data Availability
The figures and tables used to support the findings of this study are included within the article.
Conflicts of Interest
The authors declare that they have no conflicts of interest. | v3-fos-license |
2020-01-23T09:09:53.256Z | 2019-01-15T00:00:00.000 | 213969052 | {
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} | pes2o/s2orc | Contributions on the Sensorial, Physico-chemical and Nutritional Characterization of Hare Meat (Lepus europaeus pallas)
The growing demand for food worldwide, along with the increasing need for animal protein, lead to the identification of other sources of highest quality meat, then the conventional ones, such as it is the hare meat. The aim of this study was to characterize the sensorial, physico-chemical and nutritional traits of hare meat (Lepus europaeus Pallas) issued from hunting funds in North-East of Romania. The biological material consisted of 79 hares (34 males and 45 females), slaughtered by shooting at the age of about 18 months, during the regular hunting season (1 November to 31 January). Different muscle groups were collected: Longissimus dorsi (LD), Triceps brachi (TB) and Semimembranosus (SM). For physicochemical determinations (measurement of the pH at 24 and 48 hours, of water, proteins, lipids, fatty acids and of ash) were analyzed 237 samples (79 for each muscle group). The results obtained from the sensory analysis are relatively close as a score for the three muscle groups studied. The pH value was higher for TB muscles. The highest amount of protein was observed for LD muscles collected from males (21.65%), while the richest in lipids were the females TB muscles (2.38%). The fatty acids levels were predominantly higher for males (for the most of the assessed fatty acids). Very favorable ratio of PUFA:SFA was identified in LD muscles (1.695 for males and 1.531 for females), in SM muscles (1.679 for males and 1.527 for females), and in TB muscles, as well (1.885 for males and 1.820 for females). The variance analysis revealed insignificant gender related differences for the three muscle groups, concerning the sensorial traits, the pH level, ash content and energy value. However, in proteins, lipids and water levels, there were observed highly significant gender related differences in TB muscles. Also, for some fatty acids, significant statistical differences were found between genders, in all three muscle groups.
The meat products are appreciated by consumers, thus there is an increasing demand of those commodities issued from farming systems observing high standards of animal welfare [23]. The attention of farmers and meat producers has been focused on small mammals, such as domestic rabbits (Oryctolagus cuniculus), which provide high quality meat . Another leporid species, the brown hare (Lepus europaeus Pallas), has also been generating interest from meat producers [2,19,51]. The meat of these animals differs from that of poultry and other farmyard animals [52], but the consumption of their meat is not so popular as other [19,52]. Rabbit meat is healthier than other meats frequently used in human nutrition, such as chicken, beef, and pork [53], being easily digested, lean and rich in proteins (with high levels of essential amino acids), unsaturated lipids (ω3 and ω6), B vitamins, potassium, phosphorus and magnesium, low in sodium and cholesterol and very poor in uric acid [54][55][56][57][58][59][60][61].
The brown hare (Lepus europaeus Pallas) is one of the most popular small game species [20] being sometime reared for the restocking of hunting and protected areas in Europe [2,20,51]. Some authors have investigated the * email: [email protected]; [email protected] potential of adding hare meat from hunting into the human diet because of its favorable sensory characteristics, high unsaturated fatty acids [57], proteins, minerals, vitamins content and low-fat content [58,60,61] and its energetic value which is similar to other meats [51]. Hare meat is classified as red meat, mainly in terms of its high iron (Fe) content [61], but its availability is usually restricted by hunting seasons [52,57,60]. To produce high-quality meat, it is necessary to understand the characteristics of meat quality traits and the factors that control them but in wild animals is quite difficult to establish the influence of diet on meat characteristics [2,52]. Hares, like wild rabbits, are herbivorous that consume a wide variety of plants and grains that qualitatively and nutritionally differ by season, which may cause large variation in the composition of the meat [59].
There are very few data available in the literature regarding the characterization of hare meat. From our knowledge only three articles approached the quality of hare meat issued from hunting, in Austria [57], in Croatia [60], and in Slovakia [58]; another three recent studies depict the quality of hare meat collected from farmed brown hare in Italy [20,51], and Poland [2]. The lack of data on the characterization of hare meat led us to carry on this study, whose goals were to assess the sensorial, physico-chemical and nutritional traits of hare meat (Lepus europaeus Pallas) collected from hunting funds in North-East of Romania.
Materials and methods
The biological material consisted of 79 hare individuals (34 males and 45 females), slaughtered by shooting at the age of about 18 months, during the regular hunting season (1 November to 31 January). Three different muscle groups (LD -Longissimus dorsi, SM -Semimembranosus and TB -Triceps brachii muscles) were collected, due to their different physical-chemical properties, different metabolic type and in order to cover the main anatomical regions of the carcasses, as well (back -LD, hind leg-SM, foreleg -TB). The muscles on the right side of the carcass were used, to assess the physical-chemical traits (measurement of pH at 24-and 48-h post-slaughter, of water, proteins, lipids, fatty acids and ash contents), summarizing 237 samples (79 for each muscle group). They were preliminary fine grinded and homogenized using an electric shredder. The muscle groups on the left side of the carcasses (237 samples, individually packaged, vacuumed and then prepared for one hour at a constant temperature of 80°C in a water bath), were used for sensory analyses and performed by tasting. After cooling, the samples were cut and given to 23 tasters, trained in advance. The assessment sheets of the sensor y characteristics were filled in using a five-point hedonic scale (scores from 1 to 5), in which one point represented the not favourable features, while 5 points indicated the characteristics which fully satisfied the requirements of the tasters. For example: the extremely pale colour was noted with 1, while the intense red colour was noted with 5; global assessment was scored with 1 for unacceptable meat, with two points for acceptable meat, with three points for good meat, with four points for very good meat and with five points for exceptional meat. For two consecutive days after slaughter, meat pH value was measured, using the digital pH meter Hanna Electronics, type 212, on chilled samples, at 2°C. The water, protein and lipid content were determined using the Food Check Near Infrared Spectrophotometer (NIRS technology); the energy value was determined by calculation using conventional formulas and crude ash content was assessed by calcinations (at 550°C for 16 h after a preliminary carbonization) [62][63][64]. Using the FOSS 6500 spectrophotometer (NIRS technology), the assessment of fatty acids content was performed. The freshly ground samples were placed in sterile Petri dishes, weighed, then lyophilized at -110°C for 24 h , using the CoolSafe™, SCANVAC lyophilizer, weighed again and then vacuumed (in special bags) and stored in a freezer at a temperature of -80°C until the moment of their analysis. The following saturated fatty acids (SFA) were assessed: C14:0 (myristic acid), C15:0 (pentadecanoic acid), C16:0 (palmitic acid) C17:0 (heptadecanoic acid) and C18:0 (stearic acid). Among the monounsaturated fatty acids (MUFA, ω7 and ω9) there were investigated: C18:1n-7 (vaccenic acid cis isomer of oleic acid) and C18:1n-9 (oleic acid); a total of nine polyunsaturated fatty acids (PUFA, ω3 and ω6) were also assessed: C18:2n-6 (linoleic), C18:3n-3 (linolenic), C20:2n-6 (eicosadienoic), C20:3n-6 (eicosatrienoic), C20:4n-6 (arachidonic), C20:5n-3 (eicosapentaenoic or EPA), C22:4n-6 (docosatetraenoic), C22:5n-3 (docosapentaenoic or DPA) and C22:6n-3 (doco-sahexaenoic or DHA) [65][66][67][68][69][70][71][72][73][74][75][76][77][78][79][80][81]. All the achieved results were statistically processed through the main descriptors computation and analysis of variance test (Anova single factor), using the GraphPad Prism 7.0 software.
Experimental part
Following sensory appreciation of hare meat (Lepus europaeus Pallas) it could be noted a higher score of meat color (table 1), with more than 4.14 points for all muscle groups analyzed, for both females and males (4.14 for females in the LD muscles and 4.36 for males in SM muscles). The highest accumulated score for hare meat color was based on the more intense red hue specific to game meat. It is observed that the lowest color values were obtained for the LD muscles compared to the TB and SM muscles, similar results being observed in other studies: in farmed brown hares, the redness index increased at the hind leg, while it decreased in LD muscles, by age [20]. For the other analyzed sensory characteristics, a slightly higher score for males has been achieved. In SM muscles predominated a higher score for most of the evaluated characteristics, except for Fibrous appearance, where a lower score (3.00 for females and 3.07 for males) has been obtained, because muscle fibers are thinner compared to TB muscles and LD muscles. Generally speaking, the values obtained were relatively close for all studied muscles, with some small differences for certain characteristics, and the statistical analysis revealed insignificant differences between genders. Variance analysis showed not significant differences between genders related to hare meat pH level (table 2). Higher values for TB muscles were observed compared to the other muscles (6.003 at 24 h, 6.173 at 48 h in females and 6.062 at 24 h, 6.102 at 48 h in males). At 48 h after slaughter, at the level of the three muscle groups studied, it was observed that in female's pH had higher values than those measured in males, even if at 24 hours the highest pH values were recorded for males. These results are in line with other studies carried on sub-adult and adult reproducing farmed hares [19], that found the pH of hind leg (5.74 to 5.83) and LD (5.53 to 5.69) increased with age. Mertin et al., 2012 [58] found in LD muscles of hunted hares a pH value of 6.17 at 48 h. Kroliczewska et al., 2018 [2] comparing the meat of rabbit with that of hares, found the higher pH of meat for hares (average of 6.11 from: 6.15 in fore leg muscles, 6.12 in hind leg muscles and 6.07 in LD muscles); the highest pH values were observed for the fore legs and the lowest were observed for the LD, which are in accordance with our results. The high pH values in hare meat are explained as a result of the stressful conditions at slaughter and the higher levels of physical activity before the hare individuals were shot, resulting in the exhaustion of muscle glycogen stores prior to death. Furthermore, muscle glycogen level at death largely determines the ultimate pH (pH u ) [82]. Króliczewska et al., 2018 [2] specifies that the pH measurement methodology used in their conducted study (samples delivered to the laboratory within 30 min after slaughter, placed in polyethylene bags, frozen at -20°C for 30 min, and stored at -80°C until analysis), don't measure the real pH u (24 h postmortem/24 h chilling) and probably not fully depleted glycogen may cause observed higher pH values in muscles.
Hare meat was also characterized by its chemical components (proteins, lipids, fatty acids, water and mineral substances -crude ash). The proteins content has the highest average values at the level of LD muscles for males, 21.65%. For the other muscle groups, mean values were also higher for males with relatively low gender differences; the statistical significance of the differences between genders was insignificant for SM and LD muscles and distinctly significant for TB muscles (table 3).
The quantity of proteins found in this study was slightly lower than the one assessed by other authors in Croatia, Poland, Italy [59,2,19,50], but the quantity of lipids was slightly higher; other studies found higher lipids content than in ours [2]. In the meat of hare shot in the eastern region of Croatia, chemical tests showed the following chemical average content: water 75.32%, protein 23.08 %, fat 1.09%, ash 1.16% [60]. In farmed hare meat from Italy there were found on average: 74.3% water, 22% crude protein content in hind leg and respectively 23% in LD, and low ether extract content (2.1% and 1.0% in hind leg and LL, respectively), with ash content averaging 1.35% in the hind leg and 1.44% in LD [20]. Trocino et al., 2018 [20], found that the water and protein contents of hare meat decreased in the hind leg and LD with the age of hares, whereas the ether extract increased in LD only (0.92% to 1.11%). Consistent with Cobos et al. 1995 [52], Króliczewska et al., 2018 [2] wich examined the both wild leporids (rabbit and hare) they found a lower protein content for fore legs (TB muscles) than for either hind legs (SM muscles) or LD muscles. Our findings in the protein content of hare meat are in agreement with Trocino et al, 2018 [20], and lower then that found in other papers [2,51,60].
The highest lipids quantity was determined for TB muscles (2.38% for females), and the lowest was observed for LD muscles (1.52% for males). For all three muscle groups analyzed, the amount of lipid determined was higher for females compared to males. Variance analysis revealed very significant gender differences at the level of TB muscles and insignificant differences for the LD and SM muscles (table 4).
The fat content measured in this study is close to those determined by Mertin et al., 2012 [58], Skrivanko et al., 2008 [60], Vizzarri et al., 2014 [51], Trocino et al., 2018 [20], with small differences at the level of TB muscles from females where we determined slightly higher values (2.38%). Kroliczewska et al., 2018 [2], found higher lipid The lowest water content was measured in TB muscles (74.35% for females) because the amount of lipid determined was also highest for females in the same muscle group (2.38%); for the other muscle groups analyzed, the mean values were higher, exceeding 75% for both sexes, with slightly higher values for males (75.20% for SM muscles, 75.15 for LD muscles and 75.17% for TB muscles).
The water content is very close to that determined by Skrivanko et al., 2008 [60] (75.34% from muscle tissue, unspecified muscle groups), and in line with Trocino et al., 2018 [20] (that found 73.3-75.5 in adult and sub-adult hare hind leg and LD muscles); the amount of water found in present study was higher than that determined by Vizzarri et al., 2014 [51] (72.83%), Kroliczewska et al., 2018 [2] (73% in foreleg, 74% in hind leg and 73.4% in LD muscles) and Mertin et al., 2012 [58] (72.83%). Following the variance analysis test, insignificant differences were found between genders for LD and SM muscles, and very significant differences were found for TB muscles (table 5).
The ash content in the analyzed muscle groups was relatively close, with higher average values for males (1.271% for LD muscles, 1.251% for SM muscles and 1.267% for TB muscles), and following the variance analysis test were observed insignificant gender differences (table 6).
Higher values of ash content were determined in Poland (Kroliczewska et al, 2018 [2] found 2.30% ash in foreleg, 2.43% ash in hind leg and 2.44% ash in LD muscle), and close values were determined in other studies from Croatia The gross energy value supplied through hare meat consumption, showed the highest level for TB muscles collected from males (168.5 Kcal/100 g). For the other muscle groups, the mean values determined were lower but close (144.24-147.13 kcal/100 g), and statistical differences between the genders were insignificant (table 7). Skrivanko et al., 2008 [59] found lower energy value for hare meat from Eastern Croatia (105.07 Kcal/100 g), and Mertin et al., 2012 [58] found 112.13 kcal/100 g at the level of LD muscles collected from hare in South-West of Slovakia.
The average fatty acids content for all the three muscle groups studied shows predominantly higher values for males (table 8). Highest value for saturated fatty acids SFA was highlighted at the level of palmitic acid (339.02 mg/100g for LD muscles collected from females). For MUFA, the highest average values were recorded for oleic acid: in LD muscles 352.51 mg/100g for males and 278.67 mg/100g for females, in SM muscles 307.58 mg/100g for males and 241.18 mg/100g for females, and in the TB muscles were registered 389.08 mg/100g for males and 264.45 mg/100g for females, with very significant statistical differences (p < 0.001). Concerning the PUFA level, the highest values were determined for linoleic acid C18:2 n-6, with more than 500 mg/100g for LD muscles (593.23 mg/100g for males and 501.63 mg/100g for females), for SM with 556.63 mg/100g for males and 413.78 mg/100g for females (p < 0.001); also, for TB muscles was found 674.30 mg/100g for males and 568.53 mg/100g for females (p < 0.01).
After the evaluation of the statistical significance of differences on fatty acid content for TB and SM muscles, significant, distinct significant and ver y significant differences were observed for the majority of the fatty acids determined, and for LD muscles were found insignificant differences for 10 fatty acids from all 16 determined.
In this study, very favorable ratio of PUFA:SFA was found (in LD muscles 1.695 for males and 1.531 for females, in SM muscles 1.679 for males and 1.527 for females, and in TB muscles 1.885 for males and 1.820 for females). The PUFA:SFA ratio was higher than that determined by Króliczewska et al., 2018 [2] (1.17 in LD muscles, 1.20 in hind leg muscles and 1.40 in fore leg muscles) and by Trocino et al., 2018 [20] (0.83 for hind leg of subadult and 1.70 for adult, and 1.16 for females and 1.18 for males, which are in line with the present study, that found for males' higher values).
After Kroliczewska et al., 2018 [2], which determined the quality and fatty acid profile of meat from hare and domestic rabbit, the high PUFA/SFA ratio in hare meat can be perceived as favorable for human health. Also, due to the high PUFA level in hare meat, the atherogenic index was significantly lower for hare meat than for rabbit meat, which should be attractive to consumers by the role of PUFA in reducing cardiovascular diseases; the thrombogenic index tended to be lower for hare meat than for rabbit meat. The low values of both the atherogenic and thrombogenic indices may be features specific to the species.
From the nutritional point of view the consumption of hare meat had a positive contribution in human health, by its high content of protein and low content of fat, with Table 8 THE FATTY ACIDS CONTENT (mg/100 g) FOR LD, SM AND TB MUSCLES FROM HARES favourable index of nutritive value of fat, i.e. proportion of essential amino acids and unsaturated fatty acids. Hare meat is full-value meat, easily digestible with typical aroma for the given species, and has finer muscle fibers than the meat of slaughter animals. Thanks to its relatively low-fat content venison ranks among the richest proteinaceous meat along with fish meat, being higher than in farm animal [52].
Game meat usually comes from hunting, but according to results of recent papers [2,20,52], hares farmed for restocking purposes may be used for meat production due to their favourable slaughter results (high dressing percentage) and carcass traits (high proportion of meat in hind legs and loins), as well as the high nutritional value of meat and favourable fatty acid composition. This fact would offer additional commercial opportunities, in addition to restocking, to hare farmers [20]. Under controlled conditions may be an alternative method for producing a high-quality meat that could increase the health of consumers [2]
Conclusions
The results obtained from the sensory analysis are relatively close as a score for the three muscle groups studied. The pH value was higher for TB muscles. The highest amount of protein was observed for LD muscles collected from males (21.65%), that of lipids for TB muscles from females (2.38%), and that of fatty acids were predominantly higher for males (for the most of the fatty acids determined). Very favorable ratio of PUFA:SFA was determined (in LD muscles 1.695 for males and 1.531 for females, in SM muscles 1.679 for males and 1.527 for females, and in TB muscles 1.885 for males and 1.820 for females). The variance analysis revealed insignificant differences by gender for the three muscle groups studied at the level of sensory analysis, of the pH, of ash content and energy value, except for proteins, lipids and water content where only in the TB muscles, were observed very significant gender differences. Also, for some fatty acids, significant statistical differences between genders were observed, in all three muscle groups. | v3-fos-license |
2019-09-04T05:19:54.953Z | 2019-08-30T00:00:00.000 | 201829599 | {
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} | pes2o/s2orc | In Vitro Analysis of the Effects of ITER-Like Tungsten Nanoparticles: Cytotoxicity and Epigenotoxicity in BEAS-2B Cells
Tungsten was chosen as a wall component to interact with the plasma generated by the International Thermonuclear Experimental fusion Reactor (ITER). Nevertheless, during plasma operation tritiated tungsten nanoparticles (W-NPs) will be formed and potentially released into the environment following a Loss-Of-Vacuum-Accident, causing occupational or accidental exposure. We therefore investigated, in the bronchial human-derived BEAS-2B cell line, the cytotoxic and epigenotoxic effects of two types of ITER-like W-NPs (plasma sputtering or laser ablation), in their pristine, hydrogenated, and tritiated forms. Long exposures (24 h) induced significant cytotoxicity, especially for the hydrogenated ones. Plasma W-NPs impaired cytostasis more severely than the laser ones and both types and forms of W-NPs induced significant micronuclei formation, as shown by cytokinesis-block micronucleus assay. Single DNA strand breaks, potentially triggered by oxidative stress, occurred upon exposure to W-NPs and independently of their form, as observed by alkaline comet assay. After 24 h it was shown that more than 50% of W was dissolved via oxidative dissolution. Overall, our results indicate that W-NPs can affect the in vitro viability of BEAS-2B cells and induce epigenotoxic alterations. We could not observe significant differences between plasma and laser W-NPs so their toxicity might not be triggered by the synthesis method.
Introduction
Thermonuclear fusion could potentially represent an unlimited carbon-free source of energy. The International Thermonuclear Experimental fusion Reactor (ITER) project is-at present-exploiting this hypothesis. Based on the current configuration of the ITER fusion plant, tungsten (W) will be the Altogether, the literature suggests that W can impair the integrity and the normal functioning of in vitro cellular systems representative of different organ compartments. Nevertheless, the genotoxic and epigenetic effects of W-NPs have not yet been fully investigated.
In this study, we used the bronchial human-derived epithelial BEAS-2B cell line [13,14] to describe the epigenotoxicity of bench synthesized plasma sputtering and laser ablation ITER-like W-NPs. In order to mimic the particles generated by the ITER fusion reactor, W-NPs produced by plasma sputtering and laser ablation were hydrogenated and tritiated. Moreover, the size of our ITER-like W-NPs resulted in the HEPA filters escape range, further enhancing the importance of our study in an occupational risk perspective. Cell viability, evaluated via the quantification of the adenosine triphosphate (ATP) by luminescence, and cytostasis, evaluated via the cytome version of the Cytokinesis Block MicroNucleus assay (CBMN-cyt), showed that both plasma and laser ITER-like W-NPs are toxic to BEAS-2B cells and that the effect was enhanced upon exposure to hydrogenated/tritiated particles. Laser ablation-derived ITER-like W-NPs seem to exert a slightly enhanced micronuclei formation and primary DNA damage, as shown by CBMN-cyt and alkaline comet assay, respectively. Pancentromeric staining revealed that plasma and laser ITER-like W-NPs induced both clastogenic and aneugenic effects. Epigenetics, in contrast, showed that laser and plasma ITER-like W-NPs had no effects on the DNA methylation of BEAS-2B cells. Finally, oxidative stress, which has been proposed as the factor triggering the cytogenotoxicity of other types of W compounds, has been investigated and a significant alteration of the oxidized/reduced glutathione content was detected. Since more than 50% of W exhibits oxidative dissolution, the role of W 6+ species must be considered in the biological effects.
Particles Synthesis and Suspensions Preparation
Two synthesis methods were used to produce ITER-like W-NPs with different physico-chemical properties: magnetron plasma sputtering and gas condensation (plasma W-NPs), and laser ablation (laser W-NPs). Detailed description of the production set-up and morphology, composition, and crystalline structure were already described [15][16][17]. Given the characteristics of the tokamak that will operate at ITER, particles of irregular shape and of smaller size than those produced by other already existing W-based tokamaks are expected. Plasma sputtering and laser ablation were thus believed to be the two synthesis methods that can produce W-NPs that most likely will resemble those generated during ITER operation.
Plasma-derived ITER-like W-NPs were produced in a cluster source by condensation of the metal vapors supplied into argon gas flow by radio-frequency (13.56 MHz) magnetron sputtering discharge [18]. The metallic clusters produced were collected on a dedicated substrate.
Laser-derived ITER-like W-NPs were produced using laser power density deposition on a tungsten target, a dust production technique with relative simplicity and flexibility was developed in preliminary projects [19] and upgraded for the specific requirements of this study. Absorption of the laser energy in the W metallic structure triggers the excitation of free electrons, and this energy is relaxed in the matrix through a thermic wave conveyed by phonons. The brief and intense heat source at the surface of an ITER grade W sample submitted to the laser impulses allows the formation of particles by two processes: ejection of melted material or accretion of particles in the plasma formed in the heated Nanomaterials 2019, 9, 1233 4 of 19 volume. We used a picosecond laser of wavelength 1064 nm, frequency 10 Hz, pulse energy density 5 J/cm 2 , and pulse duration 50 ps to produce laser W-NPs [15].
The physical and chemical stability of the W-NPs in biological media were assessed by mean of Dynamic Light Scattering (DLS) using the Mastersizer S (Malvern Panalytical; Orsay, France) and dissolution experiments [17]. The chemical analysis of W in the suspension was performed within inductively coupled plasma mass spectrometry (ICP-MS) (Perkin Elmer Nexion 300; Villebon-sur-Yvette, France). H 2 O 2 solutions at room temperature for 24 h were used to digest the solid forms of W solid forms via a total oxidation. The dissolved W species were isolated by combining two filtration stages at 25 and 20 nm [17].
In the facility where radioactive species were manipulated, all the equipment necessary to investigate the physico-chemical properties and the cyto-epi-genotoxic potential of tritiated W-NPs were not available. We therefore included in our study both the pristine and the hydrogenated forms of laser and plasma W-NPs. Pristine is the form in which W-NPs were bench produced; hydrogenated W-NPs were investigated because they are equivalent to the tritiated ones, but not radioactive. Their lack of radioactivity allowed us to thoroughly study them even in the absence of a dedicated facility.
For the experiments with tungsten, BEAS-2B cells were exposed for 2 h and/or 24 h to increasing concentrations (0-150 µg/mL) of plasma and laser ITER-like W-NPs.
Cytotoxicity Assessment
Intracellular ATP. The viability of BEAS-2B cells was indirectly evaluated via the in vitro quantification of intracellular ATP produced by metabolically active cells upon exposure to W-NPs (1-150 µg/mL). The CellTiter-Glo ® Luminescence Cell Viability Assay was performed as previously described [20]. Data were acquired using a GloMax ® Explorer Multimode Microplate Reader (Promega; Charbonnières-les-Bains, France). For each experimental point, three independent assays were performed, each of them in triplicate (n = 9). The percentage of cellular viability was compared to the unexposed control cells.
Cytostasis and cellular replication. By performing the cytome version of the Cytokinesis Block MicroNucleus (CBMN-cyt) assay [21], adapted to the use with nanoparticles [22], cytostasis and cellular replication were also evaluated. To assess cytostasis, the Cytokinesis Block Proliferation Index (CBPI) was calculated by scoring mononucleated, binucleated, and multinucleated cells in the first 500 living cells analyzed in each sample. CBPI, which indicates the average number of cell divisions completed by the cells, was calculated as follows: The percentage of cytostasis was calculated as recommended by OECD in the test guideline 487 [23]: {100 − 100 × [(CBPI exposed cells-1)/(CBPI control cells-1)]}. The replication index, which represents the proportion of cell division cycles completed in a treated culture during the exposure period and recovery, was calculated as described in the OECD test guideline 487 [23]: [(number of binucleated cells) + (2 × number multinucleated cells)]/total exposed cells/ ([(number binucleated cells) + (2 × number multinucleated cells)]/total control cells) × 100. (3)
Genotoxicity Studies: Micronucleus and Alkaline Comet Assay
Cytokinesis-Block MicroNucleus assay. To identify chromosome breakage or chromosome loss following exposure W-NPs, the Cytokinesis-Block MicroNucleus assay (CBMN) was performed as described before [20].
Briefly, BEAS-2B cells were seeded onto a two-well Lab-Tek TM II Chamber SlideTM System (Nalgene Nunc International, Villebon sur Yvette, France) and treated with increasing concentrations (0-20 µg/mL) of plasma and laser ITER-like W-NPs. After 24 h exposure, cells were washed and 3 µg/mL cytochalasin B (Sigma Aldrich Chimie Sarl; St. Quentin Fallavier, France) was added to the cultures to block cytokinesis; BEAS-2B cells were kept in culture for an additional 28 h and then cells were fixed with 4% (v/v) paraformaldehyde in PBS. Mitomycin C (MMC, 0.1 µg/mL) served as a positive control, whereas LHC-9 culture medium as the negative one. Upon permeabilization, the cytoskeleton was stained with phalloidin-tetramethylrhodamine B isothiocyanate (Sigma Aldrich Chimie Sarl; St. Quentin Fallavier, France), while nuclei with DAPI ProLong ® Gold antifade reagent (Fisher Scientific; Illkirch, France).
CBMN was performed in duplicate, and slides were blindly scored using an Axio Imager. A2 fluorescence microscope (Carl Zeiss S.A.S; Marly Le Roi, France) at 400× magnification. Micronuclei (MN) were only assessed in binucleated (BN) cells that had completed one nuclear division following exposure to the test compounds [20]. For each experimental condition, the number of binucleated micronucleated (BNMN) cells was scored in 1000 BN cells.
Comet assay. To detect the primary DNA damage induced by plasma and laser ITER-like W-NPs in BEAS-2B cells the alkaline comet assay was performed as previously described [20]. BEAS-2B cells, seeded onto pre-coated 12-well plates (BD Falcon; Le Pont de Claix, France), were exposed to W-NPs (1-20 µg/mL) for 2 and 24 h. At the end of the treatment period, cells were washed, trypsinized, resuspended in low melting point agarose, and then spotted onto glass slides pre-coated with 1.6% and 0.8% normal melting point agarose. Cells were then lysed, and the DNA denatured in a MilliQ water solution containing NaOH and EDTA. After electrophoresis (25 V and 300-315 mA), samples were neutralized and dehydrated. Air-dried slides were stained with propidium iodide (PI) right before imaging and data acquisition. Cells incubated only with LHC-9 medium served as negative control, while hydrogen peroxide (110 µM) was used as positive control. Slides, which were prepared in duplicate for each experimental condition, were analyzed under a fluorescence Axio Imager. A2 microscope (Carl Zeiss S.A.S; Marly Le Roi, France) at 400× magnification using the Komet 6.0 software (Andor Bioimaging, Nottingham, UK). DNA damage was expressed as mean % tail DNA ± standard error of the mean (SEM).
Oxidative Stress Evaluation
The induction of oxidative stress in BEAS-2B cells upon exposure to W-NPs (1-20 µg/mL) was detected by performing the GSH/GSSG-Glo™ Assay (Promega; Charbonnières-les-Bains, France), a luminescence-based system suitable to determine the ratio between the reduced glutathione GSH and its oxidized form GSSG in cultured cells. According to the manufacturer instructions, cells were cultured in a 96-well plate and then exposed for 30 min to plasma or laser W-NPs. After exposure, ITER-like W-NPs solutions were removed, and cells were lysed with total glutathione or oxidized glutathione lysis reagent. Luciferin detection regent was then added, and plates were further incubated for 15 min before luminescence was read at a GloMax ® Explorer Multimode Microplate Reader Nanomaterials 2019, 9, 1233 6 of 19 (Promega; Charbonnières-les-Bains, France). Data were analyzed by subtracting the GSSG reaction signal from the total glutathione to obtain the value of reduced glutathione in the sample. Data were expressed as % GSH/GSSG ratio ± SEM. Exposed cells were compared to the untreated ones.
Epigenotoxic Effects of W-NPs
We investigated if pristine plasma and laser ITER-like W-NPs induced epigenetic effects, such as variation in the DNA methylation, in BEAS-2B cells. Cells were seeded onto pre-coated T25 flasks and cultivated under standard conditions until they reached 80% confluency. The BEAS-2B cultures were then exposed for 24 h to 1 and 5 µg/mL pristine plasma and laser ITER-like W-NPs, and untreated controls were also included in the experimental set-up. The collection of the samples was performed at different time points (Table 1). BEAS-2B cells were collected as follows: cell culture medium was removed and cells were washed in PBS before being trypsinized and centrifuged (1200 rpm, 5 min, RT). The pellets were dried by aspiration and then stored at −20 • C before DNA methylation was investigated.
We analyzed the methylation of the most frequent repetitive DNA sequences found in the human genome. Alu and LINE are, respectively, short or long interspersed repeats found throughout the genome, i.e., either in highly condensed heterochromatin and transcriptionally inactive loci but also in less condensed regions containing expressed genes. Given this broad distribution, epigenetic status of these two types of repeats is considered as a surrogate marker of the global profile for a given cell. We also analyzed DNA methylation of Satellite 2 and 3 (Sat 2 and Sat 3) repeats, corresponding mainly to heterochromatin associated with centromeric regions and sensitive to exogenous stress. DNA methylation was determined after sodium bisulfite modification of genomic DNA. This method is based on the oxidative desamination of cytosines. Unmethylated cytosines are converted to uraciles while methylation cytosines are not converted, allowing the analysis of individual CGs in any given sequence after PCR amplification and deep sequencing. DNA was extracted from the different types of samples using the Qiagen DNA prep kit following manufacturer's instructions. The DNA methylation protocol, data acquisition, and analysis has already been described [24]. Data are presented as % DNA methylation at different time points.
Statistical Analysis
Cell viability, primary DNA damage, and oxidative stress were statistically analyzed by one-way ANOVA with Sidak post-hoc test. Cytostasis, cellular replication, and micronuclei frequency were analyzed by Chi-square test. Statistical analysis was performed using GraphPad Prism version 8.1.2 for Windows (GraphPad Software; San Diego, CA, USA).
ITER-Like Plasma and Laser W-NPs Physico-Chemical Characterization
A detailed physico-chemical characterization of the ITER-like plasma and laser W-NPs, in their powder form, was already presented [15,16,19] and is here summarized in Table 2. Table 2. Summary of the physico-chemical properties of plasma and laser tungsten nanoparticles (W-NPs) powders.
Synthesis
Magnetron Sputtering The laser W-NPs with a smaller average size were also more oxidized than plasma ones (82% of W 4+ + W 6+ compared to 10% of W 6+ in the case of plasma W-NPs).
The ITER-like plasma and laser W-NPs suspensions were prepared from stock suspension at the initial concentration of 2.5 mg/mL in tris(hydroxymethyl)aminomethane (TRIS) medium. The particle size (Table 3) was determined using Dynamic Light Scattering (DLS). In the case of plasma-derived W-NPs, the particle size in TRIS did not significantly change after hydrogenation and tritiation. When dispersed in LHC-9, plasma-derived W-NPs at t = 0 h showed a slight increase in their mean size and PDI after hydrogenation and tritiation. In the case of laser ITER-like W-NPs dispersed in LHC-9, no significant differences were detected between the three types of laser W-NPs investigated, not in terms of size or in particle stability (PDI and Z pot). While dispersed in TRIS at t = 0, the average size of the pristine laser ITER-like W-NPs is much larger than the TEM size ( Table 2). This can suggest an aggregation effect in TRIS medium. The hydrogenation and tritiation tends to decrease the average size of the laser-derived W-NPs. Such size decrease is difficult to interpret but it might be due to a partial oxidative dissolution that, already within 2 h, took place when W-NPs were suspended in LHC-9 culture medium. Indeed, even if it was impossible to quantify the amount of dissolution in the case of laser ITER-like W-NPs due to a low amount of raw material, in the case of pristine plasma ITER-like W-NPs, the dissolved W fraction in mass corresponded to 9%, 23%, 48%, and 58% for t = 0, 2, 4, and 24 h, respectively, for a 100 µg/mL suspension. When diluted at 10 µg/mL, dissolution further increased at 10%, 50%, 60%, and 66% for t = 0, 2, 4, and 24 h, respectively. A detailed W-NPs dissolution protocol (using ICP-MS after filtration at 0.02 µm) and data analysis is available elsewhere [19].
Cytotoxic Effects
At 2 h exposure none of the tested particles were able to significantly reduce the viability of BEAS-2B cells, whereas the behavior was different after 24 h incubation ( Figure 1).
Cytotoxic Effects
At 2 h exposure none of the tested particles were able to significantly reduce the viability of BEAS-2B cells, whereas the behavior was different after 24 h incubation ( Figure 1). (Figure 1c), severely affected the metabolic activity of BEAS-2B cells. The cytotoxic effect of pristine plasma W-NPs was statistically significant at concentrations ≥40 μg/mL and increased at the highest tested condition (150 µ g/mL). Hydrogenated plasma W-NPs exhibited a statistically significant impairment of ATP production in BEAS-2B already at 5 µ g/mL, which augmented in a concentration related manner.
While upon exposure to pristine laser W-NPs (Figure 1b) no cytotoxic effects were detected neither at 2 h nor at 24 h incubation time, the hydrogenated ones ( Figure 1d) severely impaired the metabolism of BEAS-2B at 24 h exposure: 35% cytotoxicity was observed already at the lowest tested concentration of 1 µ g/mL, and the effect was increased up to 60% cytotoxicity at 150 µ g/mL.
Overall, our results show that at short exposures (2 h) W-NPs do not impair BEAS-2B cell viability, while longer incubations (24 h) have more significant effects. In addition, while at 24 h plasma W-NPs impaired the ATP production in BEAS-2B cells independently of their surface properties, the presence of hydrogen on laser W-NPs is necessary to induce cytotoxic effects. The more severe cytotoxicity observed upon exposure to hydrogenated particles could thus be due to a surface-effect. The presence of hydrogen enhances the cytotoxicity of ITER-like W-NPs and this effect seems time-related, since it does not appear at short exposures (2 h). Tritiated plasma and laser W- (Figure 1c), severely affected the metabolic activity of BEAS-2B cells. The cytotoxic effect of pristine plasma W-NPs was statistically significant at concentrations ≥40 µg/mL and increased at the highest tested condition (150 µg/mL). Hydrogenated plasma W-NPs exhibited a statistically significant impairment of ATP production in BEAS-2B already at 5 µg/mL, which augmented in a concentration related manner.
While upon exposure to pristine laser W-NPs (Figure 1b) no cytotoxic effects were detected neither at 2 h nor at 24 h incubation time, the hydrogenated ones ( Figure 1d) severely impaired the metabolism of BEAS-2B at 24 h exposure: 35% cytotoxicity was observed already at the lowest tested concentration of 1 µg/mL, and the effect was increased up to 60% cytotoxicity at 150 µg/mL.
Overall, our results show that at short exposures (2 h) W-NPs do not impair BEAS-2B cell viability, while longer incubations (24 h) have more significant effects. In addition, while at 24 h plasma W-NPs impaired the ATP production in BEAS-2B cells independently of their surface properties, the presence of hydrogen on laser W-NPs is necessary to induce cytotoxic effects. The more severe cytotoxicity observed upon exposure to hydrogenated particles could thus be due to a surface-effect. The presence of hydrogen enhances the cytotoxicity of ITER-like W-NPs and this effect seems time-related, since it does not appear at short exposures (2 h). Tritiated plasma and laser W-NPs could not be tested because of the lack of an appropriate plate reader in the facility where radioactive species are manipulated.
The cytotoxicity data allowed us to select some concentrations of interest for further experiments on the epigenotoxic effects of W-NPs. We thus chose 1-5-10-20 µg/mL as test concentrations because they do induce cytotoxicity without exceeding the levels (55% ± 5%) recommended for the genotoxicity tests [23]. Figure 2 shows the cytostasis of BEAS-2B cells in the presence, for 24 h, of plasma (Figure 2a,b) and laser (Figure 2c,d) W-NPs in their pristine, hydrogenated, and tritiated forms. Cytostasis was evaluated via Cytokinesis-Block Proliferation Index (CBPI) and Replication Index (RI). Figure 2 shows the cytostasis of BEAS-2B cells in the presence, for 24 h, of plasma (Figure 2a,b) and laser (Figure 2c,d) W-NPs in their pristine, hydrogenated, and tritiated forms. Cytostasis was evaluated via Cytokinesis-Block Proliferation Index (CBPI) and Replication Index (RI).
Effects on Cytostasis and Cellular Replication
Both types of W-NPs impaired CBPI; nevertheless, no differences were observed comparing the results obtained upon plasma ( Figure 2a) and laser (Figure 2c) exposure.
RI was also impaired by plasma and laser W-NPs. Pristine and hygdrogenated plasma ( Figure 2b) and laser (Figure 2d) ITER-like W-NPs seem to behave similarly, while tritiated plasma W-NPs seem to exert damage to BEAS-2B cells at lower concentrations than their laser counterparts. (c) CBPI following incubation with laser W-NPs; (d) RI following incubation with laser W-NPs. Independently of the presence or absence of hydrogen and tritium, both plasma and laser W-NPs reduced CBPI and RI of BEAS-2B cells. As expected, the positive control (Mitomycin C (MMC), 0.1 μg/mL) was both cytostatic and cytotoxic. Data are expressed as mean value ± SEM of three independent experiments, each in duplicate. Statistically significant differences from the untreated cells (0 µ g/mL) were determined by Chi-square test: * p < 0.05, ** p < 0.01 and *** p < 0.001. RI was also impaired by plasma and laser W-NPs. Pristine and hygdrogenated plasma ( Figure 2b) and laser (Figure 2d) ITER-like W-NPs seem to behave similarly, while tritiated plasma W-NPs seem to exert damage to BEAS-2B cells at lower concentrations than their laser counterparts.
Micronuclei Formation
The potential chromosomal damage in BEAS-2B cells was evaluated by CBMN assay (Figure 3). Dose-related and statistically significant increased MN frequency was measured following exposure to plasma ( Figure 3a) and laser (Figure 3b) W-NPs in their pristine, hydrogenated, and tritiated forms. The presence of hydrogen and tritium did not enhance the genotoxic effect of plasma and laser W-NPs, as already observed for cytostasis and cellular replication (Figure 2).
The potential chromosomal damage in BEAS-2B cells was evaluated by CBMN assay (Figure 3). Dose-related and statistically significant increased MN frequency was measured following exposure to plasma (Figure 3a) and laser (Figure 3b) W-NPs in their pristine, hydrogenated, and tritiated forms. The presence of hydrogen and tritium did not enhance the genotoxic effect of plasma and laser W-NPs, as already observed for cytostasis and cellular replication (Figure 2). To investigate more in detail the type of chromosomal damage exerted by the ITER-like plasma and laser W-NPs, a pancentromeric staining was performed ( Figure S1). The aim was to distinguish between centromeric positive (CMpos) MN, issued by a whole chromosome loss exerted by an aneugenic compound, and centromeric negative (CMneg) MN, resulting from chromosome fragmentation following exposure to a clastogenic product.
As shown in Figure S1, plasma and laser W-NPs exert both CMpos and CMneg micronuclei. Compared to their respective negative controls, statistically significant CMpos and CMneg MN formation was observed. Since from the graphical representation it was not possible to clearly distinguish and understand which effect (clastogenic or aneugenic) was predominant in laser and plasma W-NPs, the CMpos and CMneg fold increase were calculated. In the presence of plasma W-NPs ( Figure S1a,b) CMpos seem to be more induced than CMneg, suggesting a slight but not significant predominant aneugenic potential compared to the clastogenic one. Similarly, a slight predominance of CMpos MN can be observed in BEAS-2B cells upon exposure to laser (Figure S1c,d) W-NPs. Overall, the ITER-like plasma and laser W-NPs display both aneugenic and clastogenic potential.
DNA Damage Quantification
To evaluate primary DNA lesions, the comet assay was performed following a conventional alkaline protocol. As shown in Figure 4, all the tested ITER-like W-NPs induced DNA fragmentation compared to the untreated BEAS-2B cells. While no differences in the quantification of the DNA damage were observed between tritiated plasma (Figure 4a (Figure 4b,d) exposure, the behavior of pristine and hydrogenated W-NPs was different. In fact, short incubation times generated more severe DNA damage than long exposures. This is very clear in cells exposed to pristine and hydrogenated plasma W-NPs and to pristine laser To investigate more in detail the type of chromosomal damage exerted by the ITER-like plasma and laser W-NPs, a pancentromeric staining was performed ( Figure S1). The aim was to distinguish between centromeric positive (CMpos) MN, issued by a whole chromosome loss exerted by an aneugenic compound, and centromeric negative (CMneg) MN, resulting from chromosome fragmentation following exposure to a clastogenic product.
As shown in Figure S1, plasma and laser W-NPs exert both CMpos and CMneg micronuclei. Compared to their respective negative controls, statistically significant CMpos and CMneg MN formation was observed. Since from the graphical representation it was not possible to clearly distinguish and understand which effect (clastogenic or aneugenic) was predominant in laser and plasma W-NPs, the CMpos and CMneg fold increase were calculated. In the presence of plasma W-NPs ( Figure S1a,b) CMpos seem to be more induced than CMneg, suggesting a slight but not significant predominant aneugenic potential compared to the clastogenic one. Similarly, a slight predominance of CMpos MN can be observed in BEAS-2B cells upon exposure to laser (Figure S1c,d) W-NPs. Overall, the ITER-like plasma and laser W-NPs display both aneugenic and clastogenic potential.
DNA Damage Quantification
To evaluate primary DNA lesions, the comet assay was performed following a conventional alkaline protocol. As shown in Figure 4, all the tested ITER-like W-NPs induced DNA fragmentation compared to the untreated BEAS-2B cells. While no differences in the quantification of the DNA damage were observed between tritiated plasma (Figure 4a,b) and laser (Figure 4c,d) at 2 h (Figure 4a,c) and 24 h (Figure 4b,d) exposure, the behavior of pristine and hydrogenated W-NPs was different. In fact, short incubation times generated more severe DNA damage than long exposures. This is very clear in cells exposed to pristine and hydrogenated plasma W-NPs and to pristine laser W-NPs, a bit less pronounced is the difference following incubation with hydrogenated laser W-NPs. The hypothesis to explain these results might be given by the formation of reactive oxidative species (ROS) that participate, as already reported in the literature, in DNA damage. W-NPs, a bit less pronounced is the difference following incubation with hydrogenated laser W-NPs. The hypothesis to explain these results might be given by the formation of reactive oxidative species (ROS) that participate, as already reported in the literature, in DNA damage.
Oxidative Stress
To verify our hypothesis that ROS play a role in the induction of DNA damage, we measured oxidative stress in BEAS-2B cells exposed to plasma ( Figure 5a) and laser (Figure 5b) W-NPs. Already after 30 min exposure a highly statistically significant imbalance of the GSH/GSSG ratio was detected. With the exception of hydrogenated plasma W-NPs, which exerted the less severe reduction of GSH/GSSG ratio, pristine plasma W-NPs and the two tested types of laser W-NPs showed a doserelated oxidative stress. These data confirmed thus that oxidative stress took place upon exposure to ITER-like W-NPs and that it might trigger DNA damage.
Tritiated plasma and laser W-NPs could not be tested because of the lack of an appropriate plate reader in the radioactive facility. Nevertheless, because the hydrogenated particles can be considered as the non-radioactive equivalent of the tritiated ones, we could suppose that these latter ones have the same behavior than the hydrogenated.
Oxidative Stress
To verify our hypothesis that ROS play a role in the induction of DNA damage, we measured oxidative stress in BEAS-2B cells exposed to plasma ( Figure 5a) and laser (Figure 5b) W-NPs. Already after 30 min exposure a highly statistically significant imbalance of the GSH/GSSG ratio was detected. With the exception of hydrogenated plasma W-NPs, which exerted the less severe reduction of GSH/GSSG ratio, pristine plasma W-NPs and the two tested types of laser W-NPs showed a dose-related oxidative stress. These data confirmed thus that oxidative stress took place upon exposure to ITER-like W-NPs and that it might trigger DNA damage.
Tritiated plasma and laser W-NPs could not be tested because of the lack of an appropriate plate reader in the radioactive facility. Nevertheless, because the hydrogenated particles can be considered as the non-radioactive equivalent of the tritiated ones, we could suppose that these latter ones have the same behavior than the hydrogenated. Nanomaterials 2019, 9, x FOR PEER REVIEW 12 of 18
DNA Methylation Changes Analysis
Epigenetic variations were evaluated at different time points and in different BEAS-2B generations (F0 and F1) upon 24 h exposure to 1-5 µ g/mL pristine plasma ( Figure 6a) and laser (Figure 6b) ITER-like W-NPs. Compared to the untreated control, for the different sequences analyzed, no significant changes in DNA methylation were observed at any time point tested, with none of the sequences we used in our analysis. Additionally, no differences were observed in the % of methylated DNA in cells exposed to plasma or laser ITER-like W-NPs, confirming again that the biological effects of these two types of particles are similar despite their physico-chemical differences.
DNA Methylation Changes Analysis
Epigenetic variations were evaluated at different time points and in different BEAS-2B generations (F0 and F1) upon 24 h exposure to 1-5 µg/mL pristine plasma ( Figure 6a) and laser (Figure 6b) ITER-like W-NPs. Compared to the untreated control, for the different sequences analyzed, no significant changes in DNA methylation were observed at any time point tested, with none of the sequences we used in our analysis. Additionally, no differences were observed in the % of methylated DNA in cells exposed to plasma or laser ITER-like W-NPs, confirming again that the biological effects of these two types of particles are similar despite their physico-chemical differences.
DNA Methylation Changes Analysis
Epigenetic variations were evaluated at different time points and in different BEAS-2B generations (F0 and F1) upon 24 h exposure to 1-5 µ g/mL pristine plasma ( Figure 6a) and laser (Figure 6b) ITER-like W-NPs. Compared to the untreated control, for the different sequences analyzed, no significant changes in DNA methylation were observed at any time point tested, with none of the sequences we used in our analysis. Additionally, no differences were observed in the % of methylated DNA in cells exposed to plasma or laser ITER-like W-NPs, confirming again that the biological effects of these two types of particles are similar despite their physico-chemical differences.
Discussion
When thermonuclear fusion reactors become operational, generating tritiated tungsten particles, they could represent a potential risk for the environment and human health as they might be released in case of LOVA. Since the reactor is still under construction, we can only gather preliminary information from the current literature, which is mainly related to different types and forms of W and not to ITER-derived W-NPs.
The aim of our study was thus to provide an evaluation of the cytotoxic, genotoxic, and epigenetic in vitro effects that ITER-like W-NPs might have on human health, in relation with the possible W-NPs transformation in biological media. In particular, we focused our attention to an in vitro 2D model of the lung, the BEAS-2B cell line, that was chosen because inhalation is the main route of exposure to W. Similarly, a significant number of toxicity studies on W were performed on lung-derived cell lines [4,5,7,8,10,12].
The main outcome of the current literature is that W is cytotoxic and induces apoptosis. In addition, W can exert MN formation or DNA damage as well as alter gene expression and arrest the cell cycle. Generally, the production of ROS is considered as the main cause triggering W genotoxicity. Analogously, our data showed that plasma and laser ITER-like W-NPs exert cytotoxic and genotoxic effects in BEAS-2B cells, and the increased oxidative stress might be one of the reasons explaining our results.
Even if we can identify similarities between the outcome of previous studies on W and our data, there are some important differences that we think are pivotal in understanding our results and the mechanisms by which W results toxic. First of all, our bench produced plasma and laser ITER-like W-NPs display peculiar physico-chemical properties [15][16][17]. More in detail, although they are metallic W-NPs, XAS and helium pycnometer analysis have shown that a significant fraction of tungsten oxide (WO x ) is present and it could participate in the toxicity of the particles. WO 3 represented only a small fraction (10%) of plasma ITER-like W-NPs, while more oxidation was observed on laser ITER-like W-NPs powders (78% WO 3 by helium pycnometer and 50% WO 3 + 32% WO 2 by XAS analysis). Additionally, ITER-like W-NPs are highly soluble. ICP-MS, in fact, revealed that the dissolved W fraction in 100 µg/mL suspensions corresponded, in LHC-9 culture medium, to 9% at t = 0 h and it increased up to 23% and 58% at, respectively, 2 and 24 h incubation [17]. As expected, further dilution of W suspension to 10 µg/mL enhanced their dissolution kinetic: 10% was the soluble W fraction measured at t = 0 h, while 50% and 66% was dissolved at t = 2-24 h [17]. All together these data indicate that the ITER-like W-NPs we investigated were characterized by oxidation and dissolution. These findings bring thus a new perspective on the toxicity of ITER-like W-NPs and they make the plasma and laser particles we investigated unusual.
Not much information is reported in the literature on the solubility of W-containing particles, on their oxidation, and on the involvement of ionic W and W oxides in exerting toxicity. In contrast, studies on the soluble Na 2 WO 4 and the on WO 3 NP have already been reported. Sodium tungstate was applied to BEAS-2B cells at concentrations ranging from 50 to 250 µM in order to investigate the carcinogenicity of W via several endpoints such as cell transformation, cell migration, and the activation of multiple cancer-related pathways [4]. After 6 weeks of treatment, results clearly showed that Na 2 WO 4 increased BEAS-2B colonies formation at all tested doses; in addition, cell migration was tested via the cell scratch assay and the transformed clones were able to heal the wound within less than a day after the scratch was made. Changes in gene expression due to tungstate exposure were observed in BEAS-2B transformed clones: RNA sequencing showed that more than 16,000 genes were altered and the majority of them were related to lung cancer and leukemia, inflammation, and tumor morphology. Overall, the results of Laulicht and coauthors showed that ionic W has carcinogenic in vitro potential [4].
In freshly isolated human peripheral blood lymphocytes (hPBL) Na 2 WO 4 was applied in the range 0.1-1-10 mM for 24-96 h and apoptosis, cell cycle, and cytokines secretions were investigated [3]. Early apoptosis increased in tungstate exposed hPBL whereas the number of lymphocytes entering the cell cycle was reduced. Already at relatively low concentration (1 mM), W in its ionic form significantly increased the G 0 /G 1 fraction and impaired the number of cells in S-or G 2 /M-phase. Moreover, when 5-ethynyl-2 -deoxyuridine (EdU) was used to test DNA synthesis, tungstate was observed to reduce, up to 50%, the EdU-positive cells, also those in G 0 /G 1 -phase. Likewise, the production of cytokines such as TNF-α, IL-10, and IL-6 resulted significantly lowered, probably due to the high rate of apoptotic hPBL upon exposure to Na 2 WO 4 . Osterburg and coauthors suggested that the mechanism by which ionic W induces toxic effects depends on its ability to inhibit phosphatase and alter the phosphorylation of some intracellular molecules [3].
From the literature, ionic W seems thus to induce a variety of effects on different in vitro cellular systems. This effect is confirmed by our results on the soluble plasma and laser ITER-like W-NPs that, at least after 24 h exposure, decreased viability and, even if not in a significant manner, the cytostasis of BEAS-2B cells. If we only consider the ionic W released by plasma and laser W-NPs as the key factor triggering cytotoxicity, we should keep in mind that the differences in cell viability observed at 2 and 24 h exposure might depend on the kinetic of dissolution of the particles. In fact, the amount of ionic W measured by ICP-MS was low at 2 h (9%-10%), but much higher (58%-66%) at 24 h. We thus suggest this is one of the reasons that allowed us to observe more cytotoxic effects at long than at short exposures ( Figure 1).
Since the presence of WO 2 and WO 3 we detected during powders characterization is not negligible [24], oxidation should also be considered when analyzing plasma and laser ITER-like W-NPs toxicity. Ex vivo bone cells and hepatocytes of 8 weeks old male Sprague-Dawley rats were used to investigate the cytotoxicity, the genotoxicity, and the mutagenic potential of WO 3 NPs [25,26]. Upon 30 days of intraperitoneal injections of WO 3 NPs at the dose of 25-50-100 mg/kg b.w., bone cells were collected from tibia. While chromosome aberration test did not show any difference in bone cells from exposed rats compared to the untreated controls, mitotic index, a marker for cytotoxicity, and MN frequency, an indicator of genotoxic damage, were affected by WO 3 NPs. Mitotic index was significantly decreased at all the tested doses; MN frequency, conversely, was enhanced only in rats exposed to 50-100 mg/kg b.w. [25]. Turkez and coauthors further investigated the effects of WO 3 NPs in rat hepatocytes [26]. After performing an ex vivo culture of hepatocytes, cells were exposed for 72 h to WO 3 NPs concentrations ranging from 5 to 1000 ppm. The cytotoxic potential was detected by observing a decrease in cell viability, assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test, and an increase in the levels of extracellular lactate dehydrogenase at fairly high concentrations (≥300 ppm). Surprisingly, no MN formation was observed at any of the tested WO 3 NPs concentrations, while oxidative stress and secondary oxidative DNA damage were reported [26]. From the publications of Turkez and coauthors [25,26] it seems that WO 3 NPs are feebly cytotoxic and could induce chromosome losses. They also generate significant oxidative stress that can cause indirect DNA damage via the formation of adducts, as observed via 8-oxo-2-deoxyguanosine quantification or impair mitotic spindle.
In contrast, plasma and laser ITER-like W-NPs showed significant cytotoxicity on BEAS-2B cultures, they resulted genotoxic by significantly enhancing the MN frequency and by inducing DNA strand breaks. In agreement with previous studies [25,26], we equally observed oxidative stress that might have triggered DNA damage. Nevertheless, the differences between our results and those of Turkez and coauthors [25,26] might be due to the fact that only a fraction of our ITER-like W-NPs was oxidized. We think that both oxidation and dissolution played a synergistic role in the initiation of ITER-like W-NPs toxicity. Since the current literature on WO 3 NPs does not provide information on the particle dissolution, further comparison cannot be done between the studies of Turkez et al. [25,26] and ours.
While we do not have any physico-chemical information on the WO 3 NPs used in the Sprague-Dawley ex vivo studies presented just above [25,26], Chinde and colleagues have presented a thorough characterization of the WO 3 NPs they have tested on the alveolar A549 cell line [5]. In milliQ water, by TEM, the mean size of WO 3 NPs corresponded to 54 ± 29 nm, while DLS corresponded, respectively, to 170 ± 2 nm and 224 ± 5 nm in milliQ and in DMEM cell culture medium. WO 3 NPs are thus slightly bigger than the plasma and laser ITER W-NPs we investigated, whose size range, via DLS and in LHC-9 cell culture medium, was set to 103-135 nm and to 155-174 nm, respectively.
Chinde and coauthors have also investigated the dissolution of WO 3 NPs in cell culture medium. Particles were suspended for 24-48 h in DMEM and the nominal WO 3 concentration was compared to total W measured by ICP-MS. While the nominal WO 3 concentration of 200 and 300 µg/mL corresponded to 170 and 330 µg/mL total W concentration before ultracentrifugation, only a small fraction of soluble W (0.9 and 2.4 µg/mL) was detected in ultracentrifuged solutions [5]. These results clearly indicate that Chinde et al. used non-soluble WO 3 NPs; conversely, our ITER-like W-NPs showed a significant dissolved W mass fraction already at t = 0 h in cell culture medium which further increased up to 24 h ( Table 2).
Our results on BEAS-2B cells confirm the in vitro lung toxicity of WO 3 NPs observed by Chinde and coauthors, although some differences in the experimental set-up and in its outcome are noticeable. In A549 cells, the cytotoxicity was observed at high concentrations (≥200 µg/mL WO 3 NPs; 24-48 h exposure duration) [5], whereas in BEAS-2B cells and at 24 h exposure, plasma and laser ITER-like W-NPs resulted cytotoxic already at 1 µg/mL. Similarly, DNA damage, MN frequency, apoptosis, and oxidative stress were only enhanced in A549 cells exposed at nominal concentrations ≥200 µg/mL WO 3 NPs [5]. Moreover, WO 3 NPs uptake quantification showed that only 0.3% of particles were internalized by A549 cells, and that the most significant fraction (70%-75%) of W was diluted in the supernatant.
The nominal concentrations tested and the oxidation state of the particles represent the greater differences between the study of Chinde et al. and ours. Nominal WO 3 NPs concentrations of at least 200 µg/mL, in fact, differ by 10-fold compared to the maximal plasma and laser ITER-like W-NPs concentration we tested (20 µg/mL). Additionally, plasma and laser ITER-like W-NPs contained, respectively, 10% WO 3 and 32% WO 2 /50% WO 3 , which further decreases the nominal concentration of WO x we applied to BEAS-2B cells. Since only a fraction of the plasma and laser ITER-like W-NPs powders is oxidized on their surface, our hypothesis is that the oxidation and the oxidation state are not the unique factors inducing cytotoxic and genotoxic effects in BEAS-2B cells.
To our knowledge, very few epigenetic investigations have been performed on various forms of W. Verma and co-workers investigated by Western blot the posttranslational histone epigenetic modifications, such as acetylation of histone 3 (H3), phosphorylation of Ser10 (H3-Ser10), and the trimethylation of histone H3 on lysine 4 (H3K4me3) in the tail of histone H3 in human embryonic kidney (HEK293), human neuroepithelioma (SKNMC), mouse myoblast (C2C12), and hippocampal primary neuronal cultures exposed to 50-184 µg/mL metal W and W-alloys (91% W, 6% Ni, 3% Co) for 1 day or 1 week, depending on the cell type [27]. H3K4me3 showed no changes in any of the cell cultures following metal exposure, indicating absence of modification in transcriptional activation of genes. Whereas W induced dephosphorylation of H3-Ser10 exclusively in HEK293 cells, tungsten-alloy reduced H3-Ser10 phosphorylation in C2C12 cells and hippocampal primary neuronal cultures, probably taking advantage of the synergistic toxic effects of W with Ni and Co [27]. H3K4me3, as well as the demethylation of histone H3 at lysine residue 9 (H3K9me2), were further investigated in BEAS-2B and A549 cells exposed, in vitro, to 1-2.5-5-10 mM Na 2 WO 4 for 48 h [28]. Via Western blot analysis Laulicht-Glick and coauthors observed that global levels of both H3K4me3 and H3K9me2 increased compared to the untreated controls, and that the methylation was not reversible when the treatment solutions were removed and cells further incubated with cell culture medium. Further investigation revealed that the degradation of the demethylases JMJD1A and JARID1A caused the increased H3 methylation in BEAS-2B and A549 [28].
Conversely to Verma et al. [27] and to Laulicht-Glick et al. [28], we quantified the variations in DNA methylation of BEAS-2B cells upon 24 h exposure to 1-5 µg/mL plasma and laser ITER-like W-NPs. Under our experimental conditions we did not detect changes in DNA methylation compared to the control cells, not at any of the days post-exposure nor in sub-cultured BEAS-2B cells (F1 generation).
Additional tests might be required, extending the exposure period or increasing the test concentrations, to verify if and at which time point epigenetic effects are induced by ITER-like W-NPs.
Conclusions
Despite its use, W still represents a potential occupational or accidental risk. Even if, upon intake, the human body rapidly excretes W, traces could be found in kidney, liver, bones, and spleen [29]. The Occupational Safety and Health Administration (OSHA) has established that 5 and 1 mg/m 3 are the permissible exposure limits for, respectively, insoluble and soluble W compounds employed in the construction and shipyard industries [30,31]. Nevertheless, no information is yet available on ITER-derived W-NPs, their morphology, their physico-chemical properties, and their biological profile. All together, these facts highlight the importance of our study attempting at fulfilling the gap of knowledge on ITER W-NPs. Under our experimental conditions ITER-like plasma and laser W-NPs induced, in a 2D in vitro model of the respiratory compartment, cytotoxic and genotoxic effects as well as strong oxidative potential. Our data represent thus a first, although not exhaustive, multi-endpoint characterization of the biological profile and of the potential risk that ITER-released W-NPs might induce on human health.
Supplementary Materials:
The following are available online at http://www.mdpi.com/2079-4991/9/9/1233/s1, Figure S1: Pancentromeric staining in BEAS-2B cells exposed to W-NPs: (a) CMpos and CMneg formation upon exposure to plasma W-NPs; (b) CMpos and CMneg fold increase compared to untreated cells upon exposure to plasma W-NPs; (c) CMpos and CMneg formation upLHCon exposure to laser W-NPs; (d) CMpos and CMneg fold increase compared to untreated cells upon exposure to laser W-NPs. Independently of the presence/absence of hydrogen and tritium, ITER-like plasma, and laser W-NPs induced both CMpos and CMneg MN formation compared to the untreated cells (0 µg/mL). MMC (0.1 µg/mL) was used as positive control. Data are expressed as mean value ± SEM of two independent experiments, each in duplicate. Statistically significant differences from the untreated cells were determined by Chi-square test: * p < 0.05, ** p < 0.01 and *** p < 0.001. | v3-fos-license |
2017-05-02T19:26:32.988Z | 2012-06-01T00:00:00.000 | 27587998 | {
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} | pes2o/s2orc | Inhibition of corrosion of carbon steel in well water by arginine-Zn 2 + system
The environmental friendly inhibitor system arginine-Zn, has been investigated by weight-loss method. A synergistic effect exists between arginine and Zn system. The formulation consisting of 250 ppm of arginine and 5 ppm of Zn offers good inhibition efficiency of 98 %. Polarization study reveals that this formulation functions as an anodic inhibitor. AC impedance spectra reveal that a protective film is formed on the metal surface. The FTIR spectral study leads to the conclusion that the FeDL-arginine complex, formed on anodic sites of the metal surface, controls the anodic reaction. Zn(OH)2 formed on the cathodic sites of the metal surface controls the cathodic reaction. The surface morphology and the roughness of the metal surface were analyzed with Atomic Force Microscope. A suitable mechanism of corrosion inhibition is proposed based on the results obtained from weight loss study and surface analysis technique.
Introduction
The principles and practices of corrosion inhibition have begun in recent years to take into account the health and safety considerations.The use of hazardous chemicals has been restricted to no contact with the environment.Hence, there is a search for non-toxic, eco-friendly corrosion inhibitors.The use of inhibitors is one of the most practical methods to protect metals from corrosion.Corrosion inhibitor is a chemical substance which, when added to the corrosive environment at an optimum concentration, significantly decreases the corrosion rate of metals (or) alloys.Unfortunately, many common corrosion inhibitors are highly toxic and health-hazardable, such as chromates [1], nitrite [2] aromatic heterocyclic compounds [3] etc. Therefore, it is better to look for environmentally safe inhibitors [4][5][6].Some researchers investigated the inhibition effect of environment friendly inhibitors like amino acids on metal corrosion [6][7][8][9][10][11][12][13].This is due to the fact that amino acids are non-toxic, biodegradable, relatively cheap, and completely soluble in aqueous media and produced with high purity at low cost.The environmental friendly arginine is chosen as the corrosion inhibitor for this present work.The literature presents some studies involving amino acids having the ability to prevent the corrosion of iron [14], steel [15][16][17], aluminum [18,19], nickel [20] and copper [21][22][23][24][25].The electrochemical studies such as polarization and AC impedance spectra [26][27][28][29][30] and cyclic voltammetry [19] have been studied by using amino acids.The adsorption of amino acids on carbon steel in acidic environment has been investigated by Akiyama et al. [31].
Preparation of specimens
Carbon steel specimens (0.0267 % S, 0.067 % P, 0.4 % Mn, 0.1 % C and the rest iron) of the dimensions 1.0 cm × 4.0 cm × 0.2 cm were polished to mirror finish and degreased with trichloroethylene and as such used for weight-loss method and surface examination studies.
Weight -loss method
Relevant data on the well water used in this study are given in Table 1.Carbon steel specimens, in triplicate were immersed in 100 ml of well water and various concentrations of arginine in the presence and absence of Zn 2+ (as ZnSO 4 ×7H 2 O) for a period of seven days.The corrosion products were cleaned with Clarke's solution [32].The weight of the specimens before and after immersion was determined using Shimadzu balance AY62.The corrosion inhibition efficiency (IE) was calculated using the equation (1).
where W 1 is the corrosion rate in the absence of the inhibitor and W 2 is the corrosion rate in the presence of inhibitor.From the weight loss, the corrosion rate (mm year -1 ) was calculated using the equation ( 2): where CR is corrosion rate in mm year -1 ; LW is the weight loss in mg; A is surface area of the specimen in dm 2 ; t is period of immersion in days and ρ is density of the metal in g cm -2 (7.86).
Potentiodynamic polarization study
Potentiostatic polarization studies were carried out using a CHI electrochemical impedance analyzer, model 660 A. A three-electrode cell assembly was used.The working electrode was a rectangular specimen of carbon steel with one face of the electrode (1 cm 2 area) exposed and the rest shielded with red lacquer.A saturated calomel electrode (SCE) was used as the reference electrode and a rectangular platinum foil was used as the counter electrode.Polarization curves were recorded using iR compensation.The results, such as Tafel slopes, and I corr , E corr and linear polarization resistance (LPR) values were calculated.During the polarization study, the scan rate was 0.01 V s -1 ; hold time at E f was 0 s and quiet time was 2 s.
AC impedance measurements
A CHI electrochemical impedance analyzer (model 660A) was used for AC impedance measurements.A time interval of 5 to 10 minutes was given for the system to attain its open circuit potential.The real part Z' and the imaginary part Z" of the cell impedance were measured in ohms at various frequencies.The values of the charge transfer resistance R t , double layer capacitance C dl and impedance value were calculated.
where R s = solution resistance where f max = maximum frequency AC impedance spectra were recorded with initial E = 0 V; high frequency = 10 5 Hz; low frequency = 10 Hz; amplitude = 0.005 V; and quiet time = 2 s.
FTIR spectra
The structure of arginine is shown in Fig. 1.The carbon steel specimens immersed in various test solutions for seven days were taken out and dried.The film formed on the metal surface was carefully removed and thoroughly mixed with KBr, so as to make it uniform throughout.The FTIR spectra were recorded in a Perkin-Elmer 1600 spectrophotometer.
Scanning electron microscopy (SEM analysis)
SEM provides a pictorial representation of the surface.It helps to understand the nature of the surface film in the absence and presence of inhibitors and the extent of corrosion of carbon steel.The scanning electron microscopy photographs were recorded at various magnifications using Hitachi scanning electron microscopy machine S-3000 H.
Atomic force microscopy
Atomic force microscope (AFM) is an exciting new technique that allows surface to be imaged at higher resolutions and accuracies than ever before [33][34][35].The microscope used for the present study was VEECO, Lab incorporation.Polished specimens, prior to the initiation of all corrosion experiments, were examined through an optical microscope to find out any surface defects such as pits or noticeable irregularities like cracks, etc.Only those specimens, which had a smooth pit free surface, were subjected to AFM examination.The protective films formed on the carbon steel specimens after immersion in the inhibitor systems for different time durations were examined for a scanned area of 05 × 05 µm at a scan rate of 6.68 µm s -1 .The two dimensional and three-dimensional topography of surface film gave various roughness parameters of the film.
Analysis of the weight loss method
Corrosion rates (CR) of carbon steel immersed in well water in the absence and presence of inhibitor (DL-arginine) are given in Tables 1 to 3. The inhibition efficiencies (IE) are also given in these Tables.The corrosion rates of the DL-arginine -Zn 2+ systems as a function of concentrations of DL-arginine are shown in Fig. 2. It is observed from Table 1 that DL-arginine shows some inhibition efficiencies.50 ppm of DL-arginine has 48 percent IE.As the concentration of DL-arginine increases, the IE decreases.This is due to the fact that as the concentration of DL-arginine increases, the protective film (probably iron DL-arginine complex) formed on the metal surface diffuses into solution.That is, the system goes from passive region to active region [36].
Influence of Zn 2+ on the inhibition efficiencies of DL-arginine
The influence of Zn 2+ on the inhibition efficiencies of DL-arginine is given in Tables 2 and 3.It is observed that as the concentration of DL-arginine increases the IE increases.Similarly, for a given concentration of DL-arginine the IE increases as the concentration of Zn 2+ increases.It is also observed that a synergistic effect exists between DL-arginine and Zn 2+ .For example, 5 ppm of Zn 2+ has 15 percent IE; 250 ppm of DL-arginine has 34 percent IE.Interestingly their combination has a high IE, namely, 98 percent.
In the presence of Zn 2+ more amount of DL-arginine is transported towards the metal surface.This is due to fact that when Zn 2+ is added to the environment, Zn 2+ -DL-arginine complex is formed, it diffuses towards metal surface.On the metal surface, iron-amino acid complex is formed and Zn 2+ is released.This combines with OH -to give insoluble Zn(OH) 2 formed on cathodic sites which is controlled the cathodic reaction.On the metal surface Fe-DL-arginine complex is formed on the anodic sites of the metal surface and anodic reaction is controlled.Thus, the anodic reaction and cathodic reaction are controlled effectively.This accounts for the synergistic effect existing between Zn 2+ and DL-arginine.
Analysis of potentiodynamic polarization study (pH = 8)
Polarization study was used to confirm the formation of protective film formed on the metal surface during corrosion inhibition process [37][38][39][40][41][42].If a protective film is formed on the metal surface, the linear polarization resistances value (LPR) increases and the corrosion current value (I corr ) decreases.
The potentiodynamic polarization curves of carbon steel immersed in well water in the absence and presence of inhibitors are shown in Fig. 3 and the corrosion parameters are given in Table 4.When carbon steel was immersed in well water the corrosion potential was -668 mV vs SCE.When DLarginine (250 ppm) and Zn 2+ (5 ppm) were added to the above system, the corrosion potential shifted to the noble side, i.e. to -626 mV vs SCE.This indicates that a film is formed on anodic sites of the metal surface.This film controls the anodic reaction of metal dissolution by forming Fe 2+ -DL-arginine complex on the anodic sites of the metal surface.The formation of protective film on the metal surface is further supported by the fact that the anodic Tafel slope (b a ) increases from 104 to 173 mV.Further, the LPR value increases from 5.630×10 4 ohm cm 2 to 6.253×10 4 ohm cm 2 ; the corrosion current decreases from 5.775×10 -7 A cm -2 to 5.125×10 -7 A cm -2 .Thus, polarization study confirms the formation of a protective film on the metal surface.It is observed that when the inhibitors [DL-arginine (250 ppm) + Zn 2+ (5 ppm)] are added, the charge transfer resistance (R t ) increase from 1212 Ω cm 2 to 16240 Ω cm 2 .The C dl value decreases from 3.8424×10 -9 to 3.1373×10 -10 F cm -2 .The impedance value log(Z/Ω) increases from 3.120 to 4.181.These results lead to the conclusion that a protective film is formed on the metal surface.
Analysis of AC impedance spectra
It is observed that weight loss study gives very high IE.But this is not the case with the electrochemical studies.This may be explained by the fact that weight loss study is the result of 7 days whereas electrochemical studies are an instantaneous study.Similar observations were already reported [37][38][39][40][41][42][43][44][45].
Analysis of FTIR spectra
FTIR spectra have been used to analyze the protective film formed on the metal surface [39,[46][47][48][49][50][51][52][53].The FTIR spectrum (KBr) of pure DL-arginine is shown in Fig. 7a.The C=O stretching frequency of carboxyl group appears at 1610 cm -1 .The CN stretching frequency appears at 1098 cm -1 .The NH stretching frequency of the amine group appears at 3182 cm -1 .The FTIR spectrum of the film formed on the metal surface after immersion in the solution containing well water, 250 ppm of DL-arginine and 5 ppm Zn 2+ is shown in Fig. 7b.The C=O stretching frequency has shifted from 1610 to 1619 cm -1 .The CN stretching frequency has shifted from 1098 to 1005 cm -1 .The NH stretching frequency has shifted from 3182 to 3285 cm -1 .This observation suggests that DL-arginine has coordinated with Fe 2+ through the oxygen atom of the carboxyl group and nitrogen atom of the amine group resulting in the formation of Fe 2+ -DL-argi-nine complex on the anodic sites of the metal surface.The peak at 788 cm -1 corresponds to Zn-O stretching.The peak at 3442 cm -1 is due to OH -stretching.This confirms that Zn(OH) 2 is formed on the cathodic sites of metal surface [46,[51][52][53].Thus the FTIR spectral study leads to the conclusion that the protective film consists of Fe 2+ -DL-arginine complex and Zn(OH) 2 .
SEM Analysis of Metal Surface
SEM provides a pictorial representation of the surface.This helps to understand the nature of the surface film in the absence and presence of inhibitors and the extent of corrosion of carbon steel.The SEM micrographs of the surface are examined [54][55][56].The SEM images of different magnification (500 x, 1000 x) of carbon steel specimen immersed in well water for 7 days in the absence and presence of inhibitor system are shown in Figs.8a and 8b and 8c respectively.
The SEM micrographs of polished carbon steel surface (control) in Figs.8a and 8b show the smooth surface of the metal.This shows the absence of any corrosion products (or) inhibitor complex formed on the metal surface.The SEM micrographs of carbon steel surface immersed in well water (Figs.8b) show the roughness of the metal surface which indicates the highly corroded area of carbon steel in well water.However, Figs. 8c indicate that in the presence of inhibitor (250 ppm DL-arginine and 5 ppm Zn 2+ ) the rate of corrosion is suppressed, as can be seen from the decrease of corroded areas.The metal surface is almost free from corrosion due to the formation of an insoluble complex on the surface of the metal [54].In the presence of DL-arginine and Zn 2+ , the surface is covered by a thin layer of inhibitors which effectively controls the dissolution of carbon steel.
All atomic force microscopy images were obtained in a VEECO Lab incorporation AFM instrument operating in contact mode in air.The scan size of all the AFM images are 0.5 µm × 0.5 µm areas at a scan rate of 6.835 µm s -1 .
The two dimensional (2D), three dimensional (3D) AFM morphologies and the AFM crosssectional profile for polished carbon steel surface (reference sample), carbon steel surface immersed in well water (blank sample) and carbon steel surface immersed in well water containing the formulation of DL-arginine 250 ppm and 5 ppm of Zn 2+ are shown as Figs.9a, 9d and 9g, Figs.9b, 9e and 9h and Figs.9c, 9f and 9i, respectively.Root-mean-square roughness, average roughness and peak-to-valley value AFM image analysis was performed to obtain the average roughness, R a (the average deviation of all points roughness profile from a mean line over the evaluation length), root-mean-square roughness, R q (the average of the measured height deviations taken within the evaluation length and measured from the mean line) and the maximum peak-to-valley (P-V) height values (largest single peak-to-valley height in five adjoining sampling heights) [63].R q is much more sensitive than R a to large and small height deviations from the mean [64].
Table 6 is the summary of the average roughness (R a ), RMS roughness (R q ) maximum peak-tovalley height (P-V) value for carbon steel surface immersed in different environments.The value of R RMS , R a and P-V height for the polished carbon steel surface (reference sample) are 5 nm, 3 nm and 50 nm respectively, which show a more homogeneous surface, with some places in which the height is lower than the average depth [57].Figs.9a, 9d and 9g display the uncorroded metal surface.The slight roughness observed on the polished carbon steel surface is due to atmospheric corrosion.The RMS roughness, average roughness and P-V height values for the carbon steel surface immersed in well water are 28 nm, 21 nm and 137 nm respectively.These data suggest that carbon steel surface immersed in well water has a greater surface roughness than the polished metal surface.This shows that the unprotected carbon steel surface is rougher and this is due to the corrosion of the carbon steel in well water.Figs.9b, 9e and 9h display the corroded metal surface with few pits.
The presence of 250 ppm of DL-arginine and 5 ppm of Zn 2+ in well water reduces the R q by a factor of 6 nm from 28 nm and the average roughness is significantly reduced to 4 nm when compared with 21 nm of carbon steel surface immersed in well water.The maximum peak-tovalley height also was reduced to 26 nm from 137 nm.These parameters confirm that the surface appears smoother.The smoothness of the surface is due to the formation of a compact protective film of Fe 2+ -DL-arginine complex and Zn(OH) 2 on the metal surface thereby inhibiting the corrosion of carbon steel.Also the above parameters observed are somewhat greater than the AFM data of polished metal surface which confirms the formation of the film on the metal surface, which is protective in nature.
When carbon steel immersed in well water the R q , R a and maximum peak-to-valley (P-V) height are very high.In the presence of inhibitor system, these values are very close to those of polished carbon steel.This indicates that the formation of protective film starts even before the starting of corrosion process.Suppose the film is formed on the iron oxide surface then roughness would be greater than the R q , R a , maximum peak-to-valley (P-V) height of the carbon steel surface immersed in well water.But this is not the case.Hence this is concluded that the protective film is formed in the initial stage itself.
Mechanism of Corrosion inhibition
The results of the weight-loss study show that the formulation consisting of 250 ppm DLarginine and 5 ppm of Zn 2+ has 98% IE in controlling corrosion of carbon steel in well water.A synergistic effect exists between Zn 2+ and DL-arginine.Polarization study reveals that this formulation functions as anodic inhibitor.AC impedance spectra reveal that a protective film is formed on the metal surface.FTIR spectra reveal that the protective film consists of Fe 2+ -DLarginine complex and Zn(OH) 2 .In order to explain these facts the following mechanism of corrosion inhibition is proposed [65][66][67][68][69][70][71].
• When the solution containing well water, 5 ppm Zn 2+ and 250 ppm of DL-arginine is prepared, there is formulation of Zn 2+ -DL-arginine complex in solution.
• When carbon steel is immersed in this solution, the Zn 2+ -DL-arginine complex diffuses from the bulk of the solution towards metal surface.• Zn 2+ -DL-arginine complex diffuses from the bulk solution to the surface of the metal and is converted into a Fe 2+ -DL-arginine complex, which is more stable than Zn 2+ -DL-arginine [66].• On the metal surface Zn 2+ -DL-arginine complex is converted in to Fe 2+ -DL-arginine on the anodic sites.Zn 2+ is released.
• The SEM micrographs and AFM images confirm the formation of protective layer on the metal surface.
Conclusions
Inhibition of corrosion of carbon steel in well water by arginine-Zn 2+ system has been evaluated by weight-loss method.A synergistic effect exists between arginine and Zn 2+ system.The formulation consisting of 250 ppm of arginine and 5 ppm of Zn 2+ offers good inhibition efficiency of 98%.Polarization study reveals that this formulation functions as an anodic inhibitor.AC impedance spectra reveal that a protective film is formed on the metal surface.The FTIR spectral study leads to the conclusion that the Fe 2+ -DL-arginine complex formed on anodic sites of the metal surface controls the anodic reaction.Zn(OH) 2 formed on the cathodic sites of the metal surface controls the cathodic reaction.The SEM micrographs and AFM images confirm the formation of the protective layer on the metal surface.
Figure 8 .
Figure 8. SEM analysis of: a) Carbon steel (Control); b) Carbon steel immersed in well water (Blank); c) Carbon steel immersed in well water + 250 ppm of DL-arginine + 5 ppm of Zn 2+
Figure 9 .
Figure 9. a), b) c) 2D AFM images; d), e), f) 3D AFM images and g), h), f) Cross sectional profile which are corresponding to as shown lines on 2D AFM images of
Table 1 .
Corrosion rates (CR) of carbon steel immersed in well water in the presence and absence of inhibitor system at various concentrations and the inhibition efficiencies (IE) obtained by weight loss method.Inhibitor system: DL-arginine -Zn 2+ (amount of Zn 2+ = 0 ppm); Immersion period: 7 days; pH 8
Table 2 .
Corrosion rates (CR) of carbon steel immersed in well water in the presence and absence of inhibitor system at various concentrations and the inhibition efficiencies (IE) obtained by weight loss method.Inhibitor system: DL-arginine -Zn 2+ (amount of Zn 2+ = 5 ppm); Immersion period: 7 days; pH 8Amount of DL-arginine, ppmCR mm year -1 IE / %
Table 3 :
Corrosion rates (CR) of carbon steel immersed in well water in the presence and absence of inhibitor system at various concentrations and the inhibition efficiencies (IE) obtained by weight loss method.Inhibitor system: DL-arginine -Zn 2+ (amount of Zn 2+ = 10 ppm); Immersion period: 7 days; pH 8
Table 5 .
Corrosion parameters of carbon steel immersed in well water in the absence and presence of inhibitor system obtained from AC impedance spectra (pH 8).
Table 6 .
AFM data for carbon steel surface immersed in inhibited and uninhibited environments | v3-fos-license |
2018-04-03T04:12:09.163Z | 2017-02-14T00:00:00.000 | 16563155 | {
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} | pes2o/s2orc | Mechanical Stress Regulates Osteogenesis and Adipogenesis of Rat Mesenchymal Stem Cells through PI3K/Akt/GSK-3β/β-Catenin Signaling Pathway
Osteogenesis and adipogenesis of bone marrow mesenchymal stem cells (BMSCs) are regarded as being of great importance in the regulation of bone remodeling. In this study, rat BMSCs were exposed to different levels of cyclic mechanical stress generated by liquid drops and cultured in general medium or adipogenic medium. Markers of osteogenic (Runx2 and Collagen I) and adipogenic (C/EBPα, PPARγ, and lipid droplets) differentiation were detected using Western blot and histological staining. The protein levels of members of the phosphatidylinositol 3-kinase (PI3K)/Akt/glycogen synthase kinase 3β (GSK-3β)/β-catenin signaling pathway were also examined. Results showed that small-magnitude stress significantly upregulated Runx2 and Collagen I and downregulated PPARγ and C/EBPα expression in BMSCs cultured in adipogenic medium, while large-magnitude stress reversed the effect when compared with unloading groups. The PI3K/Akt signaling pathway could be strongly activated by mechanical stimulation; however, large-magnitude stress led to decreased activation of the signaling pathway when compared with small-magnitude stress. Activation of β-catenin with LiCl led to increased expression of Runx2 and Collagen I and reduction of C/EBPα and PPARγ expression in BMSCs. Inhibition of PI3K/Akt signaling partially blocked the expression of β-catenin. Taken together, our results indicate that mechanical stress-regulated osteogenesis and adipogenesis of rat BMSCs are mediated, at least in part, by the PI3K/Akt/GSK-3β/β-catenin signaling pathway.
Introduction
Mesenchymal stem cells (MSCs) have multipotent differentiation capabilities and have been confirmed to differentiate into various lineages, including osteoblasts, adipocytes, chondrocytes, myocytes, and fibroblasts [1,2]. Thus they hold great potential for regenerative medicine and tissue-engineering applications. It has been demonstrated that differentiation of MSCs into these various lineages is controlled by their particular biochemical microenvironment [2]. Additionally, mechanical stress has also been demonstrated to aid MSCs differentiation into various mature cells [3,4]. Tension [5] and fluid shear stress [6,7] have both been reported to promote osteogenic lineage commitment, while compression [8,9] and hydrostatic pressure [10] increase both chondrogenic and osteogenic differentiation.
In bones, osteoblasts and adipocytes are derived from a common progenitor cell, the bone marrow MSCs (BMSCs) [11]. In particular, osteogenic differentiation of BMSCs is essential for the maintenance of bone quality and quantity. However, reduced osteogenic potential and relatively increased adipogenic differentiation are associated with various pathological conditions such as osteoporosis, resulting in reduced bone mass, increased bone fragility, and increased susceptibility to fracture [12]. Therefore, the commitment of BMSCs to the adipocyte and osteoblast lineages appears to be of great importance in the regulation of bone remodeling.
It has been widely accepted that physiological loading is beneficial in maintaining skeletal integrity and increasing bone strength [13], while osteoporosis or bone resorption is often observed under unloading conditions such as in bedridden patients, in conditions of microgravity, following fracture of a limb and in other conditions of skeletal unloading [14,15]. Concentration of excessive mechanical stress often induces fatigue fractures and periprosthetic fractures. Physiological mechanical stress enhances the proliferation, differentiation, and extracellular matrix formation of osteoblasts [16,17], while pathological mechanical loading has been reported to exert an apoptotic effect on osteoblasts [18][19][20], leading to different outcomes of bone metabolism and bone strength.
The commitment of MSCs to different lineages in response to mechanical stress results from different types, magnitudes, and/or frequencies of forces [21]. Intensive studies using different types of stress such as tensile stretch, fluid flow, compression, and cyclic hydrostatic pressure have been reported to induce bone formation and limit adipogenesis of MSCs, which occur as a concerted response [22]. However, much more remains to be discovered about the effect of different levels of mechanical stimulation on the adipogenic and osteogenic differentiation of MSCs.
Over decades of study, it has become more and more clear that the adipogenic differentiation and osteogenic differentiation of MSCs are competing and reciprocal [23]. Our previous research has demonstrated that the apoptotic or antiapoptotic effect of mechanical stress on the osteoblast depends on the levels of the stress loading on it [20]. Just as different apoptotic effects on osteoblasts are observed in response to different levels of mechanical stress, we hypothesized that osteogenic and adipogenic differentiation of MSCs may reverse with changes in the levels of mechanical loading. The purpose of the present study was to investigate the effects of different magnitudes of cyclic mechanical stress on the osteogenic and adipogenic differentiation of BMSCs and to clarify the underlying mechanism. Markers of osteogenic and adipogenic differentiation were observed in MSCs stimulated by cyclic compression stress generated by liquid drops, cultured in both general and adipogenic medium in vitro. Furthermore, changes in the phosphatidylinositol 3kinase (PI3K)/Akt/glycogen synthase kinase 3 (GSK-3 )/catenin signaling pathway were investigated to illustrate the mechanism. Our present study thus provides a preliminary insight into the mechanical stress-induced changes of osteogenic and adipogenic differentiation in BMSCs and illustrates the role played by the PI3K/Akt/GSK-3 / -catenin signaling pathway in the process.
Isolation, Expansion, and Identification of BMSCs.
Animal study was approved by the animal research committee of PLA Second Military Medical University. Neonatal Sprague-Dawley rats (7 days) were used for the isolation of BMSCs. Bone marrow was isolated aseptically by flushing the femurs and tibias of rats and then suspended in DMEM/F12 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in a humidified atmosphere incubator containing 95% air and 5% CO 2 at 37 ∘ C. After 4 days, nonadherent cells were removed and medium was changed. Cells were passaged as the first passage when they were 90-95% confluent by trypsinization and seeded at a density of approximately 10 4 cells/cm 2 on round glasses ( = 25 mm, Thickness = 0.17 mm) covered with polylysine. BMSCs phenotypes were confirmed by flow cytometry and analysis of cell surface molecules. Briefly, cells at passage one were washed in PBS and incubated with PE-labeled anti-CD29 (1 : 5), anti-CD34 (1 : 100) anti-CD44 (1 : 50), and FITClabeled anti-CD45 (1 : 50) for 30 minutes. Then the cells were analyzed on a Flow Cytometer (BD Biosciences). Apoptotic cells were excluded from analysis using propidium iodide (PI).
Groups and Parameter
Settings. According to absence or presence of different levels of stress intervention and different medium cells cultured in, the cells were divided into experimental and control groups, respectively. Stress parameters were set as follows: 0.15 Hz × 8 cm as smallmagnitude and 0.6 Hz × 8 cm as large-magnitude for 30 min. The general medium was DMEM/F12 supplemented with 10% FBS and 1% penicillin-streptomycin, while the adipogenic medium consisted of basic high glucose DMEM supplemented with 1.0 M dexamethasone, 0.5 mM IBMX, 200 M indomethacin, 10 M insulin, 10% FBS, and 1% penicillinstreptomycin.
Cyclic Mechanical Stress Application.
Cells on glasses of experimental groups were exposed to cyclic mechanical stress when they were 90-95% confluent. Briefly, thin cover glass plates were placed over the confluent cell layers on the glasses. The cyclic mechanical stress was adjusted by drops of phosphate buffer solution (PBS) to the glass plates. Cells were subjected to different frequency (Hz) (controlled by drops per second) and height (cm) of mechanical stress ( Figure 1). Control cells were covered with a thin glass plate without any liquid dropping on it.
PI3K/Akt/GSK-3 / -Catenin Signaling Modulation.
For inhibition of PI3K/Akt signaling, cells were seeded on glasses as described and when they were 90-95% confluent, the medium was changed by DMEM/F12 with or without 10 M LY294002 for 1 h followed by mechanical stimulation (0.15 Hz × 8 cm) for 30 min. Cells without stress intervention were used as controls. Then cells were collected and protein expression levels of signaling pathway (p-Akt, Akt, GSK-3 , and -catenin) were measured by Western blot. For activation of -catenin, cells were cultured with either 10 mM LiCl or 10 mM sodium chloride (NaCl) (control) in adipogenic After washing with TBST, blots were then incubated at room temperature for 1 h with HRP-conjugated secondary antibodies diluted to 1 : 5000 in 5% nonfat milk/TBST. Protein bands were visualized using ECL reagents according to the manufacturer's instructions.
The intensity values of each phosphorylated kinase were normalized to the corresponding total protein bands. Unless otherwise stated, -actin was used as an internal control.
Histological
Staining. Accumulation of lipid droplets was used to denote adipogenic differentiation of BMSCs. After fixation in 4% paraformaldehyde, cells were rinsed with 60% isopropanol, followed by freshly prepared Oil Red O solution (Sigma, 0.3% in isopropanol mixed 3 : 2 with deionized water).
Statistical
Analysis. All data were presented as means ± standard deviation (SD). Statistical significance was determined using single-factor analysis of variance (ANOVA) followed by post hoc Bonferroni test to compare between groups. < 0.05 was considered statistically significant.
Effects of Mechanical Stress on BMSCs Differentiation.
The adipogenesis of BMSCs was confirmed by Oil Red O staining of the droplets in cells. Significant development of lipid droplets was observed in BMSCs after exposure to adipogenic medium for 7 days compared with control groups. Application of small-magnitude mechanical stress reduced intracellular lipid droplets while large-magnitude mechanical stress led to an increasing of lipid droplets compared with unstressed groups (Figure 3(a)). The expressions of Runx2, Collagen I, C/EBP , and PPAR in BMSCs that were cultured for 7 days with or without mechanical stress were determined by Western blot. The results showed that compared with that of control, Runx2 and Collagen I were upregulated ( < 0.05) while C/EBP and PPAR were downregulated, respectively ( < 0.05) after BMSCs were cultured in adipogenic medium for 7 days (Figures 3(b) and 3(c)).
After culture in adipogenic medium for 7 days, smallmagnitude stress stimulation led to a significant downregulation of C/EBP and PPAR and upregulation of Runx2 and Collagen I, respectively ( < 0.05) in BMSCs compared with unstressed groups. However, as the mechanical stress increased, the effect was reversed. Expressions of C/EBP and PPAR in BMSCs increased significantly ( < 0.05) which were even higher than that of unstressed groups. Additionally, large-magnitude stress resulted in decreased expressions of Runx2 and Collagen I when compared with small-magnitude stress groups and unstressed groups, respectively ( < 0.05) (Figures 3(b) and 3(c)).
Effect of Mechanical Stress on Activation of PI3K/Akt/GSK-3 / -Catenin
Signaling. The activation of PI3K/Akt/GSK-3 / -catenin signaling was evaluated by Western blot analysis. The results showed that small-magnitude stress significantly upregulated the levels of p-Akt and -catenin and inhibited the expression of GSK-3 compared with control cells ( < 0.05). In contrast, with the increasing of mechanical forces, levels of p-Akt ( < 0.05) and -catenin decreased and GSK-3 ( < 0.05) increased, respectively (Figure 4). These results reflected that the manner of PI3K/Akt/GSK-3 / -catenin signaling activation in BMSCs depended on the levels of mechanical stress loaded on them.
-Catenin Induces Osteogenesis and Inhibits Adipogenesis of BMSCs.
Firstly, activation of -catenin in BMSCs was investigated after treatment with LiCl. Western blot revealed that levels of -catenin significantly increased after cells were treated with LiCl for 24 h ( < 0.05), suggesting thatcatenin could be activated by LiCl. To explore the influence of -catenin on the differentiation of BMSCs, cells were 92.9% cultured in adipogenic medium for a 7-day period in presence or absence of LiCl and adipogenic and osteogenic marker proteins in BMSCs were analyzed by Western blot. As shown in Figure 5, the results demonstrated that expressions of Runx2 and Collagen I increased with LiCl treatment ( < 0.05), accompanied by reduction of C/EBP and PPAR expressions in BMSCs ( < 0.05). In all experiments, NaCl acted as a negative control for LiCl, which had no effect on -catenin levels or differentiation of BMSCs [24].
-Catenin-Regulated Different Differentiation in BMSCs
Is PI3K/Akt-Dependent. Pretreatment with 10 M LY294002 significantly blocked the mechanical stress-induced phosphorylation of Akt compared to the stress loaded cells without inhibitor ( < 0.05). Furthermore, inhibition of PI3K/Akt signaling partially blocked the expression ofcatenin by inactivation of GSK-3 ( < 0.05) under mechanical stress. However, LY294002 itself did not necessarily lead to any significant changes of p-Akt, -catenin, and GSK-3 in the absence of mechanical stimulation ( Figure 6). Taken together, the above findings indicated that mechanical stress regulated -catenin via a PI3K/Akt-dependent signaling pathway.
Discussion
Although factors such as magnitude, frequency, and application time varied between studies, different types of mechanical stimuli, including tension [25][26][27], compression [8,28], fluid shear stress [6], and hydrostatic pressure [29] have .05 versus small-magnitude stress + adipogenic medium group. Each value was presented as mean ± SD of three separate experiments and data of the treatment group was expressed as fold change versus that of control group (labeled as "1.00"). S represented small-magnitude stress; L represented large-magnitude stress. Cells cultured in general medium without mechanical loading as control.
been confirmed to induce osteogenic differentiation with or without inhibition of adipogenesis of MSCs. However, MSCs experiencing tension at high levels exhibit reduced expression of osteogenic markers such as Runx2, alkaline phosphatase (ALP), and Collagen I [30]. In previous studies, mechanical loading was reported to inhibit the adipogenic commitment of MSCs. Findings of Yanagisawa et al. demonstrated that a high compressive force converts the differentiation pathway of C2C12 cells into that of the adipogenic lineage by upregulating the expression of PPAR [28]. However, this interesting phenomenon was just reported without discussion of any mechanism and to date little information is available regarding the effect of mechanical stress on the enhanced adipogenesis of MSCs. The results of the present study confirmed the hypothesis that the osteogenic and adipogenic features of BMSCs changed as the levels of mechanical stress increased and the effect was partially mediated by the PI3K/Akt/GSK-3 / -catenin signaling pathway. Adipogenesis is driven by a complex signaling pathway consisting of several key transcription factors including PPAR and C/EBP [11]. PPAR is regarded as the central regulator of adipogenesis because no other factors have yet been reported to induce adipogenesis without it [31]. Several critical transcription factors such as Runx2, Osterix, and DLX2 play important roles in osteogenesis [23]. Of these, Runx2 is essential for the commitment of MSCs to the Each value was presented as mean ± SD of three separate experiments and data of the treatment group was expressed as fold change versus that of control group (labeled as "1.00").
osteoblast lineage as Runx2 controls other osteoblast-related genes such as Osterix and Collagen I [32] as well as autoregulating itself [33]. Therefore, we investigated the commitment of MSCs to the osteoblast and adipocyte lineages by detecting expressions of PPAR , C/EBP , Runx2, and Collagen I in the present study. In monolayer culture, BMSCs tend to differentiate into osteoblasts spontaneously instead of adipocytes when cultured in general medium. In order to investigate the effect of mechanical loading on the adipogenic differentiation of BMSCs, cells were cultured in adipogenic medium after mechanical application. By examining the specific markers, we demonstrated that large-magnitude mechanical loading increased adipogenesis and inhibited osteogenesis of MSCs, which was in accord with the findings of Yanagisawa et al. [28]. Cytoskeletal and focal adhesion have been reported to play an important role in mechanotransduction and to contribute to MSCs differentiation [1]. Cellular tension is generated through the interface of the external cellular environment with the internal actin cytoskeleton via integrins [34,35]. Stimulation such as mechanical stress activates integrins, which results in phosphorylation of focal adhesion kinase and activation of a series of signaling pathways including PI3K/Akt, mitogen-activated protein kinases (MAPKs), protein kinase C, and GTPases of the Rho family [36]. Of the several signaling pathways, the PI3K/Akt pathway attracted our attention since we have previously examined the response of this pathway to different levels of cyclic mechanical stress and demonstrated that Akt is activated by smallmagnitude mechanical stress, while phosphorylation of Akt is significantly reduced when large-magnitude mechanical stress is applied [20]. In the present report, we chose to subject the cells to two representative levels of mechanical stress, 0.15 Hz × 8 cm as small-magnitude and 0.6 Hz × 8 cm as large-magnitude. Our results showed the same trend of phosphorylation of Akt as our previous study.
BioMed Research International
The differentiation of MSCs is an intensely complex process, involving a large number of signaling pathways, cytokines, and transcription factors. Studies in recent years have demonstrated that a number of critical signaling pathways mediate MSCs differentiation, including transforming growth factor-beta (TGF-)/bone morphogenic protein (BMP), MAPKs, wingless-type MMTV integration site (Wnt) signaling, Hedgehogs (Hh), Notch, and RhoA/ROCK pathways [22,37,38]. Wnt signaling is an important pathway that regulates osteogenesis. In the canonical Wnt pathway,catenin acts as a key transcriptional coactivator and transmits extracellular signals to the nucleus to activate target genes [39]. When the Wnt signaling pathway is inactivated, GSK-3 is active and -catenin is constitutively phosphorylated by a destruction complex composed of Axin, APC, and GSK-3 , resulting in low cellular levels of -catenin. Inactivation of GSK-3 induces the accumulation of cytosolic -catenin and translocation to the nucleus to activate downstream target genes such as Runx2 and PPAR [39][40][41]. In MSCs, increased osteogenesis and repression of adipogenesis are dependent on activation of -catenin by inhibition of GSK-3 . Either GSK-3 inactivation or -catenin activation block the ability of mechanical signals to induce adipogenesis [42]. It has been reported that inactivation of -catenin in MSCs results in decreased osteogenic differentiation in vivo and in vitro [43]. Results of a study by Sen et al. showed that inhibition of GSK-3 is essential for mechanical inhibition of adipogenesis [44]. Our results revealed that upregulation of -catenin enhanced osteogenesis, while inhibiting adipogenesis of MSCs, even under adipogenic culture conditions, which is in accordance with their studies.
The PI3K/Akt pathway is reported to be activated by multiple types of mechanical stress [16,45], which can inhibit GSK-3 and activate -catenin [46]. Our present study showed that the changes of GSK-3 and -catenin under different levels of mechanical loading were in response to changes in Akt. Akt activation was responsible for the consequent GSK-3 inhibition, as the expression of GSK-3 increased significantly in the presence of PI3K/Akt inhibitor.
Taken together, these results suggest that osteogenic and adipogenic differentiation of BMSCs induced by mechanical stress is regulated by the PI3KAkt/GSK-3 / -catenin signaling pathway. However, we hypothesized that the effect of changes from the osteogenic to the adipogenic differentiation of BMSCs when mechanical stress increased is partially mediated by the pathway. In fact, decreased activation of -catenin by large-magnitude mechanical stress could indeed lead to increased adipogenic differentiation relative to osteogenesis, but the phenomenon that largemagnitude mechanical stress induced more adipogenesis and less osteogenesis than that in the control group indicated that there were some other signaling pathways participating in the process. Specifically, mechanical loading leads to activation of the MAPKs, resulting in increased Runx2 and subsequent expression of osteoblast-specific genes [47,48]. Furthermore, it is reported that several pathways mediate osteogenic differentiation of MSCs, including activation of ERK, PI3K/Akt [44], and insulin-like growth factor 1 (IGF1) signaling [49]. Our future studies will focus on other signaling pathways such as MAPKs and TGF-/BMP signaling and related downstream key factors that may mediate the regulation of stress-induced differentiation of MSCs, which will promote our understanding of the networks of signaling pathways involved in this process and thus make beneficial use of appropriate mechanical stress and avoid the deleterious effects of pathological forces. | v3-fos-license |
2019-02-21T05:14:07.762Z | 2019-02-01T00:00:00.000 | 73484616 | {
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} | pes2o/s2orc | Lateral Flow Aptasensor for Simultaneous Detection of Platelet-Derived Growth Factor-BB (PDGF-BB) and Thrombin
Here we report a lateral flow aptasensor (LFA) for the simultaneous detection of platelet-derived growth factor-BB (PDGF-BB) and thrombin. Two pairs of aptamers, which are specific against PDGF-BB and thrombin, respectively, were used to prepare the LFA. Thiolated aptamers were immobilized on a gold nanoparticle (AuNP) surface and biotinylated aptamers were immobilized on the test zones of an LFA nitrocellulose membrane. The assay involved the capture of PDGF-BB and thrombin simultaneously in sandwich-type formats between the capture aptamers on the test zones of LFA and AuNP-labeled detection aptamers. AuNPs were thus captured on the test zones of the LFA and gave red bands to enable the visual detection of target proteins. Quantitative results were obtained by reading the test band intensities with a portable strip reader. By combining the highly specific molecular recognition properties of aptamers with the unique properties of lateral flow assay (low-cost, short assay time and a user-friendly format), the optimized aptasensor was capable of simultaneously detecting 1.0 nM of PDGF-BB and 1.5 nM of thrombin in association with a 10-min assay time. The biosensor was also successfully applied to detect PDGF-BB and thrombin in spiked human serum samples. The LFA shows great promise for the development of aptamer-based lateral flow strip biosensors for point-of-care or for the in-field detection of disease-related protein biomarkers.
Introduction
The detection and the quantification of proteins play pivotal roles in basic discovery research and clinical applications [1]. Thrombin is an extracellular serine protease that plays a crucial role in the blood coagulation cascade, thrombosis, and hemostasis [2,3]. Its concentration in blood varies from nanomolar to low micromolar levels. The concentration of thrombin is connected to various coagulation abnormalities [4]. Platelet-derived growth factor (PDGF) is an important growth factor protein in human platelets that regulates cell growth and division toward fibroblasts, smooth muscle cells, and glial cells [5]. PDGF has been implicated in the pathogenesis of angiogenesis, and it is widely used as a biomarker for tumor types of hepatic fibrosis, liver cancer, and gastrointestinal stromal tumors [6]. Thus, the precise and sensitive evaluation of these proteins in biological samples will be substantial for disease diagnosis and biomedical applications. The gold standard method for the detection of PDGF and thrombin is enzyme-linked immunosorbent assay (ELISA), which involves antibodies and an enzyme label [7,8]. However, the utilization of antibodies may encounter some drawbacks with their production, stability, and modification.
Results and Discussion
The lateral flow aptasensor assay involved the capture of PDGF-BB and thrombin simultaneously in sandwich-type formats between the capture aptamers pre-immobilized on the test zones of LFA and AuNP-labeled detection aptamers on the conjugate pad. Typically, the sample solution containing PDGF-BB and thrombin was applied to the sample pad. Subsequently, the solution migrated by capillary action and rehydrated the AuNP-detection aptamer conjugates on the conjugate pad. Detection aptamers bind with targets and form PDGF-BB-aptamer-AuNP and thrombin-aptamer-AuNP complexes. Upon reaching the test zones, the complexes were captured by their respective capture aptamers immobilized on the test zones, giving two red bands ( Figure 1). The capillary action caused the liquid sample to migrate further. Once the solution passed through the test zones, excess gold NP-detection aptamer conjugates were captured on the control zone via the hybridization events between the control DNA probes (pre-immobilized on the control zone) and the capture aptamers, thus forming a third red band. In the absence of targets, only one red band was observed in the control zone and no red band was observed in the test zones. In this case, the red band in the control zone (control line) showed that the LFA was working properly. For the control zone, we designed a DNA probe with a sequence complementary to the capture aptamer sequencesof PDGF-BB and thrombin. Qualitative analysis was simply performed by observing the color change of the test zones, and quantitative analysis was realized by reading the optical intensities of the red bands with a portable strip reader ( Figure 1). The peak areas were proportional to the amounts of captured AuNPs on the test zones, which were proportional to the concentrations of PDGF-BB and thrombin in the sample solution. Figure 2 displays typical photo images and the corresponding responses of LFAs in the presence of (A) 0 nM PDGF-BB + 0 nM thrombin, (B) 50 nM PDGF-BB, (C) 50 nM thrombin, and (D) 50 nM PDGF-BB + 50 nM thrombin. One can see that PDGF-BB and thrombin do not cause interference for each other and can be detected simultaneously.
One can see that PDGF-BB and thrombin do not cause interference for each other and can be detected simultaneously.
Optimization of Experimental Parameters
The analytical parameters, including the concentrations of capture aptamers on the test zones, the dispensing cycles of the AuNP-aptamer conjugates, and the components of the running buffers, were optimized using PDGF-BB and its aptamers as a model. The intensity of the LFA test zone was affected by the concentration of the capture aptamer ( Figure 3A). One can see that the intensity of the LFA test zone increased with the increase of capture aptamer concentration from 1 OD/mL to 4 OD/mL; a further concentration increase resulted in signal saturation. As a result, 4 OD/mL of captured aptamer was used to prepare the test zone. The volume of AuNP-aptamer conjugates loaded on the conjugate pad affected the intensities of both test and control zones greatly. To obtain a maximum response using a minimal amount of AuNP-aptamer conjugates, the amount of AuNPaptamer conjugates on the conjugate pad was optimized by increasing the dispensing cycles of the One can see that PDGF-BB and thrombin do not cause interference for each other and can be detected simultaneously.
Optimization of Experimental Parameters
The analytical parameters, including the concentrations of capture aptamers on the test zones, the dispensing cycles of the AuNP-aptamer conjugates, and the components of the running buffers, were optimized using PDGF-BB and its aptamers as a model. The intensity of the LFA test zone was affected by the concentration of the capture aptamer ( Figure 3A). One can see that the intensity of the LFA test zone increased with the increase of capture aptamer concentration from 1 OD/mL to 4 OD/mL; a further concentration increase resulted in signal saturation. As a result, 4 OD/mL of captured aptamer was used to prepare the test zone. The volume of AuNP-aptamer conjugates loaded on the conjugate pad affected the intensities of both test and control zones greatly. To obtain a maximum response using a minimal amount of AuNP-aptamer conjugates, the amount of AuNPaptamer conjugates on the conjugate pad was optimized by increasing the dispensing cycles of the
Optimization of Experimental Parameters
The analytical parameters, including the concentrations of capture aptamers on the test zones, the dispensing cycles of the AuNP-aptamer conjugates, and the components of the running buffers, were optimized using PDGF-BB and its aptamers as a model. The intensity of the LFA test zone was affected by the concentration of the capture aptamer ( Figure 3A). One can see that the intensity of the LFA test zone increased with the increase of capture aptamer concentration from 1 OD/mL to 4 OD/mL; a further concentration increase resulted in signal saturation. As a result, 4 OD/mL of captured aptamer was used to prepare the test zone. The volume of AuNP-aptamer conjugates loaded on the conjugate pad affected the intensities of both test and control zones greatly. To obtain a maximum response using a minimal amount of AuNP-aptamer conjugates, the amount of AuNP-aptamer conjugates on the conjugate pad was optimized by increasing the dispensing cycles of the conjugate solutions. Figure 3B displays the histogram for the peak area of test line with an increasing cycle. It can be seen that the peak area from the test line of LFA increased up to four cycles. Therefore, a four-cycle procedure was employed as the optimal dispensing cycle in the following experiments.
Molecules 2019, 24, x FOR PEER REVIEW 4 of 11 conjugate solutions. Figure 3B displays the histogram for the peak area of test line with an increasing cycle. It can be seen that the peak area from the test line of LFA increased up to four cycles. Therefore, a four-cycle procedure was employed as the optimal dispensing cycle in the following experiments. Four kinds of buffers were used for the fabrication and assay of the LFA: (1) 0.05 M Tris-HCl buffer containing 0.25% Triton X-100, 5% Tween, and 0.15 M NaCl (pH 8.0) was used to treat the sample pad. This treatment facilitates the transportation and reduces the amount of PDGF-BB and thrombin trapped on the sample pad. (2) A buffer containing 20 mM Na3PO4, 5% BSA, 0.25% Tween, and 10% sucrose was used to disperse the pellet of the AuNP-aptamer conjugates. This buffer stabilizes the nanoparticles, reduces the nonspecific adsorption of conjugates on the nitrocellulose pad, and facilitates the release of the conjugates from the conjugate pad. Four kinds of buffers were used for the fabrication and assay of the LFA: (1) 0.05 M Tris-HCl buffer containing 0.25% Triton X-100, 5% Tween, and 0.15 M NaCl (pH 8.0) was used to treat the sample pad. This treatment facilitates the transportation and reduces the amount of PDGF-BB and thrombin trapped on the sample pad. (2) A buffer containing 20 mM Na 3 PO 4 , 5% BSA, 0.25% Tween, and 10% sucrose was used to disperse the pellet of the AuNP-aptamer conjugates. This buffer stabilizes the nanoparticles, reduces the nonspecific adsorption of conjugates on the nitrocellulose pad, and facilitates the release of the conjugates from the conjugate pad. shown in Figure 3C. The best response was obtained with the PBST (1% BSA) buffer. Thus, PBST (1% BSA) was chosen as the running buffer for the entire study.
Analytical Performances
Under optimal experimental conditions, we examined the performance of the LFA with the samples containing different concentrations of PDGF-BB and thrombin. The intensities of the test zones were recorded with a portable strip reader and plotted as a function of different concentrations of PDGF-BB and thrombin (Figure 4). One can see that the peak areas of test lines increased with the increases of both PDGF-BB and thrombin concentrations ( Figure 4A). Linear relationships between the peak areas and the logarithm of the target concentrations were obtained in the range of 1 nM-200 nM for both PDGF-BB ( Figure 4B) and thrombin ( Figure 4C). The detection limits were estimated to be 1.0 nM for PDGF-BB and 1.5 nM for thrombin (signal to noise (S/N) equals 3). The detection limits are comparable with those obtained with fluorescent aptasensors [20,21] and colorimetric aptasensors [24], and higher than those obtained with electrochemical aptasensors [25,26] and chemiluminescent aptasensors [22,23]. The signals of the LFA were saturated when the concentrations of PDGF-BB and thrombin were more than 250 nM. The specificity of LFA was assessed by testing the responses of other proteins (HSA, casein, BSA, IgG, and IgM) at 250 nM, as well as the mixture of target proteins (25 nM for both PDGF-BB and thrombin) and non-target protein (250 nM). Negligible signals were obtained in the absence of PDGF-BB and thrombin, and in the presence of 250 nM of non-target protein (results not shown). It is noted that the responses of target proteins (25 nM) were not affected in the presence of high concentrations of non-target proteins (250 nM). Therefore, the LFA shows excellent specificity for both PDGF-BB and thrombin. Since quantitative analysis relies on the stability of the analyte signal and the reproducibility of the assay, six LFAs were tested with 25 nM of PDGF-BB and thrombin to determine the reproducibility of the LFA. The relative standard deviation (RSD) for PDGF-BB and thrombin were 8.9% and 8.6%, respectively, indicating a sufficient reproducibility.
Detecting PDGF -BB and Thrombin in Human Serum
The LFA was also successfully applied to detect PDGF-BB and thrombin in spiked human serum samples. The sample solutions were prepared by spiking different concentrations of standard PDGF-BB and thrombin solution into the serum. We studied the matrix effect of serum on the signals of LFA test lines by using different volumes of serum (1 to 50 µL). It was found that there was no matrix effect observed when the serum volume was less than 15 µL ( Figure 5). Thereby, 15 µL of serum was applied in the following experiments. The serum samples spiked with different concentrations of PDGF-BB and thrombin were prepared to obtain the calibration curves in a complex biological matrix. The tests were performed under the best experimental conditions, as shown in Figure 5. Each sample was tested three times with three LFAs and the average responses were used to plot the calibration curves of PDGF-BB and thrombin. The response histogram of the LFA test lines in the presence of thrombin and PDGF-BB in spiked serum and corresponding calibration curves is similar to that shown in Figure 4 (results not shown). Detection limits of 1 nM for PDGF-BB and 2 nM for thrombin were obtained (S/N = 3).
Detecting PDGF -BB and Thrombin in Human Serum
The LFA was also successfully applied to detect PDGF-BB and thrombin in spiked human serum samples. The sample solutions were prepared by spiking different concentrations of standard PDGF-BB and thrombin solution into the serum. We studied the matrix effect of serum on the signals of LFA test lines by using different volumes of serum (1 to 50 μL). It was found that there was no matrix effect observed when the serum volume was less than 15 μL ( Figure 5). Thereby, 15 μL of serum was applied in the following experiments. The serum samples spiked with different concentrations of PDGF-BB and thrombin were prepared to obtain the calibration curves in a complex biological matrix. The tests were performed under the best experimental conditions, as shown in Figure 5. Each sample was tested three times with three LFAs and the average responses were used to plot the calibration curves of PDGF-BB and thrombin. The response histogram of the LFA test lines in the presence of thrombin and PDGF-BB in spiked serum and corresponding calibration curves is similar to that shown in Figure 4 (results not shown). Detection limits of 1 nM for PDGF-BB and 2 nM for thrombin were obtained (S/N = 3).
Reagents and Materials
Glass fibers (GFCP000800), cellulose fiber sample pads (CFSP001700), laminated cards (HF000MC100), and nitrocellulose membranes (HFB24004) were purchased from Millipore All other chemicals were of analytical reagent grade. All buffer solutions were prepared using ultrapure (>18 MΩ cm) water from a Millipore Milli-Q water purification system (Billerica, MA, USA).
Preparation of Gold Nanoparticles (AuNPs)
Citrate reduction of HAuCl 4 was used to synthesize AuNPs with an approximate diameter of 15 ± 3.5 nm [1]. All glassware used in this preparation was thoroughly cleaned in aqua regia (three parts HCl/one part HNO 3 ), rinsed with double distilled H 2 O, and oven-dried prior to use. A 250-mL aqueous solution of 0.01% HAuCl 4 was heated to boiling and vigorously stirred in a 500-mL round-bottom flask; 4.5 mL of 1% trisodium citrate was added quickly to this solution. The color changed from deep blue to wine red in about 60 s. Boiling was continued for an additional 10 min. The solution was cooled to room temperature with a continuous stirring for another 15 min. The AuNPs were stored in dark bottles at 4 • C.
Preparation of AuNP-Aptamer Conjugates
Thiolated aptamers (detection aptamers) were used for conjugation with AuNPs. The aptamers were first activated following the procedure: 100 µL of thiolated aptamer (1.0 OD) was mixed with 2 µL of TEA and 7.7 mg of DTT to react for 1 h at room temperature, then the excess DTT was removed by extraction with 400 µL of ethyl acetate solution. The activated aptamer was then added to 1 mL of 5-fold concentrated AuNP solution. After standing for 24 h, the conjugate was aged with the addition of PBS until a final concentration of 0.01 M was reached. The solution could stand for another 24 h at 4 • C, followed by centrifugation for 20 min at 12,000 rpm to remove the excess reagents. After discarding the supernatant, the red pellets were washed, recentrifuged, and resuspended in 1 mL of an aqueous solution containing 20 mM Na 3 PO 4 ·12H 2 O, 5% BSA, 0.25% Tween 20, and 10% sucrose.
Preparation of Lateral Flow Aptasensors (LFAs)
An LFA consists of four components: sample application pad, conjugate pad, nitrocellulose membrane, and absorption pad ( Figure 6). The components were mounted on a common backing layer (typically, an inert plastic, e.g., polyester). Two pairs of aptamers that bind to PDGF-BB and thrombin, respectively, were used to prepare the LFA. Test zones were prepared by dispensing the streptavidin-biotinylated capture aptamers of PDGF-BB and thrombin onto the nitrocellulose membrane at different locations. The detection aptamers of PDGF-BB and thrombin were labeled with AuNPs, the conjugates were mixed at 1:1 ratio (volume) and dispensed on the conjugate pad. The sample application pad (17 mm × 30 cm) was made from cellulose fiber (CFSP001700, Millipore) and was soaked with a buffer (pH 8.0) containing 0.25% Triton X-100, 0.05 M Tris-HCl, and 0.15 mM NaCl. Then it was dried and stored in a desiccator at room temperature. The conjugate pad was prepared by dispensing a desired volume of AuNP-aptamer conjugate solution onto the glass fiber pad (8 mm × 30 cm) using an Airjet AJQ 3000 dispenser. The pad was dried at room temperature and stored in a desiccator at 4 • C. The test zone and control zone on the nitrocellulose membrane (25 mm × 30 mm) were prepared by dispensing the capture aptamer and biotinylated DNA (control probe, complementary with one of the detection aptamers) solutions, respectively. The distance between each zone was around 2 mm and there were a total of three zones (test zone 1 for PDGF-BB; test zone 2 for thrombin; and control zone) on the biosensor. To facilitate the immobilization of the probes, streptavidin was used to react with the biotinylated aptamers (capture aptamers) and the biotinylated control DNA probe. Briefly, 42 µL of 10 OD biotinylated aptamer was mixed with 200 µL of 2 mg/mL streptavidin. After incubating for 1 h at room temperature, 508 µL of PBS was added to the mixture. The excess aptamer was removed by centrifuge for 30 min with a filter (cutoff 30,000, Millipore) at 6000 rpm. The conjugate was washed twice with 500 µL of PBS in the same centrifugal filter. The remaining solution in the filter was collected, and the solution was diluted to 500 µL by adding PBS. Following the same procedure, the biotinylated DNA with a concentration of 10 OD was used to prepare the streptavidin-biotinylated DNA complex for the control zone. The streptavidin-biotinylated aptamer and streptavidin-biotinylated DNA were dispensed on the nitrocellulose membrane to form the test zones and control zone, respectively. The membrane was dried at room temperature for 1 h and stored at 4 • C. Finally, all the parts were assembled on a plastic adhesive backing layer (typically, an inert plastic, e.g., polyester) using the clamshell laminator (Biodot, CA). Each part overlapped 2 mm to ensure that the solution migrated through the LFA during the assay. LFAs with a 3-mm width were cut using the guillotine cutting module CM 4000.
that the solution migrated through the LFA during the assay. LFAs with a 3-mm width were cut using the guillotine cutting module CM 4000.
Assay Procedure
A sample solution of 80 μL containing the desired concentration of PDGF-BB and thrombin in the running buffer (PBST containing 1% BSA) was applied to the sample pad. The test zone and control zone were evaluated visually within 10 min (qualitative measurement). For quantitative measurements, the optical intensities of the red bands were recorded with a portable 'strip reader'. For detecting PDGF-BB and thrombin in serum, sample solutions were prepared by mixing 65 μL of running buffer with 15 μL of plasma, which were spiked with different quantities of PDGF-BB and thrombin, and then the sample solution was applied to the sample pad. Qualitative and quantitative measurements were the same as described above.
Conclusions
We have developed a lateral flow aptasensor (LFA) for the simultaneous detection of PDGF-BB and thrombin in a complex biological matrix. The performance of the LFA shows great promise for the development of aptamer-based lateral flow strip biosensors for point-of-care or for the in-field detection of disease-related protein biomarkers. The multiplex capability of the proposed LFA can be realized with an LFA array by spotting multiple capture aptamers on the test zone. Future work will aim to improve the sensitivity of the LFA and apply it for the detection of cancer protein biomarkers.
Author Contributions: L.G. conceived, designed the experiment and elaborated the manuscript. A. G. did the experiments and wrote the draft; Q.W. reviewed and analyzed the data; and all authors approved the final paper.
Conflicts of Interest:
The author(s) declare that they have no competing interests. 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
Assay Procedure
A sample solution of 80 µL containing the desired concentration of PDGF-BB and thrombin in the running buffer (PBST containing 1% BSA) was applied to the sample pad. The test zone and control zone were evaluated visually within 10 min (qualitative measurement). For quantitative measurements, the optical intensities of the red bands were recorded with a portable 'strip reader'. For detecting PDGF-BB and thrombin in serum, sample solutions were prepared by mixing 65 µL of running buffer with 15 µL of plasma, which were spiked with different quantities of PDGF-BB and thrombin, and then the sample solution was applied to the sample pad. Qualitative and quantitative measurements were the same as described above.
Conclusions
We have developed a lateral flow aptasensor (LFA) for the simultaneous detection of PDGF-BB and thrombin in a complex biological matrix. The performance of the LFA shows great promise for the development of aptamer-based lateral flow strip biosensors for point-of-care or for the in-field detection of disease-related protein biomarkers. The multiplex capability of the proposed LFA can be realized with an LFA array by spotting multiple capture aptamers on the test zone. Future work will aim to improve the sensitivity of the LFA and apply it for the detection of cancer protein biomarkers.
Author Contributions: L.G. conceived, designed the experiment and elaborated the manuscript. A.G. did the experiments and wrote the draft; Q.W. reviewed and analyzed the data; and all authors approved the final paper.
Conflicts of Interest:
The author(s) declare that they have no competing interests. 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 |
2019-09-17T03:08:52.311Z | 2019-09-05T00:00:00.000 | 202885811 | {
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} | pes2o/s2orc | Oxidative Degradation of Trichloroethylene over Fe 2 O 3 -doped Mayenite: Chlorine Poisoning Mitigation and Improved Catalytic Performance
: Mayenite was recently successfully employed as an active catalyst for trichloroethylene (TCE) oxidation. It was e ff ective in promoting the conversion of TCE in less harmful products (CO 2 and HCl) with high activity and selectivity. However, there is a potential limitation to the use of mayenite in the industrial degradation of chlorinated compounds—its limited operating lifespan owing to chlorine poisoning of the catalyst. To overcome this problem, in this work, mayenite-based catalysts loaded with iron (Fe / mayenite) were prepared and tested for TCE oxidation in a gaseous phase. The catalysts were characterized using di ff erent physico-chemical techniques, including XRD, ICP, N 2 -sorption (BET), H 2 -TPR analysis, SEM-EDX, XPS FESEM-EDS, and Raman. Fe / mayenite was found to be more active and stable than the pure material for TCE oxidation, maintaining the same selectivity. This result was interpreted as the synergistic e ff ect of the metal and the oxo-anionic species present in the mayenite framework, thus promoting TCE oxidation, while avoiding catalyst deactivation.
Introduction
Trichloroethylene (TCE) is a chlorinated volatile organic solvent belonging to the class of dense non aqueous phase liquids (DNAPL) pollutants [1][2][3]. Several strategies have been considered for TCE remediation, including the use of CaO [4], bioremediation [5,6] and adsorption processes with activated charcoal or zeolites [7,8]. In addition, as TCE is highly volatile, it can be easily stripped from the remediation media (water, surfactant solutions, removed soils, etc.) with air flux and directed to further treatments in gas phase [9,10]. In this respect, catalytic heterogeneous oxidation is becoming a popular alternative to thermal incineration for treating exhausted gases rich in TCE, as catalysts lower operative temperatures and improve selectivity of the reaction towards less harmful products, with high benefits in terms of energy consumption and environmental impact.
Several heterogeneous catalysts have been developed and tested for gaseous TCE oxidation. Catalytic systems based on noble metals, particularly Pt and Pd, have been extensively employed, showing good results in terms of activity and selectivity, as reported by Gonzalez-Velasco and co-workers in a recently published review [11]. Less-expensive catalysts, based on metallic oxides, have also been prepared as uniform catalyst or supported on high surface materials (e.g., γ-Al 2 O 3 ) [12,13]. Blanch-Raga et al. reported the oxidation of TCE over different mixed oxides derived from hydrotalcites [14], with the Co(Fe/Al) catalyst being the most active (T 50% = 280 • C and T 90% = 340 • C at Gas Hourly Space Velocity, GHSV = 15,000 h −1 and [TCE] = 1000 ppm) due to its acidic and oxidative properties. Zeolites also represent an important type of active catalysts for the oxidation of TCE and many papers have reported on the synergic effect of acidic sites in zeolites [15] with metal catalysts in order to improve the performance of the whole catalytic system. Romero-Saez et al. [16], studied the performance of iron-doped ZSM-5 zeolite for TCE oxidation, finding that a ZSM-5 containing 2 wt% of Fe quantitatively oxidizes 1000 ppm of TCE at 500 • C and GHSV = 13,500 h −1 . This paper shows that the formation of active iron (III) species, as Fe 2 O 3 nanoparticles, was most likely responsible for the enhanced catalytic performance of the zeolite. Nevertheless the catalyst suffers some deactivation after 16 h of reaction due to the formation of FeCl 3 [16]. Recently, Palomares et al. reported a remarkably high selectivity towards CO 2 during TCE oxidation with Cu and Co-doped beta zeolites. The best results (T 50% = 310 • C and T 90% = 360 • C at GHSV = 15,000 h −1 and [TCE] = 1000 ppm) were obtained by using Cu-doped zeolite, which combined the acid sites of the zeolite with the redox properties of the copper ions [17]. Notwithstanding, zeolites-based catalysts suffer some drawbacks, which include coke formation, deactivation, and formation of chlorinated by-products [14].
In previous works, we reported about the oxidation of TCE by using the mesoporous calcium aluminate mayenite (Ca 12 Al 14 O 33 ) as a catalyst [18][19][20][21][22]; mayenite had a good overall performance, showing high activity and selectivity towards nontoxic compounds, and fair thermal stability and recyclability. As a main drawback, the material shows a certain tendency towards chlorine poisoning, leading to slow deactivation of the catalyst. Mayenite has a zeolite-type structure with interconnected cages and a positive electric charge per unit cell that is balanced by O 2− ions (free oxygen ions) [23]. The free oxygen ions can be substituted by other species (Cl − , H − , NH 2 − , etc.) [24,25] and can migrate from the bulk to the surface at temperatures higher than 400 • C [26], thus conferring to the mayenite oxidative properties exploited for many applications [27][28][29]; for instance, as Ni support for the catalytic reforming of tar [30][31][32].
With the aim of further improving mayenite activity for TCE oxidation and mitigating deactivation of the material, in this work, we use a mayenite containing iron that is employed for the catalytic oxidation of TCE. The performance of the system has been evaluated by means of the light-off curve and the structural properties of the material have been characterized, before and after the reaction, by means of different physico-chemical techniques, including XRD, ICP analysis, N 2 -sorption (BET), H 2 -TPR analysis, SEM-EDX, FESEM-EDS, XPS, and Raman spectroscopy. We have prepared catalysts with different iron content and compared the activity and stability of this material with those of pure mayenite. The Fe/mayenite catalyst clearly maintains the crystalline structure of mayenite. Furthermore, no peaks associated with iron oxides were observed in the Fe/mayenite samples; this is due to the low metal loading in the mayenite and their good dispersion on the mayenite support [34]. Table 1 shows the metal loading and specific surface area of the catalysts. The BET surface area of mayenite was 11.7 m 2 /g, in line with data reported for this type of material [27]. The Fe/mayenite samples had BET surface area values similar to that of mayenite, showing that the incorporation of iron does not modify its textural properties. ICP analysis confirmed that the iron content was close to the nominal value. Fe/mayenite is a porous material (view SI, Figure S1), characterized by large pores with dimensions of µm (macropores) and nm (mesopores) composed of calcium, aluminium, oxygen, and iron with an approximate content of 34%, 40%, 26%, and 2 wt.%, respectively. The structure and composition of the synthetized materials were also characterised by FESEM-EDS analysis, which yielded similar results, but allowed a detailed distribution of the atomic content. Figure 2 shows the results obtained, observing the most abundant elements in mayenite, i.e., aluminium, calcium, and oxygen. Iron atoms appear in low quantity and with a homogenous distribution in the mayenite, demonstrating good iron dispersion on the mayenite support. Moreover, the FESEM images of Fe/mayenite and pure mayenite (not shown) showed no significant differences in terms of morphology, maintaining the typical morphology of mayenite in both cases. TPR study of the catalysts is reported in Figure 3. All samples have similar hydrogen consumption, but present different TPR profiles. As can be seen, two peaks are observed for mayenite: the first one is a smaller band that appears around 550 • C, while the second is more intense and has the maximum at 620 • C. The first corresponds to the dissociative adsorption of H 2 in a heterolitic fashion [35] and the second to the reaction of these species with extra framework O x − and O 2 2− anions [36]. Iron-containing mayenites show a different profile with a unique band centred at 550 • C for the sample with 1.5% Fe or at 530 • C for the sample containing 2% Fe. These bands are assigned to the reduction of extra framework O x − and O 2 2− anions that in these catalysts are coincident with the band assigned to the dissociative adsorption of H 2 with consequential water formation, as previously reported for iron oxide-based catalysts [37]. Also, a small shoulder at 400-450 • C can be observed in the sample with higher iron content. This shoulder is assigned to the reduction of Fe 3+ to Fe 2+ , as reported by Romero-Saez et al. for Fe/zeolite samples [16]. Quantification of the hydrogen consumption shows similar results for the different samples, as the main species reduced in all the catalysts are the anionic oxygens present in the mayenite. The low iron content of the Fe/mayenite and the only partial reaction of the Fe 3+ species results in a negligible consumption of hydrogen compared to that necessary for the oxygen species reduction. The results also show that there is a relationship between the content of iron and the shift towards lower temperatures of the extra framework O x − and O 2 2− reduction peak.
Results and Discussion
This indicates that there is an interaction between iron species and anionic oxygen, which improves the redox properties of the catalysts containing iron. The synthetized catalysts have been evaluated for oxidation of trichloroethylene by monitoring the conversion percentage calculated by means of Equation (1) as a function of the temperature (light-off curve). As shown in Figure 4, 2.0% Fe/mayenite has the best conversion rate among the different catalysts tested. Blank experiments performed without a catalyst showed no significant TCE conversion below 550 • C. T 50 and T 90 (temperature at which 50% and 90% of the TCE was depleted, respectively) were found significantly lower for Fe/mayenite, with respect to pure mayenite ( Table 2). In particular, pure mayenite, used as a reference, showed T 50 = 410 • C and T 90 = 550 • C; 2.0% Fe/mayenite has a T 50 = 300 • C and T 90 = 460 • C and for 1.5% Fe/mayenite T 50 was found slightly higher (375 • C), whilst T 90 was the same as 2.0% iron-loaded mayenite. Conversion rates at 365 • C were also calculated according to Equation (2) for the three catalysts and reported in Table 2. Preliminary experiments revealed that an iron loading higher than~2% in the mayenite did not significantly improve the conversion performance. The results of selectivity tests showed that, for all the catalysts, CO 2 and CO were the main oxidation reaction products (CO/CO 2 ratio was 50:50 for all the investigated temperatures and catalysts), while only HCl was detected as a chlorinated product.
2% Fe/mayenite stability was evaluated for several hours (12 h) at stationary conditions (T = 460 • C, 1700 ppm TCE, GHSV = 6000 h −1 , 0.8 g of catalyst). As highlighted in Figure 5, the presence of iron on the mayenite surface drastically improved the stability of the material. Indeed, after 2 h of reaction, pure mayenite showed a significant loss in activity, whilst 2.0% Fe/mayenite retained catalytic activity. Only a partial deactivation of the 2% Fe/mayenite catalyst was observed after 12 h of reaction, decreasing the conversion from 95% to 80%. These results are interesting because stability of the catalyst is essential for possible commercial use of the material. As reported in previous works, the deactivation mechanism of mayenite catalyst is related with the displacement of active oxygen species by chloride ions [19,20,24,25]. In particular, the formation of HCl during the reaction caused a partial irreversible substitution of the anionic oxygen species (O x x− ) by chloride ions forming chloromayenite [19,22], with consequent deactivation of the catalyst. It is observed that the presence of iron in mayenite modifies this mechanism, probably because the Fe species catalyze the interaction of the atmospheric O 2 to form O 2− and O 2 2− , making reversible the substitution of chloride ions by oxygen and avoiding or diminishing the formation of chloromayenite. In order to understand the role of iron in improving the stability of the material, XRD, SEM-EDX, FESEM-mapping, XPS, and Raman analyses of fresh and used catalysts were performed. XRD patterns and SEM images did not show important differences before and after the reaction, indicating a high structural stability of the material both in the absence and in the presence of iron (see Supplementary Materials for details, Figures S2 and S3a). Nevertheless, the EDX spectrum ( Figure S3b) of the 2% Fe/mayenite sample after the stability test shows the presence of chloride. This is clearly observed in Figure 6 with the atomic mapping of Fe/mayenite after reaction. Comparing these images with those of the catalyst before reaction (Figure 2), it is observed that after the 12 h reaction, mayenite has the same morphology and distribution as Ca, Al, O and Fe atoms, revealing high stability of Fe/mayenite. It is observed that iron atoms do not agglomerate after reaction, indicating high stability of the metal supported on the mayenite. The main difference with the material before reaction is the presence of Cl atoms (orange points). The Cl mapping shows that this element has the same distribution as Ca atoms, suggesting that Cl is mainly interacting with Ca, because the basic properties of this element favour interaction with an acidic molecule as HCl. Different distribution of Cl and Fe atoms suggests that FeCl 3 was not formed, as it occurs in zeolites [16], indicating a high stability of the Fe species not poisoned by the chloride present on the catalyst surface. The full XPS spectra of the mayenite containing iron before and after reaction are shown in Fig S4, where a peak at 200 eV is observed in the sample after reaction corresponding to the binding energy of Cl (2p) with two contributions at 200 and 189.7 eV, characteristic of Cl (2p 3/2 ) and Cl (2p 1/2 for ionic chlorine (Cl − ) [38]. The presence of this peak in the sample after reaction coincides with a decrease in the relative intensity of the O peak at 530.9 eV due to the oxygen surface species consumption during the reaction. On the other hand, Figure 7 shows a complex multiplet-split Fe 2p XPS spectra.
At least two contributions are observed: the first one is centred at 709.5 eV and the second, which is more intense, is centred at 711.8 eV. These peaks have been assigned to Fe 2+ (2p 3/2 ) and Fe 3+ (2p 3/2 ), respectively, indicating the formation of Fe 2 O 3 , probably together with Fe 3 O 4 [38,39]. After reaction, an increase in the band centred at 711.8 eV, together with a small shift to the highest eV, is observed; it could be related with the disappearance of the Fe 2+ contribution in the Fe 3 O 4 phase or the formation of FeCl 3 [39]. These results reveal that after 12 h of reaction, part of the HCl formed in the reaction interacts with the mayenite and some chloride species are formed on the catalyst surface. Nevertheless, these species are not irreversibly adsorbed on the ionic vacancies (as in the case with pure mayenite [19,22]) and oxidant centres (anionic oxygen) are present after reaction. This is due to a synergic action of the iron present in the mayenite surface, which catalyses O x x− regeneration with the gas-phase oxygen present in the reaction media. To understand this process better, Raman spectroscopy studies were conducted; the results are given in Figure 8. It is shown that in Fe/mayenite, the signal for oxygen O 2 − at 1075 cm −1 is preserved after the catalytic test. Similar results were obtained in the XPS analysis of the samples after reaction (see Supplementary Materials). This does not occur with pure mayenite [19,22], demonstrating that the redox properties of iron catalyze the regeneration of anionic oxygen by the O 2 present in the gas feed and avoid the irreversible chlorine poisoning of mayenite. These results clearly show that the presence of well-dispersed iron on the mayenite surface improves the redox properties of the mayenite together with its stability, due to the ability of iron to catalyse the regeneration of the anionic oxygen responsible for TCE oxidation.
Catalyst Preparation and Characterization
Details on the experimental methods are reported in the Supplementary Materials; here, we briefly sketch the basic procedures employed in this work. Mayenite was prepared by following the ceramic method described by Li et al. [40], starting with a mixture of calcium and aluminium hydroxide. Successively, mayenite was loaded with two different amounts of iron (1.5 and 2.0%) by using a solution of Fe(acac) 3 [41].
Diffraction patterns were recorded on a Bruker D8 Advance automatic diffractometer, operating with nickel-filtered CuKα radiation.
The iron content of Fe/mayenite catalyst was determined by ICP-OES analysis using a Perkin Elmer Optima 7000 DV after digestion of the sample in HNO 3 . Three replicates for each sample were made.
The BET surface areas were determined with an 11-point BET analysis, after a degassing procedure in vacuum at 200 • C, by using a Nova Quantachrome 4200e instrument.
The morphological and elemental analysis of the catalysts was performed by a scanning electron microscope (SEM, Tescan Vega LMU) equipped with a X-Ray energy dispersive microanalysis of elements (EDX, Bruker Quantax 800). Additionally, Fe/mayenite was characterized by field emission scanning electronic microscope (FESEM, ZEISS ULTRA 55).
X-ray photoelectron spectroscopy (XPS) was performed by using a SPECS spectrometer equipped with a Phoibos 150MCD-9 multichannel analyser. CasaXPS software was used for spectra treatment.
Raman spectra were recorded at RT with a 514 nm laser excitation on a Renishaw Raman Spectrometer (in via) equipped with a CCD detector. The laser power on the sample was 25 mW and a total of 20 acquisitions were taken for each spectra.
Experimental Setup and Catalytic Reactions
The experiments were performed in a stainless-steel fixed bed reactor, with the catalysts (0.8 g) placed between two quartz fiberglass stoppers. The cylindrical reactor (150 mm × 18 mm i.d., 22 mm o.d.) was placed in a furnace and the temperature was controlled via a K-thermocouple located inside the reactor.
Catalytic oxidations were monitored in the temperature range of 150-550 • C and the catalysts were kept at an operative temperature in air for 30 min before flow of TCE. The inlet gas was prepared by flowing a stream of air through pure TCE at room temperature; the final gas composition was [TCE] = 1700 ppm in wet air (RH = 60%). The Gas Hourly Space Velocity (GHSV) was set to 6000 h −1 by flowing the inlet gas at 110 mL/min. The residence time, based on the packing volume of the catalyst, was 0.87 s. Blank experiments were performed in the same conditions to evaluate the thermal oxidation.
Analytical Methods
Post-reaction gases were collected in a Tedlar sampling bag (SKC Inc., Eighty Four, PA, USA) for further analyses. To evaluate the conversion yield, the organic species were determined by means of GC-MS (Agilent 7890A) equipped with a DB 17-MS column (30 m × 0.25 mm, 0.25 µm).
[CO] and [CO 2 ] were measured with an NDIRS on line system (Q-Track Plus IAQ Monitor, TSI), placed at the reactor outlet [23]. [Cl 2 ] and [HCl] were determined by tritation and ion chromatographic (IC) analyses of two aqueous solutions obtained by bubbling the reactor effluent gases into two water solutions (6 × 10 −2 M solution of KI and 0.1 M of H 2 SO 4 for Cl 2 and 2.6/0.76 mM solution of NaHCO 3 /Na 2 CO 3 for Cl − ). The production of Cl 2 was also assessed by SIM mode GC-MS analysis, using the same operative conditions described for the organic compounds [42]. The reproducibility of the results was checked by triplicate analyses (error < 5%). More details about products characterization are reported in our previous work [19].
The conversion yield was calculated as the ratio between the reacted TCE over the total TCE introduced into the reactor (1): where m i TCE are the moles of TCE introduced into the reactor and m o TCE are the number of moles measured in the outlet gases; the reaction rate was calculated in terms of converted mass of TCE with respect to the catalyst mass and the residence time of the gas in the reactor (2): conversion rate mol g × s = moles of converted TCE catalyst mass × residence time (2)
Conclusions
Iron-doped mayenite catalysts were prepared with different metal loading, obtaining active and selective catalysts able to quantitatively oxidize TCE in the gas phase. All the synthesized catalysts showed good performances for TCE oxidation, totally converted in CO 2 , CO, and HCl. Mayenite loaded with 2% iron was found to be the best catalyst in terms of T 50 (300 • C). This result was correlated with an optimum combination of the oxidative properties of the mayenite active support with the redox properties of iron, as TPR, XPS, and Raman results have shown.
2.0% Fe/mayenite showed good stability in terms of TCE conversion, respect to pure mayenite. The results only show some deactivation after 12 h of reaction, with a small decrease in the TCE conversion from 95% to 80%. In contrast, pure mayenite had significant deactivation (TCE conversion decrease from 80% to 40%) after 4 h of reaction. The atomic mapping of the samples shows that iron is well dispersed on the mayenite surface, minimizing catalyst deactivation and improving mayenite oxidant activity.
In conclusion, Fe/mayenite was found to be a promising catalyst due to several advantages such as: (i) total conversion of TCE in less-harmful products (CO 2 , CO and HCl), (ii) absence of noble and heavy metals, thus reducing costs and the environmental impact, (iii) low cost of catalyst precursors and low deactivation. | v3-fos-license |
2018-12-12T06:42:18.399Z | 2013-03-28T00:00:00.000 | 55121925 | {
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} | pes2o/s2orc | Combination satellit and in-situ data for the determination of evapotranspiration over heterogeneous landscape of the Tibetan Plateau
Key Laboratory of Tibetan Environment Changes and Land Surface Processes, In titute of Tibetan Plateau Research, Chinese Academy of Sciences, Beijing 100101, China Qomolangma Station for Atmospheric Environmental Observation and Research, Chinese Academy of Sciences, Dingri 858200, Tibet, China University of Chinese Academy of Sciences, Beijing 100049, China Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, Lanzhou 730000, China School of Earth and Space Sciences, University of Science and Technology of China, Hefei 230026, China
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Abstract
In this study, a new parameterization method based on MODIS (Moderate Resolution Imaging Spectroradiometer) data, AVHRR (Advanced Very High-Resolution Radiometer) data and in-situ data is constructed and tested for deriving the regional evaporative fraction (EF) over heterogeneous landscape.As a case study, the methodology was applied to the Tibetan Plateau area.Eight images of MODIS data (17 January 2003, 14 April 2003, 23 July 2003and 16 October 2003;30 January 2007, 15 April 2007, 1 August 2007and 25 October 2007) and four images of AVHRR data (17 January 2003, 14 April 2003, 23 July 2003and 16 October 2003) were used in this study for the comparison among winter, spring, summer and autumn and the annual variation analysis.The derived results were also validated by using the "ground truth" measured in the stations of the Tibetan Observation and Research Platform (TORP) and the CAMP/Tibet (CEOP (Coordinated Enhanced Observing Period) Asia-Australia Monsoon Project (CAMP) on the Tibetan Plateau).The results show that the derived EF in four different seasons over the Tibetan Plateau area is in good accordance with the land surface status.The EF show a wide range due to the strong contrast of surface features over the Tibetan Plateau.Also, the estimated EF is in good agreement with the ground measurements, and their absolute percent difference (APD) is less than 10 % in the validation sites.The results from AVHRR were also in agreement with MODIS, with the latter usually displaying a higher level of accuracy.It is therefore concluded that the proposed methodology is successful for the retrieval of EF using the MODIS data, AVHRR data and in-situ data over the Tibetan Plateau area, and the MODIS data is the better one and it should be used widely for the evapotranspiration (ET) research over this region.
Introduction
As the most prominent and complicated terrain on the globe, the Tibetan Plateau, with an elevation of more than 4000 m on average above mean sea leave (msl) makes up approximately one fourth of the land area of China (Fig. 1).Due to its topographic character, the plateau surface absorbs a large amount of solar radiation energy, and undergoes dramatic seasonal changes of surface heat and water fluxes (e.g.Yanai et al., 1992;Ye and Wu, 1998;Hsu and Liu, 2003;Sato and Kimura, 2007).Evapotranspiration (ET) between the land surface and atmosphere of the Tibetan Plateau play an important role in the Asian Monsoon system, which in turn are major components of the energy and water cycles of the global climate system.Some interesting detailed studies concerning the ET (or latent heat flux) have been reported in the different sites and land surface types over the Tibetan Plateau in the past years (e.g.Tanaka et al., 2001;Ma et al., 2005;Li et al., 2006;Zhong et al., 2009).These researches were, however, on point-level or a local-patch-level.Remote sensing from satellites however offers the possibility to derive ET regional (or areal) distribution over heterogeneous land surface in combination with sparse field experimental stations.And the regional ET distributions have been reported over the Tibetan Plateau in the past years (e.g.Ma et al., 2003;Ma et al., 2009), but the results were still in a meso-scale area.To understand the effect of the Tibetan Plateau on the climatic change over China, East Asia, and even the globally, the regional distribution of ET distribution of over whole Tibetan Plateau must been determined.Our purpose in this study is to estimate the regional distribution of ET and its seasonal variation over the whole Tibetan Plateau with the aid of MODIS data, AVHRR data and in-situ data.It is because the ET from land is essential for understanding climate dynamics and ecosystem productivity (e.g.Churkina et al., 1999) and it also has applications in areas such as water resource management.We will introduce "evaporative fraction (EF)" as an index for ET after Shuttleworth et al. (1989).EF is defined Introduction
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Full here as: Where H is the sensible heat flux, λE the latent heat flux, R n the net radiation flux and G 0 the soil heat flux.Our goal is not estimation of ET but rather EF in whole Tibetan Plateau area.This is due to two reasons.First, EF is more suitable as an index for surface moisture condition than ET.Because ET itself is a function not only of the land surface conditions (e.g.soil moisture and vegetation) but also surface available energy R n −G 0 (= H+λE ), ET cannot be easily interpreted for soil moisture condition or drought status.On the contrary, EF can be more directly related to these land surface conditions.Secondly, EF is useful for scaling up instantaneous observations to longer time periods.As one knows, a satellite (except for geostationary satellite) observes each land surface very briefly during a day.ET, however, can generally change drastically during a day mainly due to changes in sun angle and cloud coverage.Therefore, even if we can accurately estimate ET at the moment of satellite overpass, it cannot be directly related to daily or daytime average ET.On the contrary, EF is well known to be nearly constant during most of daytime (e.g.Shuttleworth et al., 1989;Sugita and Brutsaert, 1991;Crago, 1996a, b).
We also find that EF is also nearly constant from sunrise to sunset during the clear days in BJ station (31.37 Full 2).Therefore, if we can estimate daytime average R n − G 0 or H + λE , daytime average ET will be estimated from Eq.( 1) by using instantaneous EF derived by satellite.There are some cases for the estimation by using the satellite and in-situ data.A similar spatial variation of broadband albedo and surface temperature was proposed to estimate EF (Su, 1999;Roerink et al., 2000).Jiang and Islam (2001) estimated EF by interpolating the Priestley-Taylor parameter.Venturini et al. (2004) did the comparison of EF estimated from AVHRR and MODIS sensors over South Florida of USA.Verstraeten et al. (2005) estimated EF from NOAA-imagery at satellite overpass time over European forests area.Wang et al. ( 2006) estimated EF from a combination of day and night land surface temperatures and NDVI.But no any study of EF over heterogeneous landscape of whole Tibetan Plateau till now.
The regional distribution of EF over the Tibetan Plateau will be estimated and validated in this study.We first propose the methodology in Sect. 2. The using satellite data and in-situ data for the validation are shown in Sect.3. The application of the methodology to the Tibetan Plateau area is presented in Sect.4, where the distribution of EF are estimated for four different phases, spring, summer, autumn and winter by using eight scenes of MODIS data and four scenes of AVHRR data.Discussions are given in the Sect. 5.
Theory and scheme
The general concept and procedure of the methodology are shown in a diagram (Fig. 3).The surface reflectance for short-wave radiation r 0 (x,y) is retrieved from MODIS data by using Zhong's method (Zhong , 2007) and AVHRR data by using the method of Ma et al. (2003) with the atmospheric correction, using land surface and aerological observation data.The land surface temperature T sfc (x,y) is also derived from MODIS data by using the method of Zhong et al. ( 2010 1989) computes the downward short-wave and long-wave radiation at the surface by using satellite data, land surface and aerological observation data and the atmospheric correction (Ma and Tsukamoto, 2002).With these results the regional surface net radiation flux R n (x,y) is determined by using the surface radiation energy budget theorem for land surface.The regional soil heat flux G 0 (x,y) is estimated from R n (x,y) and field observations over the Tibetan Plateau (Ma et al., 2002).It means that based on the field observation data from the key stations over the Tibetan Plateau, a good relationship between G 0 and R n can be found over the area.And the relationship will be used for the determination of G 0 (x,y) from R n (x,y ).The regional sensible heat flux H(x,y) is estimated from T sfc (x,y), surface and aerological data with the aid of so-called "tile approach" (Ma et al., 2010).The procedure to determine R n (x,y ) and H(x,y) in detail will be found in Ma's paper (Ma et al., 2011).The regional latent heat flux λE (x,y ) can be derived as the residual of the energy budget theorem for land surface.If the evaporative fraction (EF) defining Eq.( 1) is used to the MODIS and AVHRR pixel scale, it will be become: EF value is between 0.0 and 1 Λ equaling 0.0 means that the surface is very dry, and no ET from surface.Λ equaling 1.0 means that surface is very wet, and there are maximum ET from the surface.2003, 14 April 2003, 23 July 2003and 16 October 2003) are used here to find which satellite data is better for the determination of EF over heterogeneous landscape of the Tibetan Plateau.More four images of MODIS data in 2007 is used here for the more validations of methodology due to more validation sites set up in 2007 over the Tibetan Plateau (Ma et al., 2008).
The most relevant in-situ data, collected at the key stations over the Tibetan Plateau to support the parameterization of EF and analysis of the MODIS and AVHRR images, consist of surface radiation budget components, surface radiation temperature, surface reflectance, vertical profiles of air temperature, humidity, wind speed and direction measured at the PBL towers, Wind Profiler and RASS, radiosonde and tethersonde, turbulent fluxes measured by eddy-correlation technique, soil heat flux, soil temperature profiles, soil moisture profiles, and the vegetation state.The key stations in the TORP are including BJ station (31.37 • N, 91.90 Full shown here due to their key position in the procedure of determination of EF (Fig. 3).
The R n and EF distribution maps are based on 2875 × 1487 pixels with a size about 1 × 1 km 2 .The derived R n and EF can be validated by the field measurements.In-situ data observed in nine stations of Haibei, Maqu, D105, Amdo, NPAM, BJ, NAMOR, QOMS and SETS in the TORP (Ma et al., 2008) are used for the validation in 2007 and four stations of D105, NPAM, ANNI and BJ of the CAMP/Tibet ( Ma et al., 2005) are used for the validation in 2003.In Fig. 6, the derived results are validated against the measured values in the stations.The absolute percent difference (APD) was quantitatively measures the difference between the derived results (H derived(i ) ) and measured value (H measured(i ) ) here, and H measured(i ) . (3) The results show that: (1) the derived R n and EF in four different months over the Tibetan Plateau area are in good accordance with the land surface status.The Tibetan Plateau includes variety of land surfaces such as a large area of grassy marshland, some desertification grass-land areas, sparse grass-Gobi, sparseness meadow, many small rivers and lakes, snow (glacier) mountains, forest, and farmland etc.Therefore, these derived parameters show a wide range due to the strong contrast of surface fea- The mean EF from MODIS data over the Tibetan Plateau area is increasing from January to April and August, then decreasing from October (Fig. 5b and Fig. 5c).They are 0.275, 0.415, 0.567 and 0.406 for 2007 and 0.271, 0.342, 0.569 and 0.347 for 2003.
The mean EF from AVHRR data over the Tibetan Plateau area is also increasing from January to April and July, then decreasing from October.They are 0.267, 0.335, 0.502, and 0.331(Fig.5a).( 4) Because of the land surface cover and property in spring (April) is much complex (there are ice, snow, seasonal and long-living permafrost, grass land and lakes etc existing in this month), therefore the EFs distribution in this month is also complicated (Fig. 5).( 5) The most derived regional EFs with APD less than 10.0 % at validation sites in the Tibetan Plateau are in good agreement with field measurements, except EFs derived from AVHRR in ANNI (APD = 13.0 %) and D105 (APD = 11.0 %) in 23 July 2003 (Fig. 6).The reason is that the radiation transportation processes, using MODTRAN model, land surface and aero-logical data and the process of atmospheric boundary layer, such as using "tile approach" and the measuring and calculating accurately of wind speed u, air temperature T a and specific humidity q at the reference height, zero-plane displacement d 0 , aerodynamic roughness length z 0m and thermodynamic roughness length z 0h , the excess resistance for heat transportation kB −1 etc. in each tile were considered in more detail in our method (Fig. 3).It is pointed that our proposed parameterization methodology for EF is reasonable, and it can be used over the Tibetan Plateau area.( 6) The retrieval EF and net radiation from MODIS are supe-Introduction
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Full rior to those from AVHRR even the same methodology are used .It means that the derived values from MODIS are more closed to the "ground truth" measured in the validation stations (Fig. 6).Many reasons might account for this, such as the image quality itself, the different Split Window Algorithms (SWAs), or the different algorithms for water vapor.It is therefore concluded that the MODIS data is the better one and it should be used widely for the determination of surface heat fluxes and ET over the Tibetan Plateau area.
Conclusions
Concluding remarks In this study, the regional distributions of evaporative fraction (EF) over heterogeneous landscape of the Tibetan Plateau are derived with the aid of MODIS data, AVHRR data and the in-situ data.And the MODIS data is the better one than AVHRR data for the determination of EF over heterogeneous landscape.Compared with the field measurements, the proposed EF has been proved to be a better index to getting related evapotranspiration (ET) over heterogeneous landscape.
Regionalization the evapotranspiration (ET) over heterogeneous landscape is not an easy issue.The parameterization methodology presented in this research is still in developing stage due to only a single set of values at a specific time of specific day are used in this research.To reach more accurate regional ET and seasonal and annual variations of evapotranspiration over the Tibetan Plateau area, more field observations, more accurate radiation transfer models to determine the surface reflectance and surface temperature, more MODIS data, and other satellites such as ASTER (Advanced Spaceborne Thermal Emission and Reflection radiometer), Landsat-5 TM, Landsat-7 ETM, GMS (Geo-stationary Meteorological Satellite), and ATSR (Along Track Scanning Radiometer) have to be used.These research works will be done in the next step.Introduction
Conclusions References
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Tibetan Plateau
Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | et al., 2008) (see Fig.
) and AVHRR data by using the method of Ma et al. (2003) with the atmospheric correction, using land surface and aerological observation data.The radiative transfer model MODTRAN (Berk et al., Discussion Paper | Discussion Paper | Discussion Paper |
3
Satellite data and field observation data Eight images of MODIS data (17 January 2003, 14 April 2003, 23 July 2003 and 16 October 2003; 30 January 2007, 15 April 2007, 1 August 2007 and 25 October 2007) and four images of AVHRR data (17 January 2003, 14 April 2003, 23 July 2003 and 16 October 2003) was chosen in this study for the comparison among winter, spring, summer and autumn.Same day images of AVHRR and MODIS data in 2003 (17 Discussion Paper | Discussion Paper | Discussion Paper |
Figure 4
Figure4and Fig.5shows the distribution maps of net radiation flux R n and evaporative fraction EF over the Tibetan Plateau area.The distribution maps of net radiation flux are shown here due to their key position in the procedure of determination of EF (Fig.3).The R n and EF distribution maps are based on 2875 × 1487 pixels with a size about tures over whole Tibetan Plateau.(2) The derived pixel value (Fig. 5) of EFs in summer (1 August 2007 and 23 July 2003) and autumn (25 October 2007 and 16 October 2003) are higher than that in winter( 30 January 2007 and 17 January 2003) and spring (15 April 2007 and 14 April 2003).EFs in summer are mostly from 0.70 to 1.0, EFs in autumn are mostly around 0.60, EFs in spring are mostly around 0.50, EFs in winter are mostly around 0.30 (some of the EFs are 1.0 in the distribution maps indicating cloud-Discussion Paper | Discussion Paper | Discussion Paper |covering).The differences between 2007 image and 2003 image seem to be large is due to day-to-day variability.It means that there are much more evapotranspiration in summer and autumn than it in winter and spring in the Tibetan Plateau area.The reason is that most the land surface is wet and covered by green grass and growing vegetations in summer and autumn and it is dry and most the mountain ranges is covered by snow and ice during winter and spring on the Tibetan Plateau area.In other words, sensible heat and latent heat fluxes play different roles in the partition of net radiation flux in different month in the Tibetan Plateau: sensible heat flux plays the main role in winter and spring and latent heat flux plays the main role in summer and autumn.(3) Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | EU-FP7 projects of "CORE-CLIMAX"(313085) and "CEOP-AEGIS"(212921).The in-situ data of Haibei station and Maqu station was come from Yingnian Li, Xinqaun Zhao and Yu Zhang.Discussion Paper | Discussion Paper | Discussion Paper | Wang, K., Li, Z., and Cribb, M.: Estimation of evaporative fraction from a combination of day and night land surface temperatures and NDVI: a new method to determine the Priestley-Taylor parameter, Remote Sens. Environ.,102,293-305, 2006.Yanai, M., Li, C., and Song, Z.: Seasonal heating of the Tibetan Plateau and its effects on the evolution of the Asian summer monsoon, J. Meteorol.Soc.Jpn., 70, 319-351, 1992.Ye, D. and Wu, G.: The role of the heat source of the Tibetan Plateau in the general circulation, Fig.1The location and landscape of the Tibetan Plateau.
Fig. 1 .
Fig. 1.The location and landscape of the Tibetan Plateau.
. Zhong, L.: Measurement and satellite remote sensing of land surface characteristic parameters over the Tibetan Plateau area, Ph.D. Thesis, Graduate University of Chinese Academy of Sciences, Beijing, China, 124 pp., 2007.Zhong, L., Ma, Y., Su, Z., Lu, L., Ma, W., and Lu, Y.: Land-atmosphere energy transfer and surface boundary layer characteristics in the Rongbu Valley on the northern slope of Mt.Everest, Arct.Antarct.Alp.Res., 41, 396-405, 2009.Zhong, L., Ma, Y., Salama Suhyb, Mhd., and Su, Z.: Assessment of vegetation dynamics and their response to variations in precipitation and temperature in the Tibetan Plateau, Clim. | v3-fos-license |
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} | pes2o/s2orc | INFLUENCE OF MICROBIAL POLYSACCHARIDES ON THE FORMATION OF STRUCTURE OF PROTEIN-FREE AND GLUTEN-FREE FLOUR-BASED PRODUCTS
The formation of a structure of certain dietary bakery products and flour confectionery products made without wheat flour is difficult due to the absence of gluten. There is a constant search for effective structure-forming agents to replace gluten proteins. We investigated an influence of microbial polysaccharides (MPS) of xanthan, enposan, and gellan on the formation of a structure of gluten-free and protein-free bread and gluten-free muffins in the study. The ability of a model protein-free system based on corn starch was studied to form dough with addition of xanthan, enposan, and gellan in the amount of 0.1...0.5 % by weight at Brabender farinograph. It was found that presence of the investigated microbial polysaccharides in the amount of 0.3...0.5 % enabled the formation of dough with indicators that ensured formation of the necessary structure of dough without gluten. We investigated an influence of MPS on resilient-elastic and plastic-viscous properties of gluten-free dough. It was found that reopex properties disappeared in protein-free dough due to addition of xanthan. Viscosity of protein-free dough with addition of 0.3...0.5 % of xanthan by the mass of starch reached the values, which are characteristic for wheat bread dough. The amount of MPS of 0.1 % by weight of finished products is sufficient in gluten-free confectionery dough for muffins. Effective viscosity increased by 2...3 times for all investigated MPS and provided the desired consistency of dough for formation by the sedimentation method in this case. We studied quality indicators of baked products with addition of the investigated MPS. It was shhown that their use in certain quantities leads to an increase in the specific volume and to ensuring of the porous structure of baked products. Crumbling of products decreased during storage, which indicated a slowdown of hardening processes in gluten-free systems with xanthan, enposan, and gellan. All studied MPS exhibited the same nature of influence on certain indicators, but xanthan had the greatest effect, and gellan ‒ the least one
Introduction
Gluten-free products form a separate group in bakery and flour confectionery products.They are necessary for diet and
One can influence a structure of protein-free and gluten-free products using different types of flour [14,15].Starch is the main raw material in protein-free products usually, and it is possible to add flour in the amount of 5…10 % by weight of starch only.Flour has no significant effect on a structure of products and it is more a flavoring ingredient in this case.A share of gluten-free flour in a formulation can be 5.0…90.0% by weight of starch or even 100 % in exchange of starch in gluten-free products.Studies [15][16][17] prove that each type of gluten-free flour causes certain structural-and-mechanical properties of dough and products.Creation of a structure of gluten-free products, unlike protein-free ones, becomes much easier due to the use of structure-forming protein-containing raw materials, such as egg, dairy, and fish products.
There is also instability in a structure of protein-free dough and finished products due to the use of flour raw materials and starches with different properties.
Studies [18,19] show that improvement of structural-and-mechanical properties of dough and baked goods occurs due to selection of thickeners and gelling agents that play a role of the basic structure-forming agents in gluten-free systems.Such thickeners such as pectin, carboxymethylcellulose, hydroxypropylmethylcellulose, è-glucan [20,21], guar gum, and xanthan [22,23] affect rheological properties of dough based on rice flour, corn, rice and other starches significantly.
An option to overcome difficulties in structure formation of gluten-free dough systems is optimization of the quantitative content of structure-forming agents [20].Authors of work [24] show that presence of various hydrocolloids, such as hydroxypropyl methylcellulose (HPMC), carboxymethylcellulose (CMC), plantain gum, carob, guar gum and xanthan contribute to resistance to deformation and elasticity of gluten-free dough.Studies prove that xanthan has the greatest influence on viscous-elastic properties of dough.It causes its hardening.Studies established a line of positive influence of thickeners on a structure of dough: xanthan>C-MC>pectin>agar>β-glucan.
Taking into account changing properties of structure-forming raw materials, it is necessary to study each composition to substantiate quantitative and qualitative ratios of hydrocolloids in formulations of gluten-free and protein-free products.
One should note that formulations of flour products include fats, baking powder, and flavor components, which also influence formation of their structure.Therefore, it is important to study indices of structure formation taking into account an ingredient composition of products.
Protein-free products are necessary for patients with disorders of protein metabolism, in particular phenylketonuria.There are about 1.5 thousand people suffering from phenylketonuria in Ukraine today.The incidence of the disease is 1:10,000 of newborns on average in the world.There is a limitation for protein-free products for the total protein content, which is usually from 0.6 to 2.2 % in terms of solids [1].Gluten-free products are necessary to feed patients with celiac disease, people with allergies to wheat flour and its intolerance.The exact number of celiac patients in Ukraine is not known, because it has large number of forms, different for children and adults, and it is difficult to diagnose.According to various data, the incidence of celiac disease ranges from 1:51 to 1:250 in different countries.There is also an increased public awareness of gluten intolerance and people choose gluten-free foods as an attribute of healthy eating.Consumption of gluten-free products is becoming the norm of life in western countries.Therefore, there is a growth of their production.Gluten-free products are quite popular in the USA, Europe, and Russia [2,3].
The main difference between protein-free and gluten-free products is a type of flour and its quantity in a formulation.The main raw materials for protein-free products are various types of native starch with 5.0 to 10.0 % of flour added to enhance taste and aroma.The amount of protein introduced with flour is negligible.If flour is gluten-free, then such products will be suitable not only for a protein-free diet, but also for a gluten-free one.It is possible to add gluten-free products to a protein-free diet if they are low in protein.Conversely, protein-free products may also be suitable for a gluten-free diet, but only if they do not contain flours with high gluten content (wheat, rye, barley, and oat).However, protein-free products are poor in protein and they are not adequate products for daily consumption during a gluten-free diet.
Promising raw materials for production of gluten-free products may be oilcake and solvent cake (low fat residues of oil raw material, which remains after removal of the oil by pressing or extraction, respectively).It is possible to use the mentioned raw materials to enrich production with dietary fiber, vitamins, and minerals, or as a major ingredient.If oilcake and solvent cake are made of gluten-free raw materials, then their products are suitable for a gluten-free diet.
The main structure-forming agents are thickeners and gelling agents of polysaccharide nature in absence of gluten.The search for effective structure-forming agents, which would provide formation of dough structure and finished product, is constantly underway.Today, new types of thickeners and gelling agents are emerging on the market and we need research on their use in gluten-free products.
Literature review and problem statement
Authors of papers [3,4] note that replacement of gluten is a serious technological task, since it plays a major role in formation of a dough structure and baked products.Formation of a structure of fibrin and protein free products and gluten-free products coincides largely.But it is more difficult to obtain a structure of protein-free bread in comparison with other products from a technological point of view, since requirements of the diet limit the protein content strictly and exclude all ingredients with its high content [5].
All formulation components take part in formation of a structure in one way or another.Studies on the influence of dif-In addition, formulations also include dietary fiber, vitamins, mineral premixes, amino acid mixtures, [25,26], fruit and berry raw materials obtained using fundamentally new equipment [27][28][29] to improve the nutritional value of products.Authors of studies propose to use certain technological methods and raw materials, which slows down hardening processes to increase shelf life and reduce crumbling [14,17,30,31].
Table 1 illustrates an analysis of the role of formulation ingredients that influence the formation of a structure of protein-free and gluten-free bakery products The most important factor in formation of a structure of protein-free and gluten-free products, in our opinion, is the use of hydrocolloids of different nature with substantiation of their quantitative ratio.
Researchers consider xanthan (other names: xanthan, xanthan gum) an effective thickener of microbiological origin, and papers [32,33] emphasize that it is a key ingredient for formation of a structure of gluten-free products.Xanthan exists as an extracellular heteropolysaccharide in nature.It is made of D-glucose, D-mannose and D-glucuronic acid.It appears during the life-cycle of Хаnthoтопаsсаmpеstris bacteria.Its molecular weight is 1,000… 2,000 kDa.
However, there are other microbial polysaccharides and new ones emerged recently in the market.It is possible to use them in protein-free and gluten-free technologies due to their structure and properties.They include enposan and gellan.They are relatively new microbial thickeners and are less studied.
Enposan (another name is polymixan) is a heteropolysaccharide produced by Bacilluspolymyxa.Its molecules consist of D-glucose, D-mannose, D-galactose, and D-galacturonic acid.They have a molecular weight of 1,000...1,500 kDa.Enposan is very close to xanthan by its physicochemical properties [34,35].
Gellan is a heteropolysaccharide produced by Sphingomonas Elodea (formerly Pseudomonas Elodea).It has a linear structure.Its molecular weight is approximately 500 kDa.The molecules consist of repeating tetra-saccharide units linked by pyranose rings of D-glucose, D-glucuronic acid, and L-rhamnose.It is capable of forming a jelly with almost all ions, including hydrogen ones (acidic media), but its affinity for divalent ions is much stronger than for monovalent ions [36,37].
An analysis of information sources shows that there are not enough studies into a possibility of using enposan and gellan in technologies of dietary protein-free and gluten-free products.Enposan and gellan have structures and properties close to xanthan.However, there is no full assessment of their technological potential to stabilize a structure of dough systems, especially in gluten-free dough systems.The issues of studies on product quality indicators in presence of the mentioned microbial polysaccharides remain unresolved.There is no systematic understanding of an influence of enposan and gellan, comparing with xanthan, on processes of dough structure formation and formation of such structural-and-mechanical parameters as ability to form dough, resilience, and elasticity.This is a prerequisite for further research in these directions.
The aim and objectives of the study
The objective of the study is to substantiate a use of microbial polysaccharides (MPS) of xanthan, enposan, and gellan as structure-forming agents in protein-free bread and gluten-free muffins.
We set the following tasks to achieve the objective: investigation of the ability of a model protein-free system with addition of MPS of xanthan, enposan, and gellan to form dough on Brabender farinograph; studying of an influence of MPS of xanthan, enposan and gellan on structural-and-mechanical properties of protein-free bread dough and gluten-free muffin dough; investigation of a quality of baked protein-free bread and gluten-free muffins with addition of the studied MPS.
1. Subjects of the study
The subjects of the study were: -a model system of protein-free dough, which consisted of components in the following weight ratios: corn starch -100.0,rye flour of peeled grinding -5.0 and MPS -0.1...0.5; we took the system of protein-free dough without addition of MPS as the control one; protein-free bread dough, which consisted of components in the following weight ratios: corn starch -100.0,rye flour of peeled grinding -5.0, table salt -2.5, sugar -4.0, sunflower oil -5.0 and MPS -0.1…0.5;we took the protein-free dough made according to the same formulation without addition of MPS as control; gluten-free dough for muffins, it consisted of components in the following weight ratios: wheat germ meal -37.0, white sugar -18.0, margarine -17.0, chicken eggs -8.0, kefir -18.0, vanilla sugar -1.0, baking powder -0.2, salt -0.5, and MPS -0.1...0.3; we took gluten-free muffin dough made according to the same formulation without addition of MPS as control; samples of baked protein-free bread and gluten-free muffins with addition of the investigated MPS; we took samples of baked products without addition of MPS as control.
2. Methods for determining the structural-and-mechanical properties of dough for protein-free bread and gluten-free muffins
We determined capability of the model protein-free system to form protein-free dough using Brabender farinograph.The capability was defined by such parameters as duration of formation, resistance to mixing, resistive capacity, stability, degree of dilution and elasticity [38].
We measured structural-and mechanical properties of protein-free bread dough and gluten-free muffin dough at the Tolstoy flat-parallel elastoplastometer [39] defining the modulus of momentary resiliency, modulus of elasticity, and plastic viscosity.The fixed load value for all protein-free dough systems was 50 g, and it was 20 g for muffin dough.
We determined the shear stress (τ, Pa) from formula , m g F where m is the weight of load, kg; g is the acceleration of free fall, m/s 2 ; F is the area of a plate, m 2 .The tangent shear stress was 327.0 Pa for all samples of protein-free dough, and 50.0 Pa for muffin dough samples.We maintained the same temperature of samples (20 °C) during the experiment, the height of samples of protein-free dough was 8 mm, the muffin dough -6 mm.
We determined momentary resiliency modulus from formula: where τ is the tangent shear stress, Pa; γ 0 is the relative conditional moment deformation.We determined the modulus of elasticity from formula: , where τ is the tangent shear stress, Pa; γ hе is the relative high-elastic deformation.
We determined the plastic viscosity from formula: * 0 , tg where * 0 η is the plastic viscosity, Pa•s; tgα is the tangent of an angle of inclination of a finite linear section of a curve to the axis of abscissa.
We determined viscosity of dough for bread and muffins at the "Reotest-2" rotary viscometer (Germany) with an extended rotor speed range of 0.001...100 s -1 [39].The liquid limit or a transition point was determined from the plastic state to the fluid state of substance at low revolutions of the measuring cylinder.
3. Methods for determining the quality indicators of protein-free bread and gluten-free muffins
We determined the quality of products by indicators of specific volume and crumbling after 24 hours of storage according to the methods described in a work [40].The organoleptic characteristics of products were also defined.
We calculated the specific volume of baked product samples from formula where V is the volume of a sample, cm 3 ; m is the weight of a sample, g.We calculated a crumbling indicator of baked products from formula where C is crumbling, %; a is the weight of a portion of cut cubes, g; b is the weight of crumbs formed due to friction of cubes, g.The error value for all studies was s=3…5 %, the number of experiment repetitions was n=5, the probability was P≥0.95.We processed the experimental data statistically by the Fischer-Student method at a reliability level of 0.95.We calculated the results of studies as an average of at least five repetitions.The MS Office software package was used, including MS Excel, and the standard Mathcad software package to process the experimental data.
1. Results of studies on the influence of microbial polysaccharides on structural-and-mechanical parameters of protein-free dough
We established the ability of a model protein-free system to form dough at Brabender farinograph at the beginning of the research.The model system, which consisted of starch, rye flour and microbial polysaccharides in the mentioned ratios, played a role of "protein-free flour".We investigated its ability to form dough under an influence of different amounts of MPS.
The results of studies showed that the obtained farinograms did not have a typical appearance compared to farinograms of wheat flour.They were more similar to farinograms of rye flour, especially in the part of a curve, which characterized stability of dough.Table 2 gives properties of the model systems of protein-free dough according to indicators of farinograms.
Table 2 shows that addition of MPS leads to a decrease in duration of formation of dough.Thus, xanthan has the greatest effect.The addition of 0.3 % by weight of starch reduces duration of dough formation by 44.0 %, and in the amount of 0.5 % by weight of starchby 56.0 % comparing to the control sample without MPS.Gellan demonstrates the least effect comparing to xanthan and enposanduration of formation of dough at its amount of 0.5 % by weight of starch reduces by 17.0 %.It should be noted that we did not observe stability of protein-free dough samples to mixing in dough samples with the MPS content less than 0.3 % by weight of starch, such dough did not keep consistency and, after reaching a peak, began to subside.Instead, addition of xanthan in the amount of 0.3 % led to the emergence of system resistance to mixing for 0.75 min, and in the amount of 0.5 % -for 1 min.Addition of enposan and gellan had the same effect on the system's resistance to mixing, which was slightly less comparing to xanthan.
The degree of dilution of dough, which corresponded to the value of the fall of the curve after 12 min.after start of dilution, decreased at addition of MPS.We established that introduction of additives in the amount of 0.1 % by weight of starch did not reduce the degree of dilution of dough significantly comparing with the control dough without MPS.However, one can see from Table 2 that larger quantities reduced the degree of dilution significantly.For example, introduction of xanthan in the amount of 0.3 % by weight of starch led to a decrease in the degree of dilution of dough by 23.0% and in the amount of 0.5% by weight of starchby 40.0 %.Xanthan in the amount of 0.5 % by weight of starch had the best effect on reduction of the degree of dilution of dough.
Further mixing of the protein-free dough system resulted in formation of new consistency with certain elasticity.Introduction of MPS increased elasticity of dough.For example, addition of xanthan led to an increase in the elasticity parameter from 3 mm (a control sample) to 10 mm (a sample with addition of xanthan in the amount of 0.5 % by weight of starch).One can see that addition of enposan provided values of elasticity close to xanthan.Addition of gellan had the least effect on elasticity comparing to xanthan and enposan.
An indicator of preservation of dough consistency level characterizes stability of dough.Table 2 shows that the stability of dough with addition of MPS increased.Thus, introduction of additives in the amount of 0.5 % by weight of starch led to an increase in the stability of dough comparing with the control sample.When we added xanthan, it increased in 3.6 times, enposan -in 3.4 times, gellan -in 2.8 times.
We should note that such indicators of farinograms were similar to indicators of farinograms of rye dough and provided for formation of a certain susceptible structure in absence of gluten.
Protein bread dough contains other formulation components usually.Therefore, we investigated an influence of MPS on the structure of protein-free bread dough, which contained salt, sugar, oil and MPS in the ratios specified in paragraph 4, in further research.
Resilient-elastic and plastic-viscous properties determine structural-and-mechanical indicators of dough traditionally.
Studies of resilient-elastic properties of protein-free dough at the Tolstoy elasto-plastometer showed that the momentary resilience modulus increased with addition of MPS at the constant tangential shear stress.Thus, addition of xanthan in the amount of 0.5 % by weight of starch led to an increase in the modulus of momentary resilience by 11.0 % comparing with the control sample (Table 3).Addition of enposan and gellan in the same amount increased the momentary resilience modulus by 9.3 % and 6.3 %, respectively.Addition of MPS led to an increase in the modulus of elasticity.Thus, the indicator for protein-free dough with addition of the investigational preparations in the amount of 0.5 % by weight of starch was 3.2 times higher than the sample without additive for xanthan and enposan, and 1.5 times higher for gellan.
Moreover, the value of the modulus of elasticity was an order of magnitude higher than the value of the modulus of resilience, which indicated the dominance of resilient properties over elastic ones in the dough.
One can also see that as the amount of MPS increased, the plastic viscosity of the protein-free dough increased.Thus, comparing to the control sample, the dough with addition of structure-forming agents in the amount of 0.5 % by weight of starch led to a three-fold increase in the plastic viscosity in case with xanthan and enposan, and in 2.5 times in case of using of gellan.An indicator of effective viscosity characterizes plastic-viscous properties of dough usually.Effective viscosity is the main characteristic of structural-and-mechanical properties of dispersed systems.This indicator describes the equilibrium state between processes of restoration and destruction of a structure in a fixed flow [33].Studies on an influence of all investigated MPS on the effective viscosity showed that the dependences are the same.We present the data on xanthan as an example (Fig. 1).Based on data shown in Fig. 1, one can say that we observed reopex properties of dough in the study of the control sample of the protein-free dough without xanthan.The viscosity decreased at low shear rates (from 1 to 10 s -1 ), and the viscosity increased as the rotor speed increased.The test sample with the amount of xanthan of 0.1 % by weight of starch behaved similarly.The properties of this dough are quite close to the control dough, but the reopex properties began to show up at higher shear rates than in the control sample (greater than 10 s -1 ).The protein-free dough with 0.3 % and 0.5 % by weight of starch behaved as a non-Newtonian fluid.We did not observe rheopexy in these dough samples.
One can see in Fig. 1 that the highest effective viscosity in the protein-free dough sample with addition of xanthan in the amount of 0.5 % by weight of starch (1.5•10 4 Pa•s) was at the shear rate of 0.0028 s -1 .The dough sample with 0.3 % of xanthan by weight of starch had lower viscosity and reached a value of 3.0•10 3 Pa•s at the same shear rate.We know that the structure of wheat bread dough of high-grade flour has the viscosity of 10 3 …10 4 Pa•s at a small gradient of shear rate (0.003 s -1 ), and the structure of rye doughthe viscosity of 105 Pa•s.Based on the above data, one can say that the viscosity of protein-free dough approaches the viscosity of wheat dough at low shear rate.
2. Results of studies on the influence of microbial polysaccharides on structural-and-mechanical properties of gluten-free muffin dough
We substantiated the use of microbial polysaccharides as structure-forming agents in gluten-free flour confectionery products on the example of muffins.Cupcakes, butter biscuits, biscuit cookies, and others have a structure similar to muffins.Wheat germ meal (WGM) was used as gluten-free flour in muffin formulations.
Muffins with full replacement of wheat flour with wheat germ meal have good organoleptic quality indicators.However, the products have a small volume, inelastic and too fragile crumb due to absence of gluten proteins and starch of wheat flour, which are responsible for a structure of products.Therefore, we proposed using microbial polysaccharides such as xanthan, enposan and gellan as structure-forming agents to give dough necessary properties.Previous laboratory baking showed that muffins with full replacement of wheat flour with wheat germ meal with addition of experimental additives in the amount of 0.1 % by weight of a finished product had the best organoleptic and physical-and-chemical quality indicators.
Table 4 gives the results of measurement of structural-and-mechanical properties of gluten-free dough for muffins with addition of 0.1 % of MPS by weight of a finished product.We used two control samples to compare structural-and-mechanical properties of gluten-free dough and gluten-containing dough in the experiment.Control No. 1 was the dough mixed with wheat flour without additives, and control No. 2 was the dough with wheat germ meal without investigated additives.Table 4 data shows that the wheat germ meal dough (control No. 2) had a momentary elasticity modulus by 5.4 times, an elastic modulus by 4 times, and a plastic viscosity indicator by 8 times, higher than parameters of the wheat dough (control No. 1), which contributed to formation of excessively elastic-viscous dough and complicates development and formation of dough pieces by distribution into muffin molds.
Addition of all investigated MPS to the dough with meal contributed to decrease in the value of the modulus of momentary resilience by 1.6…1.7 times, the modulus of elasticity by 1.3…1.4times and the plastic viscosity by 2.0…3.0 times comparing to control No. 2. We could distribute the dough for muffins with all MPS in the amount of 0.1 % by weight of a product into molds easily.We can state that the use of structure-forming agents can improve structural-and-mechanical properties of gluten-free dough, bringing them closer to the properties of wheat dough typical of muffins.γ, s Fig. 2 shows results of research into the viscous-plastic properties of gluten-free confectionery dough with full replacement of flour with wheat germ meal based on effective viscosity.Fig. 2 shows that the curves of the effective viscosity of the investigated samples of muffin dough as well as the curve of the control sample are typical of non-Newtonian fluids.A sharp drop in the effective viscosity of dough with a slight increase in a shear rate, and a further increase in a shear rate (greater than 2.0 s -1 ) resulted in a slight decrease in its values.The use of xanthan increased the viscosity of the dough samples by 3.0 times, and addition of enposan and gellan increased the indicator almost by 2 times comparing to the control sample.An increase in the effective viscosity by 2...3 times in the presence of all investigated MPS contributed to formation of the necessary structural-and-mechanical properties for formation of dough by distribution.
3. Results of studies of the influence of microbial polysaccharides on the quality indicators of baked protein-free bread and gluten-free muffins
Table 5 gives the results from studies on the influence of microbial polysaccharides on quality indicators of baked protein-free bread.As one can see in Table 5, addition of MPS in the amount of 0.3 % by weight of starch increased the specific volume of products comparing to the control sample.Xanthan had the greatest influencethe indicator increased its value by 36 %.Gellan had the smallest influencethe indicator increased its value by 33 %.The products with addition of all microbial polysaccharides had well-developed porosity and larger volume comparing with the control sample.They had no cracks on a surface.The bread crumb was similar to the wheat bread crumb in organoleptic terms.The product without addition of MPS had poorly developed porosity and large cracks on its surface.Addition of MPS affects also storage of products.We can characterize storage by crumbling of crumb after 24 hours of storage.We can see that the crumbling decreased with addition of MPS comparing to the control sample.Thus, the crumbling decreased by 2.3 times with addition of xanthan, by 2.2 times -with addition of enposan and by 2.0 times -with addition of gellan.The data indicated a slowdown in hardening due to the addition of microbial polysaccharides.
Table 6 gives the results from studies of the influence of microbial polysaccharides on the quality indicators of baked gluten-free muffins with wheat germ meal.One can see in Table 6 that the addition of MPS in the amount of 0.1 % by weight of a finished product increased the specific volume of a product and reduced the crumbling value.Thus, addition of xanthan increased the specific volume of products by 14.3 %, enposan -by 10.7 %, gellan -by 3.5 %, and the crumbling decreased by 50.9 %, 50.4 % and 49.1 %, respectively.Muffins with MPS had an excellent appearance, which is characteristic for these products, cracks on their surface characteristic for this type of products and soft, elastic crumb, which was not brittle and did not crumble.Xanthan in gluten-free muffin dough had also the greatest influence, and gellanthe least one.
Discussion of results of studies on properties of dough and quality indicators of finished products
Studies on the model protein-free dough system (Table 2) at a farinograph showed that addition of MPS leads to a decrease in duration of dough formation.MPS has good moisture-binding and moisture-retaining properties of MPS, so dough interacts immediately with water in the first minutes of dough mixing and creates a stable colloidal system into which starch grains are embedded during further mixing.
Addition of MPS in quantities greater than 0.3 % by weight of starch leads to an increase in resistance of protein-free dough samples to mixing.At the same time, there is no resistance to mixing at all at addition of MPS in the amount of 0.1 %.Therefore, small quantities of all tested MPS do not form the desired structure of protein-free dough The degree of dilution of dough, which corresponds to the magnitude of the fall of the curve after 12 min after the beginning of dilution, decreases significantly with an increase in the amount of MPS, apparently, due to the branched structure of biopolymer molecules, especially xanthan.Addition of them causes structuring of a protein-free dough system and dilution decreases.
Further mixing of a protein-free dough system leads to formation of new consistency with a certain elasticity due to swelling and insignificant action of hydrolytic enzymes.The elasticity increases with an increase in the amount of MPS (Table 3).An increase in the elasticity indicator with an increase in the amount of additives is obviously related to formation of an elastic gluten-like structure in dough and appearance of elasticity and resilience in it.Therefore, we can increase stability of dough by introduction of structure-forming agents in the process of mixing of a protein-free starch-based dough system, which differs from the traditional one by absence of gluten.
It is clear that the dough forming process occurs due to binding of water by dry components of a protein-free dough system.Addition of MPS accelerates formation of protein-free dough due to their good hydration ability.Elastic protein-free dough will promote formation and preservation of its porous structure during its fermentation, separation, and baking.
The study on the effective viscosity of protein-free dough (Fig. 1) showed that it exhibits rheopexy properties without addition of xanthan.A sharp increase in the shear rate leads to a sharp increase in the dough viscosity, which can lead to overload and equipment failure.Addition of xanthan in the amount 0.3 % and 0.5 % by weight of starch will eliminate reopex properties.Dough will behave like a non-Newtonian liquid.We examined such samples of protein-free dough at low shear rates (γ<1 s -1 ).The samples exhibited viscous-plastic properties, which corresponds to the Ostwald dependence.An increase in the shear rate leads to a gradual destruction of the structure and achievement of a constant final viscosity of the Newtonian fluid.The viscosity of protein-free dough at low shear rate approaches the viscosity of wheat dough at addition of xanthan in such quantities.One can predict that protein-free dough will exhibit the same properties as wheat dough when processing it at the bakery.Almost parallel displacement of the curves of dependencies of the effective viscosity on the shear rate for protein-free dough samples with xanthan in the amount of 0.5 % and 0.3 % indicates that there is almost constant coefficient between these dependencies, which is approximately 4.7±0.2.This can indicate identical mechanisms of structure formation in the dough samples.
Studies (Fig. 1) showed that addition of xanthan in the amount of 0.3...0.5 % by weight of starch strengthens significantly a protein-free system under conditions of the same shear rate.And the effective viscosity of dough increases significantly.Protein-free dough with xanthan exhibits properties of non-Newtonian liquids, so it can refer to highly concentrated dispersed systems with a coagulation structure.Interaction between elements (starch grains, flour particles, etc.) occurs through a thin layer of dispersion medium (swollen xanthan) and it is conditioned by van der Waals forces in such structures.
According to ideas about structure formation in gluten-free systems, we can say that xanthan acts as a hydrocolloid in dough.It envelops starch grains and forms a stable structure similar to the gluten structure of dough.The reason for an increase in viscosity may be that xanthan can reduce the amount of free moisture in protein-free dough, since it has a strong moisture-binding and moisture-retention capacity.In general, we can say that MPS take part in formation and maintenance of the spatial structure of dough and thus provide formation of rheological properties of protein-free dough similar to traditional bread wheat dough.
Muffin dough is significantly different from protein-free bread dough in structure.It contains more moisture, includes protein-containing dairy and egg products that are involved in formation of the spatial structure of gluten-free confectionery dough.Studies showed that a sufficient number of MPS is 0.1 % by weight of a finished product.The effective viscosity (Fig. 2) increases by 2...3 times when using the investigated MPS at the same shear rate in this case.Thus, we obtain the desired consistency of dough for molding by distribution from a confectionary bag.The results of studies on elasticity and resilience of gluten-free dough for muffins with MPS confirmed the above (Table 4).
We know the ability of gluten and other proteins included in a dough system to preserve the swelled structure during baking determines the specific volume of flour products.As addition of MPS improves the specific volume of products (Tables 5, 6), we can assume that swollen microbial polysaccharides together with proteins of other components act as gluten and provide formation of a swollen structure for products in the gluten-free dough system.
Crumbling of flour products occurs usually due to formation of air layers, because of reduction of the volume of starch grains due to their crystallization.Air layers are more noticeable in stale bread.Most likely, the ability of microbial polysaccharides to reduce crumbling of protein-free bread (Table 5) and muffins (Table 6) may be related to enveloping of partially gelatinized starch grains and slowing down of their compaction due to crystallization of amylose and amylopectin during storage.We can explain the effect by slow formation of air layers between a hydrocolloid and partially gelatinized starch grains.
The obtained results show that enposan and gellan exhibit a similar effect on formation of a gluten-free dough structure as xanthan does.However, different types of dough require different amounts of MPS, depending on availability of protein-containing raw materials, which can influence formation of the structure significantly.However, the mechanism of linkage formation in the spatial structure of dough in presence of microbial polysaccharides and their influence on formation of physical-and-chemical parameters of baked products remain not fully studied.Therefore, further studies on the influence of MPS on organoleptic quality indicators, and structural-and-mechanical properties of baked goods, including storage period, are promising.
Conclusions
1. We established that the presence of xanthan, enposan and gellan in the amount of 0.3…0.5 % by weight of starch in dough leads to formation of a susceptible dough structure in the absence of gluten.Duration of dough formation decreases by 17...56 %.System resistance to mixing and elasticity increase, and a degree of dilution of dough decreases by 23...43 %.The stability of dough increases also by 2.4...3.6 times comparing to dough without addition of MPS.
2. It was found that an increase in the amount of MPS from 0.1 to 0.5 % by weight of starch leads to improvement of resilient-elastic and plastic-viscous properties of protein-free bread dough.We established that addition of xanthan to a protein-free dough system influences a change in the effective viscosity indicator significantly.Reopex properties of dough disappear due to introduction of additives.It acquires properties of a non-Newtonian fluid, and the viscosity of protein-free dough with xanthan content of 0.3…0.5 % by weight of starch reaches the values typical of traditional wheat dough.
The amount of MPS of 0.1 % by weight of a finished product is sufficient for gluten-free confectionery dough for muffins.In this case, the effective viscosity increases by 2...3 times for all studied MPS, which provides the required consistency of dough for its formation.
3. The study showed that the use of xanthan, enposan, and gellan leads to an increase in the specific volume of products and to a porous structure during baking.We established that crumbling decreases during storage, which indicates slowing of hardening processes in gluten-free systems with addition of MPS.We obtain products with good organoleptic characteristics.They have well-developed porosity.They have a surface, taste and aroma, which are characteristic for the products.
All the investigated MPS have the same influence on certain indicators, but xanthan exhibits the greatest effect, and gellan -the least one.
Table 1
Ingredients of formulations that affect the formation of a structure of protein-free and gluten-free bakery products | v3-fos-license |
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} | pes2o/s2orc | Infrared and 2-Dimensional Correlation Spectroscopy Study of the Effect of CH3NH3PbI3 and CH3NH3SnI3 Photovoltaic Perovskites on Eukaryotic Cells
We studied the effect of the exposure of human A549 and SH-SY5Y cell lines to aqueous solutions of organic/inorganic halide perovskites CH3NH3PbI3 (MAPbI3) and CH3NH3SnI3 (MASnI3) at the molecular level by using Fourier transform infrared microspectroscopy. We monitored the infrared spectra of some cells over a few days following exposure to the metals and observed the spectroscopic changes dominated by the appearance of a strong band at 1627 cm−1. We used Infrared (IR) mapping to show that this change was associated with the cell itself or the cellular membrane. It is unclear whether the appearance of the 1627 cm−1 band and heavy metal exposure are related by a direct causal relationship. The spectroscopic response of exposure to MAPbI3 and MASnI3 was similar, indicating that it may arise from a general cellular response to stressful environmental conditions. We used 2D correlation spectroscopy (2DCOS) analysis to interpret spectroscopic changes. In a novel application of the method, we demonstrated the viability of 2DCOS for band assignment in spatially resolved spectra. We assigned the 1627 cm−1 band to the accumulation of an abundant amide or amine containing compound, while ruling out other hypotheses. We propose a few tentative assignments to specific biomolecules or classes of biomolecules, although additional biochemical characterization will be necessary to confirm such assignments.
Introduction
The perovskite methylammonium lead iodide CH 3 NH 3 PbI 3 (MAPbI 3 ) has been the subject of interest for photovoltaic (PV) applications due to its elevated light conversion efficiency [1]. However, its use is associated with the potential toxicity arising from lead [2][3][4][5], particularly because of the potential human exposure during the production process. This concern has driven the search for a replacement for Pb with reduced toxicity and lower environmental impact, without compromising the efficiency of MAPbI 3 . The structurally similar perovskite CH 3 NH 3 SnI 3 (MASnI 3 ), containing tin instead of lead, has been proposed as a viable candidate. Despite its appeal, based on its efficiency in light conversion, the compound also bears potential hazards due to the toxicity of tin [6]. For this reason, we decided to perform a systematic comparison of the effects of cellular exposure to Pb and Sn containing perovskites. Previous work by some of the authors has described the in vitro cytotoxic effects on eukaryotic cells exposed to MAPbI 3 [2]. In this follow-up work, we use infrared (IR) absorption spectroscopy as a method for the comparison of the molecular changes that accompany exposure to both MAPbI 3 and MASnI 3 . IR spectroscopy has been used for decades to characterize the molecular properties of biological samples. Molecular species absorb infrared light, giving rise to absorption patterns that reflect the composition of the sample. Spectral recordings can be obtained from samples of any composition without the need of chemical modification such as molecular labeling or staining. Analysis of complex biological samples can be performed using standard preparations such as fixed cells and tissue sections [7]. With some challenges, the measurements can be extended to living cells [7]. In some cases, the compositional information from IR spectra can be complemented by molecular information. IR absorption patterns are sensitive to structural parameters such as molecular conformation, bond length, and bond strength. One application of this capability is the study of protein secondary structure and, in some cases, of tertiary structure [8,9]. In more recent years, applications of IR spectroscopy were boosted by the coupling of interferometric technology for spectral analysis and optical microscopy configurations, leading to the introduction of Fourier transform infrared microspectroscopy (FTIRMS). FTIRMS allows for the IR spectra of single cells to be recorded with sufficient sensitivity to provide detailed analysis of molecular properties [10,11]. The applications extend beyond fundamental research in cell biology and include biomedical research and molecular diagnostics [12]. The coupling of IR microscopes to synchrotron light sources has provided improved performance, enabling high signal-to-noise measurements with diffraction-limited spatial resolution [13]. These developments have extended FTIRMS to the study of weaker spectroscopic features in cellular samples. Despite these advances, one remaining challenge is the assignment of absorption bands in complex cellular spectra to specific molecular absorbers. Assignments based on functional group identification have often been used in the recent literature. However, it has been stressed that such an approach is often unreliable for the purpose of compound identification, given the extensive number of cellular absorbers that provide overlapping bands in the same spectral region. It was proposed by one of these authors that the complexity of the problem could be reduced by using 2D correlation spectroscopy (2DCOS) to analyze the correlations between multiple bands in cellular spectra that evolve over time and thus identify bands arising from the same absorber. In the present work, we demonstrate for the first time the application of 2DCOS to the assignment of bands in static samples by using the spatial position of the measurement as the physical variable along which the spectra are aligned. We use this approach to interpret the molecular origin of the spectroscopic changes observed in the sample following metal exposure.
Results and Discussion
We studied the response of the SH-SY5Y neuroblastoma cell line and the A549 pulmonary carcinoma cell line following exposure to MAPbI 3 and MASnI 3 . Both MAPbI 3 and MASnI 3 are water-soluble compounds, and their dissolution in the culture medium provides a variety of aqueous forms of Pb 2+ and Sn 2+ ions, in addition to insoluble species such as lead carbonates, hydroxides, and phosphates. Experimental details of the preparation are provided in Section 4. In this study, we removed the precipitates before incubating the cells with the conditioned medium. Therefore, we ascribe the biological effects to soluble species such as aqueous Pb 2+ and Sn 2+ , and possibly to methylammonium cations and iodine derivatives. Mortality and overall response in cell lines exposed to either MAPbI 3 or MASnI 3 suspensions showed an increase in cellular mortality, thus underlining the toxicity of both compounds [6]. An example of the toxicity response of the two cell lines is provided in Appendix A ( Figure A1). Despite the comparable increase in cell mortality, toxicity of MAPbI 3 seems to be mediated by different mechanisms in the SH-SY5Y and A549 cell lines [2].
We tried to gain insight into the molecular events that accompany cellular response to metal exposure by performing FTIRMS measurements on fixed adherent cells following treatment with the perovskites. The cells were cultured on CaF 2 optical slides and inspected using an FTIR microscope to collect transmission IR absorption spectra and maps of single cells with diffraction-limited spatial resolution. The FTIR absorption spectrum from a SH-SY5Y cell in the energy range 1000-4000 cm −1 is shown in Figure 1A. Figure 1B shows the details of the spectrum of a single cell exposed to 100 µg/mL of MAPbI 3 (blue) or 100 µg/mL of MASnI 3 (red) and compares it to the spectrum of a non-treated cell (black).
Molecules 2020, 25, x FOR PEER REVIEW 3 of 13 shown in Figure 1A. Figure 1B shows the details of the spectrum of a single cell exposed to 100 μg/mL of MAPbI3 (blue) or 100 μg/mL of MASnI3 (red) and compares it to the spectrum of a non-treated cell (black). The main contribution to the spectral region detailed in Figure 1B arises from the absorption bands of carbonyl and imide containing compounds, although contributions from other organic molecules such as amines are also present. In cellular spectra, the dominant contribution in this region is usually assigned to polypeptide bands, termed Amide I and Amide II, around 1650 cm −1 and 1550 cm −1 , respectively, with the assumption that protein absorption is dominant. In purified proteins, the fine structure of these bands is often used to quantify the relative abundance of specific secondary structure components [14]. The use of amide bands for the study of protein conformation has also been extended to cells and tissue. Application to such samples is challenging and limited by the compositional complexity of the samples themselves. Cells and tissues, particularly live ones, are characterized by a mixture of polypeptides and other organic molecules absorbing in the same spectral region. In many cases, performing a detailed analysis of protein secondary structure is unreliable in such samples because of overlapping contributions from multiple absorbing molecules [15]. Despite these difficulties, some cases have been reported where the Amide I band has been used to track protein conformational changes within cells or tissues. One such case is the study of neurodegenerative diseases involving the formation of amyloid deposits, consisting of misfolded polypeptides that aggregate into an intermolecular β-sheet conformation. Since the deposits are compositionally nearly pure, they have been identified from the characteristic Amide I absorption at ca. 1625-1628 cm −1 [10,16,17]. In early examples, these assignments were confirmed using staining protocols for amyloids. In the case of fixed cells and tissue, the contribution of many small molecules to this spectral region is removed by the washing steps of fixation, thus simplifying the problem of interference from small molecule absorption.
To facilitate the resolution of different overlapping spectral contributions and reduce baseline effects, we analyzed the second derivative of the absorbance spectra. The 2nd derivative traces in the The main contribution to the spectral region detailed in Figure 1B arises from the absorption bands of carbonyl and imide containing compounds, although contributions from other organic molecules such as amines are also present. In cellular spectra, the dominant contribution in this region is usually assigned to polypeptide bands, termed Amide I and Amide II, around 1650 cm −1 and 1550 cm −1 , respectively, with the assumption that protein absorption is dominant. In purified proteins, the fine structure of these bands is often used to quantify the relative abundance of specific secondary structure components [14]. The use of amide bands for the study of protein conformation has also been extended to cells and tissue. Application to such samples is challenging and limited by the compositional complexity of the samples themselves. Cells and tissues, particularly live ones, are characterized by a mixture of polypeptides and other organic molecules absorbing in the same spectral region. In many cases, performing a detailed analysis of protein secondary structure is unreliable in such samples because of overlapping contributions from multiple absorbing molecules [15]. Despite these difficulties, some cases have been reported where the Amide I band has been used to track protein conformational changes within cells or tissues. One such case is the study of neurodegenerative diseases involving the formation of amyloid deposits, consisting of misfolded polypeptides that aggregate into an intermolecular β-sheet conformation. Since the deposits are compositionally nearly pure, they have been identified from the characteristic Amide I absorption at ca. 1625-1628 cm −1 [10,16,17]. In early examples, these assignments were confirmed using staining protocols for amyloids. In the case of fixed cells and tissue, the contribution of many small molecules to this spectral region is removed by the washing steps of fixation, thus simplifying the problem of interference from small molecule absorption.
To facilitate the resolution of different overlapping spectral contributions and reduce baseline effects, we analyzed the second derivative of the absorbance spectra. The 2nd derivative traces in the 1200 cm −1 -1800 cm −1 spectral region for cells unexposed and exposed to perovskite suspensions are shown in Figure 2. The traces show the 2nd derivative of the average spectra collected by mapping single cells at a diffraction-limited spatial resolution. Figure 2A shows the effect of exposure to MAPbI 3 and MASnI 3 on the spectra of A549 cells. Figure 2B shows the corresponding measurements performed on metal treated and untreated SH-SYS5 cells. Spectra in both A and B were recorded on cells fixed after five days of exposure to the metals. Figure 2C shows the difference between the average spectrum of A549 cells exposed to MAPbI 3 and unexposed cells to highlight dominant spectral changes.
For unexposed cells, the Amide I region of unexposed cells is characterized by a strong contribution at 1655 cm −1 and a shoulder at 1638 cm −1 . This pattern is common to many eukaryotic cell types and is due to the abundance of α-helical proteins absorbing around 1655 cm −1 . The contribution from β-sheet protein structures, at approximately 1635 cm −1 , is responsible for the shoulder on the lower wavenumber side. Other secondary structure components absorb in this region, together with contributions from biomolecules other than proteins, and induce small variations on this basic pattern. A weaker contribution is also observed at 1680-1690 cm −1 , which can arise from other secondary structure motifs such as β-sheet, β-turns, and other molecules including nucleic acids and sphingolipids. Due to the preparation protocol of the samples involving repeated washings following fixation, it is likely that contributions from small water soluble and alcohol soluble molecules such as metabolites have been removed, except for acyl lipids, which are at least partially retained by fixation.
Molecules 2020, 25, x FOR PEER REVIEW 4 of 13 1200 cm −1 -1800 cm −1 spectral region for cells unexposed and exposed to perovskite suspensions are shown in Figure 2. The traces show the 2nd derivative of the average spectra collected by mapping single cells at a diffraction-limited spatial resolution. Figure 2A shows the effect of exposure to MAPbI3 and MASnI3 on the spectra of A549 cells. Figure 2B shows the corresponding measurements performed on metal treated and untreated SH-SYS5 cells. Spectra in both A and B were recorded on cells fixed after five days of exposure to the metals. Figure 2C shows the difference between the average spectrum of A549 cells exposed to MAPbI3 and unexposed cells to highlight dominant spectral changes. For unexposed cells, the Amide I region of unexposed cells is characterized by a strong contribution at 1655 cm −1 and a shoulder at 1638 cm −1 . This pattern is common to many eukaryotic cell types and is due to the abundance of α-helical proteins absorbing around 1655 cm −1 . The contribution from β-sheet protein structures, at approximately 1635 cm −1 , is responsible for the shoulder on the lower wavenumber side. Other secondary structure components absorb in this region, together with contributions from biomolecules other than proteins, and induce small variations on this basic pattern. A weaker contribution is also observed at 1680-1690 cm −1 , which can arise from other secondary structure motifs such as β-sheet, β-turns, and other molecules including nucleic acids and sphingolipids. Due to the preparation protocol of the samples involving repeated washings following fixation, it is likely that contributions from small water soluble and alcohol soluble molecules such as metabolites have been removed, except for acyl lipids, which are at least partially retained by fixation. Figure 2 shows that exposure to both metal salts causes the appearance of a new strong band in the Amide I region with an apparent maximum at 1627 cm −1 . The subtraction spectrum of Figure 2C highlights the spectral changes and confirms that the main change is the appearance of a band with a maximum at 1624 cm −1 , accompanied by other minor components. The difference between the apparent band maxima in the total spectra and subtraction spectra may be due to the presence of overlapping bands that shift the apparent band maximum. Changes are also seen in the Amide II region, with some components disappearing and new components appearing after exposure to metals. The Amide II band is also sensitive to the secondary structure of polypeptides. Therefore, the observation of parallel changes in the Amide I and Amide II region favors the assignment of these changes to amide containing compounds. However, the Amide II band of Figure 2D is also unusually small compared to the Amide I in the same figure, which does not rule out a contribution from molecules other than polypeptides.
Alkyl ammonium ions can give absorption bands in the same region as the Amide I and Amide II bands. We can rule out a contribution from residual perovskite crystals because all their absorptions were below 1600 cm −1 [18]. However, we cannot exclude that the main difference bands in Figure 2C arise from the methylammonium ions themselves, which may have been adsorbed by the cell, although they should have been removed by the extensive washing steps following fixation.
The diagram in Figure 2D shows the increase of the 1627 cm −1 band over time relative to the 1652 cm −1 band, measured on samples collected at different days. The band starts appearing within two days after exposure and rapidly increases over time. The slow time course of the increase disfavors the accumulation of methylammonium iodide as the origin of the 1627 cm −1 absorption and suggests a mechanism that relies on biochemical changes in response to exposure. One important factor is the role of fixation chemistry on the final composition of the sample. While fixation preserves the morphology and part of the subcellular architecture of the cells, it also alters their chemical composition and the conformation of biological macromolecules. Molecular products of fixation should also be considered for the interpretation of spectroscopic data.
To clarify the assignments and exclude at least some possibilities, we analyzed the spectra using 2D correlation spectroscopy (2DCOS). The analysis allows us to identify groups of bands that can arise from the same compound or to exclude the ones that cannot by looking at their correlation properties. In standard applications, 2DCOS is applied to the spectra of samples that evolve as a function of time or as a function of some physical variable such as temperature or pressure [19]. We have previously applied this method to the assignment of bands in complex cellular spectra evolving in time [15,20]. In the present application, we used a recently introduced variation of the technique by reporting correlations as a function of position in the space of the measured spectrum [21]. We used such correlations to identify bands that arise from the same species as a way to confirm or disprove their assignment to specific molecules. For such purposes, we applied the 2DCOS algorithm to the set of spectra collected by mapping a single cell. The resulting synchronous and asynchronous spectra for a cell exposed to MAPbI 3 are shown in Figure 3. Bands that arise from the same molecular species or from species with the same spatial distribution must show synchronous changes (i.e., display cross correlation peaks (outside of the diagonal) with the same sign in the Synchronous plot). They must also display weak or absent correlation peaks in the Asynchronous plot, although the latter are often difficult to confirm because of band overlap. This is true for bands that arise from the same compound, but also for bands from different compounds that have the same spatial distribution. The synchronous plot in Figure 3A shows the presence of a peak multiplet at 1655, 1627, 1548, and 1515 cm −1 , marked with crosses along the diagonal, together with the corresponding correlation peaks outside of the diagonal. The positive value of the correlation peaks (red color) indicates that the peaks display an in-phase correlation. The same peak multiplet shows a lack of or minor correlation in the asynchronous plot of Figure 3B, indicating that these peaks arise from the same compound or from multiple compounds with the same spatial distribution. The correlation is consistent with, but not exclusive to, the assignment of bands in this multiplet to the Amide I and Amide II pairs [19].
In contrast, the opposite correlation was observed in the Synchronous plot of Figure 3A between the 1627 cm −1 band and the carbonyl band at 1715 cm −1 , indicating that they arise from different molecular species. The observation excluded an assignment to molecules such as glycosaminoglycans or other polysaccharides that contain both amide and ester functions (although an assignment to their hydrolysis products is possible, as discussed later). It would be useful to assess the correlation between the 1627 cm −1 band and the 2855 cm −1 /2920 cm −1 bands in the CH 2 stretching region (not shown) that are typically assigned to long alkyl chains in lipids. However, the large difference in wavelength between the two regions means that the diffraction-limited focal spots differ in size by a factor of two and effectively probe different regions of the sample, thus altering the significance of the correlations.
Molecules 2020, 25, x FOR PEER REVIEW 6 of 13 from different molecular species. The observation excluded an assignment to molecules such as glycosaminoglycans or other polysaccharides that contain both amide and ester functions (although an assignment to their hydrolysis products is possible, as discussed later). It would be useful to assess the correlation between the 1627 cm −1 band and the 2855 cm −1 /2920 cm −1 bands in the CH2 stretching region (not shown) that are typically assigned to long alkyl chains in lipids. However, the large difference in wavelength between the two regions means that the diffraction-limited focal spots differ in size by a factor of two and effectively probe different regions of the sample, thus altering the significance of the correlations. An Amide I band in the proximity of 1620-1630 cm −1 is often assigned to proteins in the amyloid fold, with a β-sheet structure supported by extended intermolecular hydrogen bonding. This fold is characterized by a two-component Amide I band, with a stronger component around 1620-1630 cm −1 and a weaker one around 1660-1690 cm −1 [22]. In the 2DCOS Synchronous plot of Figure 3A, we could not see correlation peaks involving a band in this position, suggesting that this is not a protein in the amyloid fold. A weak band is seen at 1678 cm −1 in the difference spectra of Figure 2, but the lack of a synchronous correlation and appreciable asynchronous correlation suggests that the band is unrelated to the species that absorb at 1627 cm −1 and 1655 cm −1 .
The correlation between bands at 1627, 1515, and 1548 cm −1 suggests that the band may arise from a polypeptide. However, the intensity of the band at 1627 cm −1 , relative to the one at 1655 cm −1 , is surprising. After five days of exposure, the two bands became comparable, indicating that the number of amide bonds absorbing at 1627 cm −1 is of the same order of magnitude as that of all other cellular polypeptides. This would represent a large change in the conformation of cellular proteins associated to the cell or the extensive expression of new proteins. It would be surprising for any known individual intracellular protein to account for such intensity.
An alternative assignment that is consistent with the 2DCOS spectra of Figure 3 is that the band at 1627 cm −1 may arise from the hydrolysis of amide containing molecules. Such a reaction would lead to the formation of amine groups, with absorption in the proximity of 1620 cm −1 , and of a carboxylic acid group, which would absorb approximately in the 1550 cm −1 region when deprotonated and in the 1700 cm −1 region when protonated.
IR mapping of the 1627 cm −1 band throughout an SH-SY5Y cell is shown in Figure 4. Mapping confirms that the band arises from molecules located within the cell itself or on the cell surface, and A B An Amide I band in the proximity of 1620-1630 cm −1 is often assigned to proteins in the amyloid fold, with a β-sheet structure supported by extended intermolecular hydrogen bonding. This fold is characterized by a two-component Amide I band, with a stronger component around 1620-1630 cm −1 and a weaker one around 1660-1690 cm −1 [22]. In the 2DCOS Synchronous plot of Figure 3A, we could not see correlation peaks involving a band in this position, suggesting that this is not a protein in the amyloid fold. A weak band is seen at 1678 cm −1 in the difference spectra of Figure 2, but the lack of a synchronous correlation and appreciable asynchronous correlation suggests that the band is unrelated to the species that absorb at 1627 cm −1 and 1655 cm −1 .
The correlation between bands at 1627, 1515, and 1548 cm −1 suggests that the band may arise from a polypeptide. However, the intensity of the band at 1627 cm −1 , relative to the one at 1655 cm −1 , is surprising. After five days of exposure, the two bands became comparable, indicating that the number of amide bonds absorbing at 1627 cm −1 is of the same order of magnitude as that of all other cellular polypeptides. This would represent a large change in the conformation of cellular proteins associated to the cell or the extensive expression of new proteins. It would be surprising for any known individual intracellular protein to account for such intensity.
An alternative assignment that is consistent with the 2DCOS spectra of Figure 3 is that the band at 1627 cm −1 may arise from the hydrolysis of amide containing molecules. Such a reaction would lead to the formation of amine groups, with absorption in the proximity of 1620 cm −1 , and of a carboxylic acid group, which would absorb approximately in the 1550 cm −1 region when deprotonated and in the 1700 cm −1 region when protonated. IR mapping of the 1627 cm −1 band throughout an SH-SY5Y cell is shown in Figure 4. Mapping confirms that the band arises from molecules located within the cell itself or on the cell surface, and not from a deposit in its surroundings. The band is clearly absent in cells that were not exposed to metal salts. When present, it appears to track the distribution of the 1652 cm −1 band. Unfortunately, the diffraction-limited resolution of the IR maps, approximately 7 µm at these wavenumbers, does not allow us to associate the band to any specific subcellular structure. Nonetheless, IR maps exclude the possibility that the band arises from an extracellular deposit.
Molecules 2020, 25, x FOR PEER REVIEW 7 of 13 not from a deposit in its surroundings. The band is clearly absent in cells that were not exposed to metal salts. When present, it appears to track the distribution of the 1652 cm −1 band. Unfortunately, the diffraction-limited resolution of the IR maps, approximately 7 μm at these wavenumbers, does not allow us to associate the band to any specific subcellular structure. Nonetheless, IR maps exclude the possibility that the band arises from an extracellular deposit. Identifying proteins that could give rise to the observed conformational changes is of interest in understanding the biochemical origin of Pb 2+ and Sn 2+ toxicity. Pb 2+ is known to bind tightly to Ca 2+ and Zn 2+ binding proteins under physiologically relevant conditions, thereby disrupting their function [23]. Several Zn 2+ and Ca 2+ proteins such as zinc finger proteins and calmodulin are regulatory proteins [24]. However, due to their relatively low abundance, regulatory proteins cannot account for the intensity of the observed spectroscopic changes.
Shelton et al. [25,26] and Gonick [27,28] reported the induced expression of a protein that associates with lead in forming intranuclear inclusion bodies in neuroblastoma cells exposed to lead. They also reported the constitutive expression in neural cells of a protein leading to inclusion body formation, following chronic lead exposure. The identity of the protein has not been definitively established and may also correspond to more than one polypeptide. It has been proposed that the protein or proteins accomplish a protective function by immobilizing lead in an insoluble form and prevent its interaction with other cellular components. Such a mechanism would be consistent with the de novo synthesis of large amounts of protein and explain the presence and intensity of the band at 1627 cm −1 . The inclusion bodies formed by protein overexpression in engineered bacterial cells also display the IR spectrum of amyloids [29], in agreement with the hypothesis that inclusion bodies could be the origin of the spectral changes observed in our experiments. Despite this possibility, we Identifying proteins that could give rise to the observed conformational changes is of interest in understanding the biochemical origin of Pb 2+ and Sn 2+ toxicity. Pb 2+ is known to bind tightly to Ca 2+ and Zn 2+ binding proteins under physiologically relevant conditions, thereby disrupting their function [23]. Several Zn 2+ and Ca 2+ proteins such as zinc finger proteins and calmodulin are regulatory proteins [24]. However, due to their relatively low abundance, regulatory proteins cannot account for the intensity of the observed spectroscopic changes.
Shelton et al. [25,26] and Gonick [27,28] reported the induced expression of a protein that associates with lead in forming intranuclear inclusion bodies in neuroblastoma cells exposed to lead. They also reported the constitutive expression in neural cells of a protein leading to inclusion body formation, following chronic lead exposure. The identity of the protein has not been definitively established and may also correspond to more than one polypeptide. It has been proposed that the protein or proteins accomplish a protective function by immobilizing lead in an insoluble form and prevent its interaction with other cellular components. Such a mechanism would be consistent with the de novo synthesis of large amounts of protein and explain the presence and intensity of the band at 1627 cm −1 . The inclusion bodies formed by protein overexpression in engineered bacterial cells also display the IR spectrum of amyloids [29], in agreement with the hypothesis that inclusion bodies could be the origin of the spectral changes observed in our experiments. Despite this possibility, we failed to observe inclusion body formation in our samples using transmission electron microscopy (data not shown).
The appearance of a strong band around 1625 cm −1 has been reported for eukaryotic cells undergoing apoptosis [30] and attributed to the induction of a β-sheet structure in cellular proteins. The band was formed without the exogenous addition of heavy metal ions. If the same molecular species were responsible for the appearance of this band in our samples, then the causal relationship with heavy metal ions may only be indirect. Heavy metal ions would be responsible for triggering apoptosis by inducing generally stressful conditions. The subsequent formation of the 1627 cm −1 band would be associated with the apoptotic process itself via ex novo protein overexpression or via the refolding of existing proteins.
An interesting possibility is that proteins of the pentraxin structural family may be involved in the formation of the observed aggregated β-sheets. Some of them have been detected as a component of amyloid plaques [31] and have been reported to form intramolecular β-sheet structures absorbing at 1627 cm −1 . Pentraxin expression has not been studied in the cell lines used in our study, however, their known biological and biophysical properties make them candidates as components of the intermolecular β-sheet aggregates reported in our study.
One additional possibility is that the band may arise from albumin, which is known to refold into a β-sheet structure under the effect of temperature and metal ions [32]. Albumin is a component of the culture medium, making it one of the most abundant single proteins in a cell culture. It is possible that the refolded protein associates to the cell or the membrane via an unknown mechanism, giving rise to the observed changes. However, albumin is also present in the media used for untreated cells, although heavy metals in untreated samples are limited to the nutrients present in the medium and are present only in low concentration. A hitherto undefined mechanism that involves the heavy metals must mediate its association to or its uptake by the cell to fully explain our results. The appeal of this hypothesis is that, because of its abundance in the medium used for sample preparation, albumin can fully explain the intensity of the 1627 cm −1 absorption. 2DCOS analysis excludes the presence of amyloids, implying that a different secondary structure must be at the origin of this band if the aggregation of albumin is involved.
Poly-L-lysine displays a strong band at 1627 cm -1 from the absorption of the amine group in the lysine residue. The band overlaps with the absorption from the Amide I band of the polypeptide backbone when the latter is folded into a β-sheet structure at neutral or alkaline pH [33]. The superposition of these two contributions creates the appearance that the Amide I to Amide II ratio is unusually high. Poly-L-lysine was used as an adhesion layer for cells in the preparation of our samples. It is conceivable that the chelation of metal ions may be involved in its aggregation and association to the cell surface or internalization. This explanation would rationalize both the intensity of the band at 1627 cm −1 relative to the overall cellular absorption and to the 1540 cm −1 Amide II band, where a contribution from lysine absorption is absent.
In addition to the proteins just discussed above, additional species that satisfy the constraints resulting from the 2DCOS analysis include abundant amine and amide-containing molecules that can survive the washing steps involved in cellular preparation, mostly polysaccharides, but also any smaller molecules that may be tightly associated with cellular macromolecules or be involved in crosslinking during fixation. One final possibility is that part of the multiplets in the 2DCOS spectra results from the amines (approx.1600-1630 cm −1 ) and carboxylates (approx.1500-1580 cm −1 ) produced by the hydrolysis of amide containing molecules including polypeptides, sphingolipids, or polysaccharides. Systematic biochemical tests and analysis are required to restrict the pool of candidates and reach a final identification.
Cells were plated on calcium fluoride (CaF 2 ) optical windows transparent for IR light (Crystran, Poole, UK) at a density of 20,000 cells using poly-L-lysine as an adhesion layer. Before administration to cells, MAPbI 3 and MASnI 3 powders were suspended in culture medium (concentration of 100 µg/mL). Solutions were vortexed, left for five days at Room Temperature (RT), filtered with a 0.22 µm pore size syringe filter (Merck Millipore, Burlington, MA, USA) and stored at 4 • C until used. Cells were incubated for five days with the halide perovskite medium solutions, rinsed with warm PBS (1×), and fixed using increasing concentrations of ethanol (20%, 40%, 60%, and 80%). Fixed cells were rinsed three times with MilliQ H 2 O, left to dry overnight in a laminar flow hood, then stored in a desiccator for a maximum of five days before the IR measurements.
Toxicity Studies
In vitro toxicity studies were performed by exposing plated cells to filtered solutions of MAPbI 3 (100 µg/mL) or MASnI 3 (100 µg/mL), prepared as described in the previous section. Toxicity was assessed by counting viable cells in a Neubauer chamber at given time intervals. Only attached cells displaying a clear cytoplasm were counted as viable.
IR Microscopy Measurements
We performed Fourier transform infrared microspectroscopy (FTIRMS) measurements on beamline U2B at the National Synchrotron Light Source (NSLS, Brookhaven National Lab, Upton, NY, USA), and on beamline SMIS at the SOLEIL synchrotron (Paris, France). We used a Thermo Nicolet Continuum IR microscope set-up coupled to a Thermo Nicolet Magna 860 FTIR (Thermo Nicolet Instrument, Madison, WI, USA). We used a clean area of the CaF 2 optical window for background collection, then selected cells for IR measurements using the visible optical path of the microscope. Spectra were collected in transmission mode by closing the confocal apertures to obtain a diffraction-limited spot in the mid-IR spectral region, and raster scanning individual cells. Spectra were then used individually or assembled into 2D maps representing IR absorption throughout the cell. For detailed analysis, spectra were converted to the 2nd derivative of absorbance. The averaged spectrum of a single cell was obtained by discarding spectra for which the 2nd derivative of absorbance fell outside of the range |0.00002|−|0.0002| and averaging the remaining spectra. From one to three cells were mapped for each condition under study. To obtain the plots in Figure 4D, the spectra of 100-150 single cells were averaged for each time point. The ratio of the 2nd derivative absorption at 1652 cm −1 and 1627 cm −1 ± σ (variance) is shown in Figure 4D. IR data processing and analysis were performed using the software packages Omnic version 7.2 (Thermo Fischer, Waltham, MA, USA) and OPUS version 7.5 (Bruker Optics, Ettlingen, Germany). Spectral diagrams were created using OriginPro 2018 (Origin Lab Corporation, Northampton, MA, USA). 2DCOS analysis was performed using the software package MATLAB (MathWorks, Natick, MA, USA), as described previously. [20]
Conclusions
We used FTIRMS to study the effect of exposure to MAPbI 3 and MASnI 3 on SH-SYS5 and A549 cells at the molecular level. Our observations show that the cellular response to Sn 2+ ions and Pb 2+ ions leads to similar changes in the cellular IR absorption spectra. Exposure to both compounds leads to the appearance of a strong band at 1627 cm −1 accompanied by weaker absorption bands. Our results suggest that the band at 1627 cm −1 is associated with a general response to metal cation exposure, since it is displayed by unrelated cell lines and different metal ions. The present experiments do not allow us to clarify whether this is related to a specific protective mechanism or if it arises from a non-specific response, with only an indirect causal link to the presence of heavy metals. Cellular fixation chemistry may also be contributing to the observed spectroscopic response. It is also unclear whether the changes thus detected have a direct or inverse causal relationship to the underlying molecular events that lead to cellular toxicity.
As a novel methodological tool, we used 2DCOS analysis on spatially resolved spectra to corroborate or disprove possible assignments of this band. The analysis allowed us to rule out the accumulation of polypeptides in the amyloid β-sheet fold as a possible assignment, despite the similarity of peak position with the one characteristic for the Amide I band of these structures. The same analysis also ruled out that the band may arise from acyl lipids or from polysaccharides containing both amide and ester groups.
Remaining hypotheses include the cellular accumulation of amine or amide containing molecules including some polysaccharides and smaller molecules resistant to fixation, and the metal-driven refolding and uptake of poly-L-lysine and/or albumin used in sample preparation. The aggregation and refolding of cellular proteins is not completely ruled out, although it looks unlikely in light of the extent of the changes and the need to involve a major fraction of cellular proteins. Future research involving a much larger dataset and biochemical tests will be directed to confirm or disprove these hypotheses.
While the identity of the major product of exposure, absorbing at 1627 cm −1 , has not yet been defined, excluding its assignment to an amyloid deposit is an important conclusion. The current literature on the IR microscopy of cells and tissue conventionally uses an absorption band in this position to identify the presence of amyloid deposits. We now show that such an assignment is unreliable when based only on such band and in the absence of additional biochemical characterization. Multiple alternative assignments are possible, corresponding to very different biochemical interpretations.
From the methodological point of view, these results are of interest beyond the specific biochemical issue. They provide preliminary corroboration of the usefulness of 2DCOS in simplifying the interpretation of complex biological spectra when applied to stationary samples such as fixed cells and tissue. This application of 2DCOS has to date been restricted to samples undergoing dynamic changes such as living cells. However, fixed or dried biological systems comprise most of the samples that are currently analyzed by FTIRMS, thus greatly expanding the scope of the method. Figure A1. These show a generally decreasing survival rate for both cell lines when exposed to either perovskite. Toxicity effects appear more marked for the SH-SY5Y cell line, which appear to shrink and detach following exposure. In contrast, A549 cells, while showing reduced mortality, also display morphological changes such as enlarged dimensions and multiple nuclei, and alterations of the cell cycle following exposure to both MAPbI 3 and MASnI 3 . The different response of the two cell lines has already been reported when investigating the toxicity of MAPbI 3 [2] and is now confirmed for MASnI 3 .
Molecules 2020, 25, x FOR PEER REVIEW 11 of 13 the SH-SY5Y cell line, which appear to shrink and detach following exposure. In contrast, A549 cells, while showing reduced mortality, also display morphological changes such as enlarged dimensions and multiple nuclei, and alterations of the cell cycle following exposure to both MAPbI3 and MASnI3. The different response of the two cell lines has already been reported when investigating the toxicity of MAPbI3 [2] and is now confirmed for MASnI3. Bars are means ± σ. One-way ANOVA tests followed by Tukey-Kramer post-hoc tests were performed (non-treated vs. MAPbI3 or vs. MASnI3 treated conditions), * p < 0.01, ** p < 0.005, *** p < 0.0005. Panel b is reproduced from [2] with permission from the Royal Society of Chemistry. Panels (a-d) are reproduced from [6] (I.B. doctoral thesis). Bars are means ± σ. The histograms show an average of at least three independent repeats. Bars are means ± σ. One-way ANOVA tests followed by Tukey-Kramer post-hoc tests were performed (non-treated vs. MAPbI3 or vs. MASnI3 treated conditions), * p < 0.01, ** p < 0.005, *** p < 0.0005. Panel b is reproduced from [2] with permission from the Royal Society of Chemistry. Panels (a-d) are reproduced from [6] (I.B. doctoral thesis). | v3-fos-license |
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} | pes2o/s2orc | Arrhythmogenic cardiomyopathy related DSG2 mutations affect desmosomal cadherin binding kinetics
Cadherins are calcium dependent adhesion proteins that establish the intercellular mechanical contact by bridging the gap to adjacent cells. Desmoglein-2 (Dsg2) is a specific cadherin of the cell-cell contact in cardiac desmosomes. Mutations in the DSG2-gene are regarded to cause arrhythmogenic (right ventricular) cardiomyopathy (ARVC) which is a rare but severe heart muscle disease. The molecular pathomechanisms of the vast majority of DSG2 mutations, however, are unknown. Here, we investigated the homophilic binding of wildtype Dsg2 and two mutations which are associated with ARVC. Using single molecule force spectroscopy and applying Jarzynski’s equality we determined the kinetics and thermodynamics of Dsg2 homophilic binding. Notably, the free energy landscape of Dsg2 dimerization exposes a high activation barrier which is in line with the proposed strand-swapping binding motif. Although the binding motif is not directly affected by the mutations the binding kinetics differ significantly from the wildtype. Furthermore, we applied a dispase based cell dissociation assay using HT1080 cell lines over expressing Dsg2 wildtype and mutants, respectively. Our molecular and cellular results consistently demonstrate that Dsg2 mutations can heavily affect homophilic Dsg2 interactions. Furthermore, the full thermodynamic and kinetic description of Dsg2 dimerization provides a consistent model of the so far discussed homophilic cadherin binding.
Dsg2 is a major cadherin of the cardiac desmosome and the only desmoglein expressed in cardiomyocytes. Variances in the DSG2 gene are associated with severe heart muscle diseases such as ARVC which is characterized by a progressive loss of cardiomyocytes and a fibrofatty tissue replacement predominantly in the right ventricle 13 . In this study, we focussed on the homophilic binding properties of Dsg2 wildtype and two ARVC-related mutations at the single molecule level and in a cell dissociation assay. By means of atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) we revealed a comprehensive kinetic and thermodynamic description of Dsg2 wildtype and mutant dimerization, respect\ively. Our results confirm a highly specific but low affinity binding which is affected by the mutations. Moreover the activation energy barrier ∆ ‡ G restricts a fast binding kinetic which is in full accordance with the competitive inhibition of the strand exchange binding motif. Along with the results of the cell dissociation assay both approaches merge into a detailed picture of Dsg2 dimerization kinetics and its influence on cell network stability. These data may contribute to the understanding of DSG2-related cardiomyopathy mutations.
Results
Dynamic force spectroscopy. Recombinant Dsg2 extracellular cadherin domains 1-4 (EC1-4) were covalently immobilized to AFM-tip and substrate in order to investigate the homophilic binding by means of AFM-SMFS (Fig. 1B). More than 10,000 force distance cycles were recorded at several constant pulling velocities v. Dissociation forces F and effective molecular elasticities k eff were extracted from the force distance profiles (Fig. 1C) whose characteristic distributions yield the most probable dissociation force ⁎ F and effective molecular elasticity ⁎ k eff , respectively. According to the model of Evans and Ritchie we estimated the dissociation rate constant − k 0 and the reaction length ‡ x which is the position of the activation barrier relative to the bound state, by fitting equation 2 to ⁎ F and the corresponding loading rates = ⁎ r k v eff ( Fig. 2A and Table 1). Notably, all experimental series exposed rather small dissociation forces (<40 pN) over the full range of pulling speeds (200 nm s −1 to 5000 nm s −1 ). Especially at small velocities the dissociation forces can be close to the noise level (approx. 4 pN). Therefore, the experiments demand thorough conduction, a careful data analysis and error handling. Compared to the Dsg2 wildtype which yields a bond lifetime of approx. τ = .
3 39 s and 1.57 s, respectively. Furthermore, the mutations also reveal extended reaction lengths. Within the estimated error, complex lifetimes and reaction lengths of Dsg2 wildtype and p.D105E (p.D154E) differ significantly. The fitting errors of the binding parameters estimated for p.V343I (p.V392I) overlap at least partially with the error intervals of the two other Dsg2 species ( Fig. 2A inset). Moreover, the estimation of − k 0 and ‡ x cannot be done independently. Therefore, the binding properties of p.V343I (p.V392I) apparently differentiate less from Dsg2 wildtype.
In order to prove the specificity of the homophilic Dsg2 interaction and to rule out unspecific adhesion we conducted a series of control experiments. AFM force distance cycles were recorded in calcium-buffer and calcium-free EDTA-buffer as well as on gold substrates lacking Dsg2. Our data revealed that Dsg2 dimerization critically depends on the calcium concentration (Fig. 3A). We observed a binding frequency of 32% at a Ca 2+ concentration of 2 mM, whereas in EDTA-buffer the binding probability dropped to 9%. Furthermore, the dissociation force probability distributions of experiments conducted in EDTA-buffer yield a broad shape and multiple shallow peaks that can be attributed to multifold unspecific adhesion events. Furthermore, the binding frequency on substrates lacking Dsg2 drops significantly to 4% (EDTA-buffer) and 2% (calcium buffer) proving that the background of unspecific adhesion on substrates lacking Dsg2 was minute and therefore does not affect the results. Dsg2 ‡ modified AFM cantilever and sample substrate in 2 mM calcium-buffer (green) and EDTA-buffer (orange) expose a clear dependency of the binding frequency on the calcium concentration. The background of unspecific adhesion was tested in an experiment conducted on substrates lacking Dsg2 in calcium (red) and EDTA-buffer (gray). (B) Evolution of the Jarzynski equality. The exponentially averaged work W n plotted versus the number of dissociation events n. For wildtype Dsg2 and mutations thereof (green, red and yellow) measured in a 2 mM Ca 2+ buffer-solution the Jarzynski equality converges fast yielding a robust estimate of the free energy difference ∆G 0 . The evolution of ∆G 0 for wildtype Dsg2 (blue) in calcium-free EDTA-buffer solution does not converge for < n 2000.
SCIENtIfIC RepoRts | 7: 13791 | DOI:10.1038/s41598-017-13737-x Reconstruction of energy landscapes. Jarzynski's equality (Eq. 3) allows to relate mechanical work W measured under non-equilibrium conditions with the free energy difference ∆G 0 in thermodynamic equilibrium 14 . Here, ∆G 0 represents the free energy difference between the bound and unbound state of Dsg2 15 . We determined the work W n that was dissipated to break n individual Dsg2 dimers by calculating the area under the corresponding force curves (Fig. 1C). Subsequently, the data set W n was averaged exponentially using Jarzynski's equality. This term converges rapidly for all Dsg2 species providing a robust estimate of ∆G 0 (Fig. 3B). Notably, Dsg2 wildtype and mutants yield almost identical binding energies of approx. 18(1) kJmol −1 (Table 1) coinciding with a preserved binding motif. Moreover, we used Arrhenius' equation (Eq. 5) to estimate the activation energy ∆ + − ‡ G / of the association and dissociation path, respectively. Dsg2 wildtype revealed ∆ ≈ − ‡ G 69 kJ mol −1 whereas the data for the Dsg2 mutants p.D105E (p.D154E) and p.V343I (p.V392I) yielded a slightly higher energy of approx. 75 kJ mol −1 and 73 kJ mol −1 , respectively ( Table 1). The equilibrium constant of dissociation K d was estimated as 412 μM for the wildtype Dsg2 which proves the expected low affinity of Dsg2 dimerization. The mutants p.D105E (p.D154E) and p.V343I (p.V392I) yield an even smaller affinity of 817 kJ mol −1 and 1008 kJ mol −1 , respectively. Furthermore, the association rate constants + k 0 were estimated according to equation 4 (Table 1). In total, an approximation of the free energy landscape (Fig. 2B) together with the binding kinetics provide a detailed picture of the homophilic interaction of Dsg2.
p 0 001) of the monolayer, suggesting a stronger cellular interaction. Of note, the number of fragments in the dispase assay of the mutant p.V343I (p.V392I) was not significantly different from wildtype Dsg2 (18.0(123); Fig. 4).
Discussion
In this study we revealed a detailed picture of homophilic Dsg2 interaction kinetics and thermodynamics of the Dsg2 wildtype and arrhythmogenic cardiomyopathy related mutant protein. By means of AFM-SMFS we estimated the binding properties of Dsg2. The W2 (W51) residue and the associated hydrophobic pocket which are critical for strand swapping are highly conserved throughout desmosomal and classical cadherins 16 . Therefore, kinetic and thermodynamic binding parameters estimated for Dsg2 are expected in the range of other strand swapping cadherins. For the Dsg2 wildtype homo-complexes we found an average lifetime of 0.33s. Similarly, recent reports on the dimerization of Dsg1 and C-cadherins found complex lifetimes of 0.17s and 0.91s, respectively 9,11 . Moreover, the equilibrium constant of dissociation was estimated as = K 412 d μM which is in perfect agreement with = K 433 d μM obtained by Harrison and co-workers in an sedimentation equilibrium analytical ultracentrifugation assay 12 . In a solution NMR approach E-cadherins exhibited an equilibrium dissociation constant of = K 720 d μM 17 . Notably, Dsg2 association is apparently inhibited what can be seen by the association rate constant of = + k 7270 0 M −1 s −1 which is orders of magnitude smaller than a diffusion limited association 18,19 . Accordingly, we could determine a height of the activation energy barrier of association (∆ = + ‡ G 50 kJ mol −1 , eqs 5 and 6) which is significantly larger than the difference in free energy ∆G 0 (Table 1). This constraint binding reaction is most likely due to the competitive inhibition of the N-terminal strand exchange 6 . Our experiments furthermore demonstrate that the Dsg2 dimerization probability critically depends on the Ca 2+ concentration which is in full agreement with data published elsewhere 8,9,11,[20][21][22][23] (Fig. 3A). Like classical cadherins, Dsg2 comprises three p 0 001 versus wildtype) and p.V343I (p.V392I). Box and whiskers plot: boxes extend from the 25th-75th, whiskers from the 5th-95th percentiles, the medians are shown as lines; outliers are given as dots. Means are indicated as crosses.
calcium coordination sites between each EC domain except for the linker region between EC3 and EC4 which comprises only two binding sites 12 . Interdomain calcium binding straighten the Dsg2 ectodomains to their physiological conformation which is prerequisite to span the intercellular gap 3 . The incomplete calcium coordination between EC3 and EC4 however, might be responsible for the sharp bend found in crystal structures 12 . Moreover, calcium coordination between EC1 and EC2 provides conformational strain that releases the N-terminal W2 (W51) residue from its hydrophobic binding pocket and increases its diffusional mobility. This transformation to an open momomer is prerequisite for mutual strand exchange between Dsg2 monomers 24,25 . Still, dissociation force probability distributions obtained in Ca 2+ -free EDTA-buffer support these findings. The binding frequency drops and the force distribution exposes a rather broad shape and multiple shallow peaks which suggests that in absence of Ca 2+ Dsg2 dimerizes in an unspecific manner. Finally, the Jarzynski sum of exponentially weighted adhesion energies W n does not converge which also can be attributed to an unspecific interaction within EDTA-buffer solution (3B).
In a comparative analysis of Dsg2 wildtype and variants we generally observed prolonged bond lifetimes and increased activation barriers for the Dsg2 mutants (Table 1), which results in a significantly reduced affinity of , respectively. Even though Dsg2 affinity and kinetics are affected by the mutations, the binding free energy is comparable to the wildtype coinciding with a preserved strand swapping binding motif. Interestingly, Dsg2 p.D105E (p.D154E) bears a mutation at a Ca 2+ binding site within the first interdomain linker region 3 . Recently published molecular dynamics simulations for E-cadherins revealed that calcium binding between EC1 and EC2 increases the mobility of the N-terminal strand supporting dimerization by strand swapping 24 . Therefore, we assume that an impaired Ca 2+ coordination of Dsg2 p.D105E (p.D154E) leads to a deficient release or mobility of the N-terminal strand. As a consequence the strand exchange binding is evidently inhibited, which slows down Dsg2 dimerization. We tested in parallel the cellular effects of the DSG2 mutations by a more complex dispase assay. This assay is a measure for the strength of the cellular interaction, which is based on the dissociation of a cell culture monolayer. The number of fragments resulting from the dispase assay was significantly lower when transfected with the cDNA for Dsg2 p.D105E (p.D154E), but not for p.V343I (p.V392I) in comparison to wildtype Dsg2. Thus, these experiments suggest that the resulting cell-cell contacts were indeed stronger for the mutant Dsg2 p.D105E (p.D154E), which is in agreement with the AFM-SMFS data on the highly increased lifetime of this mutant (τ = .
3 39 s) in comparison to the wildtype (τ = . 0 33 s). Nevertheless, due to the retarded association + k 0 of Dsg2 p.D105E (p.D154E), these homocomplexes expose an increased equilibrium constant of dissociation K d (Table 1) indicating a smaller number of intact bonds at a time. This result evidently disagrees with the increased adhesion observed for p.D105E (p.D154E) cellular monolayers. However, this at the first sight contradictory behavior can be consistently explained when regarding the conditions within the cell-cell contact. Desmosomes are highly ordered adhesive interfaces with a cadherin density of 17,500 μm −2 in case of epidermal desmosomes leaving a single cadherin a cell surface area of less than × 8 8 nm 2 2,3 . This compact arrangement evidently leads to a spatial confinement within the desmosome which constrains diffusion of unbound cadherin ectodomains. Moreover, Dsg2 p.D105E (p.D154E) exhibits the longest reaction length of all tested Dsg2 variants ( ≈ . ‡ x 1 1 nm; Table 1) which can be interpreted as a larger reach of the potential molecular interaction promoting fast (re-) association. Opposing Dsg2 p.D105E (p.D154E) monomers can therefore effectively bind at larger intermolecular distances (Fig. 2B) resulting in an apparently enhanced association rate. As a consequence, association rate constants estimated in solution or single molecule assays do not apply for membrane bound cadherins within dense desmosomes. Instead, the spatial restriction and an increased interaction length most likely force fast (re-) association of Dsg2 p.D105E (p.D154E) homocomplexes and an increased cell network stability. However, although we also identified aberrant binding parameters for the mutation p.V343I (p.V392I) (τ = . 1 73 s) we could not find a significant difference from the wildtype Dsg2 in the cellular assay. Of note, the dispase assay is not restricted to the homophilic interaction of Dsg2, but also reflects potential interactions with other cell-cell contact proteins like i.e. the desmosomal cadherin desmocollin 2, which is also a binding partner of Dsg2 and coexpressed in HT1080 cells. As a consequence, the homophilic molecular interaction determined by AFM-SMFS reflects only one potential molecular interaction within the desmosome. Thus, although we found a difference for the binding parameters of this mutant, these molecular properties of p.V343I (p.V392I) do not converge in the disturbance of the cellular interaction in vitro. Both mutations are listed as variants of unknown significance (VUS; http://www.arvcdatabase.info), but solid calculations of the disease penetrance and differences in the clinical phenotypes are not available. Data from our study do not support a pathogenic dominant role of the mutation p.V343I (p.V392I). We speculate that our in vitro data on the homophilic interaction might indicate that this particular mutation may be a modifier in vivo. However, these in vitro effects for the homophilic Dsg2 and cell-cell interaction in this study might be not directly transferrable to the in vivo situation and should therefore be carefully interpreted in the clinical context. In summary, the mutant Dsg2 analyzed in vitro do not result in a weaker binding energy, but in an increased binding lifetime and aberrant association and dissociation parameters. This indicates that especially the protein integration and degradation of the desmosomal cadherins in the desmosome might be affected in cardiomyopathy related DSG2 mutations.
Nomenclature of aminoacids and mutations.
Medical and biophysical publications commonly use disparate nomenclature of aminoacids and mutations. In biophysical context it is good practice to identify specific aminoacids based on the position in the mature protein whereas in medical publications and databases they are addressed on the basis of the open reading frame (ORF). In order to avoid confusion and for a comprehensive reading of this paper we used the nomenclature of the aminoacid position based on the position in the mature protein and in parallel the numbering within the ORF which is given in brackets.
Recombinant expression of Dsg2. The extracellular regions EC1-EC4 of Dsg2 were stably transfected in HT1080 cells as previously reported 26 . Expressed Dsg2 was purified from the cell culture supernatant by affinity chromatography on HisTrap excel columns (GE Healthcare, Solingen, Germany), according to the manufacturer's instructions. The protein solutions were concentrated with Amicon Ultra-15 Centrifugal Filter Units (10,000 MWCO, MerckMillipore, Darmstadt, Germany). Proteins were identified by Western blotting and analyzed by means of SDS-Page with Coomassie-brilliant-blue staining, revealing a purity of at least 90%. The concentration of Dsg2 varied between 0.8 gL −1 1.5 gL −1 .
Functionalization of AFM tips and substrates. Glas substrates were metallized by physical vapor deposition (PVD) with a 3 nm layer of chrome followed by 120 nm gold using a commercially available PVD system (MED020 Coating System, Leica Microsystems, Wetzlar, Germany). Soft gold coated silicon nitride cantilevers (BL-RC150VB, Olympus, Tokio, Japan) with a nominal spring constant of 30 pNnm −1 were cleaned with acetone and ethanol. Both, metallized glass substrates and cantilevers were coated with a self-assembled monolayer (SAM), incubated in an ethanolic solution of 1 mM 6-amino-1-hexanethiol-HCl (Sigma-Aldrich, Saint Louis, MO, USA) for 1.5 h and washed subsequently with ethanol. Cantilevers and substrates were incubated in a 100 μM water-free dimethyl sulfoxide (DMSO) solution of different hetero-bifunctional polyethylene glycol (PEG) cross-linkers for 1.5 hours at room temperature. The force probes were functionalized with the maleimide-PEG-succinimidyl valerate 3,400 cross-linker (Laysan Bio Inc., Arab, AL, USA), whereas the gold coated glass substrates were modified with NHS 3-maleimideopropionate (Celares, Berlin, Germany). Notably, both cross-liners bear identical functional groups. However, the linker used to couple Dsg2 to the cantilevers is significantly longer (approx. 25 nm). Apart from providing a covalent link to the cantilever the linker adds steric elasticity to the molecular system and it ensures that dissociation events occur far from the surface. Both is required to align the force extension curves and to discriminate between specific and unspecific binding events. Finally, AFM probes and substrates were incubated in 20 μL Dsg2 solution without further dilution at 5 °C until measurement. The free maleimide of the cross-linkers binds to the C-terminal thiol side chain of a unique cystein C447 (C496) within the recombinant Dsg2 fragment. Both, cantilevers and substrates could be used without loss of activity for up to four days. DFS experiments. SMFS was performed with a commercially available Multimode 8 AFM system (Bruker, Santa Barbara, CA, USA). Before each experimental series the cantilevers were calibrated by the thermal fluctuation method 27 . All force measurements were conducted in calcium and EDTA buffer, respectively (2 mM CaCl2, 10 mM HEPES, 150 mM NaCl, pH 7.4 and 5 mM EDTA, 1 mM EGTA, 10 mM HEPES, 150 mM NaCl, pH 7.4). Prior to buffer changes substrates and cantilevers were extensively rinsed with washing buffer (10 mM HEPES, 150 mM NaCl, pH 7.4). Up to 20,000 force distance cycles were performed for each pulling velocity ranging from 200 nm s −1 to 5,000 nm s −1 . The characteristic non-linear force ramps were individually analyzed within the theoretical framework of the worm like chain model (WLC) to rule out multiple saw tooth-shaped rupture events. Furthermore, rupture events with a dissociation length larger than 60 nm were discarded as the whole molecular complex (linkers and Dsg2 dimer) exhibits a length of approx. 60 nm. Detected dissociation forces were accepted for analysis when they exceeded the noise level (here approx. 4 pN) by a factor of three. The most probable dissociation force ⁎ F was determined for each pulling velocity using a kernel density estimation (KDE) 28,29 . The corresponding probability density function P F ( ) n was determined as: Here, F i and F i RMS denote the i-th element in a 1 by n array of dissociation forces and the root mean square (RMS) noise of the force, respectively. The most probable molecular elasticity was calculated likewise and multiplied by the corresponding pulling velocity to yield the effective loading rate = ⁎ r k v eff . In order to estimate the dissociation rate constant in thermodynamic equilibrium under zero force − k 0 and the reaction length ‡ x we applied the model of Evans and Ritchie 30 Estimate free energy difference using Jarzynski's equality. When estimating thermodynamic binding parameters such as the free energy difference ∆G 0 between bound and unbound state or the dissociation constant K d from AFM-SMFS data it is good practice to assume a diffusion limited association rate constant + k 0 31-33 . However, this means does not apply for systems in which free diffusion is possibly restricted. Instead, Jarzyski's non-equilibrium work theorem allows calculating ∆G 0 directly from a series of non-equlibrium experiments no matter how far from equilibrium the system was probed 14,15 . To estimate ∆G 0 we used Jarzynski's equality: Here, W n is the work that is dissipated to break an individual bond ( < W 0 n ). A sufficiently large set of work measurements ( → ∞ n ) obtained far from equilibrium, exponentially averaged, converge towards the free energy difference in thermodynamic equilibrium ∆G 0 and therefore give an accurate estimate. We used a custom-made MATLAB software to validate and analyze the characteristic nonlinear force ramps that mainly result from the stretching of the PEG cross-linker. In brief, the force profiles are corrected for the molecular extension and possible position and deflection offsets. A worm-like chain fit is applied to the data yielding the contour length of the molecular complex. We estimated the overall length of a Dsg2 dimer ( . 31 5 nm) from crystal structure experiments whereas the length of the PEG cross-linker is well-known (approx. 25 nm) [ 12,34 , pdbID: 5ERD]. Filtering the dataset for a distinct contour length (here, . 56 5 nm) results in a set of well-aligned force profiles. Subsequently, we estimated a set of W n by determining the area under the force profiles 14,15 . Finally, W n is weighed by applying the Jarzynski equality and solved for ∆G 0 . The equilibrium constant K d and the association rate constant + k 0 in thermodynamic equilibrium are calculaded as: In order to estimate the activation energy of the dissociation path ∆ − ‡ G we used the Arrhenius equation [35][36][37] : where N A is the Avogadro constant and ν = = × − − 6 10 s is the attempt frequency for the crossing of the transition state. Accordingly, the activation energy of association is estimated as: Cell-cell adhesion assay. Cell-cell adhesion was investigated using a dispase-based cell dissociation assay 38 .
Stably transfected full-length-DSG2 (wildtype and mutants p.D105E (p.D154E) and p.V343I (p.V392I))-pEYFP/ DSC2b-pLPCX HT1080 cells were seeded in 12-well plates and grown to confluence in the presence of 2 mM CaCl 2 . After two washes with phosphate-buffered saline, the adherent cells were incubated at 37 °C for 20 minutes with 0.3 ml dispase II (2.4 U/ml, Sigma Aldrich, Darmstadt, Germany) resulting in a non-adherent cell monolayer. Released monolayers were rotated on an orbital shaker (150 rpm) for 20 minutes. Finally, cell dissociation was calculated by counting monolayer fragments. Every experimental condition was performed in quadruplicate and repeated five times. Values are given as means ± SD; statistical analysis was performed with one-way ANOVA and Dunnett's Multiple Comparison Test using GraphPad Prism (GraphPad Software, La Jolla, CA, USA).
Conclusion
Using AFM-SMFS we estimated the dimerization kinetics and thermodynamics of Dsg2 wildtype and two mutations. Furthermore, we tested the impact of these mutations on the stability of cellular networks by means of a dispase based cell-cell adhesion assay. In combination, our results provide a fascinating glimpse into the complex molecular interplay within the cardiac desmosome. Briefly, we found that Dsg2 wildtype and both tested mutations differed with respect to their binding kinetics. Dsg2 wildtype exhibited the smallest complex lifetime (τ = . 0 33 s) whereas p.D105E (p.D154E) and p.V343I (p.V392I) exhibited considerably increased lifetimes of τ = .
1 73 s, respectively. Estimating the difference in free energy ∆G 0 by means of Jarzynski's equality allowed us to estimate the equilibrium constant of dissociation K d and the association rate + k 0 . Interestingly, Dsg2 wildtype homodimers exhibited the highest affinity ( µ = K 412 M d ) among the analyzed Dsg2 species. As a consequence the association rate of Dsg2 wildtype is one order of magnitude higher compared to both mutations. Despite the fact that Dsg2 wildtype exhibits the highest affinity cellular networks bearing the very same cadherin should expose higher resistance against tensile stress than p.D105E (p.D154E) and p.V343I (p.V392I). Nevertheless, cell cluster stability was increased for p.D105E (p.D154E) which contradicts to our single molecule results.Taking into account spatial restrictions within the dense highly ordered desmosome, we can conclude, that due to limited diffusion unbound cadherin ectodomains can (re-) associate considerably faster leading to an increased cluster stability as observed for p.D105E (p.D154E). The stability of Dsg2 homocomplexes within the desmosome is apparently governed by their bond life times as varying association rates in solution are compensated by confined diffusion and dense configuration within the intercellular gap. Evidently, the integrity of the cardiac muscle is based on the weak but cooperative binding between cadherin ectodomains. The fast binding kinetics of Dsg2 wildtype raises the question of the physiological reason. Our results imply that the cardiac desmosome can be regarded as a tight but highly dynamic connection between individual heart muscle cells. This well-balanced molecular interaction suggests that the cardiac desmosome is able to continuously tune its configuration to the alternating contractions of the heart muscle. | v3-fos-license |
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} | pes2o/s2orc | Unraveling the Early Events of Amyloid-β Protein (Aβ) Aggregation: Techniques for the Determination of Aβ Aggregate Size
The aggregation of proteins into insoluble amyloid fibrils coincides with the onset of numerous diseases. An array of techniques is available to study the different stages of the amyloid aggregation process. Recently, emphasis has been placed upon the analysis of oligomeric amyloid species, which have been hypothesized to play a key role in disease progression. This paper reviews techniques utilized to study aggregation of the amyloid-β protein (Aβ) associated with Alzheimer’s disease. In particular, the review focuses on techniques that provide information about the size or quantity of oligomeric Aβ species formed during the early stages of aggregation, including native-PAGE, SDS-PAGE, Western blotting, capillary electrophoresis, mass spectrometry, fluorescence correlation spectroscopy, light scattering, size exclusion chromatography, centrifugation, enzyme-linked immunosorbent assay, and dot blotting.
Introduction
Protein aggregation leads to the formation of insoluble fibrous aggregates, termed amyloids, which are commonly associated with disease. However, understanding of the mechanism by which proteins aggregate has remained elusive. Although larger aggregates, including fibrils, remain important for clinical determination [1,2], small oligomeric aggregates are of interest due to their potentially toxic nature and hypothesized role in disease progression. However, the study of oligomers is complex due to the fact that these early aggregates are highly unstable, present at low concentrations, and difficult to isolate.
Among the diseases to which amyloids contribute are Alzheimer's disease (AD), Parkinson's disease, prion diseases, Type II diabetes mellitus, Huntington's disease, as well as many others [3]. The clinical presentation of each amyloid disease is very different, yet the presence of amyloid fibrils is a common characteristic of each disease. These amyloid fibrils exhibit a cross β-sheet structure in which the β-strands are oriented perpendicular to and hydrogen bonding is oriented parallel to the long axis of the fibril [4][5][6][7][8][9]. In addition, it has been shown that the amyloidogenic proteins amyloid-β (Aβ), α-synuclein, huntingtin, prion, and islet amyloid polypeptide (IAPP) form structurally similar soluble oligomeric species, which share an epitope recognized by oligomer-specific antibodies [10,11]. The commonalities shared by each amyloid disease protein suggest that studying the aggregation of one amyloid protein could provide insight into the general aggregation mechanism of other amyloid proteins.
AD is the most common cause of dementia and the most prevalent neurodegenerative disorder [12,13]. The neurodegenerative effects of AD are hypothesized to arise from Aβ, a partially folded protein that aggregates during the disease process. Aβ was first identified by Masters et al. as the aggregated protein [14] deposited within plaque cores found in AD brain. In its monomeric form, this protein may be harmless [15]. However, Aβ monomer can self-assemble via a nucleation-dependent pathway into Aβ oligomers, larger Aβ aggregation intermediates, and eventually the fibrillar aggregates that deposit in the brain ( Figure 1) [5,[16][17][18]. Steps within the Aβ aggregation pathway are reversible, such that deposited fibrils could give rise to soluble oligomers and intermediates. Soluble aggregate species that appear between monomer and insoluble fibrils have been termed within the literature as oligomers [19], micelles [20], amyloid-derived diffusible ligands (ADDLs) [21,22], βamy balls [23], amylospheroids (ASPDs) [24], and protofibrils [25,26], and the aggregate sizes associated with these definitions overlap in range. Smaller species are most commonly referred to as oligomers, including both low molecular weight and high molecular weight species, while larger intermediates are often referred to as protofibrils. Controversy exists concerning the exact size of the nucleus formed within the rate-limiting step of the aggregation pathway; however, most reports speculate that the nucleus is oligomeric in nature [27][28][29]. In addition to oligomers formed along the aggregation pathway, off pathway oligomers and higher order assemblies, which fail to give rise to an organized fibril structure, have also been identified [29,30]. weight oligomeric species that can give rise to either off undetermined size. Nuclei, which arise within the rate pathway, will increase in size to form hig aggregation intermediates, and finally the fibrillar aggregates that deposit in AD brain to yield amyloid plaques.
Aβ proteins comprised of either 40 or components found in amyloid plaq aggregates, while Aβ 1-40 is more soluble and is the main circulating form in normal plasma and cerebrospinal fluid (CSF) [32]. neurodegeneration, but it is theorized fibrils, may be responsible for the cellular supported by experimental observations synthetic Aβ 1-40 and Aβ can induce in vivo where Aβ dodecamers (Aβ shown to induce memory deficits drastically inhibit hippocampal long models show a poor correlation between the levels of insoluble A It is now more widely accepted that to synapse loss, correlate most accurately with the stage of neurological impairment However, the progression from monomer understood. Therefore, it is important to develop a Aβ aggregation process. A range of techniques are available to study the These techniques fall into three main categories: monomeric and oligomeric Aβ siz oligomeric Aβ species; (3) Methods imminent need to understand oligomerization events, t first and second categories, which give in aggregation. Accumulating evidence suggests aggregation process. Aβ monomer self-assembles into low molecular weight oligomeric species that can give rise to either off-pathway oligomers or nuclei of an Nuclei, which arise within the rate-limiting step of the A pathway, will increase in size to form high molecular weight oligomers, soluble aggregation intermediates, and finally the fibrillar aggregates that deposit in AD brain to either 40 or 42 amino acids, termed Aβ 1-40 and A components found in amyloid plaques [31]. Aβ has implications for the formation of initial is more soluble and is the main circulating form in normal plasma and Controversy currently exists over the direct effect A theorized that soluble aggregates of Aβ, rather than monomers or inso the cellular pathology associated with AD [33supported by experimental observations in vitro which show that soluble aggregates formed by induce cellular dysfunction and toxicity in cultured cells (Aβ*56) have been isolated from the brains of transgenic mice shown to induce memory deficits [38]. In addition, soluble Aβ aggregates generated drastically inhibit hippocampal long-term potentiation in rats [39]. Furthermore models show a poor correlation between the levels of insoluble Aβ fibrils and disease severity It is now more widely accepted that soluble Aβ oligomers impair cognitive function and to synapse loss, correlate most accurately with the stage of neurological impairment However, the progression from monomer to oligomer to insoluble Aβ aggregates is not w understood. Therefore, it is important to develop an analytical tool that is suitable for analysis of available to study the different stages of the Aβ These techniques fall into three main categories: (1) Methods for the quantitative detection of zes; (2) Methods for the qualitative detection and characte Methods for the qualitative detection of Aβ fibrils. imminent need to understand oligomerization events, the focus of this review is which give information about Aβ oligomeric species formed during Accumulating evidence suggests that these Aβ oligomeric species play a assembles into low molecular pathway oligomers or nuclei of an limiting step of the Aβ aggregation h molecular weight oligomers, soluble aggregation intermediates, and finally the fibrillar aggregates that deposit in AD brain to and Aβ 1-42 , are the major the formation of initial is more soluble and is the main circulating form in normal plasma and xists over the direct effect Aβ has on rather than monomers or insoluble -35]. This hypothesis is which show that soluble aggregates formed by toxicity in cultured cells [21,36,37] and have been isolated from the brains of transgenic mice and aggregates generated in cell culture Furthermore, data from mouse fibrils and disease severity [40]. oligomers impair cognitive function and, in addition to synapse loss, correlate most accurately with the stage of neurological impairment [11,[41][42][43].
aggregates is not well is suitable for analysis of the different stages of the Aβ aggregation process.
for the quantitative detection of for the qualitative detection and characterization of β fibrils. As a result of the is on techniques from the oligomeric species formed during oligomeric species play a role in AD progression and severity. Therefore, it is important to gain a better understanding of the formation of smaller Aβ species in order to halt the progression of AD. The ability to identify and quantify the size of these Aβ oligomeric species without disrupting their structure is of utmost importance in order to effectively study the aggregation process and develop treatments that target these pivotal oligomerization events. Accordingly, this review focuses primarily upon techniques that have been employed in the study of in vitro aggregation of Aβ. Currently, a commonly used technique for the quantification of Aβ oligomer sizes within in vitro studies is polyacrylamide gel electrophoresis (PAGE). Other techniques that have been applied for determining the size of Aβ oligomers include Western blotting, capillary electrophoresis, mass spectrometry, fluorescence correlation spectroscopy, light scattering, centrifugation, and size exclusion chromatography (SEC). Furthermore, techniques including enzyme-linked immunosorbent assay (ELISA) and dot blot have been applied to identify Aβ oligomers, but give no size estimates. In the subsequent sections, we will discuss the application of each of these techniques to study Aβ oligomers.
SDS-and Native-PAGE
SDS-PAGE is the most common electrophoretic technique used for Aβ oligomer size determination in protein aggregation studies. Furthermore, a review by Bitan et al. cited SDS-PAGE as the most common method used to characterize toxic protein oligomers [44]. SDS-PAGE relies on the ability of SDS, a negatively charged detergent, to bind to the protein of interest. This binding typically results in the removal of secondary, tertiary, and quaternary structures from the protein. The SDS groups attach to the protein in a nearly uniform manner that gives the protein a charge approximately proportional to its length, thereby allowing for size based separations. Following the gel electrophoretic separation of proteins, the gel may be stained with a dye such as Coomassie Brilliant Blue or silver stain.
Many research groups have utilized SDS-PAGE, as a standalone technique, to study the evolution of Aβ species over time. A study by Ying et al. used SDS-PAGE to separate oligomers formed by 100 µM Aβ 1-42 incubated at 4 °C for 1 day [45]. SDS-PAGE revealed bands for monomer (4.5 kDa), trimer/tetramer (16.5 kDa), and higher molecular weight intermediates (>83 kDa) that appeared as a smear. The oligomer pattern of freshly dissolved Aβ peptides and Aβ peptides after a 7 day incubation have been observed by Satoh et al. [46]. Both Aβ 1-40 and Aβ 1-42 peptides incubated for 7 days as well as the freshly dissolved Aβ 1-42 peptide exhibited a range of species from 5-20 kDa ( Figure 2). However, the resolution of these species was low due to gel smearing. Smearing in these gels may be due to the resolution limitations of the gel or could be due to continuous associations and disassociations of the aggregating species occurring during the electrophoresis analysis. Whatever the cause, gel smearing interferes with the ability to identify a particular species and is often overcome by combining SDS-PAGE with another technique (see Sections 2.2 and 2.3).
Figure 2.
Tricine-SDS-PAGE analysis of the aggregation states of Aβ peptides freshly dissolved or incubated for 7 days. Aβ 1-42 exhibits bands at 5-20 kDa in both freshly prepared samples and samples incubated for 7 days. Aβ 1-40 incubated for 7 days also exhibits a smear at higher molecular weights, which is absent in freshly prepared samples. Reprinted from [46], with permission from Elsevier.
Although the anionic micelles formed by SDS enhance separation, they can also induce non-native behavior. SDS has been reported to accelerate the generation of Aβ fibrils. Sureshbabu et al. have shown that Aβ 1-42 freshly prepared in phosphate buffered saline exhibits monomer, trimer (~13.5 kDa), and tetramer (~18 kDa) bands when analyzed via Western blotting [47]. The addition of 1.5 mM SDS to the sample produced bands at 20 and 50 kDa. They proposed that the addition of 1.5 mM SDS causes Aβ 1-42 to develop a partial helical structure whose hydrophobicity induces aggregation. One way to counter this phenomenon is to add urea to the sample to further denature the peptide and prevent aggregation. However, the migration behavior of Aβ peptides in urea SDS-PAGE is inconsistent. A study by Kawooya et al. showed that the Aβ peptide exhibits an unusual electrophoretic mobility in urea SDS-PAGE that is proportional to the sum of the hydrophobicity consensus of the peptide rather than the number of amino acids in the peptide [31]. Therefore, under these conditions SDS-PAGE may provide information about the hydrophobicity of the peptide and not the size. The drawbacks of SDS-PAGE may be overcome by using native-PAGE to separate various Aβ sizes under conditions that allow the protein to remain in a native state.
Native or "non-denaturing" gel electrophoresis is similar to SDS gel electrophoresis, except this technique is run in the absence of SDS. With native-PAGE, protein mobility depends on both charge and hydrodynamic size. This differs from SDS-PAGE, where protein mobility depends primarily on molecular mass. Since Aβ aggregation is a process that involves changes in protein conformation, native-PAGE is often a suitable technique to detect various sizes of Aβ species. A study by Iurascu et al. used both SDS-PAGE and Tris-tricine PAGE to analyze the species formed by a solution of Aβ 1-40 solubilized in fibril growth buffer at pH 7.5 for 5 days at 37 °C [48]. They found that SDS-PAGE was able to detect Aβ 1-40 monomeric species, Aβ 1-40 oligomeric species of 20 kDa, and high molecular weight aggregates >98 kDa. In contrast, Tris-tricine PAGE was able to separate these Aβ oligomers into monomer, dimer, trimer, and high molecular weight Aβ sizes. Klug et al. have also compared native and SDS-PAGE analyses of Aβ aggregation [49]. They observed the presence of oligomers and high molecular weight species using native-PAGE with the majority of Aβ species observed in the high molecular mass region of the gel. In contrast, SDS-PAGE showed lower molecular weight species (<14 kDa) with only trace amounts of high molecular weight species (>50 kDa), suggesting that the removal of higher order protein structures by SDS may destabilize aggregates. The differences between native-PAGE and SDS-PAGE highlight the importance of examining more than one method for studies of the various Aβ aggregate sizes formed throughout the aggregation process.
SDS-PAGE in Combination with Western Blotting
Western blotting is a popular technique used to further process samples after electrophoretic separation. This technique provides a more sensitive detection of separated proteins. This detection is achieved by transferring separated proteins to a membrane where they are detected using antibodies specific to the protein of interest. Antibodies may be either monoclonal or polyclonal and are typically specific for a particular part of the Aβ sequence or a particular amyloid conformation. Some common antibodies and their recognition motifs are listed in Table 1. Selecting the proper antibody is an important consideration in order to achieve detection of the desired Aβ species or aggregation state. [52]. The band intensity for monomer, trimer, and tetramer bands was similar for both methods. However, 46 and 56 kDa intermediate sized oligomers were more apparent in the immunoblot analysis. Moore et al. have also found that immunoblot stains of Aβ 1-42 oligomers yield better results than silver stains [54].
SDS-PAGE with Western blotting has also been used to monitor the formation of Aβ oligomers in cell culture. A study by Walsh et al. employed SDS-PAGE followed by Western blotting to probe the formation of Aβ oligomers in APP-expressing Chinese hamster ovary (CHO) cells [50]. Bands corresponding to ~4, 6, 8, and 12 kDa were obtained using the monoclonal antibody 6E10. However, it was necessary to concentrate the Aβ protein via immunoprecipitation with an Aβ-specific antibody prior to performing electrophoretic separation.
Within in vitro studies of Aβ aggregation, Aβ is typically solubilized in 1,1,1,3,3,3-hexafluoro-2propanol (HFIP) to break up any residual aggregates that may be present in solution [61]. The HFIP is allowed to evaporate, and the peptide film is either resuspended in an organic solvent such as dimethyl sulfoxide (DMSO) and diluted into culture media or resuspended in a buffer solution such as phosphate buffered saline (PBS). Following incubation, samples are analyzed to detect the presence of oligomeric species. Dahlgren et al. utilized such an Aβ 1-40 oligomer preparation employing DMSO and F12 culture media with incubation at 4 °C for 24 h [53]. Western blot analysis using the 6E10 antibody showed bands corresponding to monomer and tetramer. Similar results were obtained by Stine et al. using the same sample preparation [51]. Walsh et al. utilized an Aβ 1-40 oligomer preparation in PBS (pH 7.4) at 37 °C [55]. After 5 days, Western blot analysis using the antibody 2G3 showed bands corresponding to monomer, dimer, and tetramer. However, intermediate sizes of oligomeric species >20 kDa were not obtained.
In addition to Aβ 1-40 , oligomeric Aβ 1-42 species formed in vitro have been well characterized using Western blot analyses. Stine et al. studied the formation of Aβ 1-42 oligomers using two different antibodies, 6E10 and 4G8 [51]. At 0 h, bands for monomer, trimer, and a faint tetramer band were obtained. After 24 h, these bands were more intense and a smear corresponding to oligomeric species ranging from 30 to 70 kDa was present. Furthermore, no differences in the band patterns obtained using the 6E10 and 4G8 antibodies were observed. Dahlgren et al. obtained comparable 24 h incubation results using the same oligomer preparation as Stine et al. [53]. In addition, similar 0 and 24 h results were obtained by Ryan et al. using a monomer preparation with dilution into PBS and an oligomer preparation with dilution into cold PBS + 0.05% SDS [52]. Stine et al. also examined the effect of temperature and ionic strength on the oligomeric band pattern obtained after incubation of 100 µM Aβ 1-42 for 24 h. An increase in temperature from 4 to 37 °C resulted in a decreased intensity of monomer and trimer bands and an increased intensity of the tetramer band. In addition, a smear for oligomeric species ranging from 30 to 70 kDa appeared at 25 °C with increased intensity at 37 °C. The effect of ionic strength was probed using the oligomer preparation at 37 °C with incubation for 24 h in either 10 mM Tris (pH 7.4) or 10 mM Tris supplemented with 150 mM NaCl. Both preparations yielded bands for monomer, trimer, and tetramer. However, the oligomer preparation in 10 mM Tris gave an intense oligomer smear from 30 to 97 kDa while the preparation in 10 mM Tris supplemented with 150 mM NaCl showed a less intense oligomer smear from 40 to 50 kDa. Ying et al. have also utilized the same Aβ 1-42 oligomer preparation as Stine et al. but employed for detection the monoclonal antibody A8, which is specific for oligomers [45]. A smear for oligomeric species ranging from 16.5 to 25 kDa was observed with antibody A8 (Figure 3, lanes 2 and 3). A poorer resolution of oligomers and larger species were obtained using the 6E10 antibody ( Figure 3, lane 4). These results show that 6E10 may be reacting more strongly with higher molecular weight oligomers or that these antibodies bind preferentially to different sizes of Aβ 1-42 oligomers. While Western blotting does facilitate detection of intermediate Aβ oligomers, the presence of a gel smear in many of the studies outlined above indicates that this technique does not allow quantification of individual sizes of oligomers in this range. sample was prepared in DMSO and diluted to 100 µM in Ham's F12 medium without phenol red. Oligomer mixture was separated by 15% SDS-PAGE, transferred to nitrocellulose membranes, and probed with monoclonal antibody A8 (Lanes 2 and 3) or 6E10 (Lane 4). Sample in Lane 2 was heat denatured prior to analysis, while sample in Lane 3 was untreated. Reprinted from [45] with permission. The publisher for this copyrighted material is Mary Ann Liebert, Inc. publishers.
SDS-PAGE in Combination with Other Techniques
SDS-PAGE has been used in combination with oligomer stabilization techniques. One such technique that has been applied by Bitan et al. is Photoinduced Cross-Linking of Unmodified Proteins (PICUP) [62]. PICUP was developed in the Kodadek laboratory in 1999 to study proteins that naturally form stable homo-or heterooligomers [63]. This technique provides a snapshot of different oligomer species present in solution at different times. Protein cross-linking is achieved via the visible light excitation of a tris(2,2'-bipyridyl)dichlororuthenium(II) complex which, through a series of steps, leads to the generation of a free protein radical [62,64]. This radical can attack an unmodified neighboring protein and form a covalent bond. Therefore, PICUP can be used to covalently freeze components of the sample, and these components may be separated and analyzed via techniques such as SDS-PAGE [62].
Bitan et al. have applied PICUP to compare low molecular weight fractions of Aβ 1-40 and Aβ 1-42 , where these fractions were isolated by SEC and analyzed via SDS-PAGE [65]. Aβ 1-40 exhibited bands for monomer, dimer, trimer, and tetramer with more faint bands for pentamer and heptamer ( Figure 4, lane 2). A distinctly different low molecular weight Aβ 1-42 oligomer size distribution, consisting of three groups of oligomers of varying band intensity, was obtained ( Figure 4, lane 4). This pattern led to the conclusion that the initial phase of Aβ 1-42 oligomerization involves the formation of pentamer/hexamer subunits which then associate to form larger oligomers and intermediates, or protofibrils [65]. Furthermore, they found that for Aβ 1-40 , monomer through tetramer were preexisting species in solution, while pentamer through heptamer were formed via a diffusion-dependent reaction of these preexisting species with free monomer. Their results verified that PICUP was capable of "freezing" preexisting oligomers but was also capturing oligomeric species which were not formed under typical aggregation conditions, thereby misrepresenting the true Aβ 1-40 oligomerization pattern. In addition, this study examined samples that were not cross-linked via PICUP before separation by SDS-PAGE. A single monomer band was obtained for Aβ ( SDS-PAGE has also been combined with SEC to investigate Aβ aggregation [25,66,67]. A study by Podlisny et al. used SDS-PAGE and SEC to observe the aggregation process of Aβ 1-40 secreted from CHO cells [66]. Soluble, SDS-stable aggregates of 6-25 kDa, were detected during the first 4.5 h of incubation at 37 °C via added radioiodinated synthetic Aβ 1-40 at low nanomolar concentrations. These 6-25 kDa Aβ oligomers represented ~18% of the total Aβ signal via SDS-PAGE and ~31% of the total Aβ signal via SEC. This low conservation of the Aβ gel signal over time to oligomeric species again indicates that SDS-PAGE underestimates the amount of aggregation. A study by Walsh et al. compared size estimations via SEC to those obtained by analyzing these SEC fractions by SDS-PAGE [25]. Aβ and Aβ 1-42 were dissolved in Tris-HCl (pH 7.4) and incubated for 48 and 6 h, respectively, at room temperature. SEC fractions corresponding to Aβ 1-40 dimers, protofibrils, and fibrils produced a single band at ~4 kDa on SDS-PAGE. The SEC fraction for Aβ 1-42 dimers produced a single SDS-PAGE band at ~4 kDa, while the SEC fraction for Aβ 1-42 protofibrils and fibrils produced a ladder of sizes ranging only from monomer to pentamer. These results suggest that SDS-PAGE may not accurately detect Aβ aggregate sizes produced throughout aggregation.
Summary of SDS-Based Methods
As a standalone technique, SDS-PAGE is able to detect Aβ 1-42 species ranging from monomer to tetramer. Native-PAGE has been used to separate Aβ 1-40 species ranging from monomer to pentamer. However, for higher order oligomers, these techniques only give a range of sizes that appear as a smear on the gel. SDS-PAGE is often coupled with other techniques such as Western blotting and PICUP to enhance the resolution of Aβ sizes. By coupling SDS-PAGE to these techniques, a better resolution of Aβ 1-40 species which appear as individual gel bands corresponding to monomer, dimer, trimer, and tetramer and Aβ 1-42 species which appear as individual gel bands corresponding to monomer, trimer, tetramer, and hexamer has been obtained. However, the resolution of intermediate sized Aβ oligomers ranging from 30-70 kDa by PAGE remains a significant challenge. The addition of SDS may also lead to complications including the acceleration of aggregation and the increased instability of oligomers, thereby misrepresenting the distribution of Aβ oligomeric species.
Capillary and Microfluidic Capillary Electrophoresis
Capillary electrophoresis (CE) is another electrophoretic technique employed for size based separations of Aβ. CE offers a fast and highly efficient separation of molecules with a broad range of properties thereby making it well suited for the separation of different sizes of protein aggregates [68]. CE separates molecules based on electrophoretic mobility, which results from differences in charge, shape, and/or size, and may be used either with or without SDS. Thus, CE allows a highly efficient separation and resolution of native forms of Aβ species, thereby overcoming the problem of gel smearing in many SDS and native-PAGE gel separations. CE detection typically uses either ultraviolet (UV) absorbance or laser induced fluorescence (LIF) to detect proteins. UV can detect proteins without any additional labeling, but typically has a lower sensitivity than LIF. LIF usually requires labeling of the molecules, but is highly sensitive, with previous reports of CE-LIF detection of double-stranded DNA down to the pg/µL range [69,70]. The ability to detect biomolecules at these low concentrations is necessary for the analysis of physiologically relevant protein concentrations.
CE with UV detection has been utilized by various researchers to detect Aβ species from monomers to large aggregates. Verpillot et al. used CE-UV to separate monomeric Aβ ranging in size from 37-42 residues and differing in length by a single residue, however they did not examine Aβ aggregation [71]. A study by Sabella et al. applied CE-UV with an SDS rinse for the detection of Aβ 1-40 and Aβ 1-42 oligomers formed in PBS (pH 7.4) at room temperature [72]. At 0 h, peaks for Aβ 1-42 oligomers in a size range from monomers to undecamers (~50 kDa)/dodecamers (~54 kDa) and larger aggregates were obtained ( Figure 5, t 0 ). A similar peak pattern was obtained over an incubation time period of 24 h with an increase in intensity of the higher molecular mass (>50 kDa) oligomer peak ( Figure 5, t = 1440 min). However, resolution of individual species, especially in the larger aggregate peak, was not achieved. Compared to Aβ 1-42 , the peaks for Aβ 1-40 were better resolved, but a drastically different peak pattern was observed. At 0 h, three peaks ranging in size from 3 to 30 kDa were obtained. A decrease in the intensity of the 10 to 30 kDa peak was observed over an incubation period of 24 h with the disappearance of all peaks after 48 h. This result shows that CE-UV is capable of detecting small Aβ 1-40 species and intermediate oligomeric Aβ species. In addition, the CE electrophoretic profiles of Aβ 1-40 and Aβ 1-42 differ significantly, supporting observations by PAGE that these two proteins differ in their early stages of aggregation. Picou et al. also observed substantial differences in the CE-UV electrophoretic profiles of Aβ 1-40 and Aβ 1-42 [73]. Two different preparations typically employed to form Aβ monomer or fibril were used. The Aβ 1-40 monomer preparation yielded a single monomer peak with a molecular weight of 4.3 kDa. In contrast to Aβ 1-40 , the Aβ 1-42 monomer preparation gave peaks for both monomer and fibrillar species. A peak pattern similar to the Aβ 1-42 monomer preparation was also obtained for the Aβ 1-40 fibril preparation. The Aβ 1-42 fibril preparation produced multiple aggregate peaks and no monomer peak. Although this study was able to separate Aβ monomer from mature fibrils, the detection of oligomeric Aβ species was not achieved.
LIF detection has also been utilized as a more sensitive means of identifying lower concentrations of Aβ aggregate species separated using CE. A study of the aggregation patterns of Aβ 1-42 using CE-LIF was conducted by Kato et al. [74]. The fluorescent dye thioflavin T (ThT) was used to detect two different Aβ 1-42 aggregate sizes with a 5 min analysis time [74]. In addition, this study examined the effect of seeding a freshly prepared Aβ 1-42 sample with a fibrillar Aβ 1-42 seed. For samples without a seed, a broad peak was observed with CE-LIF as opposed to seeded samples that contained both a sharp and broad peak, although no specific sizes were determined.
In addition to CE-LIF, microfluidic capillary electrophoresis (MCE) has been used to study Aβ. MCE is similar to CE except operates on a much smaller scale. The advantages of MCE over conventional electrophoresis methods include low sample consumption and a strong potential for automation and integration [75,76]. MCE has been utilized to study Aβ monomeric species. A study by Mohamadi et al. utilized MCE-LIF for the separation of five Aβ isoforms (Aβ 1-37 , Aβ 1-38 , Aβ 1-39 , Aβ 1-40 , and Aβ 1-42 ) [77]. However, MCE has yet to be applied for the study of Aβ oligomers.
CE as a technique for the detection of Aβ species formed throughout aggregation is still in its early stages. CE-UV has been utilized to detect small Aβ 1-40 species ranging from 3-30 kDa as well as to separate Aβ 1-40 monomer from fibrillar species. Aβ 1-42 species ranging from 3-50 kDa and >50 kDa have been detected using CE-UV. In addition, the separation of Aβ 1-42 monomer from fibrillar species has been achieved using CE-UV, and the separation of two different Aβ 1-42 fibrils has been accomplished with CE-LIF. The development of MCE has prompted researchers to apply this technique to the study of Aβ, with initial investigations demonstrating the separation of five Aβ isoforms differing in length by a single residue. The ability of CE to detect sizes from monomers to fibrils offers the potential to monitor the amyloid aggregation process over time, and the use of LIF provides the potential for examining physiologically relevant concentrations. However, further improvements to this technique must be made in order to enhance the resolution of intermediate sized Aβ species.
Mass Spectrometry
Mass spectrometry (MS) is a widely used technique for the detection of monomeric and oligomeric Aβ. In MS, the sample undergoes vaporization, and components are ionized by impacting them with an electron beam. Ions are separated by their mass-to-charge ratio using electromagnetic fields, and the ion signal is processed into a mass spectrum characteristic of the analyte. MS uses a variety of ionization sources depending on the sample state. For vapor samples, the most common source used to generate gas-phase ions is a radioactive ionization (RI) source [78,79]. However, other ion sources such as corona discharge ionization (CDI) [80,81], photoionization (PI) [80,82], and secondary electrospray ionization (SESI) [83][84][85][86] have been used as well. The most commonly used ionization source for liquid samples is electrospray ionization (ESI) [83][84][85][86], and for solid samples matrix assisted laser desorption ionization (MALDI) [87][88][89][90] and laser desorption ionization (LDI) [91][92][93] are widely used ionization sources. In addition, there are various types of mass analyzers that process the ion signal into a mass spectrum. These include time-of-flight, quadrupole, ion trap, Fourier transform ion cyclotron, magnetic sector, and tandem instruments as recently reviewed by Kanu et al. [94]. The most common MS techniques used for protein analyses are MALDI-MS and ESI-MS.
Matrix-Assisted Laser-Desorption Ionization (MALDI)-MS
MALDI-MS may be combined with other separation techniques such as SDS-PAGE to provide more quantitative size estimates. Iurascu et al. utilized SDS-PAGE in combination with MALDI-MS to analyze a solution of Aβ 1-40 solubilized in fibril growth buffer at pH 7.5 for 5 days at 37 °C [48]. MALDI-MS indicated that the soluble fraction contained two different ion mobilities, indicative of oligomerization. Parallel analysis using SDS-PAGE and Tris-tricine PAGE revealed the presence of oligomeric Aβ 1-40 of ~20 kDa (pentamer). A study by Maji et al. subjected wild-type and tyrosine substituted Aβ 1-40 and Aβ 1-42 to PICUP and quantified the resulting aggregate sizes via MALDI-MS and SDS-PAGE [95]. SDS-PAGE yielded wild-type Aβ 1-40 bands for monomer through hexamer. However, MALDI-MS was only able to attain masses for the monomer through tetramer bands, while masses for the pentamer and hexamer bands could not be measured. This inconsistency could be attributed to the presence of very small quantities of pentamer and hexamer. Alternatively, these species may not be desorbed from the MALDI matrix as readily as smaller oligomers. In addition, MALDI-MS spectra of tyrosine substituted Aβ 1-42 oligomers were not obtained, suggesting that either these oligomers could not be incorporated into the MALDI matrix due to their exceptional hydrophobicity or their covalent or weak noncovalent interactions were disrupted by the desorption/ionization process. These results show that although MALDI-MS may be used to quantify Aβ oligomers, this technique does have drawbacks including limited matrix interactions as well as the inability to distinguish molecules with overlapping charge-to-mass ratios, expense, and labor intensive analyses [96,97]. In addition, since MALDI is typically coupled with a pre-separation step such as SDS-PAGE, its detection capabilities may vary depending on the pre-separation technique used. (Figure 6, panels e and f). In contrast, trimers and tetramers were detected for freshly dissolved Aβ 1-40 ( Figure 6, panel c) whereas these species were not detectable for freshly dissolved Aβ 1-40 Met35(O) (Figure 6, panel d). However, after >95 min of incubation, Aβ 1-40 and Aβ 1-40 Met35(O) exhibited similar trimer and tetramer signals ( Figure 6, panels g and h). These results suggest that Met-35 oxidation slows a conformational change that may be necessary for early formation of Aβ 1-40 trimers. Although ESI-MS can be used as a way to freeze protein oligomers in time, complications arise when a protein could simultaneously populate a number of states with the same mass-to-charge ratio [97]. This complication makes it difficult to quantify different size oligomers that have the same mass-to-charge ratio.
Ion Mobility (IM)-MS
IM-MS is capable of separating ions by both their shape and charge, which has rendered it a successful technique for the separation of conformers of various shapes arising from a single protein [94,[99][100][101]. Ions are separated in time according to their cross sections by passing them through a drift cell containing helium gas under the influence of a weak electric field [102]. The flight times are combined with the drift times to yield the mass-to-charge IM distributions for all ions in the sample. The ability of IM-MS to separate species that differ in shape or size but have the same mass-to-charge ratio has made this technique a powerful tool for analyses of the early stages of Aβ oligomerization.
Various research groups have utilized IM-MS to gain a better understanding of the early events of Aβ aggregation. Aβ 1-40 conformational states in freshly dissolved and aggregated solutions have been studied by Iurascu et al. [48]. Two different conformational states were obtained for freshly dissolved Aβ 1-40 and the soluble fraction obtained by Aβ 1-40 incubation for 5 days at 37 °C and pH 7.5. Bernstein et al. used IM-MS to study the aggregation of Aβ 1-42 versus Aβ 1-42 with a Phe19→Pro19 substitution [103]. Monomers and large oligomers were produced by unfiltered Aβ 1-42 , while protein passed through a 10,000 amu filter yielded monomer, dimer, tetramer, hexamer, and an aggregate of two hexamers. In contrast, the Pro19 alloform produced monomer, dimer, trimer, and tetramer but no large oligomers. In a more recent study by Bernstein et al., a mechanism for Aβ 1-40 and Aβ 1-42 oligomerization and eventually fibril formation was postulated [104]. Using IM-MS, this group was able to determine the shape and size of Aβ 1-40 and Aβ 1-42 oligomers. Aβ 1-40 oligomerization proceeded via the formation of dimer and tetramer followed by the very slow formation of fibrils containing a β-sheet structure. In contrast, Aβ 1-42 proceeded via the formation of dimer, tetramer, and a hexameric paranucleus followed either by the formation of dodecameric species or the slow conversion into fibrils containing a β-sheet structure.
Fluorescence Correlation Spectroscopy
Fluorescence correlation spectroscopy (FCS) has also been utilized to gain information about the size of Aβ species formed throughout aggregation [24,[105][106][107]. FCS was originally developed by Eigen and Rigler in the early 1990s [108]. In FCS, unlabeled protein is combined with fluorescently labeled protein and, at various times throughout aggregation, the fluorescent dye is excited by a sharply focused laser beam. The emitted fluorescence of a small number of molecules in solution is observed. The fluorescence intensity fluctuates due to Brownian motion of the particles, and an intensity correlation function can be used to determine the average number and average diffusion time (i.e., molecular size) of molecules. Advantages of FCS include high sensitivity (nM range and below), ability to examine a wide range of molecular sizes (i.e., monomer, oligomer, fibrils) [109], fast analysis times [109], and small sample volumes (femtoliter) [110]. In addition, no pre-separation step is required for the determination of particle radius via FCS. However, assumptions must be made about the kinetics of the aggregation process as well as molecular shape in order to determine molecular weight.
Various researchers have employed FCS to monitor Aβ aggregation. A study by Matsumura et al. utilized FCS to monitor the aggregation of Aβ 1-40 and Aβ 1-42 and observed distinct aggregation pathways, dependent upon incubation conditions, that resulted in the formation of either oligomeric species or fibrils [24]. Two different site-specific labels at either the N-terminus or Lys 16 were used to monitor aggregation. One pathway involved the formation of 10-15 nm spherical Aβ 1-42 assemblies of ~330 kDa, termed amylospheroids (ASPDs), appearing after 5 h of gentle agitation of a 50 µM Aβ 1-42 solution in F12 buffer at 4 °C. These ASPDs were formed from Aβ species of ~12.7 kDa initially present in solution. In addition, the aggregation pathways were similar for the N-terminus and Lys 16 site-specific labels. An alternative pathway involved fibril formation from 100 µM Aβ 1-40 solutions in Dulbecco's PBS (pH 3.5) with gentle agitation at 4 °C. This pathway began with dimer formation at 0 h, followed by the formation of intermediate sized species of 15-40 nm after 2-9 h. Eventually, larger molecular weight fibrils (14,000 kDa) were formed after 24 h using Aβ labeled site-specifically at Lys 16 . However, much larger aggregates (120,000 and 3,900,000,000 kDa) were formed after 24 h using Aβ labeled site-specifically at the N-terminus. It was thus postulated that the Lys 16
Summary of Spectroscopic Methods
MS is capable of detecting low oligomer concentrations but is expensive and has difficulty separating species with identical mass-to-charge ratios such as Aβ aggregates [96,97]. To address this problem, MS is often coupled with an upstream separation technique such as SDS-PAGE [48,95]. In addition, IM-MS has been utilized for the separation of different sizes and conformations of Aβ 1-40 and Aβ 1-42 with promising results for small oligomers. However, the addition of a step such as IM also increases the time needed for analysis and therefore decreases the chances of detecting transient species. Consistent results for Aβ 1-40 and Aβ 1-42 oligomer sizes formed during the earliest events of aggregation have been obtained using MS techniques. However, the detection of larger Aβ 1-40 and Aβ 1-42 oligomeric species ranging from ~32-~100 kDa has not been achieved using MS. FCS does not require a pre-separation step to determine the particle radius of species in a sample. This technique has been successfully applied for the detection of small and intermediate sized Aβ oligomers as well as large Aβ fibrils. However, FCS yields average values of particle radius for a population of aggregates and not individual particle sizes or their distributions.
Light Scattering Techniques
Light scattering techniques have been used to measure Aβ aggregate sizes. Classical, or multi-angle, light scattering (MALS) employs a well collimated, single frequency light beam to illuminate a sample of macromolecules [111]. When incident light interacts with the macromolecules in solution, an oscillating dipole is induced and the light is re-radiated, or scattered [112]. Aggregated structures induce coherent scattering, and as a result the intensity of scattered light is dependent upon molar mass. Furthermore, destructive and constructive scattering that result from the independent scattering of individual molecular elements can give rise to an angular dependence of the scattered light, which is a function of the size of the molecule. Thus, the intensity of the scattered light is measured as a function of scattering angle, often referred to as Rayleigh scattering, to yield the molar mass and root mean square (rms) radius of the macromolecules [112]. MALS is ideal for characterizing larger assemblies (>10 nm). In contrast, for analyses in which smaller molecules are present in solution, dynamic light scattering (DLS), also known as quasi-elastic light scattering (QELS), is used. DLS employs a fast photon counter to measure time dependent fluctuations in scattered light at a single angle (usually 90°), which are related to the rate of diffusion of the macromolecules [112,113]. Measurement of diffusion rates allows calculation of the hydrodynamic radius (R H ) of macromolecules using the Stokes-Einstein equation [113]. When used as standalone techniques, MALS yields the weight-averaged molar mass for all molecules in solution. While DLS can distinguish populations that differ in size by a factor of five or more, individual peaks exhibit a high degree of polydispersity. Therefore, it is often necessary to utilize a pre-separation step in conjunction with light scattering to obtain an accurate estimate of the relative amounts of individual aggregates present in solution. In addition, the exponential dependence of scattering on aggregate size prohibits the detection of low quantities of small aggregates in the presence of larger species.
Various researchers have utilized MALS and/or DLS to characterize Aβ assemblies formed throughout aggregation [114][115][116][117]. Carrotta et al. utilized both MALS and DLS to monitor the aggregation of a 185 µM Aβ 1-40 sample at pH 3.1 and 37 °C [117]. DLS was used to characterize aggregate sizes formed during the early stages of aggregation up to ~38 h, as shown in Figure 9. After 5 min (Figure 9a), an average R H of 7 nm was obtained. The size distribution became more polydisperse over time and ranged from 10-52 nm after 37 h (Figure 9f). However, only average size distributions could be obtained and no information was reported about the concentrations of each aggregate species (i.e., monomer, dimer, etc.). Larger aggregates (hundreds of microns) were formed after 2 weeks as detected by MALS.
Similar to these findings, Lomakin et al. observed using DLS the initial formation of a spherocylindrical micelle with average R H of 7 nm immediately following dissolution of Aβ 1-40 at pH 2 [115]. In addition, they reported two different kinetic patterns for aggregation of Aβ 1-40 prepared at a concentration either above or below the critical micelle concentration (CMC) of 100 µM [114] Complimentary DLS and MALS studies by Murphy and Pallitto also demonstrated an effect of Aβ concentration upon aggregate formation [118]. They demonstrated that dilution of Aβ 1-40 from urea into PBS yielded larger aggregates at lower protein concentrations, while the increase in R H for aggregates was proportional to the protein concentration. In addition, MALS data indicated that the linear density of aggregates increased with protein concentration. Thunecke et al. have utilized MALS and DLS to study the aggregation of Aβ 1-40 and Aβ 1-42 in acetonitrile-water mixtures [116]. At the onset of aggregation, Aβ 1-42 was present as a 2 nm oligomer and rapidly formed fibrils with a length <50 nm within 4.5 h. In contrast, Aβ 1-40 initially exhibited large aggregates that grew 70 times slower than aggregates of Aβ 1-42 . However, the presence of these large aggregates may preclude observation of a separate population of oligomers. These findings highlight differences in the dissolution and aggregation of Aβ 1-40 and Aβ 1-42 . Distributions were determined using a constrained regularization method. Reprinted with permission from [117]. Copyright (2005) The American Society for Biochemistry and Molecular Biology.
Light Scattering in Combination with Other Techniques
Because light scattering techniques provide information about the weight-average molar mass and radius for all molecules in solution, they are often coupled with a pre-separation technique such as asymmetric field flow fractionation (AFFF) [119] or SEC [25,120,121] to better characterize individual Aβ oligomeric species. A study by Nichols et al. utilized MALS with SEC as well as DLS to characterize Aβ 1-40 protofibrils following growth by monomer elongation or lateral association [120]. They found that protofibrils isolated by SEC exhibited an average R H of 51 nm and molecular weight, determined via MALS of 30,000 kDa. Protofibrils that had grown by monomer deposition had an average R H of 143 nm and molecular weight of 57,000 kDa, while protofibrils that had grown by lateral association had an average R H of 104 nm and molecular weight of 86,000 kDa. Furthermore, SEC-MALS revealed that the mass per unit length of protofibrils was unchanged during elongation, but was increased following association. The temporal change in size of Aβ 1-40 protofibrils isolated by SEC has also been monitored via DLS by Walsh et al. [121]. The initial average R H for protofibrils isolated by SEC was ~27.8 nm, and protofibril size grew to 80.6 nm over a period of 9 days when 17 µM Aβ 1-40 in Tris-HCl (pH 7.4) containing 0.04% w/v sodium azide was incubated at room temperature.
AFFF is another technique that has been coupled with light scattering to estimate the molecular weight of individual Aβ aggregates. AFFF exploits the parabolic flow profile created by the laminar flow of a sample through a thin, parallel plate flow channel, where the lower surface is solvent permeable [122]. A perpendicular force applied to the laminar flow stream drives molecules towards the permeable boundary layer of the channel [123]. Because Brownian motion of the particles creates a counteracting force, smaller particles localize higher in the channel leading to separation of different molecular sizes, with smaller molecules eluting first [122]. Rambaldi et al. utilized AFFF-MALS to monitor the aggregation of Aβ 1-42 in PBS (pH 7.4) at room temperature over 24 h [119]. At 0 h, two major peaks were obtained corresponding to molecular weights of ~60 kDa and ~1000-100,000 kDa. In addition, the retention time of the ~60 kDa species decreased between 0 and 4 h, corresponding to an increase in aggregate size of 6.5-4.7 nm. The intensity of the two peaks also decreased over 24 h, possibly due to irreversible adsorption of the sample to the permeable surface. Although AFFF-MALS has several advantages, including gentle, rapid, and non-destructive separation, improvements to the ultrafiltration membrane are critical to enhance analysis capabilities. In addition, the smallest molecular weight cutoff for membranes is 5 kDa, making detection of Aβ monomeric species difficult.
Centrifugation
Centrifugation has also been explored as a method for determining Aβ size. Here, sedimentation coefficient (s) values can be correlated with molecular weight. Mok and Howlett provide a nice overview of sedimentation velocity centrifugation in the context of Aβ analysis [124]. Ward et al. used density gradient centrifugation to fractionate Aβ 1-40 samples incubated at pH 7.4, 35 °C for 30 min, 18 h, or 18 days [26]. Using SDS-PAGE with Western blotting to analyze sedimented samples, they found that Aβ 1-40 incubated for 18 h contained only small molecular weight oligomers (4-17 kDa), while Aβ 1-40 incubated for 18 days showed the presence of a >250 kDa band as well as significant streaking, indicating other unresolved sizes. Huang et al. used analytical ultracentrifugation to compare Aβ 1-40 samples prepared at pH 3, 5, and 7 [125]. They determined that at pH 5 there were no soluble aggregate species. At pH 7, they identified small oligomers with an average molar mass of 12.1 kDa, and at pH 3 they identified a range of aggregate sizes with an average molecular weight of 1 MDa. Nagel-Stefer et al. also used sedimentation velocity centrifugation for the analysis of Aβ 1-42 samples after 5 days of agitation at room temperature and were able to detect "globular species" ranging in size from ~270 kDa-3.8 MDa as well as even larger aggregates [126]. Interestingly, they also compared three different simulation methods for determining molecular weight from sedimentation values and obtained molar masses that differed by approximately one order of magnitude.
Size Exclusion Chromatography
SEC, a chromatographic technique, separates molecules based on molecular hydrodynamic volume or size. Molecules too large to penetrate the pores of the column packing material elute in the void volume, while smaller molecules travel through the pores and elute at later times. Globular proteins are often used as standards to estimate the size of Aβ oligomers. However, since Aβ is a linear, hydrophobic peptide, comparisons between the elution behavior of Aβ oligomers and size standards are difficult [127]. In addition, the sample is subjected to a several-fold dilution, which facilitates the dissociation of small unstable oligomers [128], thereby precluding the detection and size estimation of these species.
Although SEC is typically utilized in conjunction with another technique, SEC as a standalone technique has been employed for the study of Aβ aggregates [129][130][131]. Englund et al. used SEC to detect low molecular weight Aβ aggregates, Aβ protofibrils, and Aβ fibrils formed using different Aβ sample preparations [130]. The size of low molecular weight Aβ aggregates ranged from 4-20 kDa ( Figure 10
Summary of Additional Aβ Aggregate Size Determination Techniques
Light scattering techniques, such as MALS and DLS, have been used to detect both small and large Aβ aggregates. DLS is more suitable for the detection of smaller aggregates and gives information about aggregate size, or R H , while MALS has been utilized for the detection of larger Aβ species, including fibrils, and can provide information about molar mass. MALS and DLS, however, give a weight-average molar mass or R H for all molecules in solution and must be coupled to another technique in order to increase the resolution of individual sizes. SEC as a standalone technique has been utilized to detect low molecular weight Aβ oligomers and protofibrils, and SEC-MALS has been used to characterize protofibrils formed via different growth mechanisms. However, due to the dilutions required by SEC, small unstable oligomers are often dissociated, thereby precluding their analysis. AFFF-MALS does not require a pre-fractionation step and has been used to separate Aβ oligomers of ~60 kDa from larger species. This technique yields a gentle, non-destructive separation of molecules. However, further improvements to the ultrafiltration membranes must be made in order to reduce adsorption of the sample to the membrane. Centrifugation has also been explored for the separation of small oligomers (4-17 kDa) and larger species (>250 kDa) but requires an uncertain correlation of sedimentation coefficients with molar mass. Each of these techniques are suitable for the detection of a wide range of Aβ aggregates present throughout aggregation but present difficulties with respect to the resolution and quantification of individual Aβ aggregate sizes.
Techniques Utilized for Aβ Oligomer Identification
While this review focuses primarily on techniques capable of qualitatively determining the size of Aβ oligomers, techniques that can identify the presence of oligomers, without providing information about oligomer size, are also available. Although qualitative in nature, we have chosen to briefly discuss two of these techniques, dot blot and ELISA, as a result of their frequent use and emerging interest.
Dot Blot
Dot blots employ a protein captured upon a membrane as a spot, or dot. A primary antibody binds to the protein epitope of interest followed by the binding of a secondary antibody to facilitate detection. When dot blots are probed with antibodies that specifically recognize oligomeric Aβ, they can confirm the presence of oligomers but give no information about aggregate size. Three different Aβ antibodies, oligomer-specific A11 or sequence specific 4G8 and 6E10 (see Table 1 for Aβ binding epitopes), were employed in conjunction with a dot blot assay for detection of aggregating Aβ by Wong et al. [57]. Aβ 1-40 was diluted to 50 µM in PBS (pH 7.4) and incubated at 37 °C. At times ranging from 0-3 days, a sample was analyzed via dot blot, as shown in Figure 11. A11 binding revealed the transient appearance of oligomers in uninhibited samples, while detection via 4G8 and 6E10 remained constant until later times when signals decreased, presumably due to masking of binding sites following aggregation. Changes in these patterns in the presence of inhibitor demonstrated the ability of the inhibitor to prevent oligomer formation and slow the evolution of larger aggregates. Necula et al. used a dot blot assay to monitor the oligomerization of Aβ 1-42 dissolved in 100 mM NaOH, diluted to 45 µM in PBS (pH 7.4), and incubated at room temperature for 10 days [132]. Similar to Wong et al., they probed the specificity of three different antibodies, oligomer-specific A11 and sequence specific 6E10 and 4G8. At 0 days, 6E10 and 4G8 strongly reacted with Aβ 1-42 aliquots, while A11 reacted weakly, indicating that only monomeric species were present. A strong immunoreactivity for A11 was observed after 4 days and continued to increase in intensity over 10 days, similar to results obtained by Wong et al. Again, this was accompanied by a decrease in immunoreactivity of 6E10 and 4G8. inhibitor. Samples were taken on the indicated days and spotted on a nitrocellulose membrane. Oligomer-specific A11 antibody and Aβ-sequence specific antibodies 4G8 and 6E10 were used to detect aggregates. Reprinted with permission from [57]. Copyright (2011) American Chemical Society.
Enzyme-Linked Immunosorbent Assay
ELISA is a commonly used technique for the identification of Aβ oligomers. ELISA may be used in a traditional or sandwich assay format. In the traditional format, protein adsorbed at a surface can be detected using a primary antibody that is specific for Aβ oligomers (see Table 1). This primary antibody can be directly linked to an enzyme that converts added substrate to a detectable signal (direct ELISA) or can be coupled with a secondary antibody containing the enzyme moiety (indirect ELISA). The latter format serves to enhance the assay signal. Alternatively, in the sandwich ELISA format, a sequence-specific capture antibody (see Table 1) adsorbed onto the surface is used to capture Aβ protein, which is subsequently detected using the same sequence-specific antibody, such that only Aβ species containing multiple monomeric units, and therefore multiple epitopes, are detected [130,133]. Consequently, this sandwich ELISA will recognize only aggregated Aβ, but not Aβ monomer. Although ELISA can identify the presence of Aβ oligomers in a sample, this technique is not capable of determining sizes of these oligomeric species. Therefore, ELISA is most advantageous for the detection of oligomeric Aβ within a sample containing many different proteins.
Various researchers have utilized ELISA for the detection of Aβ oligomers [128,130,[133][134][135]. A study by Englund et al. employed a sandwich ELISA with monoclonal antibody 158 for the detection of low molecular weight oligomeric Aβ 1-40 produced by dissolving Aβ 1-40 in 10 mM NaOH with dilution to 50 µM in 2 X PBS and Aβ 1-42 protofibrils produced by dissolving Aβ 1-42 in 10 mM NaOH with dilution to 443 µM in 2 X PBS and incubation overnight at 37 °C [130]. Gonzales et al. utilized a similar ELISA assay to detect low molecular weight Aβ 1-42 formed by dissolving Aβ 1-42 in HFIP with dilution to 200 nM in PBS (pH 7.2) and incubation at 37 °C for 24 h [133]. The size of these species was confirmed with PAGE to be tetramer and pentamer; however, the bands were very faint, indicating the superior sensitivity of the ELISA assay for these oligomeric species. A detection limit for Aβ 1-40 oligomers of 80 nM was obtained in these studies.
Summary of Aβ Oligomer Identification Techniques
Dot blots and ELISAs have been employed to detect oligomeric Aβ assemblies. Dot blots have been used to observe the transient evolution of oligomers during aggregation, but provided no information about Aβ aggregate size. Low molecular weight Aβ oligomers and Aβ protofibrils have been detected via ELISA at nanomolar concentrations. However, PAGE was required to estimate the size of these species. Thus, these techniques can sensitively confirm the presence of oligomers but yield no size information.
Conclusions
This review describes a variety of techniques, summarized in Table 2, that are currently utilized to determine the size or presence of Aβ aggregates, with a focus upon oligomeric species. These techniques have been explored for the quantitative detection of different aggregate sizes with various limitations to their resolution, dependence on pre-analysis procedures, sensitivity, cost, etc. Electrophoretic techniques, such as SDS-PAGE, Western blotting, and CE, are widely used for size-based separations of Aβ aggregates. In particular, SDS-PAGE and Western blotting are suitable for the detection of monomeric and small oligomeric Aβ species. The separation of larger oligomers via SDS-PAGE is more difficult due to the sensitivity of these sizes to denaturing conditions, which can result in aggregate decomposition during analysis. The recent development of antibodies specific for Aβ oligomers has led to an increase in the application of Western blotting, dot blotting, and ELISA to study Aβ aggregation. However, the detection limits of Western and dot blotting prohibit study of physiologically relevant Aβ concentrations. While more sensitive, ELISA is better suited for the identification of specific analytes, such as Aβ oligomers, present within a mixed population but cannot distinguish individual oligomer sizes. CE with LIF detection offers a highly sensitive detection of physiologically relevant concentrations, but the application of CE to amyloid aggregation analyses is still in the early stages. MS is another commonly used technique for Aβ aggregate size-based separations. MS has been successfully used to detect small oligomeric species (especially IM-MS) but quantitative analyses of aggregate size may be limited by the pre-separation step, the ability to differentiate species with highly similar charge-to-mass ratios, and high equipment costs. FCS, MALS, and DLS may be utilized for determination of Aβ aggregate size, but yield a weight-averaged molecular weight of species, thereby limiting the resolution of individual Aβ aggregate species. Centrifugation has been used to examine small oligomeric species up to large fibrils; however, selection of the method for determination of molar mass from sedimentation coefficients can play an important role in size estimation. SEC may be coupled with these approaches or used as a standalone technique; however, SEC is complicated by dilution of the analyte during separation, inadequate resolution of intermediate oligomeric species, and limited utility of size standards. [57,132] Although each of the methods discussed in this review has the capability to determine Aβ aggregate size, the pathogenic events that initiate the misfolding of Aβ and formation of aggregate species remain elusive. Hence, there is a continued need for improvement of these techniques in order to realize the effective detection of small size differences in Aβ oligomers. In order to leverage the advantages of each Aβ detection method, a combination of approaches must be utilized, allowing validation of findings from different techniques and a better understanding of the early events of the Aβ aggregation process. | v3-fos-license |
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} | pes2o/s2orc | Direct spectroscopic speciation of the complexation of U(VI) in acetate solution
Abstract As a result of systematic UV–Vis absorption spectroscopy studies in the U(VI) acetate system, the single component spectrum of [UO2CH3COO]+ with characteristic parameters was evaluated and applied in quantitative deconvolution of multicomponent spectra. Free acetate concentrations were obtained by the use of geochemical and probabilistic modelling codes. A total of 51 UV–Vis spectra were collected in a wide range of experimental conditions where coordination of U(VI) by acetate ion was indicated by characteristic variations in the spectra structure as compared to UO2 2+. Using chemometric data analysis, the resulting factor structure was evaluated to obtain a subset of 14 spectra holding only one coordinated species next to UO2 2+ (aq). The molar absorption coefficient for the U(VI) monoaceto species was estimated as ε 418 = 17.8 ± 1 dm3 mol−1 cm−1. Spectral deconvolution was used to obtain an estimate of the species concentrations which allowed to calculate for each sample the free acetate concentration, the total U(VI) amount and, eventually, to estimate the formation quotient lg β 11 = 2.8 ± 0.3 of UO2(CH3COO)+. Graphical Abstract
Introduction
Acetate, CH 3 COO -, is the salt of the monoprotic acetic acid, CH 3 COOH. The protonation constant K A of acetic acid is reported to be pK A = 4.76 [1,2] in diluted aqueous solutions at 298 K. Under those conditions, hexavalent uranium occurs exclusively as linear dioxo entity, UO 2 2? [3]. This dioxo cation is almost always coordinated in the plane equatorial to the axial uranyl oxygens by four, five, or six neighbours. Coordination of the uranyl(VI) ion by acetato ligands may occur in a rather limited range between pH 1.9 and pH 5.0. At values below pH 1.9, the free acetate concentration is too low for significant U(VI) coordination, even in acetate brines. At values above pH 5, the solubility of U(VI) with respect to UO 3 Á2H 2 O is limiting the U(VI) concentration except at total acetate concentrations [10 -2 mol dm -3 . Here, however, complexation by atmospheric CO 2 may interfere because of the very high coordination tendency of U(VI) and CO 3 2- [4]. Experimental speciation of U(VI) should consider the onset of hydrolysis at about pH 3 [5], which may interfere. To circumvent, both precipitation and hydrolysis of U(VI) high concentrations of acetate are required in solution.
Considering the narrow field of stability of U(VI) acetate species, a surprising amount of work has been devoted to the study of the U(VI) acetate interaction, both on the experimental [6][7][8][9][10][11][12] and the theoretical level [13][14][15][16][17]. The literature given is but a selection from the past decade and the abundance available. Three groups can be distinguished: crystallographic studies [6][7][8], analytical studies often involving very advanced equipment like synchrotron sources [9][10][11][12], and quantum chemical numerical calculations [13][14][15][16][17]. Considering the wide range of experimental techniques applied to the study of U(VI) acetate interaction in aqueous solutions, e.g. infrared and Raman spectroscopy [18,19], potentiometry and calorimetry [20], capillary electrophoresis [12], X-ray absorption [16,21], mass spectrometry [13,15], ion exchange chromatography [22], a standard method of U(VI) speciation in aqueous solution is almost missing: UV-Vis absorption spectroscopy. Some early work is available [23][24][25], however, without detailed information on single component characteristics like band position and molar absorptions. Görrler-Walrand and de Jagere [26] report a single component spectrum of UO 2 CH 3 COO ? without further information on the determination and characteristics of this spectrum. Recently, single component UV-Vis absorption spectra of U(VI) acetato species have been proposed from a factor analysis study, again without further information even on very basic characteristics of the spectra, e.g. molar absorptions. The wavelength range was limited to a rather narrow region of 400-470 nm, thus omitting the characteristic absorptions of U(VI) in the range 340-400 nm [27]. The onset of the strong absorption towards the UV is a characteristic feature for each U(VI) species and a crucial proof for the reliability of proposed single component spectra in peak deconvolution studies. Successful applications of single component U(VI) spectra in the U(VI)-acetate system are not to our knowledge.
Therefore, we have studied the U(VI) acetate system systematically by UV-Vis absorption spectroscopy. For the first time, we report the single component spectrum of UO 2 CH 3 COO ? together with its characteristic parameters and show its applicability to the quantitative deconvolution of mixed spectra from the U(VI) acetate system. This data is of basic importance to compare U(VI) acetate complexation with other U(VI) carboxylate interactions up to naturally occurring organic materials where carboxylate groups are considered as functional groups relevant for U(VI) binding [14].
Results and discussion
A set of 14 U(VI) absorption spectra are given in Fig. 1, normalised to the total U(VI) concentration. This presentation reveals a systematic increase in the molar absorption from 9.7 dm 3 mol -1 cm -1 at 413.8 nm to about 16.5 dm 3 mol -1 cm -1 at 416.9 nm. A molar absorption of 9.7 dm 3 mol -1 cm -1 at 413.8 nm is known for the free UO 2 2? ion in aqueous solutions. The increase in molar absorption in the characteristic band of U(VI) correlates with the ratio of free acetate and total U(VI) concentration, that is, with increasing coordination of UO 2 2? by acetate. The concentration ratio given in Fig. 1 does not vary systematically with the change in the absorption (e.g. at the wavelength of 413.8 nm). Such a systematic change should not be expected. First, the ratio holds the total U(VI) concentration, not the free U(VI) concentration. Second, both the calculated free acetate concentration and the total U(VI) concentration are experimental quantities which are associated with a measurement uncertainty. Forming the ratio of uncertain data further enhances the uncertainty. Nevertheless, the observed absorption and the concentration ratio are strongly correlated (Pearson correlation r = 0.8).
No isosbestic point is observed. Coordination of U(VI) by acetate causes a weak band shift to longer wavelengths, barely visible at the most intense spectra in Fig. 1. A hyperchromic effect is intensifying the absorption. Increasing coordination by acetate also causes a bathochromic shift of the steep absorption band in the region below 350 nm.
The UV-Vis spectroscopic study of the U(VI) acetate system is limited by four constraints. The first constraint is the low molar absorption of UO 2 2? (aq) in the characteristic absorption bands about 413.8 nm. The second constraint is the onset of U(VI) hydrolysis at values between pH 3.3 and pH 3.7 (depending on the total U(VI) concentration). Third, the acetate ligand is formed from acetic acid with a pK A = 4.76 [1,2].
Fourth, the solubility of U(VI) in solution is limited by the solid UO 3 Á2H 2 O. Figure 2 illustrates these constraints, where stability regions of various species are given on basis of geochemical modelling using data from Table 1.
The diamond-shaped symbols indicate the 14 samples where coordination of the free uranyl ion, presumably by acetate, was observed. Figure 2, which is deduced from literature data, suggests that the monoacetato species of U(VI) forms only at quite acidic pH and rather high total acetate concentrations without interference from other species. The stability fields for the acetato species have been selected to represent conditions where the species concentrations are calculated to be above 10 % of the total species concentrations. For the hydrolysis species, the stability fields enclose conditions where species concentrations are above 1 %.
The distinction is due to the difference in molar absorptions. While the oligomeric hydrolysis products (UO 2 ) 2 (OH) 2 2? and (UO 2 ) 3 (OH) 5 ? have molar absorptions of about 100 dm 3 mol -1 cm -1 (421.8 nm) [28] and about 475 dm 3 mol -1 cm -1 (429.0 nm) [29], respectively, the acetato species have much lower molar absorptions in the range of the characteristic absorption band of U(VI). Hence, even minor amounts of oligomeric U(VI) hydrolysis products in a sample will affect an experimental UV-Vis spectrum. The respective stability field of a U(VI) diacetato species is represented in Fig. 2 by the region enclosed in dashed lines. In this region of the lg[acetate]-pH diagram, all acetato species considered, UO 2 CH 3-COO ? , UO 2 (CH 3 COO) 2°, and UO 2 (CH 3 COO) 3 -, are calculated to occur in solution with relative amounts above 10 %. Hence, direct spectroscopic speciation in such complex media requires accurate knowledge of the respective single component spectra of U(VI) acetato species to have a chance to be meaningful. In the crosshatched region in the upper right corner of Fig. 2, the monoacetato and the triacetato species of U(VI) are prevalent. At values above pH 5, all acetic acid is dissociated and further shift of pH does not significantly increase the amount of acetate ligand at a given total acetate concentration. Furthermore, under atmospheric conditions, carbonate will form in solution from CO 2 dissolved in the solutions to become a potent competitor for U(VI) [30][31][32]. Thus, Fig. 2 summarises the essential features of the interaction of acetate with U(VI), against which the experimental findings from this study will be probed.
While concisely illustrating the U(VI)-acetate system, Fig. 2 is not considered as a guide for this investigation. Data evaluation is based, as far as possible, on model-free numerical and statistical models. No decision in this study is made with respect to Fig. 2.
From a total of 51 spectra collected at random in a wider range of conditions, 51 indicated coordinated U(VI) due to significant difference of the observed UV-Vis absorption spectrum from the well-known spectrum of UO 2 2? . From these 51 spectra, a set of spectra had to be found holding only UO 2 2? and one further component. A larger number of spectra could readily be eliminated for their physicochemical state suggesting either influence of hydrolysis or formation of higher U(VI) acetato species. The remaining spectra were selected by either submatrix analysis [33] or computer-assisted target factor analysis (CAT) [34]. Thus, a data set with 14 spectra was obtained with the resulting factor structure given as a SCREE test [35] in Fig. 3. The first factor alone explains more than 90 % of the observed variance. The second factor explains just 7 % of the variance-that is the additional signal caused by the hyperchromic shift due to coordination by acetate.
The remaining factors explain random noise in the spectra contributing in sum less than 2 % of the spectroscopic signal. It has been noted previously that a larger number of statistical criteria have been forwarded to identify the number of significant factors from a principal component analysis [36]. Due to the unavoidable presence of random noise in experimental data, none of these criteria Table 1. Diamond-shaped symbols indicate locations of experimental samples. The varying U(VI) concentrations of these samples does not play a role except in case of oligomeric hydrolysis product formation Direct spectroscopic speciation of the complexation of U(VI) 1691 can be unambiguous. The researcher's choice must be judged on basis of the outcome of the overall analysis. In case of UV-Vis spectroscopic investigations, factor analysis forwards information which may serve as criteria. An example is the total U(VI) concentration in a sample. This concentration is known to the experimenter (within experimental uncertainty). Spectral deconvolution and factor analysis provide an estimate for this concentration by the sum of all U(VI) species quantitatively estimated in solution for all solutions based on the experimental spectra and the single component spectra included into the data analysis. Thermodynamic parameters, e.g. formation quotients of the species formed in solution, may serve as additional criterion. The single component spectrum of UO 2 CH 3 COO ? resulting from these analyses is shown in Fig. 4, together with the spectrum of UO 2 2? (aq) for comparison. The monoacetato species shows an absorption maximum at 418.0 nm with a molar absorption coefficient e 418 = 17.8 ± 1 dm 3 mol -1 cm -1 . The spectrum is given in Fig. 4 together with 0.68 % (dashed) and 0.95 % (dotted), obtained from a moving block bootstrap analysis [36]. Within the limits of precision, the UV-Vis absorption spectrum of UO 2 CH 3 COO ? does not show a shift in the absorption maximum or the bands/shoulders in the characteristic absorption region of U(VI) compared to the absorption spectrum of UO 2 2? except in the absorption maximum. The small difference in the position of the absorption maximum of the characteristic band of U(VI) causes the shift observed with the experimental spectra in Fig. 2.
The single component spectrum of UO 2 (CH 3 COO) ? (Fig. 4) is applied to the experimental spectra given in Fig. 1. The physical and chemical parameters of these samples are summarised in Table 2 together with results of a spectral deconvolution using the mean value spectra of UO 2 2? and UO 2 CH 3 COO ? from Fig. 4. Examples of this deconvolution are given in Figs. 5, 6 and 7 for three spectra with widely varying ratio of the U(VI) species concentrations. These spectra illustrate the power of the single component spectrum given in Fig. 4 to quantitatively interpret U(VI) solutions with widely varying physicochemical conditions.
These spectra demonstrate further that the bathochromic shift of the absorption edge to the UV region can be well interpreted quantitatively. These three spectra are the first examples to our knowledge where a single component Table 2 also presents mean value results for the species concentrations from the deconvolution, the calculated free acetate concentrations, the total U(VI) concentrations, the respective U(VI) concentrations as obtained by summing the species concentrations, and a comparison of both U(VI) concentrations in per cent differences.
The quotient of species concentrations is obtained from the quantitative spectral deconvolutions, while the free acetate concentrations may be estimated on basis of the known total acetate concentration, the pH, and the pK A of acetic acid. These quantities are given in Table 2. A graphical summary is given in Fig. 8 where parameters from Table 2 are interpreted by a trend with fixed slope 1 according to Eq. (1). The formation quotient lg b 11 for UO 2 CH 3 COO ? is defined as where [A] denotes molar concentrations of species A. The intercept lg b 11 is obtained as 2.8 5 ± 0.0 5 , where the uncertainty is given on the 95 % level. The evaluation of a formation quotient has not been a main focus of this study because lg b 11 has been previously determined by a variety of methods rather consistently. Respective presentations are available in literature (e.g. recently [12]). We note that the evaluation of this parameter on basis of the derived single component spectrum Fig. 4 falls within this range of literature data.
In the ionic strength range used here (0.01 \ I \ 0.2), values in the range 2.4 \ lg b 11 \ 3.0 have been reported. Notwithstanding a complete uncertainty analysis of the thermodynamic data, the reliability of lg b 11 from this study is tentatively estimated as lg b 11 = 2.8 ± 0.3. While the dashed lines in Fig. 8 give the uncertainty of the slope, the dotted lines give the estimated uncertainty (0.95 %) of predicting a further measurement value on basis of the existing ones. All experimental data points are found within that limit.
Conclusions
The 51 UV-Vis spectra of U(VI) acetate in the concentration range from 9 9 10 -4 mol dm -3 to 1 9 10 -2 mol dm -3 and the pH range from 1.9 to pH 5 were registered. From these, a subset of 14 spectra holding only a single complexed species was obtained using submatrix analysis or computerassisted target factor analysis (CAT) [33,34]. This procedure is routinely applied to resolve UV-Vis absorption data of complex systems, e.g. [37][38][39].
The single component spectrum is assigned to the UO 2 CH 3 COO ? species. It is the only species to consider under given experimental conditions. In the given range of pH, hydrolysis is the only possible interfering reaction. Great care has been taken to avoid hydrolysis species with amounts above 1 %.
The derived spectrum of the UO 2 CH 3 COO ? species has a molar absorption of 17.8 ± 1.0 dm 3 mol -1 cm -1 at the absorption maximum of the characteristic absorption band of U(VI) at 418 nm. This spectrum is able to interpret the Direct spectroscopic speciation of the complexation of U(VI) 1693 14 spectra from which it was derived with only minor residuals. The total U(VI) concentrations in each of the 14 solutions is reproduced within a few percent. From the relative species concentrations of UO 2 CH 3-COO ? and UO 2 2? , the formation quotient could be derived as 2.85 ± 0.05, whereby the figure given after the '±' symbol is a 0.95 % range but only accounting for the misfit of the regression line. It is not a complete measurement uncertainty budget.
The deconvolution of the spectra into the species' individual amounts has been done by a least square residuals criterion. The residuals contribute about 2-3 % of the maximum absorption. With the exception of the spectrum at pH 2.96 given in Fig. 7, the residuals are unspecific. This single spectrum's residuals show a certain fine structure potentially indicative of a small contribution of another species-even if at the edge of detectability. Clearly, the availability of the UO 2 CH 3 COO ? single component spectrum opens the chance to interpret more complex spectra, e.g. those recorded in a range of physical conditions where interference by hydrolysis is possible. Characterisation of hydrolysis components is relevant to avoid misinterpretation as higher acetate complexation. This will be the focus of our future activities.
Experimental
Total U(VI) concentrations were in the range 9 9 10 -4 mol dm -3 to 1 9 10 -2 mol dm -3 . The range of pH was varied between 1.9 and 5.0. Total acetate concentration was varying between 0.003 and 0.14 mol dm -3 . From these information, free acetate concentrations varying between 7.7 9 10 -5 and 5.8 9 10 -2 mol dm -3 are obtained from numerical speciation. U(VI) perchlorate solutions were prepared from UO 2 (CH 3 COO) 2 Á2 H 2 O solid (CHEMAPOL/LACHEMA Co., Warsaw, Poland) by dissolution in water and re-precipitation with H 2 O 2 (20 %). The yellow precipitate was filtered, washed, and heated in a furnace at 200°C (4 h) and 400°C (8 h). The resulting UO 3 solid was redissolved in a stoichiometric amount of perchloric acid (70 %, Fluka Co., Switzerland). A more detailed description of the procedure is given in [40]. The acetate medium was prepared from a standard solution of 0.3 M NH 4 CH 3 COO (POCH S.A. Co., Gliwice, Poland).
Numerical modelling of the sample solutions indicates that ionic strength of the samples varies between I = 0.01 and I = 0.3, hence is found outside the range of diluted
Apparatus and data collection
A double-beam UV-Vis spectrometer (UV-2401 PC, Shimadzu Co., Japan) was used for collecting absorptions in the UV-Vis range. Spectra were recorded quadruply and averaged for noise reduction. Samples were placed in quartz cells with 20 mm path length and recorded digitally in the wavelength range 345-570 nm in 0.1 nm steps with a slit width of 1 nm. Determination of pH was made by a glass combination electrode (ELMETRON pH-meter Cp-315 Co., Zabrze, Poland) following the 5-point calibration scheme described by IUPAC [41,42]. The calibration pH standard solution was traceable material (Merck Co., Darmstadt, Germany).
Data analysis
Speciation was made with the geochemical code PRE-EEQC [43] and the probabilistic speciation code LJUNGSKILE [44]. Geochemical modelling is based on the parameters given in Table 1. Thermodynamic data, if not given otherwise, are taken from the JESS Thermodynamic Database [45,46]. Spectral deconvolutions are made with custom-made codes based on the sequential Simplex [47] using least-squares criteria. Variance-covariance matrices and uncertainties of the spectral curves are estimated from quadratic forms in the minimum of the numerical optimum [48]. If not stated otherwise, uncertainties are given as 68 % confidence intervals. For data derived from small sample sizes, the necessary corrections have been made to transform standard deviations into confidence regions. The uncertainties in spectral decompositions (cf. Figs. 5, 6, 7) are given on the 0.95 % range. None of the uncertainties represents a complete measurement uncertainty budget since only statistical contributions to uncertainty (e.g. misfit) are considered.
Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. | v3-fos-license |
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} | pes2o/s2orc | The use of extracellular DNA as a proxy for specific microbial activity
The ubiquity and relevance of extracellular DNA (exDNA) are well-known and increasingly gaining importance in many fields of application such as medicine and environmental microbiology. Although sources and types of exDNA are manifold, ratios of specific DNA-molecules inside and outside of living cells can give reliable information about the activity of entire systems and of specific microbial groups or species. Here, we introduce a method to discriminate between internal (iDNA), as well as bound and free exDNA, and evaluate various DNA fractions and related ratios (ex:iDNA) regarding their applicability to be used as a fast, convenient, and reliable alternative to more tedious RNA-based activity measurements. In order to deal with microbial consortia that can be regulated regarding their activity, we tested and evaluated the proposed method in comparison to sophisticated dehydrogenase- and RNA-based activity measurements with two anaerobic microbial consortia (anaerobic fungi and syntrophic archaea and a microbial rumen consortium) and three levels of resolution (overall activity, total bacteria, methanogenic archaea). Furthermore, we introduce a 28S rRNA gene-specific primer set and qPCR protocol, targeting anaerobic fungi (Neocallimastigomycota). Our findings show that the amount of actively released free exDNA (fDNA) strongly correlates with different activity measurements and is thus suggested to serve as a proxy for microbial activity. Electronic supplementary material The online version of this article (10.1007/s00253-018-8786-y) contains supplementary material, which is available to authorized users.
Introduction
Extracellular just like intracellular DNA is a ubiquitous component of environmental samples. In the scientific literature, there are several definitions and abbreviations addressing this fraction of DNA. While some authors abbreviated extracellular DNA to eDNA Pietramellara et al. 2009;Gómez-Brandón et al. 2017a;Gómez-Brandón et al. 2017b), others refer to eDNA as environmental DNA (e.g. Taberlet et al. 2012) or simply as extracellular DNA Agnelli et al. 2007), whereas in medicine, freely circulating nucleic acids are defined as cirDNA or CNA (Ziegler et al. 2002). To avoid any misunderstanding, we will introduce the acronym exDNA to refer to extracellular DNA as distinct from intracellular DNA (iDNA). ExDNA has mostly been studied in soils and marine sediments (Torti et al. 2015), where it is accumulated through the lysis of dead (proand eukaryotic) cells (Levy-Booth et al. 2007), active release by living (prokaryotic) cells, allochthonous input of biogenic matter, or transducing phages (Torti et al. 2015;Nielsen et al. 2007;Paget 1994). After being released, easily degradable exDNA can persist, e.g., in the soil environment due to its interaction (adsorption vs. binding) with surface-active colloids of mineral soil or sediment particles, being in that case partially physically protected from enzymatic degradation (Agnelli et al. 2004(Agnelli et al. , 2007Pietramellara et al. 2006;Ceccherini et al. 2009). Due to the additional phylogenetic information related to iDNA, it was proposed that exDNA can be used to improve the evaluation of the soil microbial community composition ) via comparative genetic fingerprinting of the extra-and intracellular Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00253-018-8786-y) contains supplementary material, which is available to authorized users. fraction of the total DNA pool (Agnelli et al. 2004;Ascher et al. 2009;Chroňáková et al. 2013) or via quantitative PCR (Gómez-Brandón et al. 2017b); recently, the two DNA fractions have also been screened in the dead wood environment to monitor deadwood decay (Gómez-Brandón et al. 2017a), and evidence was found that small exDNA molecules exert a specific inhibitory effect on individuals of the same species (Cartenì et al. 2016). exDNA has also been detected and studied in various biofilms, representing one of the major components of the extracellular polymeric substances (EPS) (Wu and Xi 2009). In the EPS matrix, exDNA appears in free form or bound to extracellular proteins, polysaccharides, and other polymers. It is released by autolysis (Montanaro et al. 2011) but also through active release by microorganisms (Nielsen et al. 2007). It performs a number of tasks such as the support of bacterial adhesion, the facilitation of horizontal gene transfer, the enhancement of antimicrobial resistance, and the structural stabilization of biofilms (Okshevsky and Meyer 2015). These properties make exDNA an interesting target to increase the susceptibility of biofilms to antimicrobial treatment offering novel strategies to combat biofilms.
In medicine, exDNA predominantly of endogenous origin plays a role in tissues such as blood vessels, forming complexes with lipids and proteins, occurring within membranebearing particles or bound weakly or tightly to the cell surfaces. In these ways, it is protected from nuclease degradation or recognition by immune cells. It is usually present in low, yet stable, concentrations in healthy subjects but will increase in abundance in subjects with cancer and autoimmune disorders, making it an interesting study object for non-invasive early diagnostics (Rykova et al. 2012).
Thus, each habitat is characterized by specific conditions and thus allows for varying functions and fates of extracellular DNA. However, this exDNA is always either (i) free and thus easily degradable (fDNA), (ii) weakly bound to various particles via inorganic cation bridges with the DNA phosphate group and thus more stable (wbDNA), or (iii) tightly bound to cell membrane proteins through bivalent cations (tbDNA) and well-protected from degradation (Crecchio et al. 2005).
As stated above, the main source of exDNA is thought to be the lysis of dead cells (Levy-Booth et al. 2007) but can also be an active secretion by living cells or the indirect entrance into the environment via, e.g., partly digested feces or via transducing phages (Nielsen et al. 2007). Once released from cells, the most immediate cause of exDNA degradation is through extracellular and cell-associated nucleases, which are ubiquitous in most environments such as soils, marine waters, and sediments (Torti et al. 2015). These enzymes break down the DNA into smaller molecules, facilitating the uptake of the degraded molecules by other microbial cells, where they either serve as building blocks for newly synthetized nucleic acids or are further broken down to essential nutrients (C, N, P) (Torti et al. 2015). The persistence of exDNA in soil depends on a number of factors such as the DNA composition, methylation, or conformation, as well as the prevailing environmental conditions, being slowed down for example by rapid desiccation, low temperatures, high salt concentrations, low pH, or content of expandable clay minerals Crecchio et al. 2005). The persistence of exDNA in soils was estimated to range from few days to several years (Nielsen et al. 2007;Agnelli et al. 2007), although some experiments showed that in soil microcosms, the residual amount of the target exDNA was never higher than 6% of the added amount when extraction was carried out immediately after the DNA addition to soil (Frostegård et al. 1999;Demaneche et al. 2001). In marine waters, calculated turnover rates for dissolved exDNA range from 6.5 to 25 h (Paul et al. 1989) and in surficial sediments from 29 to 93 days (Dell'Anno and Corinaldesi 2004). Furthermore, the measured exDNA survival time in the various extracellular environments strongly depends on the detection method used, e.g., qPCR or fluorometric quantification of exDNA target genes vs. total exDNA. However, while the persistence of DNA in various environments varies greatly due to various factors, the total amount of exDNA within a specific environment is believed to increase with increasing numbers of lysed (dead) cells (Levy-Booth et al. 2007;Ye et al. 2012). If this is true, the amount of exDNA (from lysed, dead cells) could be related to the amount of iDNA (from intact, alive cells), allowing a dead/ live ratio (ex:iDNA) to be calculated. If a time series of such ratios is generated, it could give information about the specific activity of the species, genus, or microbial group of interest. To our knowledge, this has not been done before but could be a relatively easy way to address some major issues present in the field of microbial ecology.
The assessment of specific activity of the entire microbiota, a certain microbial group or even of a single species in a consortium, is interesting for a broad field of applications, such as the evaluation of the functionality of soils, decay stage of deadwood, activity of biofilms, activated sludge, or the microbial consortia in biogas plants. In the latter, the presence or absence and particularly the specific activity of certain archaeal and bacterial groups are important for the generation of biogas, and the activity of certain groups can be used to monitor the performance and stability of the reactor.
So far, activity measurement of a specific microbial group or species is generally quite challenging: Live/dead staining, photometric methods for determining dehydrogenase activity (DHA), or microbial respiration measurements have the disadvantage to target the activity of the whole consortium and as such are not selective. Fluorescence in situ hybridization (FISH) targeting active cell compartments is selective but time-consuming due to the number of technical and biological replicates that have to be analyzed. Moreover, background fluorescence signals and cell cluster forming in complex environments often hamper the FISH approaches (Nettmann et al. 2010). A recent approach combining FISH with flow cytometry promises to detect process-relevant active microorganisms in samples from biogas reactors but requires extensive probe design for specific questions (Nettmann et al. 2013). The most sophisticated way to measure specific microbial activity is the quantification of rRNA applying reverse transcription PCR (RT-PCR) after extraction of RNA and has been applied in a number of recent studies (e.g. Männistö et al. 2013;Hunt et al. 2013). This method, however, has a number of limitations highlighted in a study by Blazewicz et al. (2013). In addition, it is highly error-prone, and time and cost-intensive due to the instability of the RNA and the prolonged workflow.
A specific activity assessment based on DNA would have the advantage to be (i) easily applicable to a variety of environments and species, (ii) relatively fast, (iii) cost-saving with regard to laboratory equipment and extraction kits, and (iv) adjustable in its sensitivity: If, for example, more easily degradable fractions of DNA are used for the activity measurement, changes will be detected faster than with more stable fractions.
This study aims to investigate the suitability of extra-and intracellular DNA ratios (ex:iDNA) as a proxy of specific microbial activity by the following: 1. Establishing a suitable extraction protocol, accounting for all exDNA fractions (classified by their strength of binding) and the iDNA and testing the protocol on fresh and old microbial cultures (BMethod establishment^) 2. Comparison of the ex:iDNA ratio with established activity measurement methods (DHA and RT qPCR) by time series activity assessments of two different microbial consortia (BMethod testing^)
Method establishment
Extraction of fDNA, wbDNA, tbDNA, and iDNA Several methods, such as high-speed centrifugation and membrane filtration, have been used to isolate exDNA (Wu and Xi 2009;Steinberger and Holden 2005;Bockelmann et al. 2006;Ascher et al. 2009). Own optimization experiments suggested the application of low-speed centrifugation (1000 to 5000×g) to discriminate between ex-and iDNA, as a release of exDNA by partial cell lysis through higher speed centrifugation cannot be excluded (data not shown). However, by centrifugation alone, a considerable portion of the exDNA might be lost, as exDNA is often physically or chemically associated with extracellular proteins, polysaccharides, and other polymers of the EPS (Wu and Xi 2009). Therefore, in this study, we applied a method capable of harvesting the various exDNA fractions sequentially, by modifying the method described by Laktionikov et al. (2004), avoiding the lysis of the cells by using only low centrifugation speeds and mild chemical concentrations. To do so, we used a commercial DNA extraction kit designed for soil samples, as it allows for an efficient extraction from environmental DNA samples and has been previously found to work well for the extraction of exDNA and iDNA (FastDNA Spin Kit for Soils, MP Biomedicals, Santa Ana, USA) ). However, it should be noted that DNA kits could contain contaminated DNA and should be handled with care (Vestergaard et al. 2017). Prior to the standard purification of the DNA, several steps were added to discriminate between the various fractions of exDNA and iDNA (cf. (Torti et al. 2015) due to competition between phosphate ions and the phosphate groups in the sugar-phosphate backbone of DNA (Saeki et al. 2010), thus liberating weakly bound exDNA (wbDNA). A further centrifugation (5 min, 5000×g) is applied to yield the weakly bound extracellular DNA in the supernatant (II). & (III) Intensive washing: The nucleic acids tightly bound to membrane receptors (tbDNA) in the remaining pellet were detached by hydrolysis for 5 min with trypsin (0.125% solution in PBS) and stopping the reaction for 5 min with ¼ volume 0.125% trypsin inhibitor solution (0.125% trypsin inhibitor in PBS). Subsequently, the dissolved tbDNA was yielded in the supernatant after centrifugation (5 min, 1000×g). & After this preliminary separation of the various exDNA fractions, the remaining exDNA-free cell pellet was treated according to the manufacturer's instructions in order to disrupt the cells and obtain the iDNA. & Each of the four resulting subsamples were mixed with the required volumes of sodium phosphate buffer and MT buffer from the extraction kit and then treated for 1 h at 37°C with DNAse-free RNAse (0.1 mg mL − 1 , ThermoFisher Scientific, Waltham, USA). The RNAfree subsamples were then treated according to the remaining steps of the manufacturer's protocol of the FastDNA Spin Kit for Soils (MP Biomedicals, Santa Ana, USA), i.e., proceeding with protein precipitation solution (PPS) by adding 250 μL of PPS and later on performing DNA binding using the binding matrix suspension.
Experimental setup
In order to test this protocol, we sampled co-cultures of the a n a e r o b i c f u n g u s ( A F ) C a e c o m y c e s c o m m u n i s (Neocallimastigomycota) and its archaeal symbionts (mostly Methanobrevibacter sp.), isolated from cattle rumen and subcultured in anaerobic serum flasks, filled with a modified medium containing rumen fluid (Leis et al. 2014). These cocultures allow testing for an overall activity as well as for the specific activity of the fungus. Samples were taken in triplicate from cultures of differing age: The first culture was sub-cultured and then fed and kept in the same serum flask for 8 days (fresh), the second for 3 weeks (intermediate = IM) and the third for 14 weeks (old). To refer the results to the amount of microbial biomass, total solids (TS) of the fungal samples were determined by drying at 105°C for 12 h.
In order to assess the influence of each additional extraction step, three approaches were executed, expanding the protocol by one step at a time and measuring DNA amounts before and after the purification with the extraction kit (Bcrude^vs. Bpure^DNA), with crude DNA being measured after the RNA digestion and the removal of proteins by the GENECLEAN® procedure involving protein precipitation solution within the FastDNA Spin Kit for Soils (MP Biomedicals, Santa Ana, USA). These three approaches were called (I) centrifugation approach, discriminating between fDNA and iDNA, (II) mild washing approach, additionally yielding the wbDNA, and (III) intensive washing approach, additionally yielding tbDNA. Furthermore, a normal extraction without discrimination between exDNA and iDNA, yielding total DNA directly from the whole co-culturesample (liquid and solid phase), was executed as a control (IV; classic approach).
The amounts of crude and purified DNA were measured using a Quantus™ Fluorometer and the QuantiFluor® Dye System for double-stranded DNA (dsDNA) (both Promega, Fitchburg, USA). Quality of DNA was checked on 1.5% (w/v) agarose gels using GelGreen™ (Biotium Inc., Fremont, Canada) for staining.
For the intensive washing approach, fungal DNA was subsequently quantified in all subsamples applying a real-time qPCR with primers targeting the anaerobic fungal clade of Neocallimastigomycota as described in the section Bex:iDNA method and qPCR.M
ethod testing
In order to test the ex:iDNA method, two different microbial consortia were tested for their activity by measuring/ monitoring several activity-related parameters over the timespan of Bhigh^to Blow^activity.
Cattle manure-borne consortia (CBC)
Fresh cattle manure was kept at 4°C for 5 days. To boost microbial activity, anaerobic digesters (500 mL reactor Fig. 1 Principle of the proposed method to discriminate between external DNA (exDNA) fractions and the internal DNA (iDNA) of the total DNA pool (metagenome). fDNA free DNA, wbDNA weakly bound DNA, tbDNA tightly bound DNA. Ratio-forming (e.g. exDNA:iDNA) is intended as proxy of microbial activity. Here, exDNA is the sum of the fDNA, wbDNA, and tbDNA fractions volume, n = 3) were filled with 300 mL of cattle manure and incubated anaerobically at 37°C (M_T0). After 48 h of acclimatization, when microbial consortia should be most active, reactors were thoroughly mixed and M_T1 samples were taken. M_T2 samples were taken after 8 days, when most nutrients were assumed to be depleted and activity should have started to decrease. Then, the reactors were transferred to 4°C to suppress the activity of the established mesophilic consortium until day 10 (M_T3), when they were opened and exposed to the atmosphere and left at 4°C for another 10 days to further inactivate strictly anaerobic microorganisms (M_T4). Samples for quantification of enzyme activity (dehydrogenase [DNA]) were analyzed immediately, with DNA samples stored at − 20°C and RNA samples immediately placed in liquid nitrogen until nucleic acid extraction. Overall activity measurements were conducted using the dehydrogenase (DHA) method and the proposed ex:iDNA method. Additionally, specific activity of total bacteria and total methanogens was measured via RNA quantification (RT-qPCR) and via the ex:iDNA method in combination with qPCR.
Rumen-borne consortia (RBC)
In order to further test the proposed ex:iDNA method with samples of different origins still being able to artificially manipulate their activity, an isolate of anaerobic fungus OF1 (C. communis) co-cultured with a consortium of rumenborne archaea was sub-cultured (n = 3). We used serum bottles with 40 mL modified medium M10 containing cellobiose (1.6 g L −1 ), glucose (1.6 g L −1 ), and microcrystalline cellulose (0.6 g L −1 ) plus 20% (v/v) rumen fluid and a vitamin solution (Leis et al. 2014). The cultures were incubated anaerobically at 39°C and samples with a volume of 3 mL were taken immediately (R_T0) and after 1 day (R_T1), 4 days (R_T2), and 7 days (R_T3). Subsequently, bottles were opened and stored at 4°C, in order to reduce mesophilic as well as anaerobic activity and were sampled twice, 9 (R_T4) and 15 days (R_T5) after chilling. For these samples, overall activity measurements were conducted using the DHA method and the ex:iDNA method.
ex:iDNA method and qPCR
All different DNA fractions were extracted as described in the section BMethod establishment^with the modification that tbDNA and wbDNA were not extracted sequentially, but in one single step by adding both solutions, EDTA/PBS and trypsin-solution at the same time and naming this fraction bound DNA (bDNA).
Subsequently, purified DNA of cattle manure samples was used for qPCR targeting total bacteria and total methanogens. The primers, their concentrations, and cycling conditions for all qPCR assays are listed in Supplementary Table S1. Next to established primers targeting total bacteria and methanogens, anaerobic fungi were quantified using a specially designed primer set GGNL1F (5′-CATAGAGGGTGAGAATCCCG TA-3′) and GGNL4R (TCAACATCCTAAGCGTAGGTA). Unlike most fungal primers, these primers are neither targeting the internal spacer region nor the 18S rRNA gene, but the 28S rRNA gene as proposed by Dollhofer et al. (2015). Primers were checked for specificity using Primer-BLAST (Ye et al. 2012) and were found to cover 94% of all available AF sequences (n = 247) but did not show any other target templates in the reference genome database including Opisthokonta (n = 1168). All qPCR reactions (standards and samples) were conducted in duplicate, with the SensiFAST SYBR® Hi-ROX chemistry (Bioline, London, UK) and with 1:10 diluted (methanogens DSMZ800, bacteria DSMZ21879) or undiluted (Neocallimastigomycota KF312496) DNA as a template. For standard curve construction freshly prepared, 10-fold dilutions in 1× TE buffer were used. The R 2 of standard curves was ≥ 0.99. Stock DNA was generated by endpoint PCR on the FlexCycler (Analytik Jena AG, Jena, Germany), and real-time cycling was performed on the Rotor-Gene 6000 Real-Time Thermal Cycler (QIAGEN GmbH, Hilden, Germany). Calculation of gene copy number g −1 total solids (TS) was performed with the Rotor-Gene Series Software 1.7. Amplicon quality check of qPCR via melt-curve analysis was carried out by increasing the temperature at a rate of 0.25°C min −1 , from 65 to 99°C for bacterial community, and 0.20°C min −1 , from 60 to 99°C for the archaeal methanogens, respectively.
RNA extraction, cDNA synthesis, and RT-qPCR
In order to obtain a reference value to the ex:iDNA, RNA was extracted for all time points (M_T1-M_T4) and a reverse transcription qPCR (RT-qPCR) was carried out targeting the same microbial groups with the same qPCR conditions. RNA was extracted using the FastRNA® Pro Soil Direct Kit (MP Biomedicals, Santa Ana, USA) according to the manufacturer's instructions with the following modifications: Two technical replicates of each sample with 0.5-0.8 g were mixed with 750 μL RNApro™ Soil Lysis Solution and with 500 μL of phenol/chloroform (1:1). Both technical replicates were merged again before adding RNAMATRIX® Binding Solution and RNAMATRIX® Slurry. To increase RNA yield, final elution was performed in two successive steps in 25 μL DEPC-H 2 O (AppliChem GmbH, Darmstadt, Germany). To remove residual DNA, the RNA extract was subjected to a DN ase assay w ith the TU RBO D NA-free™ K it (AMBION®, ThermoFisher Scientific, Waltham, USA) and further purified using the Agencourt AMPure XP Kit (Beckman Coulter, Inc., Brea, USA). The RNA extracts were checked for concentration and length with the Agilent RNA 6000 Pico Kit and the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA). Complementary DNA was synthesized using the SensiFAST™ cDNA Synthesis Kit (Bioline, London, UK) with 5 μL purified RNA as template. Finally, complementary DNA products were used for qPCR, targeting total bacteria and methanogens according to the procedure described above.
As internal quality and reference control, 4 μL artificial RNA (RNA Extraction control 610, Bioline, London, UK) was added to the samples prior to RNA extraction. The final yield of this internal reference was used to correct the final RNA amounts for the loss during RNA extraction, cDNA generation, and qPCR cycles.
Evaluation of the ex:iDNA fractions and ratios
Ultimately, the measured DNA amounts were used to generate eight different ex:iDNA ratios including iDNA:exDNA, i D N A : b D N A , i D N A : f D N A , i D N A : t o t a l D N A , bDNA:iDNA, bDNA:totalDNA, fDNA:iDNA, and fDNA:totalDNA. All results were normalized to a range of 0 to 1 to evaluate (i) the suitability/reliability of these ratios in terms of extraction and quantitation procedure, (ii) the various DNA fractions with regard to their accumulation or reduction during various states of activity, and (iii) the comparability of the data. In order to test for correlations avoiding the problem of spurious relationships in time series data, we tested correlations among all data by firstly calculating the magnitudes of increase or decrease between the normalized results of two consecutive time points and secondly determining the Pearson's correlation coefficient of the generated data in R (R core team 2012).
Method establishment
Generally, the more washing steps were performed, and the higher amounts of exDNA were yielded. A separation of exDNA and iDNA by centrifugation (I) and all further treatments (II and III) caused an increase in total crude DNA yields compared to the classical approach (IV).
Prior to the GENECLEAN® procedure, crude iDNA accounted for 11 to 47% of the sum of all sequentially extracted DNA fractions, while the exDNA accounted for 53-86% (fDNA), 1.7-3.2% (wbDNA), and 0.8-8.4% (tbDNA). After purification, all resulting fractions became PCR-compatible; the mentioned ratios changed, however, the highest percentage remaining for iDNA (58-97%), followed by fDNA (3-37%), tbDNA (2-13%), and wbDNA (0.3-3.6%). Furthermore, a high amount of the DNA was lost during the purification process, resulting in total DNA from 4 to 55 ng DNA mg total solids (TS) −1 after purification compared to 153-207 ng crude DNA mg TS −1 (Fig. 2a, b). The recovery rates increased with the stability of the respective DNA fractions, being highest for iDNA, followed by tbDNA, wbDNA, and fDNA (Fig. 2c). In general, the recovery rates were up to 10-fold higher for tbDNA and iDNA of the fresh cultures when compared to the intermediate and old cultures. The length of the purified DNA was 500 to 600 bp for all DNA fractions as well as for the total DNA obtained by the classical approach (IV). An additional band with very long DNA (> > 1000 bp) was present for the iDNA and for tbDNA and wbDNA in the intermediate sample (Fig. S1).
The absolute amounts of DNA in the consortium revealed that around 10% of the overall DNA is of fungal origin (Fig. 3a, b) and that the relative amounts of exDNA were greater in the whole consortium compared to fungi alone (Fig. 3c, d). Regarding the DNA deriving from C. communis and associated symbionts, summed up DNA amounts were decreasing (fresh 44.8 ng mgTS −1 , IM 9.4 ng mgTS −1 , old 3.6 ng mgTS −1 ), while relative amounts of exDNA were increasing (fresh 10%, IM 39%, old 42%) with culture age (Fig. 3a, c). The same was true for the qPCR results targeting AF (C. communis), where summed up DNA amounts accounted for 5 ng mgTS −1 (fresh), 3.8 ng mgTS −1 (IM), and 0.6 ng mgTS −1 (old) and relative amounts of exDNA range from 2.5% (fresh), to 5.5% (IM), and 17.7% (old).
Looking at the results for the activity measurements, the overall CBC reached a first maximum in activity (DHA) during anaerobic incubation at 37°C (T1), decreasing at T2 (nutrient depletion) and rising again after being set at 4°C (T3) and after oxygenation (T4) (Table 1, Fig. 4a). Similar to the overall consortium, bacterial RNA suggested that their maximum activity was reached at T1 but decreased with depletion of nutrients at T2 and after cooling at T3, while it increased with oxygen supply (T4) (Table 1, Fig. 4b). For methanogens, RNA reached a maximum at T2 and decreased at 4°C (T3) and with oxygen supply (T4) (Table 1, Fig. 4c). For the overall RBC, DHA showed an absolute maximum shortly after incubation at 39°C and decreased over time from T2 to T5 (Table 1, Fig. 4d).
When correlated to the established activity measurements, fDNA resulted in the highest correlation coefficients, with a mean positive correlation of r = 0.726 and showing consistently significant correlations for all four levels of resolution (r = 0.838 for DHA in CBC, r = 0.863 for RNA bacteria, r = 0.631 for RNA methanogens and r = 0.572 for DHA in RBC) ( Table 2). Next to fDNA yields, both ratios, fDNA:totalDNA and fDNA:iDNA, showed the best fitting ratios, resulting in a mean r of 0.665 and 0.670, respectively, but no significant correlations were found for two pairs (DHA in RBC for fDNA:totalDNA and RNA bacteria for fDNA:iDNA).
Comparing the three best fitting proxies (fDNA yield, fDNA:totalDNA, and fDNA:iDNA) to the DHA measurement, they suggested a steeper decrease in activity over time ( Fig. 4a) but showed almost the same trend, resulting in a first maximum at T1, a decrease with depletion of nutrients (T2), and an increase with temperature change (T3). At T4, however, the three proxies suggested a slight activity decrease rather than an increase. Similarly, fDNA yields, fDNA:totalDNA, and fDNA:iDNA showed the same trend as RNA for the bacterial activity from T1 to T3, but a slight decrease rather than an increase in activity at T4 (Fig. 4b).
For methanogens, fDNA yields, fDNA:totalDNA, and fDNA:iDNA gave the same activity pattern as RNA. They showed, however, a steeper decrease in activity at T3 than the decrease indicated by the RNA approach.
For the RBC, all three proxies indicated a peak of activity during T1 and a consistently low activity for all other time points except for T5, where fDNA:totalDNA showed an increase in activity, in contrast to the DHA results. Furthermore, DHA results suggested a less steep decline in activity as compared to the exDNA proxies and a phase of stable activity between T2 and T3, being only marginally visible in fDNA yields and fDNA:totalDNA.
Method establishment
The three-step washing of the cells allowed for a high yield of exDNA, the major part of which is fDNA. Although it has to be kept in mind, that minor cell lysis may occur during each of the extraction steps, the increased yields in total DNA with each additional washing step are well in line with findings of sequential extraction by Ascher et al. (2009) andWagner et al. (2015). After the DNA purification process, however, amounts of exDNA decreased remarkably, i.e., about 99% for fDNA, 97% for wbDNA, 64% for tbDNA, and 60% for Roman numbers indicate the treatment conducted to discriminate among different DNA fractions. I centrifugation, II mild washing, III intensive washing, IV classic extraction. c Mean recovery rates for the different DNA fractions after purification. TS total solids. fDNA free extracellular DNA, wbDNA weakly bound extracellular DNA, tbDNA tightly bound extracellular DNA, iDNA internal DNA iDNA and pointing to a higher recovery rate of iDNA compared to exDNA. These results suggest that (I) despite the manufacturer's claims, the crude DNA quantified by a fluorometer is not only crude DNA but is biased by fluorescent substances (such as RNA and other oligonucleotides) and/or that (II) these substances, but most probably also parts of the DNA, were washed out due to their DNA-like properties during the purification procedure. Such DNA loss within the purification procedure is unavoidable and well documented whenever PCR-compatible DNA is required for downstream analyses (Fornasier et al. 2014;Braid et al. 2003;Robe et al. 2003;Roose-Amsaleg et al. 2001). Using the FastDNA™ Spin Kit for Soils (MP Biomedicals, Santa Ana, USA), a kit highly recommended for (anaerobic) sludge (Guo and Zhang 2013), the DNA purification is performed mainly through binding to a binding matrix, utilizing the negative charge of DNA. The shorter the DNA molecule, the poorer is its binding to the matrix and the greater are the losses during the purification process. This leads to variable recovery rates of the various exDNA fractions after purification. According to their stability in the extracellular space, fDNA molecules are easily degradable and thus quickly lose their length, while wbDNA and especially tbDNA seem more protected and thus better recovered by the binding matrix during the purification process. The length of the purified DNA, however, was the same for all fractions, suggesting that fragments shorter than 600 bp are washed out (lost) during the purification process or are simply not yieldable by the FastDNA™ Spin Kit for Soil. According to the manual, the size of the extracted DNA is expected in the range from 4 to 20 kb.
The qPCR procedure targeting a specific microbial group or species was further affecting the ratios of DNA fractions, as this step is again very sensitive to short or damaged DNA fragments: In this approach, the qPCR results only represented about 10% of the total extracted DNA.
These results clearly suggest that each of the considered exDNA fractions are crucial to generate a meaningful value expressing the (relative) activity of a specific microbial group. A separate measurement of wbDNA and tbDNA, however, is not necessary as they represent only small parts of the total DNA and show similar characteristics (recovery, size).
Activities
For CBC, all activities measured with DHA and RNA showed a typical batch culture activity pattern until the time point of a change of conditions. First, activities increased due to favorable conditions, warm temperatures, and sufficient nutrients. Then, activities dropped due to a depletion of nutrients (T2).
For the overall CBC, the temperature shock after T2 from 37 to 4°C leads to increased activity, probably due to the establishment of psychrophilic microorganisms consuming the remaining nutrients. Finally, the change to aerobic conditions (after T3) enabled the growth of aerobic microorganisms and was further increasing the activity (T4).
Regarding bacteria, the temperature change led to a decrease of the activity of the established thermophilic bacterial consortium, while the change of the system to aerobic Fig. 3 a, b Mean absolute and c, d relative DNA amounts of approach III (intensive washing) (n = 3) for the total extracted DNA consisting of DNA from Caecomyces communis and associated archaeal symbionts measured photometrically (a, c) and the fungal DNA measured via qPCR (b, d). fDNA free external DNA, wbDNA weakly bound external DNA, tbDNA tightly bound external DNA, iDNA internal DNA Table 1 Mean values of various fractions of purified DNA, RNA, and dehydrogenase activity and associated standard deviations in italics (n = 3) conditions caused an increase in bacterial RNA, suggesting an increased activity of aerobic bacteria. For methanogens, activity decreased consistently at 4°C and further with aerobic conditions. Obviously, the established methanogenic archaea deriving from the cattle intestine are adapted to mesophilic conditions and are obligate anaerobes (Angel et al. 2012).
The RBC generally showed a relatively low activity pattern with regard to DHA of CBC, pointing to more poorly adapted microorganisms to the artificial rumen medium and to a stressed consortium due to various sub-culturing processes during long-term cultivation (Leis et al. 2014). The DHA shows an activity peak immediately after sub-culturing, when nutrients are abundant and conditions are most favorable. Then, activities remained stable on a lower level while incubated anaerobically at 39°C but decreased with lowered temperatures and under aerobic conditions. These results are well in line with the common observation during the cultivation of anaerobic fungi and their associated bacteria and archaea, which requires feeding or sub-cultivation every 2 to 3 days (Leis et al. 2014) and is done under anaerobic and mesophilic conditions.
Intracellular DNA (iDNA)
Regarding CBC, iDNA represents the largest fraction in all levels of investigation (overall consortium, bacteria, methanogens), and especially in qPCR results, with 10-100 times more iDNA than any of the other fractions. It is possible that this DNA is of higher quality/purity and is thus amplified more efficiently, consistent with previous studies assessing DNA yields photometrically ) and using qPCR (Chroňáková et al. 2013;Gómez-Brandón et al. 2017a, b). However, in the RBC, iDNA was not the dominant form of DNA and in some cases bDNA was up to 20 times more abundant. This is probably resulting from a higher general lysis rate due to harsh growth conditions and a higher stress rate also confirmed by the generally much lower DHA in the RBC.
Over time, iDNA increased for all levels in the CBC, deriving from either active or inactive intact cells or from dead inactive but not yet lysed cells. In the RBC, no accumulation of cells was reported over time through the measurement of iDNA, but rather a delayed activity pattern was observed, with the overall peak occurring during T2 and then decreasing until T5. Again, this time trend suggests an increased stress level in the RBC, where inactive cells lyse soon after inactivation and cause the iDNA values to form a time trend that mimics the measured activity pattern with some delay. For the CBC, however, these dynamics were not observed, probably due to the better and more favorable growth conditions found by CBC when incubated in fresh cattle manure, representing their natural substrate, providing all essential nutrients. In such an environment, accumulation and longer persistence of active as well as inactive (dormant) cells are favored, causing a stable increase of iDNA.
Bound extracellular DNA (bDNA)
Looking at the variation in bDNA, the pattern is similar for all levels of investigation in the CBC. In general, bDNA increased over time and decreased only after a change of conditions such as temperature (T3 CBC) and a shift to aerobic conditions (T5 RBC). Possible explanations could be (i) adaptation of microorganisms to the new conditions by the integration of potentially helpful DNA during horizontal gene transfer (Chen and Dubnau 2004), (ii) increased nuclease activity as a result of increased activity of the newly establishing consortium, or (iii) solubilization of bDNA to fDNA, which, however, is only visible in the results for the overall consortium and not for bacteria or methanogens alone.
Free extracellular DNA (fDNA)
fDNA is the smallest fraction when looking at the CBC and RBC overall consortia but is still an important contributor to the total DNA pool (metagenome). For CBC, it is the only fraction with a changing time pattern for different microbial levels of resolution, while for the overall consortium and for bacteria, it shows a peak at T1, methanogens peak at T2. This is a first hint that fDNA could be sensitive enough to account for activity changes of different groups of microorganisms. Interestingly, the amount of fDNA is proportional to the measured activity (DHA or RNA), even though the assumption was that fDNA would increase with less activity. Our results suggest, however, that more active cells secrete higher amounts of fDNA. This finding is also supported by the study of Draghi and Turner (2006), stating that intact cells are performing a specific secretion of DNA and that cell death alone could not always account for the levels of extracellular DNA. There is also evidence that autolysis-independent DNA release plays a role especially in biofilms and that eukaryotes can also be a donor of exDNA (Vorkapic et al. 2016). Recent studies using ex:iDNA ratio as a proxy for microbial activity (assuming that a lower ratio points to higher microbial activities due to the exDNA release after cell lysis) (Gómez-Brandón et al. 2017a, b) showed surprising results; while microbial activity (ex:iDNA) in various decay classes of wood was higher in north-facing slopes when compared to southfacing ones, reflecting thermal signals (different temperature, moisture, and pH) due to different sun exposure, no particular pattern was found for consecutive decay classes. A reinterpretation using our proposed specific fDNA as a proxy for microbial activity would still detect a higher microbial activity at north-facing slopes but would also hint to an increasing microbial activity with increasing age of the investigated deadwood. Such activity increase with progressing deadwood decay would also be confirmed by increasing nutrient contents, microbial abundances, and physical wood damages.
The high correlation of fDNA with the activity measurements was confirmed by the Pearson's correlation (Table 2), showing the highest value for fDNA for all three levels of resolution and both consortia. Linked to this high correlation, ratios including the fDNA-fraction (fDNA:iDNA and fDNA:totalDNA) showed high correlations but could not reach the correlation levels of fDNA alone. The use of total DNA alone, however, was confirmed to be not suitable as estimator/proxy of microbial activity, a finding also made by many others, given that presence does not imply activity (de Vrieze et al. 2016). In fact, in the case of total DNA, no discrimination between iDNA and the various fractions of exDNA is possible Ceccherini et al. 2009).
Furthermore, our findings suggest that general bDNA levels could be an indicator (quantitative descriptor/estimator) of the general stress level of a culture, being low with regard to , and methanogens (c) in cattle manure-(a-c) and rumen-borne consortia over time (hours). CBC cattle manure-borne consortium, RBC rumen-borne consortium, DHA dehydrogenase activity, fDNA free external DNA, total DNA (totDNA) fDNA + bDNA + iDNA, bDNA wbDNA + tbDNA. Vertical error bars show the standard deviation (n = 3) iDNA for not stressed consortia and high for stressed consortia (such as in the case of RBC).
The investigation of the most auspicious activity proxy (fDNA) and calculated ratios (fDNA:iDNA and fDNA:totalDNA) revealed some major discrepancies from the measured activities (DHA and RNA) for fDNA:iDNA and fDNA:totalDNA (Fig. 4): although showing a similar trend, both tended to underestimate the activity due to the increased iDNA of intact cells accumulated over time. fDNA alone, however, traced the normalized activity of DHA or RNA most accurately. This is most probably due to DNA that is actively released by living cells: Being different in its characteristics (Lorenz et al. 1991), it is naturally of better quality than fDNA deriving from cell lysis, which is immersed in a solution of cell components including nucleases, making the DNA fragments shorter and easily accessible for uptake by other organisms (Nielsen et al. 2007). This property makes it easier to be retrieved with the purification method used here, and it seems that only actively released fDNA is being quantified. Many bacteria, including Acinetobacter, Alcaligenes, Azotobacter, Bacillus, Flavobacterium, Micrococcus, Neisseria, and Pseudomonas, are known to actively release (extrude/secrete) DNA during growth, as do some archaea, in particular methanogens (Nielsen et al. 2007). Our results, however, strongly suggest that bacteria and archaea (methanogens) are actively releasing fDNA of high quality that is preferably detected after purification, compared to fDNA derived from lysed cells and that it is a useful proxy for the activity of DNA-secreting (i.e. growing) cells.
Some minor deviations from established activity measures have been found with increased time of experiment ( Fig. 4a, b T4, c T3) for the CBC and with increased stress (Fig. 4d T2 + 3 + 4) for the RBC. During T3 and T4, stress increased in the cattle manure environment too, as temperature was set to 4°C and anaerobic culture flasks changed to aerobic conditions: Overall activity increased due to establishment of psychrophilic (T3) and of aerobic microorganisms (T4), but the previously established CBC was decaying. During T4, less fDNA than expected was being secreted, or more fDNA was metabolized by other establishing microorganisms. In fact, DNA has been reported as a substrate for microbes to feed on (Finkel and Kolter 2001;Levy-Booth et al. 2007;Nielsen et al. 2007;Pietramellara et al. 2009). For the RBC, measured DHA was considerably higher for T2 to T4 than the normalized amounts of fDNA but was still showing the same pattern. However, as observed for CBC, less fDNA than expected was measured. With the data generated here, we cannot give a clear answer to this observation, but as stated above, they are hinting to either less production by the active microorganisms or to an increased uptake by the more active establishing consortium. This new consortium could use the secreted DNA for several purposes: (i) for genetic diversity, i.e., horizontal gene transfer; (ii) as a repair tool, where DNA of closely related microorganisms could be used to repair DNA damage; and (iii) as substrate serving as a source for phosphorous and nitrogen (Chen and Dubnau 2004). Whether this underestimation of activity during changing conditions is a general pattern or is specific for this experiment needs to be investigated in further studies.
Our findings suggest that extracellular DNA, especially free (not adsorbed) DNA, is a promising candidate to be used as a proxy for microbial activity. We are aware, however, that further experiments are needed to further elucidate possible lysis effects during the washing steps of the DNA extraction and to validate the reliability and accuracy of the proposed method in other environments and different experimental conditions. | v3-fos-license |
2020-04-01T13:57:38.152Z | 2019-11-26T00:00:00.000 | 214729988 | {
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} | pes2o/s2orc | Role of glutathione redox system on the susceptibility to deoxynivalenol of pheasant (Phasianus colchicus)
There are only a few reports on the effects of mycotoxins on pheasant (Phasianus colchicus) and the susceptibility to deoxynivalenol of these birds have never been reported before. The present experiment focuses to investigate the effects of different dietary concentrations of deoxynivalenol on blood plasma protein content, some parameters of lipid peroxidation and glutathione redox system and on the performance of pheasant chicks. A total of 320 1-day-old female pheasants were randomly assigned to four treatment groups fed with a diet contaminated with deoxynivalenol (control, 5.11 mg/kg, 11.68 mg/kg and 16.89 mg/kg). Birds were sacrificed at early (12, 24 and 72 h) and late (1, 2 and 3 weeks) stages of the experiment to demonstrate the oxidative stress-inducing effect of deoxynivalenol. Feed refusal was dose dependent, especially in the last third of the trial, but only minor body weight gain decrease was found. Lipid-peroxidation parameters did not show dose-dependent effect, except in blood plasma during the early stage of the trial. The glutathione redox system, reduced glutathione content and glutathione peroxidase activity, was activated in the liver, but primarily in the blood plasma. Glutathione peroxidase activity has changed parallel with reduced glutathione concentration in all tissues. Comparing our results with literature data, pheasants seem to have the same or higher tolerance to deoxynivalenol than other avian species, and glutathione redox system might contribute in some extent to this tolerance, as effective antioxidant defence against oxidative stress.
Introduction
Trichothecenes are a group of mycotoxins having over 180 structurally related compounds [1,2] primarily produced by Fusarium molds. All of these toxins characterized by a tetracyclic 12, 13-epoxytrichothec-9-ene skeleton and an olefinic bond with different side chain substitutions [3]. The clinical signs of trichothecene toxicity are weight loss, lower feed conversion ratio, feed refusal, emesis, bloody diarrhea, dermatitis, coagulopathy, necrosis and hemorrhage. These latter signs occur mainly in the mitotically active tissues [4]. Trichothecenes are potent inhibitors of protein synthesis due to the interaction of 60S ribosomal subunit in the eukaryotic cell [5]. In the trichothecene family deoxynivalenol (DON, vomitoxin) is the most prevalent mycotoxin in the world [6]. Among farm animal species swine is considered to be particularly sensitive to DON while poultry species followed by ruminants have lower sensitivity, possibly due to low bioavailability in these species [7]. This fact is supported by several literature data in which adverse effects of DON can be seen at low (1 ppm) dietary concentration in swine [8], while much 1 3 higher levels even at 18 ppm [9,10] and 66 ppm [11] have not been associated with impaired productivity in gallinaceous poultry and dairy cow, respectively. In poultry, the toxicity of DON is manifested through reduced growth rate, anemia, and decreased serum triglyceride level [12] increased relative gizzard weight [13] reduced feed intake and oral lesions [14]. It can negatively effect several egg quality parameters such as egg shell weight and albumen height [10]. High load of DON have a potential to impair immunity [15] as it results in ribotoxic stress response due to inhibiting the expression genes encoding pro-inflammatory cytokines [16]. In broiler chicken, DON is confirmed to be associated with formation of oxygen free radicals, therefore it can induce membrane and DNA damage [17]. It is clear that mycotoxin research is out of the scope of the experiments focuses on game bird species. Only the toxic effects of aflatoxin B1, ochratoxin A and T-2 toxin in partridge [18], pheasants [19,20] and quail [21] are described. There are information about the aflatoxin B1 and T-2 toxin tolerance of mallard ducks [22] and so far this is the only game bird where exposure to DON have been studied [23]. According to the results of Boston et al. [23], mallard ducks do not avoid DON contaminated cereal grain (5.8 ppm DON) and no adverse clinical or pathological effect could be confirmed due to the consumption of contaminated diet. Since the intensity of growth is a major factor associated with susceptibility to DON [9,12,13], hypothetically, higher tolerance can be expected in a relatively slow growing game bird. However, DON toxin tolerance of the pheasant (Phasianus colchicus) has never been reported before. The purpose of present study was to investigate the effects of short and long term DON exposure on the birds' performance, rate of lipid-peroxidation and on the antioxidant defense system of young female pheasants.
Experimental birds and diet
A total of 320 day-old female pheasants were purchased from a hatchery and transported to the experimental facility of the Department of Nutrition, Szent István University. Birds were housed in 1 m diameter rounded wall pens. Feeding was based on a commercial pheasant diet, which was supplied in mash form. During the experiment the feed and drinking water were provided ad libitum. Feed consumption was recorded daily for each group, while individual live weight was measured before the extermination of the birds. All of the birds were exterminated by cervical dislocation and exsanguination.
Mycotoxin production and experimental contamination of diet
In the experimental period feed was artificially contaminated with deoxynivalenol. DON produced by Fusarium graminearum (NRRL 5883) strain on corn substrate [24]. The basal diet (crude protein: 23.88%, crude fiber: 3.90%, crude ash: 8.30%) was artificially contaminated with ground corn containing 16,324 mg/1000 g DON toxin to reach the desired toxin concentrations. Predicted toxin levels were 5, 10, and 20 mg/kg feed in the three treatment groups, and the measured DON content of the feed was determined according to Pussemier et al. [25] by HPLC method with fluorescence detection after immunoaffinity cleanup (Table 1).
Ethical issues
The experiments were carried out according to the regulations of the Hungarian Animal Protection Act, in fulfillment with the rules of EU. The experimental protocol was authorized by the Food Chain Safety, Land use, Plant and Soil Protection and Forestry Directorate of the Pest County Governmental Office (PE/EA/1965-7/2017).
Experimental design
A total of 320 day old female pheasant were randomly assigned to four groups with different concentration of DON toxin (control, Low dose DON, Medium dose DON and High dose DON) with two replicates (8 pens with 40 birds in each pen). The intended toxin levels in feed (5 mg/ kg feed, 10 mg/kg feed and 20 mg/kg feed) were based on previous works of, Kubena et al. [9], Huff et al. [12], Prelusky et al. [26] and Xu et al. [27] as in poultry the negative effects of deoxynivalenol occurred at relatively high toxin levels (10 mg/kg or higher). From day 1 to day 9 birds were allowed to get accustomed to the environment. In this period all the animals were fed with the control feed. Treatments started with the 1 week old birds and lasted for 21 days. Both short and long term effects of DON exposure were investigated as six birds from each group were sacrificed at the early (12th, 24th and 72nd h) and the late (day 7, 14, and 21) stages of the trial. After extermination post mortem blood and liver samples were taken. Blood samples were centrifuged (1500 rpm) for 10 min in order to isolate plasma. Red blood cell haemolysate (1:9 v/v) was also prepared with adding ninefold volume of redistilled water to RBC. Whole liver was taken and weighed, and lower third of the large lobe was dissected and packed for further analyses. All the samples were stored at −70 °C. For further analysis, liver samples were homogenized in 1:9 v/v physiological saline.
Lipid-peroxidation
In the fresh liver homogenate (1:9 w/v in physiological saline) conjugated dienes (CD) conjugated trienes (CT) [28], as well as malondialdehyde (MDA) concentrations were measured to analyze the state of lipid peroxidation [29]. In blood plasma and red blood cell haemolysate only the metastable end-product of lipid peroxidation, malondialdehyde [30] concentration was determined.
Antioxidant system
To study the antioxidant system, some parameters of the glutathione redox system were investigated by measuring the reduced glutathione (GSH) content [31], and glutathioneperoxidase (GPx) activity [32] in the 10,000 g supernatant fraction of liver homogenate and in the blood plasma and RBC haemolysate. GSH content and GPx activity was calculated to protein content which was determined by biuret reaction [33] in blood plasma and RBC, while Folin-phenol reagent [34] was applied for the 10,000 g supernatant fraction of liver homogenates.
Statistical analysis
Statistical analysis of the data was carried out with the use of GraphPad InStat 3.05 software (GraphPad Software, San Diego, CA, USA). First Kolmogolov-Smirnov normality test was done and based on the results either parametric one-way analysis of variance (with Tukey's post hoc test) or non-parametric Kruskal-Wallis test (with Dunn's post hoc test) was carried out.
Feed intake, weight gain, and feed conversion ratio
Slight, clearly dose dependent feed refusal was revealed through the whole experiment ( Fig. 1) and it was especially obvious in the last third of the trial. The average individual daily feed intake in the Low, Medium And High dose DON groups was 7.2, 10.6 and 12.1% lower than that of the control, respectively. The body weight of the birds however, was not in accordance with the trends in the feed intake, thus the highest values were measured primarily in the Medium DON group (Fig. 2)
Clinical signs of toxicity and mortality
Increased mortality can not be attributed to DON toxicity as low level of mortality was seen in all four groups (Table 2). Also no clinical signs of toxicity have been found in the dissected animals.
Changes of lipid peroxidation parameters
There were no changes in the marker of lipid peroxidation, malondialdehyde, levels in the early stages of the experiment in blood plasma. However, on the second week elevating tendency was seen in the Medium dose group as compared to the control, however the difference was not significant. Later, on the third week, MDA levels were significantly higher in the Medium and High dose groups as compared to the control (Table 3). MDA levels did not changed significantly in red blood cell haemolysate in the first week of the experiment, but on the second week higher MDA levels were observable in the Medium and High dose groups as compared to the Low dose and control groups, however, the difference was significant only as compared to the Low dose group. No further changes or tendencies were seen later ( Table 3). Markers of the initial phase of lipid peroxidation, conjugated diene and triene levels, did not changed significantly through the experiment in liver homogenate, regardless of the toxin level or the time of the exposure (data not shown). After 12 h of exposure MDA content, as marker of termination phase of lipid peroxidation, was remarkably the highest in the High dose group, but the difference was statistically confirmed only for the Low and Medium dose groups. Similar tendencies were seen after 24 h of exposure, however no significant difference was found. On the third day of the experiment a marked decrease was observable in the Low dose group, however the only significant difference was found as compared to the Medium dose group ( Table 4).
Changes of a low molecular weight antioxidant, reduced glutathione, content
The elevation of GSH levels in blood plasma in the treated groups were observable both in the early and late stages of the experiment (Fig. 3a). After 24 h of DON exposure GSH levels was significantly higher in the Medium and High dose groups as compared to the control and to the Low dose groups. Later, the GSH content increased significantly in the High dose group as compared to the control after 1 and 3 weeks of exposure (Fig. 3a). Remarkable changes in the GSH levels in red blood cell haemolysate were observable only in the early stages of the experiment (Fig. 3b). After 12 h of exposure GSH concentration was the highest in the High dose group, however, the difference was statistically significant only for the Medium dose group, and in the next 12 h GSH levels decreased significantly in all three treatment groups as compared to the control (Fig. 3b). GSH level was significantly elevated in the 10,000 g supernatant fraction of liver homogenate only after 2 week of exposure in the Medium and High dose groups as compared to the control (Fig. 3c).
Changes of the antioxidant enzyme glutathione peroxidase
During the experiment the GPx activity has changed parallel with the concentration of GSH in blood plasma. Namely, GPx activity did not change significantly in the first 3 days of the experiment, but after 1 week of DON exposure elevated significantly in the Low dose group, however increasing tendency was seen in the High dose group, as compared to the control. After 3 weeks of exposure GPx activity was significantly higher in the Low and High dose groups as compared to the control (Fig. 4a). GPx activity changed similarly to GSH level also in red blood cell haemolysate, thus significantly higher GPx activity was found in the High dose group as compared to the Medium dose group, but lower enzyme activity was measured in the Low dose and the control groups (Fig. 4b). GPx activity increased significantly after 1 week of exposure in the High dose group as compared to the control in the 10,000 g supernatant fraction of liver homogenate. On the second week GPx showed increasing tendency in the two higher toxin dose groups, however no significant difference was found as compared to the control (Fig. 4c).
Discussion
Gallinaceous poultry is known to be particularly tolerant to deoxynivalenol, therefore the consumption of high dose of DON is not necessarily accompanied with decreased weight gain [14,35], and even significantly higher body weight was measured in chickens fed with DON contaminated diet [9]. Despite the high tolerance to deoxynivalenol kg T-2 toxin. At higher doses, however, T-2 toxin resulted considerably lower feed intake than DON. T-2 toxin also affected weight gain, feed conversion ratio and mortality more severely than DON, especially in the medium and high toxin groups [20]. Due to the DON exposure, some changes in the biochemical parameters were seen in all observed tissues, and their tendencies were similar. However, blood plasma was the most informative as most of the significant differences were found here. Elevation in the MDA concentration, occurred primarily in the two higher dose groups (11.68 and 16.89 mg/kg feed) and in the late stages of the experiment indicating that high dose and prolonged exposure are required to induce oxidative stress in pheasant. Trichothecenes known to inhibit protein synthesis [40] and the decreased level of plasma protein in the Low and Medium dose groups after 21 days of exposure is a possible sign of that effect. Similar results was found in broiler chicken after long term (42 days) exposure even with a relatively low level (3 mg/kg) of DON contamination [41].
GSH as a small molecular weight antioxidant was present in elevated concentration in blood plasma at the early and late stages of the experiment as well, and resulted parallel in increased GPx activity. Similar changes were revealed in the liver, but only at the end of the experimental period. These elevations in GSH content and GPx activity are possibly indicating that the consumed amount of DON was high enough to trigger and activate the antioxidant defense of the birds, however it was too low to maintain the activation through the whole period of the experiment. The increased GSH levels might be also the result of glutathione reductase activation due to oxidative stress induced by DON as it was revealed in another study [42], but it was not studied in our experiment. On biochemical level the major difference between T-2 toxin [20] and DON was the time of exposure required to trigger significant changes. T-2 toxin decreased protein concentration but increased GSH concentration and GPx activity from the beginning of the experiment meanwhile these effects primarily expressed after a long term (at least 1 week) of DON exposure.
It can be concluded that pheasant can tolerate short-term DON exposure even at high toxin level since most of its adverse effects such as reduced feed consumption, lowered blood protein concentration due to potential inhibition in protein synthesis or increased lipid-peroxidation have occurred only in the late stages of the experiment. It is also clear that these effects cannot be considered to be detrimental even at long term. | v3-fos-license |
2017-03-31T08:35:36.427Z | 2017-03-03T00:00:00.000 | 15882381 | {
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} | pes2o/s2orc | Recovery Process of Li, Al and Si from Lepidolite by Leaching with HF
: This work describes the development of a new process for the recovery of Li, Al and Si along with the proposal of a flow sheet for the precipitation of those metals. The developed process is comprised of lepidolite acid digestion with hydrofluoric acid, and the subsequent precipitation of the metals present in the leach liquor. The leaching operational parameters studied were: reaction time, temperature and HF concentration. The experimental results indicate that the optimal conditions to achieve a Li extraction higher than 90% were: solid-liquid ratio, 1.82% ( w/v ); temperature, 123 °C; HF concentration, 7% ( v/v ); stirring speed, 330 rpm; and reaction time, 120 min. Al and Si can be recovered as Na 3 AlF 6 and K 2 SiF 6 . LiF was separated from the leach liquor during water evaporation, with recovery values of 92%.
Introduction
Fluorides and fluorinated materials appear in various aspects of modern life.The strategic role of fluoride materials involves diverse research fields and applications in areas such as energy production, microelectronics and photonics, catalysis, pigments, textiles, cosmetics, plastics, domestic wares, automotive technology and the construction industry.
One of the most commonly used lithium salts is LiF, which is utilized as a flux in the ceramics and glass industries, as well as in light metals welding [1,2].It is especially used in the manufacture of optical components for analysis equipments (IR and UV spectroscopies).Also, it has recently been used in the fabrication of new cathodes for lithium-ion batteries, in the LiF-Fe ones [3].A potentially important use of LiF is represented by atomic fusion, where it would be employed as a source of 6 Li isotopes [4,5].
The fluorometalates of K, and particularly fluorosilicates, have applications in the field of Al brazing.Firstly, the corresponding reaction gives rise to the formation of elemental Si and K fluoroaluminate, which act as a fluxing agent.The elements formed combine and alloy with Al, diffusing through the base material so as to reach the corresponding eutectics mixtures, thereby locally lowering the melting point of the Al and thus acting as a metal fillet or clad, which forms the joint [6].
Synthetic cryolite is a very important fluoroaluminate regarding to its industrial applications.Probably its most important and best-known application is in the production and refining of aluminum in combination with other fluorides, the so-called Hall-Heroult process [7].Cryolite is also employed in grinding applications as an abrasive aid in the polymeric matrix of phenolics resins [4].Other uses, dealing with lower volumes, include solid lubricant for brakes in heavy-duty applications, and in the production of welding agents, pyrotechnics and metal surface treatments.
The most significant processes for the extraction of Li from lepidolite are the "sulfate acid" and "lime" methods.Acid digestion is carried out with concentrated sulphuric acid at temperatures higher than 250 °C, whereas the lime method is carried out with CaCO3 at 1040 °C.The products obtained through these methods are Li2CO3 and LiOH, respectively [1,2].
Relevant findings about the dissolution of lepidolite with a combination of both pyro and hydrometallurgical routes have been published.In such processes, lepidolite is firstly calcined together with Na2SO4, K2SO4, FeSO4 or FeS, at temperatures higher than 800 °C.Then, the obtained mixture is leached with water [10][11][12][13][14].In the whole process mentioned before, the only valuable metal recovered is Li.Al and Si are lost with the waste generated during the Li recovery process.
The aim of this paper is to describe a process to recover Li, Si and Al from lepidolite and to highlight the controlling parameters of the process, including the best operational conditions.
Materials and Methods
The leaching agent was HF (40% w/w), with analytical grade.The reagents used for the recovery assays were KOH and NaOH, both of analytical grades.The employed mineral was lepidolite, extracted from the mine "Las Cuevas", located in the department of San Martín, San Luis, Argentina.The ore was concentrated by hand sorting, then grounded in a ring mill and sieved to a particle size of <45 μm.
Characterization of the ore and products was performed by X-ray fluorescence (XRF) on a Philips PW 1400 instrument (Philips, Amsterdam, The Netherlands) and by X-ray diffraction (XRD) in a Rigaku D-Max III C diffractometer (Rigaku, Osaka, Japan), operated at 35 kV and 30 mA.The Kα radiation of Cu and the filter of Ni, λ = 0.15418 nm were used.Morphological analysis was done by scanning electron microscopy (SEM), in an equipment LEO 1450 VP (Zeiss, Jena, Germany) which was equipped with an EDAX Genesis 2000 X-ray dispersive spectrometer (EDAX, Mahwah, NJ, USA), used to determine the semi-quantitative composition of the residues obtained through the leaching of the minerals by electron probe microanalysis (EPMA).
Determination of lithium content in the ore was performed by atomic absorption spectroscopy (AAS) using a Varian SpectrAA 55 spectrometer (Palo Alto, Santa Clara, CA, USA) with a hollow-cathode lamp (analytical error 1.5%).Previously, the sample (lepidolite) was dissolved using a concentrated mixture of sulfuric acid and hydrofluoric acid, according to the method of Brumbaugh and Fanus, 1954 [15].The bulk composition of the ore is shown in Table 1, determined by AAS (Li and Na) and XRF (Si, Al, Fe, Ca, Mg, K and Ti).The results of the characterization of the ore by XRD are shown in Figure 1.The XRD patterns in Figure 1 show that the sample is mainly composed of lepidolite (ICDD 01-085-0398), with the presence of albite (ICDD 96-900-1631) and quartz (JCPDS 33-1161) as gangue.
Leaching Assays
The experimental tests were performed in a steel closed vessel of 500 mL coated with Teflon and equipped with magnetic stirring and temperature control systems.
For each test, a mass of the ore and a volume of distilled water were placed into the reactor.The mixture was subsequently heated in an oil bath, with stirring until the final work temperature was reached.Once the desired temperature was achieved, an appropriate amount of HF was added to the mixture, so as to obtain different acid concentrations.From that moment, the reaction time was calculated.Once the experiment was finished, the solid was filtered, dried at 75 °C, and then weighed.
Li was analyzed by atomic absorption to calculate the extraction percentage in all the experiments by the following equation: where: Lim is the initial amount of lithium in the mineral and Lis is the amount of the element in the leach liquor [16,17].
The experimental study of this work was performed by univariate analysis.In order to evaluate the experimental error, each test was replicated three times.The average extraction efficiency and standard deviation were calculated for each parameter studied.
The operational studied parameters were: temperature, reaction time and HF concentration.The following parameters were kept constant: particle size, <45 μm; solid-liquid ratio, 1.82% (w/v); and stirring speed, 330 rpm.
Recovery Assays
The residual Si and Al in the liquor obtained after the leaching of the mineral were removed as the compounds K2SiF6 and Na3AlF6, by using KOH and NaOH, respectively.The reactions proposed for obtaining these compounds are the following [16][17][18]: 2KOH(aq) + H2SiF6(aq) → K2SiF6(s)+ 2H2O (2) 3NaOH(aq) + H3AlF6(aq) → Na3AlF6(s) + 3H2O The recovery of Li from the resulting solution after the precipitation of Al and Si can be carried out by one of the many known methods.In this case, lithium was precipitated as LiF, evaporating the solutions above the solubility product constant (Ksp) of LiF [16,17,[19][20][21][22][23].The recovery efficiency of each element was calculated by gravimetric analysis.
In Figure 2 it is presented the process flow sheet for Li, Si and Al recovery.
Effect of Temperature
In order to investigate the effect of the leaching temperature on the Li extraction, a series of leaching experiments were performed from 75 to 220 °C.Conditions of the leaching process were as follows: solid-liquid ratio, 1.82% (w/v); HF concentration, 7% (v/v); stirring speed, 330 rpm; and reaction time, 120 min.The results are presented in Figure 3 from which it can be appreciated from the error bars that the largest standard deviation in the experimental data is about ±2.5%.
Figure 3 illustrates that the reaction temperature has an obvious effect on the leaching process.The extraction efficiency increases significantly when the leaching temperature is increased from 75 to 123 °C.This coincides with the results of Rosales et al. (2014 and2016), who determined that the dissolution of lithium aluminosilicates (β-spodumene) with HF is strongly dependent on the temperature [17,18].The extraction efficiency was 90% when the reaction temperature was 123 °C.However, a further increase of the temperature did not result in a clear increase of the extraction efficiency (92%).The optimum temperature was chosen to be 123 °C for the economic and industrial applications.
Effect of Reaction Time
To study the effect of the reaction time on the Li extraction, experiments were conducted at conditions of solid-liquid ratio, 1.82% (w/v); HF concentration, 7% (v/v); temperature, 123 °C; and stirring speed, 330 rpm.
The results are plotted in Figure 4, which shows that leaching time has a remarkable effect on the dissolution of the mineral.As the time increased from 10 to 240 min, the lithium extraction efficiency increased from 37% to 95%, due to the reaction time favoring the contact between the leaching agent and the mineral, improving the extraction efficiency.As the reaction time increased up to 120 min, the extraction percentage of Li remained almost constant.Statistical analysis of the results indicates that there is a standard deviation of 1.6%.
Effect of HF Concentration
In order to investigate the effect of the HF concentration on the Li extraction, a series of leaching experiments were performed ranging from 7% to 20% (v/v) of HF.The conditions of the leaching process were as follows: solid-liquid ratio, 1.82% (w/v); stirring speed, 330 rpm; temperature, 123 °C; and reaction time, 60 min.The results are plotted in Figure 5. From Figure 6 it can be inferred that the dissolution of the lepidolite present in the ore occurs, and even more, it enhances the intensity of the diffraction lines of albite and quartz.This indicates that, working at 123 °C, the dissolution reaction is preferential for lepidolite.Meanwhile, the dissolution of lepidolite and albite is achieved at 160 °C, leaving quartz in the leaching residue.
Recovery of Silicon as K2SiF6
The leach liquor obtained after the first filtration was treated with KOH to precipitate the residual Si according to Equation (2).The amount of KOH used was equal to the stoichiometric value calculated from Equation ( 2).The result of the XRD characterization of the obtained solid is shown in Figure 7. Figure 7 shows the appearance of diffraction lines that correspond to the structure of K2SiF6 (JCPDS 7-217), which is in agreement with the proposed Equation (2).By gravimetry, it was determined that the recovery of Si was 93%.
The K2SiF6 precipitate was analyzed by microanalysis EDS and XRF to determine the purity of the precipitate.Table 2 shows the purity of the compound.The results obtained from Figure 7 and Table 2 indicate that the precipitation reaction was highly selective to Si, seeing that there was no formation of any Al and Li compound.
Recovery of Aluminum as Na3AlF6
To precipitate Al as Na3AlF6, the liquor obtained after the recovery of Si was treated with NaOH according to Equation (3) (Rosales et al., [16,17]).The amount of NaOH used was equal to the stoichiometric value calculated from Equation (3).After the addition of NaOH, the appearance of a white precipitate corresponding to Na3AlF6 was observed.The solid was filtered and dried for characterization.
Figure 8 shows the diffractogram of the solid obtained after the addition of NaOH.The diffractogram in Figure 8 indicates that the formation of solid Na3AlF6 has taken place according to Equation (3), without other phases detected as impurities.
Table 3 shows the purity of the compound Na3AlF6, obtained by microanalysis EDS and XRF.The appearence of Na2SiF6 in the diffractogram of Figure 8 is not observed, but the presence of Si (2.5%) by EDS analysis is detected.This indicates that the Si, which was not recovered in the previous step, was precipitated as Na2SiF6 in amounts below the detection limit of XRD.For further purification the solid can be washed with solutions at different pH values [20].The achieved recovery percentage of Al was 95%.
Recovery of Lithium as LiF
The liquor obtained after the recovery of Al and Si was evaporated (at 75 °C) until the appearance of a gelatinous white precipitate that corresponded to LiF [23].The obtained solid was washed and dried at 75 °C for further characterization.The total lithium precipitation, obtained by gravimetric analisys, was about 92%.Figures 9 and 10 show the diffractogram and the SEM micrograph of the obtained LiF, respectively.Figure 9 shows that the compound LiF was obtained without other phases as impurities.Frontino et al. (2008), obtained a similar diffractogram for the precipitation of LiF from spent Li-ion batteries [23].
In Figure 10 it can be seen that the LiF particles have a well-defined crystal structure and coincided with the cubic shape of LiF particles found in the literature.
The resulting LiF was analyzed by AAS and EDS to determine the concentration of major impurities in the sample.The results are presented in Table 4.
Conclusions
In summary, a process that includes leaching with HF was employed to extract Li, Al and Si from lepidolite.The experimental results indicate that the optimal conditions to achieve a Li extraction higher than 90% are: solid-liquid ratio, 1.82% (w/v); temperature, 123 °C; HF concentration, 7% (v/v); stirring speed, 330 rpm; and reaction time, 120 min.The compounds K2SiF6 and Na3AlF6 can be obtained as subproducts of the process, with a recovery of 93% and 95%, respectively.The Li dissolved can be separated by chemical precipitation as LiF, with a recovery value of 92% and a purity of 99.1%.Furthermore, Al and Si are recovered as valuable subproducts, diminishing the generation of residues during the process.The results of the current work can provide a simple, economic and effective way to recover Li from lepidolite.
Figure 2 .
Figure 2. Generalized process flow sheet for Li, Si and Al recovery from lepidolite.
Figure 3 .
Figure 3.Effect of the temperature on the Li extraction efficiency.
Figure 4 .
Figure 4. Effect of reaction time on the Li extraction efficiency.
Figure 5 .
Figure 5.Effect of HF concentration on the Li extraction efficiency.
Figure 5
Figure5illustrates that the HF concentration has a notorious effect on the leaching process.The extraction efficiency increased significantly when the HF concentration was increased from 7% to 20% (v/v).The maximum extraction value of Li (95%) was achieved by working with HF 15% (v/v).Above 15% (v/v) of HF, the Li extraction percentage decreased, since high concentrations of HF produce a precipitation of Li alkali fluorides (e.g., Li3Na3Al2F12)[17].
Figure 6 .
Figure 6.XRD patterns of the leaching residue obtained at 123 and 160 °C.
Table 1 .
The bulk composition of the ore by AAS and XRF.
Table 4
indicates that the purity of the LiF precipitated was 99.1%. | v3-fos-license |
2019-04-05T03:28:42.083Z | 2019-04-03T00:00:00.000 | 93001329 | {
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} | pes2o/s2orc | QSAR analysis of immune recognition for triazine herbicides based on immunoassay data for polyclonal and monoclonal antibodies
A common task in the immunodetection of structurally close compounds is to analyze the selectivity of immune recognition; it is required to understand the regularities of immune recognition and to elucidate the basic structural elements which provide it. Triazines are compounds of particular interest for such research due to their high variability and the necessity of their monitoring to provide safety for agricultural products and foodstuffs. We evaluated the binding of 20 triazines with polyclonal (pAb) and monoclonal (mAb) antibodies obtained using atrazine as the immunogenic hapten. A total of over 3000 descriptors were used in the quantitative structure-activity relationship (QSAR) analysis of binding activities (pIC50). A comparison of the two enzyme immunoassay systems showed that the system with pAb is much easier to describe using 2D QSAR methodology, while the system with mAb can be described using the 3D QSAR CoMFA. Thus, for the 3D QSAR model of the polyclonal antibodies, the main statistical parameter q2 (‘leave-many-out’) is equal to 0.498, and for monoclonal antibodies, q2 is equal to 0.566. Obviously, in the case of pAb, we deal with several targets, while in the case of mAb the target is one, and therefore it is easier to describe it using specific fields of molecular interactions distributed in space.
Introduction
Triazines are herbicides which are widely used in agriculture and may accumulate in soil, as well as in food products [1].Triazines are bound in soil to solids as well as to dissolved fractions of humic and fulvic acids, which leads not only to the accumulation of triazines in soil but also to the contamination of surface and ground waters [2] since triazines are water soluble.
Triazines may undergo chemical transformations of both a biotic and abiotic nature.The parent compound atrazine is subjected to oxidation of the alkyl substituents, oxidative dealkylation, hydroxylation, and also to ring cleavage [3][4][5].The derivatives of atrazine may be toxic to a greater or lesser extent.Triazines may induce different physiological disorders in humans and animals such as immunity suppression [6] and birth defects [7].Thus, contamination of the environment by triazine herbicides is a significant risk factor.For this reason, it is necessary to monitor the triazine levels in soil, water, agricultural products and foods.
The methods of triazine detection, such as NMR, HPLC, mass-spectrometry and others, are time-and labour-consuming.The immunoassay determination of triazines is much easier, less expensive, highly sensitive and rapid.Due to its selectivity and simplicity, the immunoassay has been used to detect a wide variety of environmental pollutants including triazines [8][9][10].However, the significant variety of triazines and their derivatives makes the detailed analysis of the regulations of their immune recognition necessary.
The quantitative structure-activity relationship (QSAR) is widely used to study the immune recognition of different classes of toxic food contaminants and veterinary drugs, including pesticides, etc. [11][12][13][14].The work by Yuan and co-authors reported an immunoassay analysis of triazines; however, on only a small set of 11 compounds [9].QSAR is used for the analysis of immunoassays on quinolones and fluoroquinolones [15][16][17][18][19], organophosphorus pesticides [20][21], phenylurea herbicides [22], and sulfonamides [23].In this study, we used molecular modeling to study triazine recognition by monoclonal and polyclonal antibodies.Twenty triazines along with 2D and 3D-QSAR methodology were used to study the relationship between the antigen and antibody.The experimental data of microplate immunoenzyme assays with broad specificity for triazines were used from a classical work by A. Dankwardt and co-authors [24].The comparative immunoassay of polyclonal and monoclonal antibodies used 4-arylamino-6-amino-1,3,5-triazines (Fig 1).Antibodies were grown using atrazine as an immunizing hapten.
The antibody-binding activity of triazines from Table 1, presented in the logarithmic form (pIC 50 = -log IC 50 ), was used as the dependent variable y during QSAR calculations.In Table 1, S2 stands for polyclonal sheep antibodies, while K4E7 stands for monoclonal antibodies.
Conformational analysis and geometry optimization
The preparation of molecular geometries for 2D and 3D QSAR analysis was carried out in the Spartan v.16 program.A series of conformers for each of the 20 compounds was obtained by the systematic search method using the MMFF force field [25].The optimization of the geometry for each conformer was carried out using the semi-empirical AM1 method [26].In order to confirm the correspondence between the obtained geometry and the minimum surface of the potential energy for each conformer, the Hessian was calculated.The conformers with the lowest total energy were used for 2D QSAR analysis.
2D QSAR
Using a random number generator, triazines were divided into two samples: the training set (80%) and test set (20%).The requirements for the maximum and minimum values in the test sample were as follows: 1) the maximum value of pIC 50 should be less than or equal to the maximum value of pIC 50 in the training sample; and 2) the minimum value of pIC 50 must be greater than or equal to the minimum value of pIC 50 in the training sample.The actual limit IC 50 values given in Table 1 are equal to 500 nmol l -1 , which were experimentally tested values in the original work by Dankwardt et al., 1996 [24].When the activity IC 50 was equal to or greater than 500 nmol l -1 the pIC 50 value of 6.30103 was used.
The required linear regression equation should have the following form: where y is the dependent variable (pIC 50 ); a 1 and a n are regression coefficients; x 1 and x n are independent variables (descriptors); and c is a regression constant.We estimated the contribution of a descriptor to the model using the following equation: where α(x 1 ) is the relative contribution of the descriptor x 1 to the model with three descriptors; R 2 x 1 ; x 2 ; x 3 ð Þ is the determination coefficient of the model with all three descriptors; and R 2 x 2 ; x 3 ð Þ is the determination coefficient of the model with two descriptors: x 2 and x 3 .During the 2D QSAR analysis, different types of descriptors were used: constitutional (number of hydrogen bond donors, number of hydrogen bond acceptors, number of rings, number of chains, number of CH 3 groups, number of OH groups, etc.), electrostatic descriptors (maximum positive charge, maximum negative charge, the number of positively charged atoms, the number of negatively charged atoms, etc.), topological descriptors (kappa-indices describing the shape of the molecule, the indices of molecular bonds of Kier and Hall, etc.), 3D descriptors (volume, surface area, length and area of the projection on the coordinate axis, etc.), physico-chemical (lipophilicity (LogP), molecular refraction, polarizability, solubility in water, etc.) and quantum-chemical descriptors (energy of frontier molecular orbitals, electrostatic charges of atoms, the population of atoms according to Mulliken, etc.), amounting to a total of more than 3000 descriptors.The values of the descriptors were calculated using the program packages Spartan v. 16 and E-Dragon 1.0.
For the QSAR obtained model, the cross-validation was performed.Also, the models were tested for their predictive power using an external test set of compounds.The following statistical indicators were used: • r 2 -coefficient of determination for the training sample [27]; • The determination coefficient R 2 adj, corrected for the number of descriptors involved in the model; • q 2 -r 2 from the results of the internal cross-validation of the training sample using the leaveone-out (LOO) method [28,29]; • LOF-error of Friedman approximation (Friedman Lack of Fit); • RMSE-root-mean-square error; • Max Error-the maximum prediction error for all the compounds in the test and training set; • pred_r 2 -r 2 , which estimates the predictive ability of the model relative to the test set.
3D QSAR
Spatial alignment of molecules.The alignment of molecules plays a key role in 3D-QSAR.As a rule, the most energetically favorable conformation of the most active compound or the geometry corresponding directly to the interaction of the ligand and target (antibody), obtained on the basis of X-ray diffraction analysis or docking data, is chosen as the template for the alignment [30].In our case, the most active conformation is unknown, and a number of the most active compounds (10,14) have a large number of conformers, which makes it difficult to analyze them correctly using quantum-chemical methods of required accuracy.Compounds 5, 11 and 12 are also highly active, but with a small number of conformers compared to other triazines in the sample.All conformers of compounds 5 (12 conformers), 11 (23 conformers) and 12 (12 conformers) were optimized using the Hartree-Fock method and 6-31G(d) basis set (Fig 2).It was found that the most energetically favorable conformers of these compounds are identical, and therefore it can be assumed that this particular conformation is the most active and corresponds to the interaction with the antibody.
Triazine molecules in various conformations, optimized with the AM1 method, were superimposed on the template (compounds 5 and/or 11) using the algorithm presented in the open3DALIGN program [31].Conformations corresponding to the largest overlap with the atoms of compounds 5 and 11 were selected (Fig 3).
The aligned geometries (S1 Fig) were used to create and analyze the fields of molecular interactions using the open3DQSAR program [32].Visualization of 3D structures and contour maps of the force fields was carried out in the PyMol program.
Building of molecular interaction fields (MIF).Molecular interaction fields (MIF) were calculated to analyze the pIC 50 values of triazines and to create CoMFA models for the S2 and K4E7 systems.
Two potentials were used to create the fields of molecular interaction: the steric potential in the form of the Lennard-Jones function 6-12 between the atoms of the molecule and the sp3 carbon atom: The electrostatic field was calculated by summing the Coulomb interactions between the test atom with charge +1 and triazine molecules: MIF preprocessing.The force-field data was preliminarily processed to remove noninformative x variables: 1) The values of the variables less than 0.05 kcal mol -1 were equated to zero; 2) Cut-off values were above 30 kcal mol-1 and below -30 kcal mol -1 ; 3) x variables with a standard deviation value below <0.1 were deleted; 4) Variables that had four or fewer nonzero values within the sample were removed.Building of regression models.After all the preliminary operations were completed, we proceeded to construct the models using the partial least squares (PLS) method [33], based on the NIPALS algorithm (non-linear iterative partial least squares) [34].The predictive power of the models was evaluated using the "leave-one-out" (LOO) cross-validation method: Also, we used the "leave-many-out" cross-validation method: a sample of 20 compounds was randomly broken into the training (75%) and test (25%) parts.The q 2 parameter was calculated for the test part.The partition procedure was repeated 50 times, and the average of the 50 partitioning variants was given the value q 2 (LMO).
To estimate the internal stability and predictive power of the 3D models, the standard prediction error (SDEP) parameter was calculated: SDEP ¼ ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi where y obs is the experimental value; y pred is the predicted value; y mean is the mean value; and N is the number of molecules in the sample.
Procedures for selecting variables and methods for creating clusters of variables with common characteristics were used in order to increase the predictive ability of models: the SRD (smart region definition) method [35] groups variables based on their localization in threedimensional space.This procedure reduces the number of descriptors bearing the same information; and fractional factorial design (FFD) [36,37] allows the selection of variables that have the greatest impact on the predictive ability of models.The FFD selection was conducted based on the leave-many-out cross analysis, using the SRD of the variable group.
2D QSAR
S2 system analysis (polyclonal antibodies).For the S2 system, a series of multiple linear regression models was obtained: Model 1 It is believed that QSAR is predictive if the following conditions are satisfied: R 2 > 0.6, q 2 > 0.5 and pred_R 2 > 0.5.Table 2 shows that all the presented models are statistically reliable and possess a predictive ability.Model 5 has the highest statistical parameters among the five models.
Let us consider the descriptors involved in Model 5. Solvation_E (relative contribution, α = 66.8%) is the solvation energy calculated with the AM1 method.Compound 18 has the highest solvation energy value (-20.27kJ mol -1 ), while compound 13 has the lowest (-71.22 kJ mol -1 ) (S1 Table ).The lower the solvation energy, the higher the solubility.However, the energy of solvation has a negative value, and therefore compounds that are less soluble in water have higher pIC 50 values.
ES_Sum_sssCH (26.1%) is the electrotopological index of methantriyl groups [38].The ES_Sum_sssCH value is directly proportional to the activity of the molecule (1, 2 and similar compounds).ES_Sum_sssCH is equal to zero for compounds without >CH-groups.Obviously, the presence of the ES_Sum_sssCH descriptor in Model 5 indicates the participation of >CH-groups of triazine compounds in van der Waals interactions with antibodies.IC2 (7.1%) is the information content index (neighborhood symmetry of 2-order).This descriptor makes it possible to estimate the degree of heterogeneity of the molecular structure.Compound 1 has the lowest value of IC2 (3.431), and compound 9 has, on the contrary, the highest value of this descriptor (4.426).IC2 can be considered as a measure of the complexity of the triazine topology.IC2 makes a minor contribution to the model (α = 7.1%).
Experimental and predicted pIC 50 values for triazine interactions with polyclonal antibodies (S2) based on Model 5 are presented on Fig 4 .K4E7 system analysis (monoclonal antibodies).For the K4E7 system, a series of multiple linear regression models was obtained: Model 10 has a high predictive ability, and its statistical parameters are higher than for models 6-9 (Table 3).
Num_H_Donors_Lipinski (α = -37.9%) is the number of hydrogen bond donors (S2 Table ).The value of the descriptor is inversely correlated with the activity: the greater the number of hydrogen bond donors, the lower the activity.Apparently, the presence of amine groups (-NH-and -NH2) negatively affects the activity.
ES_Sum_sCl (34.6%) is the electrotopological index of chlorine atoms. 35The ES_Sum_sCl value is directly proportional to the activity of the molecule.Apparently, the chlorine atom, as an electron-acceptor substituent, participates in electrostatic interactions.The experimental and predicted pIC 50 values for triazine interactions with monoclonal antibodies (K4E7) based on Model 10 are presented in Fig 5 .3D QSAR S2 system study.A 3D QSAR analysis was carried out for all 20 compounds.The interaction energy of the "probe" (carbon atom with charge +1) with the target molecule was calculated at each point of the regular 3D lattice.The dimensions of the 3D cubic grid were 26 × 18 × 22 Å.The lattice spacing was 2 Å.
One can consider Table 4 to compare the accuracy of the activity prediction for individual molecules within the model.
Van der Waals interactions make the main contribution to the interactions between the triazine and antibody in the S2 system in all three models (S3 Table ).
To estimate the predictive ability of the 3D QSAR models, the following statistical parameters were used: the coefficient of determination (R 2 ), the mean-square error of the model (SDEC), the F-statistic value, the correlation coefficient of the leave-one-out (LOO) method (q 2 (LOO) ), the correlation coefficient of the LMO method (q 2 (LMO) ), and the mean-square error of prediction (SDEP).The S2 system 3D QSAE model showed satisfactory statistical results (Table 5).
As can be seen from Table 5, the model has high predictive ability.High coefficients of determination (R2 = 0.938) and LOO cross-validation (q 2 = 0.68) indicate the statistical significance of the obtained model.The high correlation between predicted and experimental values of the model can be seen in Fig 6.
Molecular interaction field (MIF) contour maps were obtained to visualize the information about the 3D-QSAR models.Contour maps are presented in K4E7 system study.A 3D QSAR analysis was carried out for all 20 compounds without splitting the sample into the training and test parts.The same alignment was used as for the S2 system.
The predicted values of triazine activity are shown in Table 6.The calculated values of the model have the best match with the experimental data.The level of correlation between the activities predicted by the CoMFA model and the experimental values is also shown in Fig 8. Table 7 shows that the model has high values of the determination coefficient (R 2 = 0.952) and LOO cross-validation parameter (q 2 = 0.637), which indicates the statistical significance of the model obtained.The contour maps of the molecular interaction for this model are presented in Fig 9.
The contributions of the van der Waals and electrostatic interactions to the model are approximately equal at 51% and 49%, respectively (S4 Table ).
Discussion
It seems reasonable to compare the most similar compounds with each other and find out which descriptors play a key role in their recognition.
Atrazine in the S2 system (polyclonal antibodies) exhibits significantly higher activity (CR 100%) than its metabolites: compounds 2, 3 and 4 (CR 15%, 0.4% and 6%, respectively).Atrazine has the highest value of the Solvation_E descriptor (see S1 Table ).The value of the Solva-tion_E descriptor is directly proportional to pIC 50 .The lower the Solvation_E, the higher the polarity of the molecule.For compounds 1-4, Solvation_E is equal to -33.49 kJ mol -1 , -49.34 kJ mol -1 , -56.07 kJ mol -1 and -58 kJ mol -1 , respectively.In general, this is consistent with our observation that the polarity of triazines is unfavorable for the interaction with the antibodies.Also, compounds with a CR higher than 1% (compounds 1, 2 and 4) possess ES_Sum_sssCH with a non-zero value, while the compound with the lowest ES_Sum_sssCH value (compound 3) has the lowest CR value-0.4%.For the K4E7 system (monoclonal antibodies), the highest pIC 50 value of compound 1 among compounds 1-4 is determined by non-zero values of its descriptors ES_Count_sssCH and ES_Sum_sCl.Also, compound 1 has the lowest value of the descriptor Num_H_Donor-s_Lipinski.The descriptor Num_H_Donors_Lipinski is inversely correlated with pIC 50 .For system S2, the highest CR value of compound 6 among compounds 6-9 (CR 126%, 109%, 74% and 9%, respectively) is determined by its lowest value of E_Solvation (-37.49kJ mol -1 ), while E_Solvation is inversely correlated with CR.
In the case of the K4E7 system and compounds 6-9, we should regard the CoMFA contour maps.The substituent at the C6 position does not differ in these compounds, and the variation of the substituent in the C4 position obviously plays the main role.The geometry of compound 7 is the most advantageous for the interaction with the antibody, and the geometry of 9 is the least advantageous (Table 1).The contour map of electrostatic charges ( that the contour map is favorable for the interaction with the nitrile located at the 2' and 3' positions of the aniline ring.Let us regard compounds 13, 14 and 15, which possess trifluoromethyl substituent (-CF3) in the ortho-, para-and meta-positions, respectively.In the S2 system, molecule 14 is more active than 13 and 15 (Table 1).In order to answer the question why these compounds exhibit different activities, it is required to consider the Solvation_E descriptor involved in 2D QSAR model 5.This descriptor is inversely correlated with pIC 50 .For compounds 13, 14 and 15, Solvation_E is equal to -20.27 kJ mol -1 , -30.06 kJ mol -1 and -27.35 kJ mol -1 , respectively.Obviously, the lower the solvation energy, the lower the activity.Also, the pIC 50 of compounds 13, 14 and 15 is correlated with IC2 values of 4.15, 4.21 and 4.09, respectively.The higher value of IC2 descriptor for compound 14 means a higher heterogeneity of 14 among compounds 13-15.
Obviously, the low activity of compounds 16, 19 and 20 in both systems (S2 and K4E7) is due to the absence of the >CH-group at the C6 position.This is confirmed both by the zero values of 2D descriptors ES_Count_sssCH, ES_Sum_sssCH, and by the contour maps of van der Waals interactions (Figs 7A and 9A).Also, it is quite obvious that compounds 17, 18 have low pIC 50 values due to the presence of the -OH group in the C2 position instead of chlorine.If we compare the 2D QSAR models for monoclonal antibodies (mAb) and polyclonal antibodies (pAb), one can see that both models (Model 4 for mAb and Model 7 for pAb) contain the polar surface area parameters Jurs_FPSA_3 and Jurs_TPSA, respectively.The polar surface area is inversely correlated with the activity, which means that the polarity of atrazines is unfavorable for the interaction with both monoclonal and polyclonal antibodies.Both mAb and pAb are affected by the amount of methantriyl groups (ES_Sum_sssCH and ES_Count_sssCH parameters, respectively), which means that, in both cases, bulk substituents are favorable for higher activity.The latter statement is supported by 3D molecular interaction field contour maps (Figs 7A and 9A).We may see that both mAb and pAb systems have a great deal in common.
Conclusions
In this study, we evaluated the efficiency of the interaction of 20 triazines with polyclonal and monoclonal antibodies grown using atrazine as the immunizing hapten.
The comparison of the two immunoassay systems showed that the system with the polyclonal antibodies (S2) is much easier to describe using 2D QSAR methodology, and the system with monoclonal antibodies can be described using the 3D QSAR CoMFA method.Thus, for the 3D QSAR model of the S2 system, the main statistical parameter q 2 (LMO) is equal to 0.498, and for K4E7, q 2 (LMO) = 0.566.Apparently, this is explained by the fact that in the case of polyclonal antibodies, we deal with several targets, while in interaction with monoclonal antibodies, the target is one, and therefore it is easier to describe it using specific fields of molecular interactions distributed in space.
There is an interesting fact: while for the S2 system the main contribution (72%) is made by steric interactions (S3 Table ), for K4E7, the fraction of van der Waals and electrostatic interactions is equal (S4 Table ).
Based on our analysis, we conclude that the effectiveness of the interaction with polyclonal antibodies significantly depends on the presence of the isopropylamino group in the C6 position of triazine, the presence of chlorine at the C2 position, and also the aniline substituent in the C4 position, whose substituents, in turn, participate in electrostatic interactions and hydrogen bonding.
The obtained data as well as the earlier-published results of QSAR technique application for immune recognition demonstrate the efficiency of this technique in the identification of key structures responsible for the distinguishing of structurally close antigens by antibodies.This information allows the theoretical formulation of criteria for molecules that could be recognized by the same antibodies (thus, extending the selectivity of one immunoassay) or requirements in different antibodies for detection (thus, realizing assays with mixtures or arrays of immunoreactants).
Fig 7 .
The steric fields presented by green contours are favorable for bulk substituents and have a positive effect on cross-reactivity (Fig 7A), while the yellow contours represent the areas where the presence of bulk substituents is unfavorable for high activity (Fig 7B).The electrostatic fields are represented by blue and red contours.The blue contour reflects the areas where positively charged atoms have a positive effect on activity (Fig 7C), while the red contours reflect the regions in which the presence of negatively charged atoms is favorable for high activity (Fig 7D).
Fig 6 .
Fig 6.Experimental vs. predicted values of triazine cross-reactivity in the S2 system according to the CoMFA method (3D QSAR).https://doi.org/10.1371/journal.pone.0214879.g006 Fig 9D) indicates that the presence of a negatively charged carboxyl group at position 2' (compound 9) is unfavorable for the binding with the antibody.Fig 9C, on the contrary, shows how the presence of negatively charged oxygen atoms of the methoxy groups of compound 7 has a positive effect on the activity.The contour map on Fig 9A shows that the aniline residue at the C4 position (namely, the benzene ring) generally has a positive effect on the activity of atrazines.Fig 9B shows that the bulky substituents localized at the meta position of the aniline have a negative effect on the activity; the methoxy substituents of compound 9 are located in these areas.Compounds 10, 11 and 12 have ortho-, meta-and para-positions of the nitrile of the aniline substituent, respectively (Fig 1).The contour maps of electrostatic interactions (Fig 7C and 7D) may explain why the activity of these compounds in system S2 is different.Fig 7D shows
Table 3 . Statistical parameters of 2D QSAR models for the K4E7 system.
ES_Count_sssCH (27.4%) is the number of methantriyl groups.Obviously, the presence of the ES_Count_sssCH descriptor in Model 10 indicates the participation of bulky substituents of triazine compounds in steric interactions with antibodies. https://doi.org/10.1371/journal.pone.0214879.t003 | v3-fos-license |
2016-03-22T00:56:01.885Z | 2004-11-30T00:00:00.000 | 37010671 | {
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} | pes2o/s2orc | Asymmetric Synthesis and Antimicrobial Activity of Some New Mono and Bicyclic Β-lactams
Reaction of the amino acid D-phenylalanine ethyl ester (4) with cinnamaldehyde gave chiral Schiff base 5, which underwent an asymmetric Staudinger [2+2] cycloaddition reaction with phthalimidoacetyl chloride to give the monocyclic β-lactam 6 as a single stereoisomer. Ozonolysis of 6 followed by reduction with lithium aluminum tri(tert-butoxy) hydride afforded the hydroxymethyl β-lactam 8. Treatment of 8 with methansulfonyl chloride gave the mesylated monocyclic β-lactam 9, which was converted to the bicyclic β-lactam 10 upon treatment with 1,8-diazabicyclo[5,4.0] undec-7-ene (DBU). Deprotection of the phthalimido group in β-lactams 6 and 10 by methylhydrazine and subsequent acylation of the free amino β-lactams with different acyl chlorides in the presence of pyridine afforded mono and bicyclic β-lactams 14a-d and 15a-d respectively. The compounds prepared were tested against Escherichia coli, Staphilococcus citrus, Klebsiella pneumanie and Bacillus subtillis. Some of these compounds showed potential antimicrobial activities.
Introduction
The β-lactam skeleton is the key structural element of the most widely used class of antimicrobial agents, the β-lactam antibiotics [1].Among the various methods for constructing this pharmaceutically important four-membered ring, the ketene-imine cycloaddition, also known as the Staudinger reaction [2] has provided useful and economical entries to β-lactams, mainly due to the ready availability of both Schiff bases and ketenes [3].The asymmetric syntheses of α-amino β-lactams can be divided into five categories: a) asymmetric induction from the imine component; b) asymmetric induction from the ketene component; c) double stereodifferentiating cycloadditions; d) carbacephem intermediates and e) 2-oxaisocephems and 2-isocephems [1].The asymmetric induction in the reaction of achiral ketenes with chiral imines has been effected from imines derived from chiral aldehydes and achiral amines and also from imines derived from chiral amines and achiral aldehydes.Although it is believed that the former case is better for diastereoselectivity, it has been reported that the reaction of the imine derived from D-glucosamine and cinnamaldehyde with phthalimidoacetyl chloride and triethylamine furnished the β-lactam 1 as a single isomer [4].It has also been found that the imine derived from the amino acid D-threonine upon treatment with azidoacetyl choride and triethylamine affords β-lactams 2 and 3 in a 95:5 stereoisomeric ratio [5].Gunda has reported the use of imines derived from chiral amines in βlactam syntheses giving various degrees of diastereoselectivity [6].The reaction of fluoroacetyl chloride with the imine derived from p-anisidine and D-glyceraldehyde acetonide afforded the corresponding cis β-lactam as a single diastereomer [7].High levels of asymmetric induction in reactions between Evans-Sjogren ketenes and imines derived from (R)-and (S)-α-amino acid esters have been reported [8].
Hashimoto and his coworkers have used chiral imines derived from erythro 2-methoxy-1,2diphenylethylamine and aromatic aldehydes to prepare β-lactams in good yields with high diastereoselectivity [9].Recently, high levels of asymmetric induction have also been reported for monocyclic β-lactam formation [10][11].Thus, we decided to synthesize some diastereoselective mono and bicyclic β-lactams using the asymmetric induction by the imine component and their antimicrobial activities against some pathogenic microorganisms have been tested.
Scheme 1
In addition, the absence of a singlet peak for the benzylic protons in 1 H-NMR spectrum of 11 confirms the formation of bicyclic β-lactam 10.The IR spectrum showed the β-lactam carbonyl absorption at 1780 cm -1 for 10 which has increased a little due to fusing two rings and subsequently the amide resonance is lowered.We next turned to the deprotection of the phthalimido protecting groups of β-lactams 6 and 10 (Scheme 2).
Scheme 2
Among the different methods available for this kind of deprotection, the one using methylhydrazine worked best in this case [19][20][21][22] and a significant improvement in the yield and in the ease of isolation of the free amino β-lactam is noted when methylhydrazine is employed instead of the usual hydrazine.This is due to the decreased acidity of the by-product N-methylphthalhydrazide with respect to phthalhydrazide and hence no complex is formed with the free amine.Consequently there is no need to heat or for acid treatment of the reaction mixture.N-methylphthalhydrazide separates from a chloroform solution of the methylhydrazine-phthalisoimide adduct leaving the free amine in solution.Therefore the methylhydrazine method is most suitable for sensitive substrates [21].Upon treatment with methylhydrazine, β-lactams 6 and 10 were converted to free amino β-lactams 12 and 13 respectively.Treatment of these mono and bicyclic β-lactams with benzoyl, phenoxyacetyl, cinnamoyl and phenylacetyl chloride in the presence of pyridine afforded β-lactams 14a-d and 15a-d in moderate yields
Biological Screening. Antimicrobial Activity Tests
The antimicrobial activity test was performed using the disk diffusion method [23] using ampicillin and gentamycin as the reference compounds.Some of the prepared compounds: 6, 7, 8, 10, 14a-d and 15a-d were tested against one strain each of a Gram positive bacteria (Staphylococcus citrus), a Gram negative bacteria (Escherichia coli), a Gram negative containing capsule (Klebsiella), and a Gram positive spore (Bacillus subtilis).As shown in Table 1, it was found that compounds 6, 14b and 14c were highly active against Bacillus subtilis and moderately active against Staphilococcus citrus.Other compounds were all inactive against these four pathogenic microorganisms.
General
Melting points were determined in open capillary tubes in a Buchi 510 circulating oil apparatus and are uncorrected.Infrared spectra were recorded on a Perkin Elmer 781 spectrophotometer. 1 H-NMR spectra were recorded at room temperature for CDCl 3 solutions, (unless otherwise stated) on a Jeol JNM-EX 90A FT NMR and Bruker Avance DPX 250 MHz spectrometers using tetramethylsilane as an internal standard.All chemical shifts are reported as δ values (ppm).Mass spectra were obtained on a GCMS-QP 1000 EX mass spectrometer (70 eV).The microorganism strains used in this study were β-lactam sensitive ones.
Table 1 .
Results of antimicrobial activity tests of the synthetic monocyclic and bicyclic β-lactams. | v3-fos-license |
2019-01-22T22:25:34.086Z | 2019-01-04T00:00:00.000 | 58022770 | {
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} | pes2o/s2orc | Development of the covalent antibody-DNA conjugates technology for detection of IgE and IgM antibodies by immuno-PCR
Immuno-PCR (iPCR) is one of the methods used for the detection of a wide range of analytes and features the high sensitivity of the polymerase chain reaction (PCR) method. iPCR uses antibodies coupled to DNA, followed by the amplification of the attached DNA using RT-PCR. Two major types of antibody-DNA conjugates are currently used, which are obtained as a result of non-covalent (biotin-streptavidin) or covalent interactions. Using a strain-promoted azide-alkyne cycloaddition (SPAAC), we synthesized covalent DNA-antibody conjugates, optimized the reaction conditions, and developed an efficient protocol for the purification of conjugates, with which all unreacted antibodies and oligonucleotides are separated. Covalent DNA-antibody conjugates were tested with iPCR assays that were previously developed for the detection of IgE and IgM antibodies with the use of the supramolecular complex of 5'- and 3'-biotinylated DNA and streptavidin. The results show that the modification of antibodies with amino groups did not allow us to obtain monolabeled antibodies or antibodies with a strictly defined number of DNA-labels. The degree of labeling determined by the dyes introduced through the azido group reflects the actual labeling degree statistically. If the average labeling degree for azido groups is 1.1, the conjugates contain 25% mono-labeled antibodies, 50% double-labeled antibodies, and 25% unlabeled ones. The specificity of the monoclonal antibody to human IgE (BE5) changed after conjugation with the oligonucleotide. The sensitivity of iPCR in the detection of IgM antibodies produced against the LeC disaccharide using a covalent conjugate was similar to that of a supramolecular complex of 5'- and 3'-biotinylated DNA and streptavidin, but the new procedure is two steps shorter.
Introduction Immuno-PCR (iPCR) is a highly sensitive method for the detection of a wide range of analytes ranging from bacterial and viral antigens and antibodies to non-protein drugs and toxins. It combines advantages of both the polymerase chain reaction (PCR) and enzyme immunoassay methods. iPCR is based on the usage of an antibody-DNA conjugate followed by the amplification of the DNA label [1]. DNA-antibody conjugates are currently used for iPCR, immuno-RCA (immunoassay with rolling circle DNA amplification) [2], proximity ligation assay (PLA) [3], and Electrochemical Proximity Assay (ECPA) [4].
The main methods for the preparation of conjugates are based on either biotin-streptavidin interaction or covalent binding. When biotin-streptavidin conjugates are used, biotin is introduced into both the antibody and the DNA label. In the case of iPCR, after applying of detecting antibodies, a solution of streptavidin is added to the wells of a plate, followed by a biotincontaining DNA label. Thus, the antibody-streptavidin-DNA conjugate is formed during analysis. This approach is known as "universal iPCR" and is popular for its simplicity [5]. However, an obvious disadvantage of the method is the three-stage assembly of the conjugate during analysis, which increases the analysis time and the number of washings.
the preparation of conjugates and their application in iPCR. However, almost none of them described the technical details of the preparation and purification. In the present study, we synthesized the antibody-oligonucleotide conjugate by a strain-promoted azide-alkyne cycloaddition for use in iPCR. We focused on the optimization of the conditions for the preparation and isolation of conjugates with a certain number of labels.
Sera, allergens, and antibodies
Monoclonal mouse antibodies to human IgM (MA2) and human IgE (BE5) were purchased from Sorbent Ltd, Russian Federation. Svirshchevskaya et al. describe the sera and the preparation of the recombinant allergen Alt a 1, as well as the biotinylation of the BE5 antibodies [17]. The BE5 binding was detected by sheep anti-mouse antibody HRP conjugate (Dako, Sweden).
iPCR using DNA-streptavidin complex iPCR was performed using DNA-streptavidin complex in accordance with previous studies for the detection of IgE [18] and the detection of antibodies to Le C [19].
Antibodies and oligonucleotide modification
A strain-promoted azide-alkyne cycloaddition was used to obtain an antibody and singlestranded 60-base oligonucleotide (ODN) conjugates (see Fig 1). Pentynoic acid sulfotetrafluorophenyl ester (STP-N3) (Lumiprobe) was used as a modifying agent for the antibody. The Nhydroxysuccinimide ester of dibenzocyclooctyne with hydrophilic linker (DBCO-PEG 4 -NHS, Jena Bioscience) was used to modify the oligonucleotide with the amino group introduced during synthesis.
Antibody modification (general scheme)
The antibody was transferred into 0.1 M sodium bicarbonate at pH 8.3 and simultaneously concentrated by washing 3 times with 650 μl of buffer solution on a Spin-X UF 30K MWCO column (Corning). Different excesses of STP-N3 were added to 25 nmol of antibody, and the reaction mixtures were incubated at room temperature for 1 h. The excess reagent was removed using CentriPure MINI PBS Z-50 Spin Columns. The activity of the antibodies after azidation was examined using ELISA. The completeness of the reaction was controlled as follows. An aliquot of modified antibody was mixed with a tenfold molar excess of dibenzocyclooctyne derivative of sulfo-Cy5 (DBCO-Sulfo-Cy5, Jena Bioscience), followed by purification using gel filtration. The labeling level was calculated by analyzing the UV-vis spectra.
Oligonucleotide modification (general scheme)
Acetone/lithium perchlorate was used to precipitate 100 nmol of ODN Ip3-am, which was then dissolved in 100 μl of buffer containing 0.1 M NaHCO 3 at pH 8.3 and 80% DMSO. Next, 50 μl of 100 mM DBCO-PEG 4 -NHS (5 μmol) was added, and the reaction mixture was incubated at room temperature for 1 h. The oligonucleotide was precipitated using acetone/lithium perchlorate and purified by gel-filtration on an EMP CentriPure N2 while simultaneously transferring it into a PBS solution. An aliquot of the reaction products was analyzed using RP HPLC (Phenomenex Luna 5u C8, linear gradient of acetonitrile in 50 mM sodium acetate; results are not provided). The reaction products were evaporated using a vacuum centrifuge and dissolved in 50 μl of PBS. The reaction progress was controlled by mixing an aliquot of modified oligonucleotide with a 10-20× molar excess of azido derivative of sulfo-Cy5 (sCy5-N3) (Lumiprobe, Russian Federation), followed by purification using RP HPLC.
Synthesis and purification of covalent DNA-antibody conjugates (general scheme)
The modified antibody and oligonucleotide were mixed at a ratio of 1:4 by active groups (the determination of the active groups' content is described below). The reaction mixture was kept at room temperature overnight. The purification was carried out in several stages. To remove the unreacted antibody, the reaction mixture was placed in a RESOURCE Q anion exchange column (GE Healthcare). Chromatography was carried out in 50 mM TrisHCl at pH 7.5 with a linear gradient of NaCl and a flow rate of 1.5 ml/min. The resulting fractions were concentrated and placed in a Superdex 200 10/30 Increase column (GE Healthcare). Chromatography was carried out again in 0.1× PBS buffer at a flow rate of 0.5 ml/min. The fractions were evaporated in a vacuum centrifuge and analyzed by SDS-PAGE in 6% gel (in non-reducing conditions without the addition of mercaptoethanol) and 10% gel (in reducing conditions with the addition of mercaptoethanol), followed by silver staining.
iPCR analysis with covalent DNA-antibody conjugate iPCR was performed with direct DNA-antibody conjugate as reported previously [18][19][20] except for the conjugate addition stage. The IgE analysis was carried out by coating plate wells with the recombinant allergen Alt a 1 in carbonate-bicarbonate buffer at pH 9.6 with a concentration of 2 μg/ml (50 μl per well). The coating was carried out overnight at +4˚C. Next, the wells were washed three times with TETBS buffer, and the sera from allergic and healthy donors were added at various dilutions to the TETBS with 20% goat serum (50 μl per well). The plate was incubated on a shaker for 1 hour at room temperature. After incubation, the plate was washed three times with TETBS buffer, and solutions of monoclonal antibody conjugates with 20% goat serum were added at various concentrations (50 μl per well). The plate was incubated on a shaker for 30 minutes at room temperature. The wells were then washed 6 times with TETBS buffer by alternating between short (10 sec) and long (5 min) washes, as well as 3 times for 10 seconds with TBS buffer. A PCR mixture was introduced into the wells (35 μl per well) and covered with mineral oil (30 μl per well). The composition of the PCR reaction was the following: 35 μl of the amplification reaction mixture Quantitative PCR (qPCR) was performed on a DTprime amplifier (DNA-Technology, Russia) using a protocol of initial denaturation for 5 min at 94˚C, followed by 40 cycles of the following: 15 sec of annealing and elongation at 60˚C and 5 sec of denaturation at 94˚C. At each cycle, the fluorescent signal from the probe was measured at 520 nm (FAM channel). Cq values were automatically determined for all reactions using amplifier software (RealTimePCR v.7.9.5.25). The average value of the threshold cycle (Cq) and standard deviation were calculated for each sample.
The detection threshold was calculated as three standard deviations for Cq, which is the value of the threshold cycle in negative samples. The threshold cycle difference (ΔCq) for different samples was calculated using the equation, ΔCqi = [average (Cq − Cqi)], where Cqi is a value of the threshold cycle in the sample being studied. Samples with a ΔCq below the detection threshold were considered as negative, and those with a ΔCq greater than or equal to the detection threshold were considered positive. The serum titer was evaluated as the maximum serum dilution at which the corresponding sample was considered positive.
iPCR was performed for the detection of IgM antibodies to Le C as described previously [19]. One-half of the plate wells was coated with the polymer derivative antigen (Le C -PAA), and the other half was coated with pure polymer (PAA). After washing, a blocking solution was added (1% BSA, 1% casein, and 1 μM oligonucleotide for blocking in TBS), followed by the addition of samples with a known content of anti-Le C IgM (3 wells with Le C -PAA and 3 wells with PAA).
In the case of streptavidin supramolecular complexes, the detection was performed using biotinylated anti-IgM antibodies, followed by interaction with DNA-streptavidin complexes and qPCR analysis. In the case of covalent conjugates, the detection was performed using the conjugates, followed by qPCR. Next, the difference between the Cq values was calculated as follows for each sample added to the wells coated with glycan-free PAA Cq (PAA)(i) (background) and Le C -PAA: Samples with ΔCq less than or equal to zero were considered negative, and those with values greater than zero were positive. Only ΔCq values were compared in different experiments.
Enzyme-linked immunoassay (ELISA)
ELISA of IgE was performed for the recombinant allergen Alt a 1 in the blood serum of an allergic patient as described previously [17].
Statistical analysis
The average and standard deviation were calculated using Excel software, which was also used to plot the Calibration curves.
Optimization of the covalent conjugate preparation technique by the example of anti-IgM antibodies
Control of the azido group introduction into antibody molecule (Ab-N3 DOL determination). The azido group introduction was controlled by reaction with DBCO-sCy5. The initial concentration of the MA2 antibody in all cases was 1 mg/ml, and the reaction was carried out in a carbonate buffer (0.1 M, pH 8.3) for 1 hour at room temperature. The excess DBCO-sCy5 was removed by gel filtration. The degree of labeling of antibody by the azido group (Ab-N3 DOL) was determined as the molar ratio of Cy5 (650 nm) to antibody (280 nm) with the following equation: A is the absorption at the corresponding wavelength, and CF 280 is a correction factor (CF 280 = 0.04). The extinction coefficients for the antibody (ε Ab , 280 nm) and sCy5-DBCO (εCy5, 650 nm) were assumed to be 210,000 L � mol -1 � cm-1 and 251,000 L � mol -1 � cm -1 , respectively. The results are presented in Table 1.
ODN-DBCO reactivity monitoring (ODN-DBCO DOL determination). As a result of analytical RP-HPLC of the oligonucleotide reaction with dibenzocyclooctyne-activated ester, two peaks of reaction products were obtained with a total yield of 80%. Spectrophotometric analysis of the conjugates of DBCO and sCy5-N3 derivatives demonstrated that each of the products is reactive with a yield of 70%. Thus, the content of reactive ODN-DBCO oligonucleotide was about 60%. Since both reaction products were reactive, we did not perform chromatographic purification of the dibenzocyclooctyne derivative of the oligonucleotide.
ODN-DBCO DOL was defined as the molar ratio of ODN to Cy5 in the ODN-DBCO reaction product with Cy5-N3 at room temperature overnight. Excess Cy5-N3 was removed by double reprecipitation of the reaction product in acetone/lithium perchlorate. The molar ratio of ODN to Cy5 was determined spectrophotometrically. The coefficients of the molar extinction of ODN and Cy5 were 613,500 L � mol -1 � cm -1 (260 nm) and 250,000 L � mol -1 � cm -1 (650 nm), respectively, and the correction factor Cy5 for 260 nm was 0.03. Thus, in a conjugation reaction with the azidated antibody, a mixture of ODN and DBCO-NHS reaction products was used after removing the excess reagent by gel filtration and determining the content of the reactive group. Optimization of ODN-DBCO excess and monitoring of conjugation reaction. The optimal excess of ODN-DBCO sufficient for conjugation was determined using an antibody concentration of 1 mg/ml and reaction at room temperature overnight. We investigated the completeness of the conjugation reaction under these conditions with different excesses of ODN-DBCO (1× (equimolar), 2×, 4×, and 8×). To achieve this, an antibody with Ab-N3 DOL = 6.4 was obtained using a 20-fold excess of STP-N3. ODN-DBCO was added at ratios of 1:1, 1:2, 1:4, and 1:8 (by active groups) to equal aliquots of the antibody.
After performing the conjugation reaction with ODN, a 3-fold excess of sCy5-DBCO was added to the reaction mixture. This substance interacts with the azido groups of the modified antibody that were unreacted in the previous step (reaction with ODN-DBCO) for 24 hours at room temperature. The conjugate was isolated on a Superdex 200 10/30 Increase column. The completeness of the reaction (η) of the antibody with ODN was calculated from the molar ratio of ODN and Cy5 in the reaction product using the following equation: η = (A260 / εODN)/ ((A260 / εODN) + (A650 / εCy5)) � 100%. The extinction coefficients for ODN (ε ODN , 260 nm) and sCy5-DBCO (ε Cy5 , 650 nm) were taken to be 613,500 L � mol -1 � cm -1 and 251,000 L � mol -1 � cm -1 , respectively. The results are presented in Table 2.
An aliquot of the 1:1 reaction mixture was placed in a RESOURCE Q anion exchange column. There was almost a complete absence of unmodified antibody, as shown in Preparation and testing of conjugates with different antibody-ODN ratios. Conjugates were obtained with different degrees of antibody labeling with oligonucleotide (mono-, bi-, tri-, etc.) in the antibody azidation by using 2-and 6-fold excesses of STP-N3. Ab-N3 DOL for these antibodies was 1.1 and 2.6, respectively. These results are in good agreement with the data in Table 1. The conjugation was carried out by mixing and incubating the solutions of the azidated antibody and oligonucleotide with DBCO overnight at room temperature. After determining the amount of active reaction groups (Ab-N3 DOL and ODN-DBCO DOL), the excess of oligonucleotide introduced into the reaction was calculated. In each case had a 4-fold excess of reactive ODN-DBCO relative to the azido groups of the antibody. The presence of unreacted azide groups of the antibody was tested by introducing a 10-fold excess of sCy5-DBCO. The chromatogram demonstrates the presence of unreacted antibody (peak 1). Fractions 2 and 3 showed no bands of free or labeled antibodies on the electrophoregram (the concentration is below the sensitivity of the detection method). Fraction 4 contains a monolabeled antibody, and fraction 5 contains mono-and double-labeled antibodies. Fraction 6 mainly contains double-and triple-labeled antibodies, but it also contains mono-, quadruple-, and even quintuple-labeled antibodies. The conjugation reaction was done using 250 μg of azidated antibody. The antibody from peak 1 did not react with sCy5-DBCO, suggesting that there were no azido groups in the antibody structure. The amount of antibody without azido groups was 60 μg, which corresponds to 25% of the amount of antibody taken in the conjugation reaction. Thus, with a labeling degree of antibody Ab-N3 DOL equal to 1.1, up to 25% of it remains unchanged. This indicates a wide distribution of the labeling degree and suggests the presence of up to 25% of the antibodies with two ODNs in the reaction mixture. Therefore, we were not able to obtain a monolabeled antibody with a high yield under these conditions.
To increase the yield of the conjugate, it would be reasonable either to obtain a mixture of mono-and double-labeled antibodies (Ab-N3 DOL nearly 1.5) or to reuse (modify) the antibody from fraction 1. After a chromatographic purification (Fig 4a) on the RESOURCE Q anion exchange column, fractions were isolated from the reaction mixture with the antibody MA2 Ab-N3 DOL = 2.6. The distribution of the number of labels per antibody varied from 1 (fraction 5, about 180 kDa by the length marker) to 6 (fractions 8 and 9) (Fig 4b). There was no peak corresponding to the unreacted antibody.
Subsequently, all fractions were purified by gel filtration on a Superdex 200 10/30 Increase column to remove the unbound oligonucleotides. The electrophoregram of the fractions obtained is shown in Fig 5. One can see that the repeated fractionation of the target peak allows for the isolation of fractions with an exact label content. Among the obtained fractions, we have chosen the following fractions for use in iPCR: fraction 5-12, which contains one label per antibody molecule according to the results of electrophoresis; fraction 7-3, which contains two labels per antibody molecule (with an admixture of 3 labels); fraction 8-3, which contains three labels per antibody molecule (with an admixture of 2 and 4 labels); and fraction 9-1, which contains four to five labels per antibody molecule.
Quantitative determination of IgM antibodies to the Le C disaccharide in serum using the iPCR method with covalent MA2-ODN conjugate. To select an optimal working dilution of conjugates for iPCR, qPCR data were first obtained for 100-fold titrations of conjugates in deionized water. qPCR was performed with dilutions of conjugates and ODN solutions with known concentrations (100 pM and 1 pM) as standards (qPCR was performed in three repeats with standards and in two repeats with conjugates dilutions). The efficiency of the PCR reaction was 92%. The concentration of the initial solutions of conjugates varied from 0.7 to 4.4 μM. Dilutions of the conjugate with one label (fraction 5-12; Fig 5) with final concentrations of 40 pM, 400 pM, and 4 nM were used for iPCR. The iPCR was performed according to the procedure described in Materials and methods (Fig 6).
The highest value of ΔCq pM, which provides a low background signal and the largest ΔCq for a sample with a high concentration of anti-Le C IgM.
Testing of conjugates with different numbers of oligonucleotides per antibody
Conjugates for the detection of anti-Le C IgM antibodies in serum. The following conjugates were selected: 5-12 with one label per antibody; 7-3 with two labels (with an admixture of 3 labels); 8-3 with three labels (with an admixture of 2 and 4 labels), and 9-1 with four to five labels. In the first stage, an attempt was made to align the conjugates by antibody content, for which the titrations of the conjugates were tested in qPCR. The quantity of oligonucleotide was converted to the quantity of antibodies based on an evaluation of the ratio presented in electrophoregrams. The following concentrations of conjugates by ODN were used in iPCR (the antibody concentration is the same): 1 label: 40 pM; 2 labels: 80 pM; 3 labels: 120 pM; 4-5 labels: 180 pM.
Taking into account the data obtained from the electrophoregram, we can consider the concentration of the antibody and antibody conjugate as a whole to be almost the same for all conjugates (approximately 40 pM). The average background value of Cq (PAA) for all conjugates during iPCR was 22.7-24.6. Fig 7 shows the results of testing the conjugates in the iPCR on a panel of samples with a known concentration of anti-Le C IgM, as well as a comparison with the DNA-streptavidin complex (DNA-Stvd) used earlier.
Conjugates with 1 and 2 labels exhibited the same sensitivity as the DNA-streptavidin conjugate (6 ng/ml). For a 3-label conjugate, the lowest detectable target concentration was 20 ng/ ml, and that for a conjugate with 4 and 5 labels was 60 ng/ml. Accordingly, the sensitivity of the method decreases drastically with the increase of the labeling degree of the detecting antibody in this case. There was no noticeable difference for conjugates with 1 and 2 labels. Bearing in mind the wide distribution of antibody labeling with the azido group, we believe that obtaining an antibody with an Ab-N3 DOL of 2 is optimal. This will ensure a 100% conversion of the antibody to the conjugate with the main product being double-labeled.
Conjugate for the detection of IgE antibodies in serum. Conjugates of ODN-BE5 antibody to human IgE were obtained as described above for the conjugate of anti-IgM antibodies, so a detailed description of the results is not provided. A conjugate with two labels was used in iPCR. In the case of the BE5-ODN conjugate, we found a non-specific binding to blood serum components of both allergic and healthy donors (Fig 8). This phenomenon was observed in iPCR analysis in both the presence and absence of the sorbed Alt a 1 allergen at concentrations of the BE5-ODN conjugate of tens of ng/ml (Fig 8a and 8b). When analyzing the allergic patient serum by ELISA, a similar nonspecific signal was also observed in the 20-fold dilution, but at higher concentrations of ODN-BE5 conjugate, the signal was on the scale of micrograms per milliliter (Fig 8c). We did not observe this phenomenon in the case of unconjugated antibody (Fig 8c) and BE5-streptavidin-DNA complex [18], as well as in the case of incubation with unconjugated oligonucleotide. Therefore, we believe that the observed nonspecific binding is a result of changes in the structure of the antibody molecule during its chemical modification.
To eliminate nonspecific binding, we tried to optimize the conditions of the immunoassay and introduced an additional blocking step between the stages of the sorption of allergen and the addition of the analyzed serum (compared to the method described for the DNA-streptavidin complex [18]). Blocking was performed with TBS buffer with various components: 1% BSA, 10% goat serum, 0.73 mM sucrose, 0.5% tween-20, 0.02% tween-20 with 1% BSA, 2%
Fig 7. Results of the different degrees of labeling conjugates tested by iPCR on a panel of samples with a known concentration of anti-Le C IgM.
The numbers of labels per AB corresponds to conjugates with the same number of ODN molecules per antibody. DNA-Stvd is a variant of iPCR using a streptavidin supramolecular complex [19].
Attainment, isolation, purification, and analysis of conjugates
Covalent DNA-antibody conjugates were obtained using the cycloaddition reaction of azides and alkynes. This necessitated the introduction of an azide group into the antibody. We chose to introduce the azido group into the antibody at the amino group via 4-sulfotetrafluorophenyl (STP) ether, which reacts with amino groups like NHS ethers and forms a strong amide bond [21]. This method is one of the simplest ones available.
Previously, we attempted to obtain a conjugate by the cycloaddition reaction of azides and alkynes catalyzed by monovalent copper (the "copper click" method). However, the complex of monovalent copper with THPTA often leads to precipitation when mixed with the antibody solution (although not for all antibodies). This is consistent with the fact that copper ions can cause protein denaturation [8]. When selecting the reaction conditions for human IgG preparation, we succeeded in identifying conditions under which the antibody did not precipitate at a low catalyst concentration. However, with a 20-nucleotide ODN model with a linear alkyne, only 30% of the antibodies were labeled. In contrast, when using the 60-nucleotide ODN with a linear alkyne, no conjugate was obtained at all if the reaction mixture was kept in argon atmosphere for the entire conjugation time.
The main problem was the isolation of the conjugate and its purification from unreacted ODN. We did not succeed in isolating the conjugate by anion-exchange chromatography on the RESOURCE Q anion exchange column. Although the retention time of unreacted azidated antibody is much less than that of ODN (9.16 and 11.7 min, respectively), the conjugate is not eluted between them as expected. Furthermore, at 11 min, mono-and double-labeled conjugates are eluted before the main peak of the ODN, and 4-5-labeled conjugates are eluted after the main peak of the free ODN (there was no peak separation at 260 nm). The chromatography was performed in a wide range of pH (4.5-7.5), but this did not improve the separation of the conjugate and ODN in a linear gradient of NaCl.
We performed a reaction with pentynoic acid sulfotetrafluorophenyl ester at pH 8.3. At this pH, both the N-terminal α-amino group (pKa 8.95) and the ε-amino group of lysine (pKa 10.53) were reactive and are referred to as reactive amino groups [22,23]. Analysis of the resulting conjugates indicated that when the azido group is introduced into the antibody using STP or NHS esters, there was a wide distribution of the labeling degree of antibody by the azido group. The average degree of antibody labeling by the azido group only statistically reflects the presence of labels on the antibody. Thus, it was shown that when Ab-N3 DOL is 1.1, 25% of the antibodies contained no azido groups, up to 25% contained 2 azido groups, and up to 50% contained one azido group (Fig 3). The selected scheme for the purification of the reaction products allows for the removal of the unmodified antibody at the anion-exchange chromatography stage and the excess ODN at the gel filtration stage. Nevertheless, it was impossible to obtain only the mono-labeled conjugate with a yield exceeding 50%. Obtaining a predominantly double-labeled conjugate in this case seems to be more rational.
Gong et al. obtained similar labeling degrees of antibody with a similar excess of DBCO--PEG5-NHS reagent [9]. The use of four molar equivalents of DBCO-PEG5-NHS resulted in 2.3 DBCO molecules per antibody after the reaction. In turn, this yielded a conjugate with 1.2 ODNs per antibody after reaction with a 4-fold excess of azidated oligonucleotide (45 bases). Unfortunately, the authors did not explain the incomplete involvement of DBCO-groups in the conjugation. They investigated the effect of the unlabeled antibody in the conjugate and showed that the 12.5% impurity of unlabeled antibody in the conjugate does not affect Cq.
It should be noted that a sharp decrease of ΔCq (a decrease of the target signal) was observed for only the content of impurity of the unlabeled antibody above 400% relative to the conjugate content. In the range of 25-400%, the decrease of ΔCq does not exceed one. We did not study the tolerance of our systems of the presence of unlabeled antibodies, despite the possibility of not performing the purification from unreacted antibody in the case of such a strong tolerance detection. Van Buggenum determined the position of the reactive lysine residues in mouse IgG upon modification by a 5-fold excess of tetrazine-NHS [10]. It turned out that there residues in the heavy chain were reactive (12 lysine residues in the molecule comprising 2 in the light chain and 10 in the heavy one). Unsurprisingly, all the reactive lysine residues were on the antibody surface and were available to the solvent.
Thus, the usage of NHS (or STP) chemistry did not allow us to produce an antibody labeled with a strictly defined number of labels or at least with a small spread in the number of labels -we always obtained a mixture. To solve this problem, it seems necessary to introduce the azido group in a different way. It is likely that methods based on the modification of glycan residues would improve the situation. Monoclonal antibodies contain one conserved N-glycosylation site at position N297 in each heavy chain. In this case, about 20% of the antibodies also contain a second N-glycosylation site in the variable region of the heavy chain [24]. Glycan motifs on antibodies are diverse (up to 30 variants), and monoclonal antibodies in culture can be synthesized in various glycoforms [25]. Nevertheless, enzyme processing can produce a homogeneous glycan composition [26]. The best solution for recombinant antibodies would be to use modified aminoacyl-tRNAs that carry amino acids with introduced functional groups that are inserted into a particular site during translation [27]. Another alternative is the SpyTag/SpyCather technique [28].
iPCR with covalent conjugates
Anti-IgM-ODN conjugate for anti-Le C antibody detection in sera. The usage of a supramolecular complex and covalent direct conjugates on the example of the anti-Le C IgM detection system resulted in similar sensitivity for mono-and double-labeled conjugates and the supramolecular complex (Fig 7). It should be noted that the protocol based on direct conjugates is shorter by several stages (1 hour of incubation and 3 wash steps). However, the same result can be achieved by using pre-prepared and gel-filtered DNA-biotin-streptavidin complexes with introduced antibodies, which were first described by Niemeyer in 1999 [29]. The increase of the labeling level of antibody in the example of the anti-Le C IgM detection system led to a decrease of the sensitivity, which was first observed at 3 ODN molecules per antibody and showed no significant difference between the conjugates with one and two labels.
Anti-IgE-ODN conjugate for anti-allergen antibody detection in sera. iPCR assay of anti-IgE conjugates showed that the specificity of the BE5 antibody changed. We used DBCO--PEG 4 -NHS for ODN modification, which is similar to what Gong et al. used for antibody modification [9]. They did not observed any changes in specificity of the antibodies. We expected that a higher solubility of the reagent and the extended linker between dibenzocyclooctyne and NHS-group would reduce the effect of ODN on the affinity and specificity of the antibodies. However, the specificity of the BE5-ODN conjugate prepared using the DBCO-PEG 4 -NHS reagent also changed.
The positions of the reactive amino groups where azido groups are introduced followed by the attachment of ODN seem to be very important. The position of the lysines available for the reaction of amino groups on the surface of the antibody is specific for each antibody. This was confirmed by the fact that we obtained conjugates with the MA2 antibody with preserved antibody specificity if the antibody amino groups were modified, and so did other authors with several other antibodies. It is should be noted that the biotinylated BE5 antibody did not change in specificity after streptavidin was added to the supramolecular complex of 5'-and 3'biotinylated ODN and streptavidin [18]. The biotinylation of antibody BE5 was carried out with the N-hydroxysuccinimide ester of biotin attached to the same reactive amino groups as STP-N3. Nevertheless, further streptavidin binding in the ODN-complex did not affect the specificity of the BE5 antibody.
Conclusions
We obtained covalent DNA-antibody conjugates using SPAAC reaction, and the method for the preparation and purification of the conjugates was optimized. The possibility of using these conjugates in iPCR was analyzed. After modification of the antibodies by amino groups, it was impossible to obtain monolabeled antibodies or antibodies with a strictly defined number of labels. The degree of labeling determined by the dyes introduced to azido groups only statistically reflected the final result.
One of the two monoclonal antibodies used in this study (anti-IgE BE5) lost its specificity after covalent (direct) conjugation with ODN. The sensitivity for the detection of IgM antibodies against Le C by iPCR using covalent conjugate proved to be similar to that measured for iPCR using the supramolecular complex of 5'-and 3'-biotinylated ODN and streptavidin, which we examined in a previous study [19]. However, the procedure became two steps shorter and at least 1 hour faster. | v3-fos-license |
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} | pes2o/s2orc | DFT Modeling of the Adsorption of Trimethylphosphine Oxide at the Internal and External Surfaces of Zeolite MFI
: The characterization of the acidity of zeolites allows a direct correlation with their catalytic activity. To this end, probe molecules are utilized to obtain a ranking of acid strengths. Trimethylphosphine oxide (TMPO) is a widely used probe molecule, which allows the sensing of solid acids by using 31 P NMR. We have performed calculations based on the density functional theory to investigate the Brønsted acid (BA) sites in zeolite MFI by adsorbing TMPO as a probe molecule. We have considered the substitution of silicon at the T2 site by aluminum, both at the internal cavity and at the external surface. The di ff erent acid strengths observed in the zeolite MFI when probed by TMPO (very strong, strong, and weak) may depend on the basicity of the centers sharing the acid proton. If the proton lies between the TMPO and one of the framework oxygen atoms binding the Al, the acidity is strong. When the framework oxygen atom is not directly binding the Al, it is less basic and a shortening of the TMPO − H distance is observed, causing an acid response of very strong. Finally, if two TMPO molecules share the proton, the TMPO − H distance elongates, rendering a weak acid character.
INTRODUCTION
Zeolites are well-known microporous materials whose frameworks are formed by corner-sharing SiO 4 tetrahedra. A negative charge is created when Al 3+ substitutes Si 4+ at the center of a tetrahedron, which is compensated by extraframework cations within the pore system of the zeolite. Brønsted acid (BA) sites are generated when the counterbalancing cation is a proton that covalently binds the O atom bridging the Al and the Si. 1 These BA sites, together with the size selectivity of the pore system, are the driving forces behind the wide range of catalytic applications of zeolites. 2−5 The characterization of the zeolite's acidity by number, density, and strength may allow a direct correlation to its catalytic activity. However, in contrast with an acid in an aqueous medium, there is no unique way to rank the acidity of solid materials. 6 In zeolites, the proficiency of each BA as a proton donor will depend on its location within the pore system and its accessibility by the adsorbed reactant, 6 which hinders the analysis of the zeolite's acidity.
The strength and number of the BA sites are usually measured through the adsorption of probe molecules, which act as bases and are protonated upon interaction with the BA sites. There are several analytical methods to evaluate the extent of the protonation of the probe molecule, 1,6 for example, the nuclear magnetic resonance (NMR), which provides high accuracy and specificity to examine the consequences of the proton transfer. 7 The movement of the acid proton from the BA site to the probe molecule may be sensed directly when recording the 1 H NMR spectrum, as in the case of the pyridine/pyridiniun system. 8 In addition, other nuclei different from 1 H may also be analyzed if their magnetic response to the applied field is affected by the protonation of the probe molecule, e.g., 13 C in acetone 9 and 31 P in trimethylphosphine oxide. 10 Trimethylphosphine oxide (TMPO, see Figure 1 for a representation of the molecule's structure) has shown to be a suitable molecule for sensing BA sites of different strength, owing to the high sensibility of the TMPO's 31 P nucleus to the intensity of the phosphine−BA interaction. 7 The 31 P chemical shift moves from 39 ppm (crystalline TMPO) toward a range of 50−105 ppm upon protonation of the TMPO's oxygen atom; the stronger the proton transfer, the larger the chemical shift. 7 Therefore, the strength of BA sites in zeolites can be classified according to the value of the 31 P chemical shift as very strong (90−80 ppm), strong (80−70 ppm), and weak (70−60 ppm), where 86 ppm is the calculated threshold of superacidity for TMPO. 7,10−12 The present work proposes a density functional theory (DFT) study of the adsorption of TMPO in zeolite MFI. Zeolite Socony Mobil-5 (ZSM-5) has the framework type of MFI, which is a material with a wide array of industrial applications and high versatility. 13−16 We have described in detail the TMPO−BA interaction at the internal and external surfaces of zeolite MFI, providing atomic level information to complement previous experimental reports. The MFI's internal and external surfaces behave differently: three types of BA sites of variable strength have been detected at the internal surface (very strong, strong, and weak), whereas the strong BA sites tend to disappear at the external surface. 10,12 Thus, to distinguish between these sites, alternative experimental methods must be used. For instance, silica chemical vapor deposition (Si-CVD) deactivates the external surface while keeping the internal sites undamaged, 10 whereas the inclusion of tributylphosphine oxide (TBPO), which is too big to diffuse into the zeolite's pore system, allows the exclusive sensing of the external surface. 10,12 We have also explored how a variable number of TMPO molecules affects the acid response of the BA site. Zheng et al. have previously reported the enhancement of the Brønsted acidity in mordenite by the intermolecular solvent effect when several TMPO molecules are confined in the micropore. 17 The authors relate this effect to van der Waals interactions among the TMPO molecules, which provoke simultaneous decrease and increase of the O−H distance of the TMPO molecules, generating in consequence superacidity and weak acidity, respectively. 6,7,17
COMPUTATIONAL METHODS
The results presented in this work were obtained using the DFT approximation as implemented in the Vienna Ab-initio Simulation Package (VASP). 18−21 The generalized gradient approximation (GGA) under the scheme proposed by Perdew, Burke, and Ernzerhof (PBE) was used in all calculations. 22 Grimme's method of the sum of pairs was added to the GGA energy to account for the long-range-interactions (DFT-D2). 23 The inclusion of dispersion corrections improves the predicted cell parameters and elastic properties of the zeolite MFI. 24 The valence electrons were treated explicitly using a basis set of plane waves, although their nodal features and interactions with the ion were included within the projected-augmented-wave method (PAW). 25,26 The number of plane waves considered during the calculations was determined by a maximum kinetic energy of 500 eV. The electronic convergence was improved using the Gaussian smearing method with a bandwidth of 0.1 eV for the zeolite and zeolite−TMPO systems and 0.01 eV for the isolated TMPO molecule. 27,28 Only the Gamma point was taken into account during the numerical integration over the Brillouin zone due to the large dimensions of the MFI unit cell. Finally, the electronic threshold was set to 10 −5 eV.
The orthorhombic unit cell of the MFI framework has 12 nonequivalent T-sites to be substituted by Al (the center of each tetrahedron is referred as T-site and numbered according to the symmetry of the zeolite's unit cell). In conjunction, the acid proton can bind four O atoms for each T-site, which produces a high number of possible combinations, making practically impossible a detailed analysis of each arrangement. We have therefore opted for Al substitution in the T2-site, binding the proton to O1 (see Figure 1), which allowed us to focus on the acid response of that BA site to the number and orientation of TMPO molecules, as well as the consequences of the BA site location at the internal or the external surface.
The T2-site was chosen for the Al substitution, and the charge-compensating proton was bound to the O1 atom, leading to the formation of the BA site (see Figure 1). This specific position was chosen because T2 is at the interception of straight and sinusoidal channels and is easily accessible by the probe molecule, allowing a fair study of the TMPO agglomeration around the acid site. We have studied the proton transfer to TMPO, accommodating up to three molecules simultaneously. The optimized cell parameters of the zeolite MFI were 20.317, 19.979, and 13.413 Å along the a, b, and c directions, respectively, 24 within 1% of the experimental measurements. 29 Using periodic boundary conditions, the external surface was described by a slab formed of two pentasil layers, 30 as shown in Figure 1, with a surface area per unit cell of 272.512 Å 2 and a perpendicular vacuum gap of 20 Å between the slabs. In this termination, each dangling Si− O bond was properly saturated by hydroxyl, thus forming silanol groups at the bottom and top surfaces of the slab; in the case of pentasil layers, there is only one dangling Si−O bond per Si atom. 24 The isolated TMPO molecule was first optimized in a 20 × 21 × 22 Å 3 cell, before it was loaded in the zeolite, varying its numbers and orientations, ahead of a full geometry optimization.
The initial geometries set for optimization were constructed by loading the TMPO molecules in close proximity to the acid proton of the zeolite. These geometries were locally optimized using a conjugate gradient algorithm until all forces acting on the ions were smaller than 0.03 eV/Å. The local relaxations were followed by short quantum molecular dynamics (MD) simulations over a simulation time of 10 ps, with a time step of 0.5 fs. A microcanonical ensemble was used for each geometry during the first 2.5 ps of MD while keeping the temperature at 300 K. A canonical ensemble centered at 300 K was simulated during the next 7.5 ps of MD, controlling the temperature fluctuation with a Noséthermostat. 31−33 We have used the last 2.5 ps to obtain the average structural parameters of interest.
All the images related to structural and charge density visualizations were obtained with the code Visualization for Electronic and Structural Analysis (VESTA 3). 34 2.1. Classification of the Acid Strength. Brønsted acid sites of different strength are detected in zeolite MFI when probed with TMPO. According to Seo et al., the acid strength can be classified as very strong (86 ppm), strong (76 ppm), and weak (68 and 66 ppm), 12 in agreement with previous The Journal of Physical Chemistry C Article experimental reports. 10 Zheng et al. reproduced the range of 31 P chemical shifts by modeling the adsorption of TMPO on zeolite MFI, which was represented by a cluster of eight Tsites. 11 The authors tuned the acid strength by varying the terminal Si−H distances of the cluster, thus controlling the extent of the proton transfer from the BA site to the TMPO's oxygen atom, hereafter referred to as O (P) . Zheng et al. reported a wide spectrum of O (P) −H distances after optimization, calculating the 31 P chemical shift for each geometry; those distances ranged from 1.368 Å (45.5 ppm) to 1.014 Å (88.9 ppm). 11 We noted that our calculated O (P) −H distances changed within the limits proposed by Zheng et al., and hence we assumed that the theoretical work in ref 11 matches the expected 31 P chemical shift for our own data. Figure 2 shows the correlation between the O (P) −H distance and the 31 P chemical shift as calculated in ref 11. From that curve, we extrapolated the O (P) −H distances corresponding to the experimental measurement of the 31 P chemical shift that classifies the BA sites by strength (see Figure 2). 12 Taking the middle points between the extrapolated O (P) −H distances, we obtained approximate ranges of O (P) −H distances to classify the effective acid strength of our model (see Figure 2). The ranges are as follows: very strong acid (from 1.001 to 1.041 Å), strong acid (from 1.041 to 1.097 Å), and weak acid (from 1.097 to 1.188 Å). In comparison, we calculated a O (P) −H distance of 0.978 Å for a fully protonated TMPO molecule loaded in the zeolite, for which the position of the P atom was fixed at the center of the pore interception of zeolite MFI to avoid any Hbonding with nearby framework O atoms. In addition, on the basis of Car−Parrinello MD of a hydronium ion in aqueous During the local relaxations, the proton was transferred from the BA site to the TMPO molecule, at both the internal and external surfaces, regardless of the initial configuration. The Bader analysis of atomic charges 36−38 confirmed the movement of the proton; the total charge obtained for protonated phosphine oxides ranged from +0.8 to 0.9 e − , and more than 80% of the corresponding negative charge left in the zeolite framework was associated with the AlO 4 tetrahedron. Figure 3 shows the final equilibrium structures with the corresponding structural parameters listed in Table 1. The O (P) −H bond length ranged from 1.045 to 1.066 Å, i.e., larger than in the MFI external silanol groups (<0.970 Å) and MFI acidic protons with intraframework H-bonds (<1.014 Å). 24 These longer bonds were due to strong H-bonds established between the protonated TMPO molecules and the O (Al) atoms of the framework, with O (P) −O (Al) distances below 2.6 Å and H− O (Al) −O (P) angles smaller than 3°. 39 According to the division 11 The experimental classification of the acid sites of the zeolite MFI according to the TMPO 31 P chemical shift is indicated by vertical dashed blue lines. 10,12 The experimental classification is used to extrapolate the expected O (P) −H distances from its interception with the theoretical curve (indicated by red circles); the corresponding distance values are written above the horizontal dashed black lines. The spectrum of O (P) −H distances is divided into three zones taking the middle points between the extrapolated O (P) −H distances. These zones are shaded alternately in light gray and white, corresponding to (top) weak acids, (center) strong acids, and (bottom) very strong acids; the limits of each range are indicated by the red numbers at the left-hand side of the graph. Table 1.
The Journal of Physical Chemistry C Article of the spectrum of possible O (P) −H distances, shown in Figure 2, the acid strength of the BA site may be classified as strong for the four different configurations after local optimization. No evidence of very strong or weak acidity was obtained at this stage. The variation of the adsorption energy could not be correlated with corresponding changes in the O (P) −H distance. Nonetheless, the average TMPO interaction energy was larger for the internal BA when compared with the external BA: −190 and −167 kJ/mol, respectively. The contribution of dispersion forces to the total adsorption energy was 47% and 38% for the internal and external BA site, respectively. However, these percentages should be viewed with reservation because the DFT-D2 method tends to overestimate attractive interactions owing to the omission of terms above the two-body contribution; the error can increase beyond 10% for supramolecular complexes. 40 The MD simulation did not result in significant changes when compared to the local optimization; Table 2 shows the average values and the standard deviations of the last 2.5 ps.
The average values of the O (P) −H bond length remained within the range 1.05−1.08 Å, with fluctuations between 0.05 and 0.08 Å, which confirms that the strength of the modeled BA site can only be classified as strong when a single TMPO is adsorbed. Furthermore, we observed the replacement of O1 (Al) by O2 (Al) as the acceptor of the H-bond for the configuration in Figure 3b (see Figure 4 for a representation of the structure after 10 ps of MD). This movement occurred during the first 2.5 ps of MD simulation, although the acid response continued to be strong.
3.2. Adsorption of Two TMPO Molecules. We next adsorbed two TMPO molecules at a short distance from the BA site to analyze their effect on the proton transfer. Figure 5 shows the most stable structures after local optimization, out of several tested configurations at the internal and external BA sites; related structural parameters are compiled in Table 3. The O (P) −H bond length of the configurations in Figure 5 decreased to 1.016 and 1.033 Å for the internal and external surfaces, respectively, suggesting an apparent stronger acidity triggered by the presence of the second TMPO molecule. In consequence, the acid strength of the internal and external acid sites can be classified as very strong according to the scheme in Figure 2.
We have calculated average adsorption energies per TMPO of −125 kJ/mol for the internal surface and −112 kJ/mol for the external surface, with the dispersion forces representing 84% and 54% of the total adsorption energy, respectively, indicating that the role of van der Waals interactions increases with the number of probe molecules.
The second TMPO is a source of steric hindrance that affects the interaction through H-bonding between the first TMPO and the BA site. Because the protonated TMPO cannot form an ideal orientation in front of the bridging O atom, the Hbond is not strong enough, which produces a further shift of the proton position toward O (P1) . We did not observe any evidence that the second TMPO interfered through direct competition for the proton: the O (P2) −H distance increased above 2.5 Å Table 3.
The Journal of Physical Chemistry C Article during local optimization, which diminishes the possibility of H-bonding.
In contrast to the adsorption of a single TMPO molecule, where only strong acids are observed after both local optimization and MD, the systems composed of two TMPO molecules continued evolving during the MD simulation, modifying the acid strength classification given by the local optimization. At the internal surface, the movement of the TMPO molecules is capped owing to the reduced space inside the pore system. Therefore, we noticed a small shift of the protonated TMPO from O1 (Al) to O6 (Al) , which was still enough to increase the separation from the second TMPO and establish a strong H-bond with O6 (Al) (see Figure 6a and Table 4). As a consequence, the average O (P1) −H bond length increased to 1.05 Å after 10 ps of MD, from an initial value of 1.016 Å. The absence of confinement effects at the external surface allowed greater movement of the second TMPO, moving away from the first TMPO, to finally establish two strong H-bonds with nearby silanol groups (see Figure 6b). The withdrawal of the second TMPO from the vicinity of the protonated phosphine reduced the steric effects and hence enabled the reorientation of the first TMPO. Without steric interference, the acid proton could be better shared between the bridging O1 (Al) and O (P1) , which increased the O (P1) −H bond length from 1.033 Å after local optimization to an average value of 1.07 Å after 10 ps of MD. Therefore, the very strong acidity predicted by the local relaxation was not conserved during the MD simulation; the BA site behaved as a strong acid instead.
3.3. Adsorption of Three TMPO Molecules. Proton transfer can be classified as weak for O (P) −H distances that range from 1.097 to 1.188 Å (see Figure 2). We observed this weak acidity when a direct O (P) −Al interaction was induced after adding a third phosphine oxide into the 2TMPO/1BA systems shown in Figure 5. Figure 7 shows two equivalent structures after local optimization, out of several tested during our work, where O (P1) −H bond lengths of 1.131 and 1.154 Å were obtained after adsorption on the internal and external surfaces, respectively; structural parameters of interest are compiled in Table 5. The proton was almost evenly shared between O (Al) and O (P1) , rendering the acid response of the BA site weak. On this occasion, the average interaction energies for the internal and external BA sites differed by only 2 kJ/mol: −99 kJ/mol (internal) versus −97 kJ/mol (external). The adsorption at the internal surface, without considering the correction for dispersion forces, was repulsive, by 12 kJ/mol on average. The Journal of Physical Chemistry C
Article
Once the van der Waals interactions were taken into consideration, the overall adsorption energy became attractive, with a value of −99 kJ/mol. At the external surface, the dispersion contribution represented 77% of an average adsorption energy of −97 kJ/mol. Our interpretation of the weakening of the acid as proton donor after the adsorption of three TMPO molecules was based on the strengthening of the O (Al) −H bond. The O (Al) −Al bond was weakened by the interaction between the Al atom and one of the nonprotonated TMPO molecules, which was reflected in O (P) −Al distances that remained around 2.0 Å for both surfaces, as shown in Figure 7 and Table 5. There was also a large displacement of the Al atom during the optimization toward the nonprotonated phosphine. Under conditions of significant agglomeration of TMPO molecules around the BA site, the Al center may show residual Lewis acidity, with subsequent repercussions on the acid strength of the BA site. We did not observe direct competition for the acid proton by the nonprotonated phosphines, emphasized by O (P2) −H and O (P3) −H distances above 2.8 Å after local optimization.
In order to observe the perturbation and reordering of the electronic charge density derived from the TMPO-Al interaction, we have calculated the charge density difference for the configurations shown in Figure 7 according to the equation The ρ zeolite+3TMPO term corresponds to the electronic density of the whole system with three TMPO molecules, while the density of the zeolite without the TMPO molecule closest to the Al atom is represented by the ρ zeolite+2TMPO term. The electronic density of the isolated phosphine oxide with exactly the same structure as when it was interacting with Al atom is denoted by the ρ 1TMPO term. We have used the same grid of points to represent the charge density and the same box size for the three calculations. The resulting distribution shows an increase of the charge density between O (P2) and Al atoms (see Figure 8), which highlights the formation of bonding interactions between both centers. Figure 9 shows the projected density of states (PDOS) onto O (P2) (2p) of the TMPO interacting with the Al atom, as represented in Figure 7a, corresponding to the interior of the zeolite. The O (P2) (2p) PDOS presents intense peaks at the upper occupied edge of the energy spectra, between −2 and 0.0 eV (see Figure 9a). These O (P) (2p) states are complemented by small peaks in the Al(3s,3p) PDOS (Figure 9b,c) which disappear once the TMPO−Al interaction is deleted by removing the phosphine molecule (Figure 9d,e). In addition, the Al(3s,3p) PDOS reports the appearance of empty states above the Fermi energy as a result of the phosphine removal (Figure 9d,e). These transformations emphasize electron donation from the TMPO into empty electronic states associated with the Al atom.
After local optimization, there were differences in the behavior of the internal and external BA sites in the presence of three TMPO molecules. The internal ones almost always Table 5. The Journal of Physical Chemistry C Article performed as weak acids, whereas the external BA sites varied in strength between strong and weak, depending on the configuration; Table 6 summarizes these tendencies. When the O (P2) atom remained at less than 2.15 Å from the Al, the proton transfer was weak. Once this distance increased and the nonprotonated TMPO moved away from the Al atom, which was the case in four out of six configurations considered for the external BA site, the acidity was classified as strong. These findings underline the importance of the TMPO−Al interaction for the presence of the weak acidity.
The locally optimized arrangements of three TMPO molecules evolved differently during the MD simulation, depending on whether the adsorption took place at the internal or external surface. The MD calculations were performed on the configurations shown in Figure 7, with the average of the main structural parameters compiled in Table 7. The internal BA site continued to behave as a weak acid after 10 ps of dynamics, mostly due to the conservation of the initial configuration given as an input, with O (P2) atom binding the Al center. However, at the external surface the agglomeration of three TMPO molecules at the acid site was not preserved; both nonprotonated TMPO molecules moved away toward nearby silanol groups to establish H-bonds. Once the O (P2) −Al link was broken, the acid strength of the external BA was again strong, illustrated by an average O (P1) −H bond length of 1.05 Å. This exemplifies how equivalent BA sites are driven to behave differently as a consequence of the confinement produced by the pore system. The same confinement may be mimicked at the external surface if a large enough number of TMPO molecules is adsorbed in the vicinity of the BA site, limiting the movement of those phosphines that are in direct contact with the acid.
3.4. Full Deprotonation of the Brønsted Acid Site. We have considered the migration of a protonated TMPO from the vicinity of the BA site by moving the phosphine to the second pore interception that is present within the MFI unit cell. The distance between the acid proton and any of the four O atoms binding the Al center was larger than 5 Å before relaxation, rendering the acid site fully deprotonated. After local optimization, the O (P) −H bond length decreased to 0.979 and 1.005 Å for adsorption at the internal and the external surfaces, respectively; Figure 10 shows the optimized geometries, with the O−H distances compiled in Table 8. Those bond lengths correspond to 31 P chemical shifts of around 86 ppm, which translates into an acid classification of very strong (see Figure 2). The framework O atoms that do not bind the Al center are not basic enough to lengthen the O (P) −H bond, even if a H-bond is established. The MD simulation did not result in further modifications, with average O (P) −H bond lengths remaining around 1.00 Å (see Table 8).
We also placed a second TMPO molecule in close proximity to the protonated phosphine in order to form the species (TMPO) 2 H + and thus to induce the sharing of the acid proton Table 8. Table 8); those values correspond to 31 P chemical shifts that classify the acid strength as weak. Therefore, full proton transfer may be probed experimentally as either very strong or weak, depending on whether the proton is binding a single TMPO or is shared between two phosphine molecules, respectively. During the adsorption of TMPO on Keggin-type 12tungstophosphoric acid (HPW), 41 TMPOH + species appear within the region 95−85 ppm, while the formation of (TMPO) 2 H + moves the 31 P chemical shift into the range 75−53 ppm, in agreement with our calculated O (P) −H distances. However, the signals associated with (TMPO) 2 H + disappear after thermal pretreatment of the sample, to emerge again when the sample is oversaturated with TMPO molecules. 41 Extrapolating to the zeolite MFI, it is more probable that the formation of (TMPO) 2 H + occurs at the external surface, where the acid sites are exposed to higher loads of TMPO molecules than at the internal surface.
The implications of the (TMPO) 2 H + formation depend on its coexistence with the other arrangements analyzed above. The acid proton may be shared between O (P) and O (Al) , or between O (P1) and O (P2) ; the former alternative produces a strong proton transfer, as it was observed above after the MD simulation, while the latter corresponds to (TMPO) 2 H + , interpreted as weak acidity in the 31 P NMR spectrum. The relative occurrence of the first or the second alternative should depend on the number and mobility of TMPO molecules throughout the framework. For instance, a second TMPO can get close to the acid site where a protonated phosphine is already interacting through an H-bond with one of the O (Al) atoms. The second O (P2) may replace O (Al) within the H-bond to form (TMPO) 2 H + (our MD simulation was too short to observe this transformation when more than one TMPO was adsorbed). Although O (P2) is more basic than O (Al) , both TMPO molecules must have a specific orientation to properly share the proton. Owing to the presence of the (CH 3 ) 3 P segment, steric effects may play against the formation of (TMPO) 2 H + , which are accentuated by the confinement along the pore system. At the external surface, the TMPO's mobility and number increase as well as the access to the acid sites. This favors the formation of (TMPO) 2 H + over the adsorption of a single TMPO at the acid site and may be the reason behind the disappearance of the 31 P signal for strong acids at the external surface of zeolite MFI. 10,12 Once the (TMPO) 2 H + is formed, an additional equilibrium between (TMPO) 2 H + and (TMPOH + + TMPO) can be established. This suggestion may explain the presence of very strong proton transfer, considering that we only observed an average O (P) −H bond length below 1.01 Å after MD simulation for the TMPOH + species in Table 8. A similar process has been proposed for the solvation of H + ions in an aqueous medium, where an interconversion between H 5 O 2 + and (H 3 O + + H 2 O) is observed using Car−Parrinello MD. 35
CONCLUSIONS
We have used DFT with dispersion corrections to study the adsorption of TMPO at the internal and external surfaces of zeolite MFI, with the T2-site substituted by Al. We initiated relaxation of the structures using local geometry optimization, followed by 10 ps of MD centered at 300 K; we considered the MD results as the final criterion to characterize the acid site. The acid should perform as strong, according to the 31 P chemical shift, when the proton is shared between the TMPO's oxygen atom and one of the framework O atoms that bind the Al. This trend holds when the number of TMPO molecules around the BA site increases, being the only exception the adsorption of three molecules at the internal surface. In that case, one of the nonprotonated TMPO molecules directly interacts with the Al center through direct contact between O (P) and Al. This interaction weakens the O (Al) −Al bond, making the O (Al) −H bond stronger and less prone to be broken; in consequence an O (P) −H bond length of 1.2 Å is calculated, which translates into an acid strength classified as weak by 31 P NMR. The TMPO−Al interaction is more likely at the internal surface, where confinement effects keep the TMPO within close proximity of the Al atom. At the external surface, the nonprotonated TMPO molecules tend to migrate from the acid site, and establish H-bonds with nearby silanol groups.
We only observed O (P) −H bond lengths smaller than 1.01 Å (acid classified as very strong by 31 P NMR) when a protonated TMPO was assumed to migrate from the acid site. In the absence of H-bonding between O (P) and O (Al) , the O (P) −H bond becomes shorter. Furthermore, if the acid proton is shared between two TMPO molecules, forming (TMPO) 2 H + , the O (P) −H bond length increases to an average value between 1.1 and 1.2 Å, producing 31 P chemical shifts that classify the acid as weak. Therefore, the three main regions in the 31 P spectrum that are used to classify the acid sites as very strong (Figure 10a The Journal of Physical Chemistry C Article | v3-fos-license |
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} | pes2o/s2orc | Tolfenamic acid inhibits GSK-3β and PP2A mediated tau hyperphosphorylation in Alzheimer’s disease models
Tolfenamic acid, a nonsteroidal anti-inflammatory drug, alleviated learning and memory deficits and decreased the expression of specificity protein 1 (SP1)-mediated cyclin-dependent kinase-5 (CDK5), a major protein kinase that regulates hyperphosphorylated tau, in Alzheimer’s disease (AD) transgenic mice. However, whether tolfenamic acid can regulate the major tau protein kinase, glycogen synthase kinase-3β (GSK-3β), or tau protein phosphatase, protein phosphatase 2A (PP2A), further inhibiting hyperphosphorylation of tau, remains unknown. To this end, tolfenamic acid was administered i.p. in a GSK-3β overactivation postnatal rat model and orally in mice after intracerebroventricular (ICV) injection of okadaic acid (OA) to develop a PP2A inhibition model. We used four behavioural experiments to evaluate memory function in ICV-OA mice. In this study, tolfenamic acid attenuated memory dysfunction. Tolfenamic acid decreased the expression of hyperphosphorylated tau in the brain by inhibiting GSK-3β activity, decreasing phosphorylated PP2A (Tyr307), and enhancing PP2A activity. Tolfenamic acid also increased wortmannin (WT) and GF-109203X (GFX) induced phosphorylation of GSK-3β (Ser9) and prevented OA-induced downregulation of PP2A activity in PC12 cells. Altogether, these results show that tolfenamic acid not only decreased SP1/CDK5-mediated tau phosphorylation, but also inhibited GSK-3β and PP2A-mediated tau hyperphosphorylation in AD models.
Introduction
The World Alzheimer Report in 2018 showed that the number of people with dementia worldwide is currently 50 million, but will reach 152 million in 2050 [1]. Alzheimer's disease (AD) is a global threat to human health [2][3][4]. The clinical hallmarks of AD are cognitive and psychiatric dysfunction. The major neuropathological hallmarks of AD are senile plaque deposition, neurofibrillary tangle formation, and neuronal cell loss [2]. The tau protein, a microtubule-associated protein that stabilizes the neuronal cytoskeleton, can be phosphorylated at specific sites [5]. However, high levels of phosphorylated tau, which destabilizes the neuronal cytoskeleton, have been found in the brains of patients with neurodegenerative disorders [6,7]. Hyperphosphorylated tau causes mitochondrial dysfunction, synapse impairment, and cognitive disorders [8].
Specificity protein 1 (Sp1) is a transcription factor, and its target genes include amyloid precursor protein (APP), beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1), and CDK5, all of which play vital roles in AD. Therefore, the regulation of SP1 has an important role in AD therapeutic strategies.
Tolfenamic acid (TA) is an SP1 inhibitor [9]. An experimental study of its safety showed that repeated oral administration of 50 mg/kg tolfenamic acid for 6 weeks did not affect mouse body weight and had no toxic effect on mouse organs [10]. Tolfenamic acid attenuated learning and memory deficits in AD transgenic mice through inhibiting the expression of APP, BACE1, and SP1-mediated CDK5/hyperphosphorylated tau [11,12]. However, whether tolfenamic acid can prevent other protein kinases or protein phosphatases from regulating tau hyperphosphorylation is still unknown.
In this study, we administered tolfenamic acid i.p. in a GSK-3β overactivation postnatal rat model and orally in ICV okadaic acid (OA)-induced mice, a model of PP2A inhibition, to evaluate the effect of tolfenamic acid on hyperphosphorylated tau. The Y-maze test, novel object recognition test, Morris water maze test, and passive avoidance test were used to test the effect of tolfenamic acid on cognitive function in ICV-OA model mice. Furthermore, we treated PC12 cells with wortmannin (WT, a specific inhibitor of PI3K)/GF-109203X (GFX, a specific inhibitor of PKC) and OA to induce tau hyperphosphorylation and confirm the effects of tolfenamic acid on neurotoxicity and tau phosphorylation and the mechanism of tolfenamic acid.
Animals
Two-month-old male and female Wistar rats and male C57BL/6 J mice (22 to 25 g) were purchased from Liao Ning Chang Sheng Biotechnology Co., Ltd. (Benxi, China). Rat pups at postnatal day 12 (P12) were used for studies of the efficacy of GSK3β inhibition. All animals were maintained in polyacrylic cages under standard housing conditions with a 12-h light/dark cycle. Food and water were provided ad libitum.
ICV injection
ICV injection was performed as described in our previous report [13]. Briefly, the mice were anaesthetized with 2.5% avertin (Sigma-Aldrich, USA). Then, the head of each mouse was held in a stereotaxis instrument. OA (100 ng in 2 μl of normal saline) or vehicle (2 μl of normal saline) was injected into the right ventricle at the following coordinates: − 0.5 mm anterior to bregma, 1.0 mm lateral, and − 3.0 mm ventral. The OA dose was based on a study by Rajasekar et al. [14].
Treatment schedule
Experiment A: tolfenamic acid (50 mg/kg) was dissolved in 5% DMSO and injected i.p. in P12 rats from the same litter. At 4 and 6 h after injection, the rats were killed, and the brain tissue was dissected.
Experiment B: C57BL/6 J mice were divided into a control group, model group (ICV-OA), a group treated with 10 mg/kg TA and a group treated with 50 mg/kg TA (n = 10/group). The mice were given tolfenamic acid or vehicle i.g. After behavioural assessment, the mice were killed, and the brain tissue was dissected. The brains from five mice in each group were used for immunohistochemical staining. The cerebral cortices from the other mice were used for ELISA and a PP2A activity assay. The hippocampi were used for western blotting. Tolfenamic acid doses were selected based on those reported by Adwan et al. [12] and those in our previous study [15]. The experimental schedule is summarized in Fig. 1.
Y-maze test
The Y-maze test apparatus contained three arms (40 cm long × 10 cm wide × 12 cm high). Each mouse was paced in one arm and allowed to explore for 5 min. The number of entries into each arm and the sequence of these entries were recorded. Entrance of the mouse into the three arms in sequence was referred to as a "successful alternation". Alternation behaviour was calculated as [number of successful alternations/(total number of arm entries-2) × 100%] [15].
Novel object recognition test
A square box (50 cm long × 50 cm wide × 15 cm high) was used as equipment for the novel object recognition test. On the first 2 days, the mice were habituated to the equipment 2 times/day for 10 min each. On the test day, the mice were allowed to explore two identical objects, A1 and A2, for 5 min. After a 1 h intertrial interval, A2 was replaced with novel object B, and the mice were returned to the box for 5 min. After a 24-h retention interval, B was replaced with novel object C, and the mice were returned to the box for another 5 min. The exploration time for each object was recorded. The preferential index (PI) was calculated as [time spent exploring a novel object/total exploration time × 100%] [15].
Morris water maze test
A circular pool (100 cm in diameter, 50 cm high) containing 30-cm-deep water at 25 ± 1 °C was used as the equipment for the Morris water maze test. The fourth quadrant contained a security platform that was 1 cm below the water level. The mice were trained twice a day for 3 days after a 3-h intertrial interval. The mice were allowed to swim for 60 s to find the platform. If the mice failed to find the platform in 60 s, they were placed on the platform for 20 s. On the fourth day, the spatial memory of the mice was tested. The platform was removed, and each mouse was allowed to swim for 60 s. The escape latency (time spent in the fourth quadrant), swimming distance, and swimming speed were recorded [16].
Passive avoidance test
The equipment used for the passive avoidance test consisted of a bright room and a dark room. A bright light bulb was hanging on the top of the bright room to encourage the mice to enter the dark room, and copper bars at the bottom of the dark room could conduct electricity at a voltage of 31 V. The experiment was carried out on 2 days. On the first day (the training phase), the mice were placed individually in the bright room without electricity and moved freely for 5 min. Then, the mice were driven into the dark room through the use of alternating current. The normal reaction of the mice was to run back to the bright room to avoid electric conduction. Some of the mice repeatedly ran into the dark room, where they were shocked, and then quickly ran back into the bright room. The same method used on the training day was used on the test day after a 24-h retention interval; however, the dark room was not electrified. The number of mice that entered the dark room within 5 min was recorded as the error time [15].
Immunohistochemical staining
Briefly, brain sections were incubated with antibodies against p-Tau S396 (1:1000) and p-PP2A (1:200) at 4 °C overnight. Then, the sections were incubated with biotin-labelled secondary antibody at 37 °C for 30 min. The sections were treated with avidin-biotin enzyme reagent and visualized using a DAB kit (Boster, Wuhan, China). The positive area in each section was quantified by using ImageJ software [15].
Measurement of PP2A activity
PP2A activity was measured with a kit according to the manufacturer's protocol (V2460, Promega, USA). Briefly, extracts of cerebral cortex tissue were centrifuged to remove particulate matter, and endogenous free phosphate was then removed with gel chromatography. Samples were incubated with a chemically synthesized phosphopeptide in a 96-well plate at 37 °C for 30 min. The reaction was stopped by the addition of 50 μl of a molybdate dye/additive mixture. The absorbance at 630 nm was measured using a fluorescence reader (Thermo Fisher Scientific, USA).
Cell viability
PC12 cells were obtained from the National Infrastructure of Cell Line Resource (Beijing, China [17][18][19], and our preliminary experiment.
Statistical analysis
The data were analysed using SPSS 21.0. Statistical significance was determined using one-way or two-way ANOVA followed by Fisher's LSD multiple comparisons test with homogeneity of variance or Dunnett's T3 test with heterogeneity of variance. Experimental data are represented as the mean ± SEMs. A value of p < 0.05 indicates statistical significance.
Tolfenamic acid attenuated tau hyperphosphorylation in postnatal rats
As reported by Selenica et al., Takahashi et al., Leroy and Brion, p-tau and p-GSK-3β levels were increased in the developing rat brain and peaked at postnatal day 12 (P12) [20][21][22]. P12 rats were used for studies of the efficacy of GSK3β inhibition [20]. Tolfenamic acid has been reported to reduce p-tau levels by inhibiting CDK5 expression [12]. In this study, 50 mg/kg tolfenamic acid was injected i.p. for 4 and 6 h. Then, we used western blotting for semi-quantification of the p-tau level in the brain. The p-tau levels were significantly decreased 4 and 6 h after tolfenamic acid treatment (p < 0.01, Fig. 2). Tolfenamic acid enhanced p-GSK-3β (Ser9) levels in the brains of the P12 rats and also increased levels of p-Akt (Ser473) (p < 0.05, Fig. 2), which is upstream of GSK-3β. We next used a specific cellular model of GSK-3β activation to test the effect of tolfenamic acid on an upstream target of p-tau.
Tolfenamic acid attenuated tau hyperphosphorylation in WT/GFX-treated PC12 cells
The combined use of WT (a PI3K inhibitor) and GFX (a PKC inhibitor) can specifically trigger GSK-3β, further inducing tau hyperphosphorylation. Therefore, we used WT/GFX-treated PC12 cells as an in vitro model to confirm the effect of tolfenamic acid on GSK-3β activationinduced neurotoxicity and tau hyperphosphorylation. Pretreatment with tolfenamic acid for 24 h significantly prevented WT/GFX exposure-induced PC12 cell death (p < 0.05, Fig. 3a). Tolfenamic acid also decreased p-tau Ser396, p-tau Thr231, and p-GSK-3β Tyr216 levels (p < 0.05, Fig. 3b-d) and enhanced p-GSK-3β Ser9 and p-Akt Thr308 levels (p < 0.05, Fig. 3e, f ), which was in accordance with the results obtained in postnatal rats. Based on the above in vivo and in vitro results, the inhibitory effect of tolfenamic acid on GSK-3β activity may be one mechanism by which hyperphosphorylation of tau is decreased.
Tolfenamic acid attenuated cognitive dysfunction induced by ICV-OA in mice
In addition to protein kinases, protein phosphatases regulate tau phosphorylation and contribute to cognitive dysfunction in AD. OA is an inhibitor of PP2A, the most important protein phosphatase. Injection of OA in the mouse brain can induce cognitive impairments for at least 4 weeks. In this study, we used an ICV-OA mouse model to test the effect of tolfenamic acid on cognitive function and PP2A activity. The Y-maze test was per- mice. Treatment with 50 mg/kg tolfenamic acid alleviated spontaneous alternation behavioural deficits in OA-injected mice (p < 0.01, Fig. 4a). The novel object recognition test was performed to evaluate recall memory and visual recognition in individual mice. The PI for novel object B was obviously decreased in the model group (p < 0.01, Fig. 4b). Similar results were observed with novel object C. That is, OA injection induced significant memory recall and visual recognition impairments. Tolfenamic acid (50 mg/kg) obviously attenuated this decrease in PI (p < 0.05, Fig. 4b).
In the Morris water maze test, during the training period, mice in the model group exhibited a longer swimming time and farther swimming distance to find the platform than mice in the control group (Fig. 5a). In the probe test, compared to the control group, the Fig. 4 Effect of tolfenamic acid on working memory, memory recall and visual recognition deficits in ICV-OA mice. Model group mice exhibited a decrease in spontaneous alternation behaviour in Y-maze test (a), a significant decrease in 1 h and 24 h PI in novel object recognition test (b), which compared to control mice. Tolfenamic acid treatment attenuated these cognitive deficits. All results are expressed as the mean ± SEM. n = 10; ## p < 0.01 versus control; *p < 0.05, **p < 0.01 versus model model group exhibited significant decreases in exploration time and exploration distance in the target quadrant (p < 0.01, Fig. 5b). Mice in the tolfenamic acid group spent more time and travelled a longer distance in the target quadrant than mice in the model group (p < 0.05, Fig. 5b). There were no significant differences in swimming speed among the animal groups (p > 0.05, Fig. 5b).
The passive avoidance test was performed in the mice to evaluate long-term memory. The error time was obviously increased in the model group compared to the other groups (p < 0.01, Fig. 6). The error time in the 50 mg/kg tolfenamic acid group was less than that in the model group (p < 0.01, Fig. 6). Thus, tolfenamic acid could attenuate deficits in working memory, spatial memory and long-term memory. We next tested the effect of tolfenamic acid on PP2A inhibition-induced tau phosphorylation.
Tolfenamic acid attenuated tau hyperphosphorylation in ICV-OA mice
The p-tau level was tested by western blotting and immunohistochemistry and found to be significantly increased in the model group (Ser202, p < 0.05; Ser396, p < 0.01, Fig. 7). Tolfenamic acid significantly decreased the level of tau phosphorylated at the Ser202 and Ser396 sites (p < 0.01, Fig. 7). In the immunohistochemical experiment, the model group mice exhibited high levels of p-tau Ser396 labelling in the hippocampal CA3 region (p < 0.05, Fig. 8a). Labelling for p-tau Ser396 was reduced after tolfenamic acid treatment (positive area (%), p < 0.05, Fig. 8a).
PP2A regulates almost 70% of phosphatase activity, and its phosphorylation inhibits its activity. OA injection increased the expression of p-PP2Ac (Tyr307) (p < 0.01, Fig. 6 Effect of tolfenamic acid on long-term memory and learning ability in ICV-OA mice. Model group mice exhibited a significant increase in error times, compared to control mice. Tolfenamic acid attenuated the cognitive deficits in passive avoidance test. All results are expressed as the mean ± SEM. n = 10; ## p < 0.01 versus control; **p < 0.01 versus model Fig. 7 Effect of tolfenamic acid on the expression of p-tau and p-PP2A in hippocampus of ICV-OA mice. Tolfenamic acid treatment significantly decreased the expression of p-tau (Ser202 and Ser396) and p-PP2A (Y307), increased the expression of PP2A. All results are expressed as the mean ± SEM. n = 5; # p < 0.05, ## p < 0.01 versus control; *p < 0.05, **p < 0.01 versus model Fig. 7). Tolfenamic acid decreased the p-PP2A level and enhanced the expression of PP2A (p < 0.01, Fig. 7). In the immunohistochemical experiment, the model group mice exhibited high levels of p-PP2A labelling in the hippocampal CA3 region, but this increase was prevented by tolfenamic acid (positive area (%), p < 0.01, Fig. 8b). We next used a kit to test whether tolfenamic acid enhanced the activity of PP2A. The activity of PP2A in the model group had decreased to 72.65% of that in the control group, but tolfenamic acid prevented this decrease (p < 0.01, Fig. 9).
Tolfenamic acid attenuated tau hyperphosphorylation in OA-treated PC12 cells
We used an in vitro model to confirm the effects of tolfenamic acid on PP2A inhibition-induced neurotoxicity and tau hyperphosphorylation. Pretreatment with tolfenamic acid for 24 h significantly prevented OA exposure-induced PC12 cell death (p < 0.01, Fig. 10a). Tolfenamic acid also decreased the expression of p-PP2A (Tyr307) and tau phosphorylated at the Ser396 and Thr231 sites (p < 0.05, Fig. 10b-d). The selection of the tolfenamic acid/OA doses and treatment times was based on reports by Adwan et al., Yang et al., and Yang et al. [17][18][19] and our preliminary experiment. Based on the above in vivo and in vitro results, tolfenamic acid increases PP2A activity, which is a mechanism to prevent hyperphosphorylation of tau and cognitive dysfunction.
Discussion
Hyperphosphorylated tau aggregates into neurofibrillary tangles (NFTs) in neuronal cells and glial cells in the brain. AD is a tauopathy induced by intraneuronal hyperphosphorylated tau. The literature has reported that NSAIDs can attenuate cognitive deficits in AD animal models and prevent AD pathologies, such as Aβ deposition, the release of neuroinflammatory cytokines and brain cell loss [23][24][25]. Therefore, some NSAIDs can be considered potential anti-AD agents.
The therapeutic effect of tolfenamic acid on AD animal models was first reported by Professor Zawia and colleagues [26]. Most therapeutic compounds need 2 months or longer to attenuate cognitive dysfunction in AD transgenic mice [11,27]. However, the use of tolfenamic acid for a very short time (34 days) significantly prevented spatial memory deficits in the Morris water maze test and working memory deficits in the Y-maze test in AD transgenic mice [27,28]. Tolfenamic acid inhibited the transcription factor Sp1, which reduced the transcription of APP and BACE1 in Pb-exposed human neuroblastoma SH-SY5Y cells and decreased Aβ accumulation in APP transgenic mice [11,17]. Tolfenamic acid also decreased hyperphosphorylated tau through inhibiting SP1-mediated CDK5 expression in transgenic R1.40 mice with AD [12]. In this study, we report for the first time that tolfenamic acid inhibited tau hyperphosphorylation through inhibiting GSK-3β and activating PP2A.
Protein kinases and phosphatases contribute to tau phosphorylation. As the most important protein kinase, GSK-3β increases p-tau levels. Phosphorylation of GSK-3β at the Ser9 site decreased GSK-3β activity, while phosphorylation of GSK-3β at the Tyr216 site increased GSK-3β activity [29]. Selenica ML et al. reported that both GSK-3β activity and p-tau levels were increased in the brains of developing rats, and this increase peaked at postnatal day 12 (P12 rats) [20]. In addition, GSK-3 inhibitors could reduce tau hyperphosphorylation in P12 model rats, which only required a treatment time of 1 to 4 h [20]. PI3K/Akt is an upstream target that negatively regulates the GSK-3β pathway. Activation of GSK-3β by WT (a PI3K inhibitor) and tau hyperphosphorylation could be reversed by activating PKC [30]. The combined use of WT and GFX (a PKC inhibitor) in the model, which activated GSK-3β, further increased p-tau levels [30]. Therefore, in this study, we used P12 rats and WT/GFX-treated PC12 cells to test the effect of tolfenamic acid on GSK-3β-induced tau hyperphosphorylation. We found that tolfenamic acid decreased the levels of tau phosphorylated at the Ser202, Thr231 and Ser396 sites. Tolfenamic acid also enhanced p-Akt and decreased GSK-3β activity in the brains of P12 rats and in WT/GFX-treated PC12 cells. Thus, tolfenamic acid can decrease hyperphosphorylated tau through inhibiting GSK-3β activity.
The most commonly reported phosphatase in AD is PP2A, a serine/threonine phosphatase. PP2A acts as a suppressor of tau hyperphosphorylation and regulates almost 70% of tau phosphatase activity. Activation of PP2A induces tau dephosphorylation at many sites, including Thr205, Thr212, Thr231, Ser199/202, Ser214, Ser262 and Ser396/404. In the AD patient brain, the activity and protein and mRNA levels of PP-2A were all decreased, and the expression of an endogenous PP2A inhibitor (Inhibitor 2 of Protein Phosphatase 2A, I2PP2A) was increased [31,32]. OA, a PP2A inhibitor, triggers tau hyperphosphorylation. The literature reports that OA significantly decreased the expression of PP2A in mice, rats, zebrafish and some cell lines in vitro [33][34][35]. AD-like pathological changes, such as neuronal apoptosis, Ca 2+ overload, brain energy metabolic abnormalities, and decreases in brain-derived neurotrophic factors, have been found in OA models [32,36,37]. ICV-OA for approximately 4 weeks induced cognitive impairments in mice. In our study, tolfenamic acid prevented impairments in working memory, recall memory, visual recognition, spatial memory, and long-term memory. Tolfenamic acid significantly decreased ICV-OA-induced tau hyperphosphorylation at three sites: Ser202, Thr231 and Ser396. By using a PP2A activity detection kit, we found that tolfenamic acid enhanced the activity of PP2A. This may be the mechanism by which tolfenamic acid decreased hyperphosphorylated tau. PP2A activity is decreased by its phosphorylation. The phosphorylation of PP2A at Tyr307 was found to inactivate PP2A and increase abnormally hyperphosphorylated tau, finally causing NFTs [38,39]. In our study, tolfenamic acid decreased p-PP2A (Tyr307) levels both in vitro and in vivo.
Conclusions
A previous study showed that tolfenamic acid inhibited protein kinase CDK5-mediated tau hyperphosphorylation in AD transgenic mice [12]. Based on our present study, the list of potential targets by which tolfenamic acid regulates tau phosphorylation has expanded. Tolfenamic acid inhibits the protein kinase GSK-3β and the activity of the protein phosphatase PP2A. As the clinical use of tolfenamic acid is known to be safe, tolfenamic acid may be a candidate for the treatment of AD. Studies have reported that PP2A can regulate GSK-3β through the PI3K/Akt pathway [40,41]. However, activation of GSK-3β also inhibited PP2A [19]. It is difficult to determine whether GSK-3β is upstream of PP2A or vice versa. In this study, we did not determine whether tolfenamic acid can fit into the binding sites of Akt, GSK-3β or PP2A or directly trigger GSK-3β or PP2A. We cannot rule out that the target of tolfenamic acid is upstream of GSK-3β and PP2A. We will use molecular docking methods, proteomics and systems biology to uncover the molecular mechanisms of tolfenamic acid in our future study. | v3-fos-license |
2018-04-03T04:44:41.485Z | 2017-02-20T00:00:00.000 | 17441658 | {
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} | pes2o/s2orc | Dual mechanisms regulate the nucleocytoplasmic localization of human DDX6
DDX6 is a conserved DEAD-box protein (DBP) that plays central roles in cytoplasmic RNA regulation, including processing body (P-body) assembly, mRNA decapping, and translational repression. Beyond its cytoplasmic functions, DDX6 may also have nuclear functions because its orthologues are known to localize to nuclei in several biological contexts. However, it is unclear whether DDX6 is generally present in human cell nuclei, and the molecular mechanism underlying DDX6 subcellular distribution remains elusive. In this study, we showed that DDX6 is commonly present in the nuclei of human-derived cells. Our structural and molecular analyses deviate from the current model that the shuttling of DDX6 is directly mediated by the canonical nuclear localization signal (NLS) and nuclear export signal (NES), which are recognized and transported by Importin-α/β and CRM1, respectively. Instead, we show that DDX6 can be transported by 4E-T in a piggyback manner. Furthermore, we provide evidence for a novel nuclear targeting mechanism in which DDX6 enters the newly formed nuclei by “hitch-hiking” on mitotic chromosomes with its C-terminal domain during M phase progression. Together, our results indicate that the nucleocytoplasmic localization of DDX6 is regulated by these dual mechanisms.
Results
Nuclear localization of DDX6 in human cells. To examine the nuclear presence of DDX6 in human cell nuclei, we performed subcellular fractionation followed by WB on the HeLa human cervical cancer cell line, the transformed human embryonic kidney cell line HEK293T, and human embryonic stem cell-derived cardiomyocytes (hESC-derived CM). We used the Gagnon-Li method 31,32 and the NE-PER reagents for nuclear/cytoplasmic fractionation (Fig. 1a). Our WB results showed that DDX6 was detectable in both cytoplasmic and nuclear extracts prepared using both methods (Fig. 1a). The specificity of anti-DDX6 WB in nuclear extract was further confirmed in DDX6-depleted cells ( Fig. S1a and b). The WB results also showed specific detection of the nuclear marker LMNA in the nuclear extracts, whereas the cytoplasmic marker GAPDH and endoplasmic reticulum (ER) marker CALR were barely detected (Fig. 1a). Notably, LMNA was expressed at a low level in hESC-derived CM, so we used POLR2A p-Ser2 as a nuclear fraction marker instead (Fig. 1a). These results confirmed that there was minimal cross-contamination between the cytoplasmic and nuclear fractions, supporting the nuclear presence of DDX6. To the same end, we also exploited an in-house HeLa nuclear/cytoplasmic proteome dataset with two biological replicates that was produced by quantitative mass spectrometry (Fig. 1b). In both replicates, DDX6 was found to be present based on at least two unique peptides in both the nuclear and cytoplasmic fractions ( Fig. 1b and Table S1). Similar to our WB results, various markers for the nucleus, cytoplasm, or ER were differentially detected in the cytoplasmic and the nuclear fractions (Fig. 1b). This result also supports the nuclear presence of DDX6. In addition, IF staining of DDX6 in HeLa cells followed by confocal microscopy (Fig. 1c) and 3D-projection ( Fig. S2) further revealed the nuclear localization of DDX6, and the detected signals of DDX6 in nuclei were less as compared with that in cytoplasm. The specificity of anti-DDX6 IF in the cell nuclei was further confirmed in DDX6-depleted cells and mock IF (Fig. S3). To circumvent the artefacts that could potentially be introduced by fractionation or fixation, we also scanned live HeLa cells expressing YFP-tagged DDX6 (YFP-DDX6 FL) by confocal microscopy and detected fluorescent signals within the nuclear area (Fig. S4). Combined, our results support the nuclear presence of DDX6 in human-derived cells.
To test whether nuclear DDX6 is functionally active for RNA binding, we examined the interaction of DDX6 with nuclear RNAs, in particular the nuclear-retained lncRNAs. To this end, we performed a standard RNA co-immunoprecipitation (RIP) by using anti-DDX6 to pull down the endogenous DDX6 associated complexes and followed by RT-qPCR to examine the DDX6 interacting RNAs. Our results showed that one of the nuclear-retained lncRNAs, MALAT1, was enriched by anti-DDX6 IP (Fig. S5a). The specificity of anti-DDX6 IP was confirmed by control IgG IP (Fig. S5a), anti-DDX6 IP in DDX6-depleted cells, and the co-IP of several known protein-protein interaction (PPI) partners (Fig. S6a). The specificity of MALAT1 co-IP was confirmed in DDX6-depleted cells (Fig. S6b). To further confirm the interaction of DDX6 with MALAT1 in vivo, we tracked the co-localization of DDX6 and MALAT1 in interphase HeLa cells with fluorescent in situ hybridization (FISH) and IF double staining. DDX6 was frequently observed co-localizing with, or docking to, MALAT1 in cells in which MALAT1 aggregates to nuclear speckles (Figs S5b and S6b,c), supporting the fact that DDX6 interacts with MALAT1 in the nucleus.
DDX6 is insensitive to LMB treatment. Because DDX6 localized to human cell nuclei, we next sought to determine how it is distributed nucleocytoplasmically. Although it was proposed that vertebrate DDX6 homologues are shuttled between the nucleus and the cytoplasm by the importin-α /β and the CRM1 pathways 1,24 , this shuttling model was challenged by conflicting data concerning CRM1-dependent export 24,28,29,33 . To clarify the nuclear shuttling mechanism of DDX6, as well as that of other P-body interacting proteins, we surveyed the LMB-sensitive P-body components. Our results showed that only PATL1 and 4E-T, but not DDX6, accumulated in the cell nuclei conspicuously after the treatment of LMB (Fig. 2a). Furthermore, even under a high dose of LMB, no grossly observable nuclear accumulation was seen for DDX6 (Figs 2b and S7). Together, our results indicate that LMB does not affect DDX6 and its nuclear export via the CRM1-dependent pathway.
The putative NES is structurally masked and is inaccessible to CRM1. To support our new evidence that DDX6 is not LMB-sensitive, we resorted to a structural analysis to obtain biochemical insight into the feasibility of CRM1-dependent export of DDX6. Inspection of the existing X-ray crystallographic structures showed that the DDX6 NTD folds into a globular structure in all three published crystal structures: DDX6 NTD (1VEC) 34 , ATPase-compatible conformation (4CT4) 4 , and the ATPase-incompatible conformation (4CT5) 4 . The putative NES proposed by previous studies (Fig. 3a) indeed folds into an L-rich NES-like structure that is characterized by α -helical folding with leucine residues protruding in the same direction 35 (Figs 3b and S8). Nonetheless, this putative NES is buried inside the core region of the DDX6 NTD, with L151 and L155 directly blocked by the 174-199 segment (Fig. 3b inset 1 and 2 and Fig. S8), while L158 and L160 protrude downward and inward to the DDX6 NTD core (Fig. 3b inset 3 and Fig. S8). In addition, the putative NES is one of the most stably (a) DDX6 is detectable in the nuclear extracts of human cell lines. Cytoplasmic and nuclear extracts from HeLa cells, HEK293T cells, and hESC-derived CM were prepared by NE-PER reagent or Gagnon-Li method. Equal amounts (proteins in μ g) of nuclear and cytoplasmic extracts were separated by SDS-PAGE and analyzed by WB. LMNA and POLR2A p-Ser2: lamin A/C and phosphorylated RNA polymerase II subunit 1, nuclear markers; GAPDH: a cytosolic marker; CALR: an ER lumen marker. (b) Quantitative MS analysis confirms the nuclear presence of DDX6. Scatter plot showing the comparison of the protein nucleocytoplasmic signal ratios between the two replicates of the quantitative MS data. The original samples were fractionated by NE-PER to yield cytoplasmic and nuclear extracts as in (a). The N/C signal ratios were calculated using the summed-up extracted ion chromatogram (XIC) intensities, an estimate of the protein abundance. Several known cytosolic (blue), ER (brown), and nuclear (red) proteins were shown side-by-side with DDX6 (green). (c) IF detects endogenous DDX6 in the nuclei of human cells. HeLa cells were analyzed by immunofluorescence using antibodies against DDX6 (shown in red) and AGO2 (shown in green). Nuclei were stained with Hoechst 33342 (shown in blue) and images were digitally merged (c, i and ii). Bottom panels (c,iii and iv) display magnified views of the boxed areas in i. Arrows indicated endogenous DDX6 in the nuclei. Scale bar = 10 μ m.
folded segments within the DDX6 NTD ( Fig. S9a and b), indicating that the putative NES is unlikely to contact other proteins at the molecular surfaces.
Despite the structural features, the N-terminal 1-164 segment of Xp54, which is homologous to the 1-166 segment of human DDX6 (Fig. 3a), was reported to be exported by the CRM1-dependent pathway 24 . One plausible explanation for this discrepancy is that the recognition and shuttling of the Xp54 1-164 segment by CRM1 results from the removal of the downstream masking peptide segments, which exposes the putative NES. That is, the CRM1-dependent export of the Xp54 1-164 segment could be a gain-of-function mutation when the protein Figure 2. The nucleocytoplasmic localization of DDX6 is insensitive to LMB treatment. (a) LMB treatment does not alter DDX6 nucleocytoplasmic distribution. HeLa cells expressing control EYFP, EYFP-or EGFP-tagged P-body components were treated with 5 ng/mL LMB for 5 hours prior to imaging. (b) DDX6 is insensitive to high concentration LMB treatment. HeLa cells transfected with YFP-PATL1, YFP-DDX6 FL, and myc-DDX6 FL, and untransfected cells were treated with 0, 5, 10, and 50 ng/mL LMB for 5 hours prior to imaging. (a and b) Same volume of solvent to highest dose LMB treatment was used as control (0 ng/mL LMB). YFP signal is shown in green. Nuclei were stained with Hoechst (shown in blue). Merge channels show overlay of YFP and Hoechst signals. Scale bar = 20 μ m. is truncated. To test this hypothesis, we examined the localization of YFP-DDX6 1-166 and YFP-DDX6 141-483, two truncated DDX6 mutants. We predicted that, whereas the 1-166 segment of human DDX6 can be exported through the CRM1-dependent pathway, the DDX6 FL, DDX6 NTD, and DDX6 141-483 segments cannot be exported through the CRM1-dependent pathway (Fig. 3c). As expected, the export of YFP-DDX6 1-166 was sensitive to LMB treatment and to mutation at the putative NES ( Fig. 3d and e). The localizations of YFP-DDX6 FL, CTD, and 141-483, however, were not altered by LMB treatment or mutation ( Fig. 3d and e). These results corroborated our prediction and confirmed that the putative NES is structurally inaccessible to CRM1 in the intact DDX6 NTD. Additionally, the comparative analysis of the homologous regions of the putative NES in other DBPs in the eIF4A-like clade revealed that this segment is highly enriched with hydrophobic amino acids (AA) arranged in NES-like sequences (Fig. S9c-e). However, no NES-like function has been reported for these homologous regions. These findings suggest that the N-terminal region containing the putative NES of DDX6 may instead provide essential hydrophobic interactions for the domain folding common to other DBPs.
The putative K/R-rich NLS, miRISC, and MALAT1 are dispensable for the nuclear entry of DDX6. The shuttling model from a previous study proposed that the vertebrate DDX6 homologues are imported to the cell nuclei by the importin-α /β complex, which recognizes the K/R-rich NLS 1,24 . However, this has not yet been supported by experimental data. To test this model, we mutated the putative NLS (mNLS) in YFP-DDX6 1-166 (Fig. S10a). Upon LMB treatment, YFP-DDX6 1-166 mNLS still accumulated in the nucleus (Fig. S10b), indicating that its ability to enter the nucleus was not determined by the putative NLS. We then used the NucPred program 36 to scan for K/R-rich NLS embedded within the peptide sequence of DDX6, but no potential target was reported (Fig. S10c). Additionally, treatment with importazole (IPZ), an inhibitor of importin-β , did not block the nuclear localization of endogenous DDX6 and YFP-DDX6 1-166 WT (Fig. S11). The putative NLS of DDX6 deviates from the consensus K/R-rich NLS 37 . These lines of evidence argue against the idea that K/R-rich NLS facilitates the nuclear import of DDX6.
It was recently discovered that the core RNAi machinery can be shuttled nucleocytoplasmically by TNRC6A 29 . Thus, we wondered whether DDX6 is co-transported as part of the miRISC. To this end, we first disrupted RISC formation by AGO2 KD (Fig. S12a) and then examined the nucleocytoplasmic distribution of DDX6 (Fig. S7b). The results showed that DDX6 levels in the cytoplasmic and nuclear extracts were not significantly altered by AGO2 depletion (Fig. S12b). We then tested whether the nuclear localization is regulated by MALAT1. However, we did not observe a significant change in DDX6 levels in the cytoplasmic and nuclear extracts when MALAT1 was depleted ( Fig. S12c and d). These data suggest that the nuclear entry of DDX6 is not determined by miRISC or MALAT1 lncRNA.
DDX6 CTD facilitates nuclear localization.
To map the DDX6 compartment responsible for nuclear entry, we first tracked the subcellular localization of the two DDX6 domains (Figs 4a and S13a). Surprisingly, YFP-DDX6 CTD, similar to YFP-DDX6 FL, was present in the nuclear extract, whereas YFP-DDX6 NTD was barely detectable (Figs 4b and S13b). To rule out the possible effect of nuclear entry driven by the EYFP tag, we verified DDX6 CTD localization with Myc-tagged DDX6 CTD (Myc-DDX6 CTD; Fig. S13a). We found that Myc-DDX6 CTD localized predominantly to the nuclei in a similar pattern to that of YFP-DDX6 CTD (Fig. S13c). Although we noticed that the nucleolar localization of DDX6 CTD could be an artefact resulting from 4% PFA fixation (Fig. S13d), fixation did not change the general nuclear localization of YFP-or Myc-tagged DDX6 CTD. Together, these results suggest that DDX6 CTD can be important for nuclear entry.
To elucidate the role of DDX6 CTD in nuclear localization, we adopted a chimeric protein approach 38,39 to examine whether DDX6 CTD is sufficient to drive nuclear entry. We constructed YFP-PKM-DDX6 CTD and YFP-PKM-DBR1 CTD by fusing DDX6 CTD and DBR1 CTD to the C-terminus of YFP-PKM, a non-shuttling cytoplasmic enzyme ( Fig. 4c and d). Unlike YFP-PKM, which predominantly localized to the cytoplasm, YFP-PKM-DBR1 CTD was highly nuclear-enriched (Fig. 4d). These data confirmed that YFP-PKM can be imported by C-terminally fused protein. Interestingly, YFP-PKM-DDX6 CTD exhibited a phenotypic continuum (Fig. 4d). We defined three subcellular localization types: Type 1 represents cells with a dominant cytoplasmic distribution; type 2 represents cells with a homogenous distribution; and type 3 represents cells with a dominant nuclear distribution. Two days after transfection, cells with an apparent nuclear signal (type 2 and 3) accounted for approximately 60% of the transfected cells. Together, these results indicate that DDX6 CTD facilitates nuclear entry.
Alternatively, we tested whether DDX6 CTD harbours one or more non-canonical NLSs. These sequences are diverse in terms of length and sequence pattern. We first constructed deletion mutants by further deleting the first 60 and the last 63 amino acids in DDX6 CTD (YFP-DDX6 CTD-Δ 1 and YFP-DDX6 CTD-Δ 2; Fig. 4e). As a result, the nuclear localizations of both YFP-DDX6 CTD-Δ 1 and YFP-DDX6 CTD-Δ 2 were abolished (Fig. 4f). The abolition of nuclear localization indicates the following: First, integral DDX6 CTD may be necessary for nuclear localization; second, both the DDX6 301-360 and the 421-483 segments alone were insufficient for nuclear entry, suggesting that these two segments do not function as stand-alone NLSs.
DDX6 CTD facilitates chromosome association during mitosis. Two other mechanisms could potentially underlie the nuclear localization of DDX6. First, diffuse DDX6 can be engulfed into the nucleus as the nuclear membrane reassembles in late telophase. However, this model is insufficient to explain the predominantly nuclear-localized phenotype of DDX6 CTD (Fig. 4b). We then considered a second possibility, that DDX6 CTD may achieve nuclear entry by "hitch-hiking" on mitotic chromosomes during M phase. Two independent proteomic studies, which detected DDX6 in the mitotic chromosome extract of extract of chicken DT40 cells 42 and chromatin extracts from human HeLa-S3 cells 43 , provided evidence for this hypothesis. To examine whether DDX6 indeed associates with chromosomes, we tracked the localization of DDX6 constructs in mitotic HeLa cells (Fig. 5a). Whereas EYFP and YFP-DDX6 NTD were diffuse throughout the cytoplasm during M phase, both YFP-DDX6 FL and CTD co-localized with chromosomes ( Fig. 5a and f). Viewed by confocal microscopy, YFP-DDX6 FL and CTD displayed an indistinguishable chromosome binding pattern (Fig. 5b). These findings indicated that DDX6 CTD could facilitate binding to mitotic chromosomes.
To confirm that DDX6 CTD is responsible for chromosome association, we used the "domain swap" approach. We first reconstituted YFP-DDX6 (YFP-DDX6 Rec) by fusing the DDX6 CTD to the C-terminus of YFP-DDX6 NTD (Fig. S15a and c). Subsequently, we swapped the DDX6 NTD for PKM. As a result, while YFP-DDX6 Rec was chromosome-positive in 40% of the mitotic cells, YFP-PKM-DDX6 CTD was chromosome-positive in 80% of the mitotic cells (Fig. 5e). This result suggests that the DDX6 NTD is dispensable, or even inhibitory, to chromosome association. Conversely, when the DDX6 CTD was removed, the resulting YFP-PKM was exclusively diffuse in mitotic cells (Fig. 5c and e), suggesting that PKM does not contribute to chromosome binding. We further tested whether the truncation in the DDX6 CTD abolishes the chromosome binding. As a result, both YFP-DDX6 CTD-Δ 1 and YFP-DDX6 CTD-Δ 2 exhibited a diffuse pattern (Fig. 5d and e), suggesting that structurally integral DDX6 CTD may be required for chromosome association. Collectively, the chromosome binding in the mitotic cells showed a striking positive correlation with the nuclear localization in interphase cells, implying a causal link between these two phenomena.
DDX6 CTD facilitates nuclear entry by hitchhiking on mitotic chromosomes. To further investigate the relationship between the chromosome binding and nuclear localization of DDX6, we monitored the chromosome-association dynamics of DDX6 constructs during M phase with time-lapse microscopy. First, we observed that cytoplasmic YFP-PKM-DDX6 CTD quickly bound the condensed chromosomes after the nuclear membrane broke down (Fig. 6a). This finding demonstrated that the binding of the mitotic chromosome occurs at early prometaphase, and the subcellular localization of YFP-PKM-DDX6 CTD in the previous cell cycle does not limit this process. We also observed that, in some cells, the interaction between YFP-PKM-DDX6 CTD and the mitotic chromosomes persisted even after cytokinesis (Fig. 6b). Consequently, the chromatin-bound YFP-PKM-DDX6 CTD became trapped in the newly formed nuclei (Fig. 6b). In other cells, the interaction between YFP-PKM-DDX6 CTD and the mitotic chromosomes declined during the mitosis (Fig. 6c), leaving a small amount of YFP-PKM-DDX6 CTD to be enclosed in the newly formed nuclei. This divergent dynamics during the mitosis was consistent with the divergent phenotypic continuum in the interphase cells (Fig. 4d). Conversely, we also observed a persistent interaction between YFP-DDX6 CTD and the mitotic chromosomes, and the subsequent entrapment of YFP-DDX6 CTD in the newly formed nuclei (Fig. 6d). For YFP-DDX6 FL, the interaction with the mitotic chromosomes declined during the mitosis (Fig. 6e). These observations were also consistent with our previous observations in the interphase cells (Figs 2, 3e and 4b). Collectively, our findings point to a novel mechanism in which DDX6 enters newly formed cell nuclei by hitch-hiking on the mitotic chromosomes using its CTD.
4E-T can mediate piggyback shuttling of DDX6.
It has been recently proposed that DDX6 may be shuttled nucleocytoplasmically in a piggyback style 33 . Among the known interacting partners of DDX6, 4E-T, PATL1, and LSM14B are indeed directly transported by the CRM1 pathway (Fig. 2a) 28,33,44 . However, several proteomic profiling efforts have shown that DDX6 has a greater average copy number per cell than these other proteins (Fig. S16), especially in HeLa cells (Fig. S16d) 33,[45][46][47] . This is consistent with the observation that the DDX6 localization was unaffected under LMB treatment (Fig. 2). To overcome this limitation imposed by the protein stoichiometry, we monitored the localization of the co-expressed CFP-DDX6 with the overexpression of YFP-4E-T, YFP-PATL1, and YFP-LSM14A under LMB treatment. Because LSM14A, unlike its paralogue LSM14B 33 , is not a shuttling protein, we observed no change in the subcellular localization of YFP-LSM14A and CFP-DDX6 under LMB treatment (Fig. 7a, xxi and xxiv). Conversely, YFP-PATL1 accumulated in the nuclei under LMB treatment (Fig. 7a, xiii-xvi). However, YFP-PATL1 displayed a diffuse nuclear pattern in nearly all the observed cells (Fig. 7a, xiv). This was drastically different from the pattern without co-expressing CFP-DDX6, which was characterized by localization to nuclear granules (Fig. 2a). In contrast, YFP-4E-T accumulated and localized to the nuclear granules under LMB treatment regardless of the co-expression of CFP-DDX6 (Fig. 7a, vi). Strikingly, CFP-DDX6 co-localized with YFP-4E-T within the nuclear granules under LMB treatment (Fig. 7a, v-viii). This result indicates that DDX6 can be exported via the CRM1-dependent pathway when complexed with 4E-T in the nucleus. Our finding also suggests that there is 4E-T-mediated DDX6 import for two reasons: First, the localization of CFP-DDX6 to nuclear granules under LMB treatment is only observed when YFP-4E-T is co-expressed. Second, there was no grossly observable nuclear accumulation of CFP-DDX6 with a diffuse pattern in other cases, thereby reducing the possibility that CFP-DDX6 was imported by other potential factors but was not recruited to the nuclear granules by 4E-T.
To further assess the role of 4E-T in the nuclear import of DDX6, we examined whether the binding between DDX6 and 4E-T, and more importantly the nuclear import of 4E-T, are crucial for the import of DDX6. To this end, we constructed a 4E-T-binding mutant of DDX6 (CFP-DDX6 mut3) 40 reduced with the mut3 mutation in CFP-DDX6 (Fig. 7c, xiii-xvi) and with the co-expression of YFP-4E-T Δ CHD (Fig. 7c, xxi-xxiv) while the localization of YFP-4E-T to the nuclear granules appeared unaffected (Fig. 7c, xiv and xxii). Furthermore, the Δ NLS mutation in YFP-4E-T abolished its nuclear entry (Fig. 7c, xxx), and the localization of CFP-DDX6 to the nuclear granules was abolished in correlation (Fig. 7c, xxix). These results confirmed that the nuclear import of 4E-T and its binding to DDX6 are both required for the localization of DDX6 to nuclear granules when 4E-T is overexpressed. This supports a model in which DDX6 can be co-transported with 4E-T in a piggyback manner.
Discussion
In this study, we used different approaches to show that human DDX6 is present in the nucleus. We used WB and MS to detect DDX6 in nuclear extracts from HeLa cells, HEK293T cells, and hESC-derived CM ( Fig. 1a and b). We also used confocal microscopy for anti-DDX6 IF (Fig. 1c) and YFP-DDX6 FL live-cell imaging (Fig. S4) to detect nuclear DDX6 in HeLa cells. Our results were consistent with previous reports that DDX6 is present in the nuclei of MKN45 gastric cancer cells and DM1-affected fibroblasts 26,27 . Taken together, the nuclear localization of DDX6 has now been verified in five distinct, human-derived cell types. Because DDX6 is ubiquitously expressed in human tissues/cells, we expect the nuclear localization of DDX6 to be widespread in different human cell types. This notion is supported by the detection of DDX6 in the nuclear extracts of hESC-derived CM (Fig. 1b). This notion is also supported by the knowledge that DDX6 family members are widely observable in the nuclei of metazoans [23][24][25]49 , including lab rodents. The fact that DDX6 family members are present in animal cell nuclei across different phyla implies that DDX6 may have conserved or specialized nuclear functions, which would require further investigation.
We can estimate the ratio of endogenous nuclear DDX6 in various cell types. In our WB and MS analysis, we often observed comparable cytoplasmic and nuclear DDX6 signals ( Fig. 1a and b). Because the protein abundance in the cytoplasmic and nuclear extracts was approximately 9:1 to 4:1, as prepared by using the NE-PER reagents, we estimated that the molar ratio of cytoplasmic and nuclear DDX6 in HeLa cells does not exceed 4:1. That is, the nuclear DDX6 is unlikely to account for more than 20% of the cellular DDX6. Although this estimate is conservative, it is comparable to the previous estimates. For instance, in stage III Xenopus oocytes, nuclear Xp54 accounts for approximately 16.7% of total Xp54 24 . In the rat testis, nuclear DDX6 accounts for ~7% of total DDX6 25 . This estimate was also consistent when we compared the anti-DDX6 WB signal in the cytoplasmic and nuclear extracts prepared from the same number of cells (Fig. S1c). Although these results are largely comparable, the exact proportion of nuclear DDX6 may be organism-, cell type-, or developmental stage-dependent and may vary depending on the means of detection. Collectively, these results indicate that a relatively minor but substantial proportion of cellular DDX6 is consistently present in the nucleus in many, if not all, cell types.
To further characterize nuclear DDX6, we demonstrated that DDX6 can bind nuclear lncRNA (Figs S5 and S6). This result aligns with previous reports that DDX6 can interact with noncoding regions of mRNAs or lncRNAs both in vitro and in vivo 9,26,27,34,[50][51][52][53][54][55][56] . This finding is also among the earliest reports of the interaction between DDX6 and the lncRNA that is highly enriched in the nucleus. We note that, while this manuscript was under revision, a study reported the interaction between DDX6 and 7SK nuclear RNA 57 . Because DDX6 generally binds RNA promiscuously 50,52,53 and nuclear lncRNA represents a substantial proportion of the human transcriptome 58,59 , we propose that DDX6 may bind to various other nuclear lncRNAs. We also speculate that DDX6 may possess regulatory functions for nuclear lncRNAs in addition to cytoplasmic mRNA silencing. Although we observed no apparent functional regulation of MALAT1 stability or subnuclear localization by DDX6 (data not shown), we cannot fully dismiss the possibility that DDX6 may participate in the structural regulation of lncRNA without further investigations, and we will pursue such investigations in the future.
We provided evidence contradicting the existing nucleocytoplasmic shuttling model of DDX6, and we revealed a novel nuclear targeting mechanism for DDX6. On one hand, our results, supported by the structural details, show that DDX6 itself is insensitive to LMB treatment; it cannot be exported via the CRM1 pathway with the putative NES (Figs 2 and 3, S7 and S8). On the other hand, our results argue against the role of the K/R-rich NLS in DDX6 nuclear import (Figs S10 and S11). This finding and our structural masking hypothesis for the putative NES together represent a departure from the existing model. Alternatively, our results show that the nuclear import of DDX6 is facilitated by its CTD (Figs 4 and S13), and demonstrate that the DDX6 CTD can mediate association with mitotic chromosomes (Fig. 5). As direct evidence, our time-lapse imaging revealed that the DDX6 CTD indeed enters the nucleus along with post-mitotic chromosomes as the nuclear membrane reassembles (Fig. 6). These findings suggest that DDX6 may enter the cell nucleus by associating with mitotic chromosomes throughout M-phase progression, and we summarized this as the "hitch-hiking" model ( Fig. 8a). Under this framework, the association and dissociation processes serve as a mechanism to balance and redistribute DDX6 throughout the cells during mitosis. Furthermore, the incomplete dissociation of DDX6 from mitotic chromosomes underlies the nuclear distribution of DDX6 observed in interphase cells (Fig. 8a). The underlying mechanism and functional relevance of the association with mitotic chromosomes/interphase chromatin and interactions with nuclear lncRNA (Fig. S5 and unpublished data) represent new directions for inquiries into DDX6 functions beyond cytoplasmic mRNA silencing.
In parallel, we also demonstrated that DDX6 can be transported in a piggyback manner through its direct interacting protein, 4E-T (Figs 7 and 8b). This finding enlisted DDX6 as another transport cargo of 4E-T in addition to EIF4E 44 , although we note that the depletion of 4E-T does not greatly alter the nucleocytoplasmic distributions of EIF4E and DDX6 in HeLa cells (Fig. S17). On the other hand, even though we had no explicit evidence for the piggyback style transportation by PATL1, we observed that co-expressed CFP-DDX6 affected the subcellular localization of YFP-PATL1 (Figs 2a and 7a, xiv). Thus, we cannot fully dismiss the possibility that PATL1 is also capable of shuttling DDX6. Moreover, it was originally proposed that Xenopus LSM14B (xRAP55B, xRAPB) may serve as a transporter of Xp54 33 . Interestingly, in developing Xenopus oocytes, the nuclear Xp54 level decreases after stage III 24 , coinciding with the decline in total xRAPB after stage III 60 . Therefore, it is possible for LSM14B to shuttle DDX6 in human cells, as it is possible for xRAPB to shuttle Xp54 in Xenopus oocytes. However, in the biological context of HeLa cells, the abundance of DDX6 greatly exceeds that of PATL1, 4E-T, and LSM14B (Fig. S16). Thus, the shuttling rate by this mechanism may be slow. It is consistent with our observation that the nucleocytoplasmic localization of DDX6 is insensitive to LMB treatment (Fig. 2) and 4E-T depletion in HeLa cells (Fig. S17). Similarly, when we increased the 4E-T levels by overexpressing YFP-4E-T, we observed co-transportation of YFP-4E-T and CFP-DDX6 (Fig. 7). The sensitivity of such co-transportation behaviour to both LMB and the mutation of 4E-T NLS supports the use of CRM1-dependent and importin-α /β -dependent shuttling pathways. Together, these results suggest that this mode of transportation is regulated by protein stoichiometry. Notably, in the oocytes and unfertilized eggs of Xenopus (Fig. S16a-c), as well as approximately 25% of the studied human cell lines (Fig. S16e), the protein stoichiometry of DDX6 and 4E-T decreases by one order of magnitude, suggesting the 4E-T-dependent transportation of DDX6 can be more pronounced in these systems.
The two mechanisms of transport were discovered along different lines. The hitch-hiking mechanism stemmed from the rejection of the shuttling model, and the piggyback mechanism was rooted in evidence that supported CRM1-dependent export. Their parallel existence therefore reconciles the conflicting ideas found in past research. Physiologically, the hitch-hiking mechanism is restricted to proliferating cells, whereas the piggyback mechanism is constrained by protein stoichiometry. Our data provide clear evidence that both mechanisms can serve to distribute DDX6 across the nucleus and the cytoplasm. Thus, they may dominate under different biological contexts, and they may complement/buffer each other's effects.
Methods
Cell culture and transfection. HeLa cells and HEK293T cells were cultured in DMEM (GE healthcare) supplied with 10% (v/v) fetal bovine serum, penicillin, streptomycin and 5% CO2 at 37 °C. The conditions for maintaining hESC culture and stimulating CM differentiation were described previously 61 The "piggyback" model refers to that 4E-T is sufficient to transport DDX6 via the importin-α /β and the CRM1 pathways in a piggyback manner. condition media to the desired concentration. Same volume of 70% MeOH or 100% DMSO to the highest dose of LMB or IPZ treatment was used as the control. Cells treated with LMB or IPZ were harvested or imaged at 5 hours post-treatment. For cell cycle synchronization at prometaphase, we adopted the thymidine-nocodazole block strategy. To arrest the cell cycle at S phase, the cells were first incubated with high concentration (2 mM) of thymidine (Sigma-Aldrich) for 24 hours. Next, the cells were released from thymidine treatment for 2 hours prior to nocodazole treatment. Cells were then incubated with 50 ng/mL nocodazole (Sigma-Aldrich) for up to 16 hours to arrest the cell cycle at prometaphase.
Cloning and constructs.
Cloning of full length DDX6, 4E-T, and AGO2 into pEYFP-C1 expression vector was described previously 3 . Deletion mutants and point mutants of DDX6 and 4E-T were generated by either PCR amplification of DNA fragments or 2-step PCR mutagenesis. Myc-tagged DDX6 CTD are PCR amplified from EYFP-tagged constructs and inserted into pKR43 expression vector containing 1 × N-terminal myc tag. Full length PKM, PATL1, EDC3 and DBR1 CTD were PCR-amplified from HeLa cell cDNA, and were subsequently inserted into pEYFP-C1 expression vector. Construction of chimeric proteins was done by inserting DNA fragments into pEYFP-C1 vectors. pEGFP-DCP1A and DCP2 were gifts from Dr. Elisa Izaurralde (Addgene plasmid #25030 and 25031).
Custom Stellaris FISH Probes recognizing MALAT1 (NR_002819) were directly labeled with Quasar 570 and were purchased from Biosearch Technologies, Inc. (Petaluma, CA). Probe set sequences utilized in the experiments have been previously described. HeLa cells were hybridized with the MALAT1 Stellaris FISH Probe set, following the manufacturer's instructions. For anti-DDX6 and anti-phosphorylated SC35 IF (ab11826, Abcam) after MALAT1 FISH, the cells were washed with 50% FISH wash buffer in PBS, PBS and 0.1% Triton X-100 in PBS sequentially before proceeding on with IF procedures from blocking with 2% BSA/0.1% Triton X-100 in PBS.
Microscopy, image processing and analyses.
For general purpose imaging, we routinely use Leica DMI3000 B wide field fluorescent microscope. Images acquired with Leica DMI3000 B were pseudo-colored. Leica DMI3000 B accounts for the images in this article unless specified. Statistics of phenotypic counting were done with ImageJ cell counter plugin. For each construct, at least 3 biological replicates were counted.
To capture high-resolution images of AGO2, nuclear DDX6, MALAT1 and SC35, cells were imaged with Leica confocal imaging system (TCS-SP2) or Zeiss LSM780 confocal microscope. Chromosome association of YFP-DDX6 FL, YFP-DDX6 CTD was also captured with Zeiss confocal microscope. Images were pseudo-colored. For Z-stacking, stacks of images were taken with spacing less than 1 μ m along the Z-axis, and were maximally projected on the XY-plane (Z-projection) or projected 3-dimensionally (3D-projection). Z-projection was done using ImageJ Z-projection function. 3D-projection was done with the mesh 3D function in ICY image analysis platform. Signal measurement was carried out using ImageJ. For LUT representation of the optical signals, the built-in Fire LUT in ImageJ was utilized.
For time-lapse imaging, we used DeltaVision Core (GE healthcare) imaging systems. The cells were kept warm (34 °C~37 °C) in heating chamber supplied with CO 2 . The cells were imaged across a 10~20 μ m range along the Z axis with 1 μ m spacing at 5 minute time interval for at least 3 hours. We utilize the DeltaVision built-in deconvolution function to improve the image resolution post-acquisition. High-resolution images were obtained from applying enhanced ratio (aggressive) mode of blind deconvolution with 10 iterations on each image in the original stacks. YFP, Hoechst and merged signals were made into Montage. The resulting image hyperstacks were Z-projected (sum slice projection) for each time point. The resulting T-stack images were cropped, adjusted, and made into Montage.
Whole cell, nuclear and cytoplasmic protein extraction. HeLa whole cell lysate, or total cell lysate (TCE), were prepared from lysing cells in RIPA buffer (0.1% [w/v] SDS, 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 1% Triton X-100). Cytoplasmic and nuclear fractions were prepared from HeLa and HEK293T cells using either NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific) or the Gagnon-Li method 31,32 . For NE-PER fractionation, the extraction procedure generally follows manufacturer's protocol, except for the additional washing of nuclei pellet with PBS before resuspending the nuclei with NER reagent. For nuclear lysis, we had two rounds of additional freeze-thawing with liquid nitrogen after four rounds of vortexing/ standing described in the original protocol. For Gagnon-Li fractionation, we followed the procedure for preparing cytoplasmic extract and total nuclear extract. In brief, the cytoplasmic fraction was released by lysing cells in hypotonic lysis buffer (HLB; 10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl 2 , 0.3% [v/v] Nonidet P-40, and 10% [v/v] glycerol). The resulting nuclei pellet was washed with HLB before resuspending the nuclei in nuclear lysis buffer (NLB; 20 mM Tris-HCl pH 7.5, 150 mM KCl, 3 mM MgCl 2 , 0.3% [v/v] Nonidet P-40, and 10% [v/v] glycerol). The nuclei were then lysed by sonication (Digital Sonifier 450, Branson) at 40% power for a total of 45 s with 1 min cooling every 15 s. The nuclear extract was then centrifuged at 1,200 × g for 5 min. The resulting supernatant was collected as total nuclear extract. RNA co-immunoprecipitation (RIP). RIP was performed as described as the previous publication 3 . TCE from HeLa cells was prepared by lysing cells in IP lysis buffer (0.2 mM EDTA, 20 mM HEPES pH 7.9, 0.35% Triton X-100, 10 mM NaCl, 1 mM MgCl 2 ) for 10 min on ice and then centrifuged at 12,000 × g for 10 min to remove cell debris. For anti-DDX6 or control IgG IP, the supernatants were subsequently incubated with Dynabeads M-280 Sheep Anti-Rabbit IgG (Life Technologies) and anti-DDX6 (A300-460A, Bethyl Labratories, Inc) or normal rabbit IgG (Santa Cruz) at 4 °C for 3 hr, respectively. After incubation, the beads were washed with IP lysis buffer and IP wash buffer (25 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.3% [v/v] Nonidet P-40) and were split into 2 fractions: one for protein elution and the other for RNA extraction. For protein elution, beads were boiled at 95 °C in SDS buffer. As for RNA extraction, beads were resuspended in TRIzol reagent.
RNA extraction, reverse transcription and quantitative real-time PCR (qPCR). Total RNA from HeLa cells and co-IPed RNA in anti-DDX6 IP experiments were extracted using TRIzol reagent (Life Technologies) and were reverse-transcribed into cDNA using SuperScript III First-Strand Synthesis System (Life Technologies) with random hexamer priming, following the suppliers' protocols. For RNA extraction from RIP samples, glycogen (molecular biology grade, Roche) was used to promote RNA precipitation according to the supplier's recommendation. The resulting cDNA were further analyzed with quantitative real-time PCR using iQ SYBR Green Supermix (Bio-Rad) and gene-specific primer sets. Fold changes in gene expression level analyses were calculated by Δ Δ C(t) method, using GAPDH mRNA as reference for normalization. Fold enrichments in RIP-qPCR were calculated by 2^[C IgG (t) − C anti-DDX6 (t)]. Primers for qPCR are listed in Supplementary Table S3.
Quantitative mass spectrometry and analysis. HeLa cells transfected with control siRNA (siDDX6 mm) or DDX6 targeting siRNA (siDDX6 pm). At 48 hours post-transfection, the transfected HeLa cells were harvested and fractionated with NE-PER cytoplasmic and nuclear extraction kit (Thermo Scientific) as described above. Both the nuclear and the cytoplasmic fractions were reduced with dithiothreitol (DTT) and S-alkylated with iodoacetic acid (IAA). Subsequently, the protein samples were digested with endoproteinase Lys-C and trypsin, followed by differential isotopic labeling 62 . The labeled samples were then fractionated with strong cation exchange (SCX) chromatography 63 . The SCX-fractionated samples were subsequently analyzed with liquid chromatography coupled to tandem mass spectrometry. Protein identification was performed with Swiss-Port or RefSeq protein database. Ambiguous peptides were aggregated with Perseus software (http://www.perseus-framework.org/). Protein quantification was performed with MaxQuant software 64 . The spectral intensities were used as estimation of protein abundance for each protein groups, and were log10-transformed and (geometric) mean-centered to give comparable intensity distributions. | v3-fos-license |
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} | pes2o/s2orc | Modifying electron transfer between photoredox and organocatalytic units via framework interpenetration for β-carbonyl functionalization
Modifying electron transfer pathways is essential to controlling the regioselectivity of heterogeneous photochemical transformations relevant to saturated carbonyls, due to fixed catalytic sites. Here we show that the interpenetration of metal–organic frameworks that contain both photoredox and asymmetric catalytic units can adjust the separations and electron transfer process between them. The enforced close proximity between two active sites via framework interpenetration accelerates the electron transfer between the oxidized photosensitizer and enamine intermediate, enabling the generation of 5πe− β-enaminyl radicals before the intermediates couple with other active species, achieving β-functionalized carbonyl products. The enriched benzoate and iminium groups in the catalysts provide a suitable Lewis-acid/base environment to stabilize the active radicals, allowing the protocol described to advance the β-functionalization of saturated cyclic ketones with aryl ketones to deliver γ-hydroxyketone motifs. The homochiral environment of the pores within the recyclable frameworks provides additional spatial constraints to enhance the regioselectivity and enantioselectivity.
C atalytic synthesis methods that work under ambient atmosphere with benign reaction environments and clean energy have been a major goal in synthetic chemistry 1 . Of the essential structural motifs that are frequently found in pharmaceutical, material, agrochemical, and fine chemicals, the carbonyls play a pivotal role as powerful building blocks in broad areas of synthetic organic chemistry [2][3][4] . The functionalization of carbonyls, one of the most fundamental transformation in organic synthesis, has evolved to a range of widely used organic reactions and synthetic protocols 5 . While direct α-carbonyl substitution is flourishing because the α-methylene position of the carbonyl group can be readily functionalized with diverse nucleophiles and electrophiles via enolate or enamine chemistry [6][7][8] , the direct β-activation of saturated carbonyls has proven to be a more cumbersome and challenging task owing to the typically unreactive β-C(sp 3 )-H bonds and competitive αsubstitution or other reactions [9][10][11] . Most primary β-carbonyl substitution is still achieved indirectly by the nucleophilic addition of α, β-unsaturated carbonyls 12,13 , despite a few examples of direct β-activation having been reported by enamine oxidation or palladium catalysis 14,15 .
On the other hand, light-promoted photocatalysis, a mild and green chemical synthesis approach, furnishes powerful molecular activation modes to enable bond constructions that are elusive or impossible via established protocols 16,17 , undoubtedly granting a new opportunity for the direct β-position bonding of saturated carbonyls. Recent investigation has also suggested that the combination of photoredox catalysis with enamine organocatalysis is a promising strategy for the β-functionalization of saturated carbonyls through a 5πe − carbonyl activation mode 18 . It is postulated that the clean energy activation mode applies to a wide range of β-carbonyl functionalization transformations and is also compliable with asymmetric catalysis; meanwhile, the long-life and high-energy radical in such homogeneous system requires the co-existence of several types of catalysts and adducts to enhance its stability and ensure effective coupling reactions.
However, in heterogeneous systems, owing the fixed catalytic sites, the modification and optimization of the rapid electron transfer between the two catalytic cycles for regioselectivity control should be given important consideration during the design of the catalysts. In this case, the major challenge goes beyond the incorporation of suitable photoredox centers and organocatalytic centers within the catalytic systems and includes the precise regulation of the electron transfer pathway between the active sites. New strategies wherein the location of all the catalytic requisites can be modified in an easy-to-control model with adjustable electron states and interactional patterns appear imminent to achieve photocatalytic transformation.
Metal-organic frameworks (MOFs) are fascinating hybrid solids with infinitely ordered networks consisting of organic bridging ligands and inorganic nodes 19,20 . The tenability and flexibility of the polymers granted by the diversity of their building blocks allow the incorporation of photoactive organic dyes and the necessary adducts into a single framework, which represents a new approach to heterogenizing several photocatalytic conversions 21 . The regular distribution of catalytic sites within the confined micro-environment benefits the fixation and stabilization of the active radicals formed under irradiation to overcome restrictions of homogeneous processes 22 . The longrange ordered structure of a MOF-based crystalline material contains precisely located catalytic centers, which provides an excellent platform to study photon capture and electron transport and delivery 23,24 . As the integration of both photocatalysis and asymmetric organocatalysis into a single MOF has been considered a promising approach to the α-functionalization of saturated carbonyls 25 , we believe the modification of electron transfer between the two active sites within the frameworks via framework interpenetration is a potential strategy for the βfunctionalization of saturated ketones and aldehydes.
Here we show that the interpenetration of the polymers allowed the appropriate adjustment of the separation and interactions between the photoredox and organocatalytic sites. With Fig. 1 The assembly and crystal structure of InP-1. The components (a) and coordination environments of the Zn(II) ions and NTB 3− ligand (b) in InP-1; Symmetry code: A, x + 1/2, y-1/2, z. 3D structures of the isolated frameworks viewed along the b-axis, before c and after e the twofold interpenetration. The intra-(d) and inter-(f) framework separations between the catalytic sites. Atoms in the pyrrolidine rings of L-PYI are marked in yellow and turquoise. H-atoms and solvent molecules are omitted for clarity the closer interframework distances, the electron transfer between the two active sites is accelerated, such that the special 5πe − βenaminyl radical is more likely to be quickly generated from the enamine intermediate to achieve β-functionalized carbonyl products before the enamine intermediate is coupled with other active species. As a heterogeneous photocatalysis for the direct βfunctionalization of saturated carbonyls, the ability to recover and reuse the photocatalysts makes our system interesting in terms of the practical applications of the photochemical transformation. Additionally, the enriched benzoate and imine groups in the polymers create a suitable Lewis acidic/basic environment to replace the complex catalytic adducts in a homogeneous system to further stabilize long-life and high-energy radicals, which makes the catalytic system simpler, milder and more environmentally friendly. The matched redox potentials of the interpenetrated polymers promoting this catalytic protocol are further advanced to allow the β-functionalization of the other hard-toreduce saturated carbonyls.
Synthesis and characterization of the interpenetrated MOF.
Solvothermal reaction of L-pyrrolidin-2-ylimidazole (L-PYI) 26 Fig. 1). Single-crystal X-ray diffraction revealed the formation of the twofold interpenetrated coordination polymer and the crystallization into the chiral space group C 2 . Within each of the identically isolated chiral framework, the Zn(II) ion was coordinated in a chiral tetrahedral geometry with three carboxylate oxygen atoms from three different NTB 3− ligands and one nitrogen atom from the PYI coligand. The propeller-like NTB 3− ligand rotated the arms with special angles to meet the requirements of the Zn(II)-tetrahedron geometries (Fig. 1b) and linked three zinc ions through the carboxylate groups to generate a 3D framework containing 1D chiral channels with a cross section of 16.7 × 17.0 Å 2 (Fig. 1c). Two symmetric related frameworks were interpenetrated to form the crystalline solid, with the opening of the channels being reduced to 14.3 × 9.4 Å 2 (Fig. 1e, Supplementary Figs. 9 and 10). Chiral PYI catalytic sites were regularly distributed on the inner surfaces of the channels with the exposed active amine groups (N−H pyrrolidine) working as accessible catalytic sites to active aldehydes or ketones. Most importantly, the interpenetration of the two frameworks enforced the close proximity between the photoredox sites (NTB 3− ) and the asymmetric organocatalytic sites (PYI), enabling the shortest intraframework N···N separation of 8.4 Å between the pyrrolidine and triphenylamine N atoms in each non-interpenetrating framework aforementioned shorten to 6.8 Å in the interpenetrated InP-1 (Fig. 1d, f). The formed structural motif will benefit the acceleration of the electron transfer between the enamine intermediate and the photosensitizer. This acceleration promotes the generation of the 5πe − β-enaminyl radical, a key step for the achievement of the β-activation of carbonyl, being more rapid than the direct coupling reaction of the enamine intermediate with other active species. Additionally, the free migration of protons around the uncoordinated nitrogen or oxygen atoms of the ligands and the chiral organocatalysts allows the enriched Lewis acid and Lewis base to possibly act as catalytic adducts to stabilize high-energy radicals. This structural motif grants a possibility of replacing the complex catalytic adducts of the homogeneous system 18 , making the reaction conditions simpler, cleaner and more environmentally friendly, and allowing separation, and recovery in potential practical applications.
The chirality of the interpenetrated frameworks was identified by the chiral space group it crystallized into and confirmed by the circular dichroism (CD) spectrum, with a positive Cotton effect centered at~263 nm and a negative Cotton effect centered at 366 nm ( Fig. 2a and Supplementary Table 1). The porosity of the polymer was determined by the PLATON software 27 with 18.8% of the whole crystal volume calculated. To evaluate the accessibility and stability of the channels to the bulky organic substrates in the solution environment, a dye-uptake assay, an increasingly common method was performed before the MOFbased catalyst was used in catalytic investigations 28,29 . In the general procedure, the as-synthesized crystalline solid InP-1 was first soaked overnight in a methanol solution containing 2′,7′dichlorofluorescein. The resulting crystalline solid was filtered and washed with methanol several times to remove the potential dye molecules adsorbed on the external surfaces of the sample. The dye-absorbed InP-1 was then dissociated by concentrated hydrochloric acid, and the resultant clear solution was diluted to 10 mL and adjusted to a pH of 1.5. The quantum dye-uptake was determined as 12.5% of the solid weight by ultraviolet-visible (UV-vis) spectroscopy ( Supplementary Fig. 6). Confocal laser scanning microscopy of the dye-adsorbed crystal showed the successful uptake of the dye molecules within the channels of the polymer ( Fig. 2b and Supplementary Fig. 7) 30,31 , which revealed the good stability of the well-maintained pores in solution and the possibility of the substrates diffusing inside the channels.
To further confirm the capability of InP-1 to transport substrate molecules through the open channels, the InP-1 was immersed in solutions of CH 2 Cl 2 containing the possible substrates 1,4-dicyanobenzene or 3-(4-methoxyphenyl)propionaldehyde. Infrared (IR) spectrum of InP-1 with adsorbed 1,4dicyanobenzene showed the characteristic signals of ν st C≡N at 2231 cm −1 (Fig. 2c), and the 1 H NMR of InP-1 with adsorbed substrates revealed that InP-1 could adsorb~1.3 equiv of 1,4dicyanobenzene or 1 equiv of 3-(4-methoxyphenyl)propionaldehyde per triphenylamine moiety ( Fig. 2d and Supplementary Fig. 20). All these results demonstrated that the pores of the MOFs are accessible for smooth substrate diffusion and suggested that InP-1 was suitable for heterogeneous catalysis. Furthermore, because the InP-1 was evolved from two of the same noninterpenetrating frameworks via interpenetration, this transformation provides a chance of adjusting the pore environment and at the same time enhancing the density of triphenylamine groups around the pores.
Photocatalytic β-functionalization of saturated aldehydes. The β-arylation reaction was initially examined by employing 1,4dicyanobenzene and propionaldehyde as the coupling partners, along with a 20 watt fluorescent lamp as the light source. A 5 mol % loading of InP-1 gives an 87% conversion to the β-functionalization under irradiation without the formation of any αfunctionalization adduct (entry 1, Table 1). Compared with the complex reaction conditions (multiple additives: base, water, acid, photosensitizer, catalyst, and 1,3-dimethyltetrahydropyrimidin-2 (1H)-one (DMPU) solvent) in the recently reported homogeneous β-functionalization of carbonyls 18 , our catalytic system afforded efficient and selective transformation in the presence of only the organic base 1,4-diazabicyclo[2.2.2]octane (DABCO) and the designed MOF-based catalysts in N,N-dimethylformamide (DMF) solution, which made our reaction conditions simpler, milder, and more manageable. Moreover, the heterogenization of the organic photoredox catalyst H 3 NTB into the frameworks replaced the noble-metal Ir(ppy) 3 in the homogeneous systems, which not only prevented heavy metal pollution on final reaction products but also saved the costs of synthesis and post-processing in mass production, rendering our system less costly and more environmentally friendly.
The InP-1 solids were easily isolated from the reaction suspension by simple filtration. The removal of InP-1 by filtration after 12 h of irradiation shut down the reaction directly, with the filtrate affording only 4% additional conversion over another 36 h of irradiation (Fig. 3a). Washing the filtered residues with DMF enabled their direct reuse in a new round of reactions. After three rounds of reuse, InP-1 solids exhibited a moderate loss of reactivity (87~82% conversion; Fig. 3b and Supplementary Table 5). The PXRD pattern of the InP-1 filtered from the reaction mixture matched well with the simulated one based on the single-crystal simulation, suggested the maintenance of the MOF framework ( Supplementary Fig. 1). Additionally, further confirming the maintenance of the pores of the catalysts, dyeuptake studies for the recycled InP-1 exhibited an 11.7% dyeuptake corresponding to the framework weight. The comparable adsorption ability before and after the catalytic reaction confirmed the chemical stability of the well-preserved pores (Fig. 2b). The results suggested the successful execution of our structural design and demonstrated that InP-1 was a heterogeneous catalyst with advantages of easy separation and recovery 32 that utilizes mild and more environmentally friendly reaction conditions and has potential practical applications.
Control experiments showed that no transformation occurred in the presence of only the 5 mol% H 3 NTB, the 5 mol% PYI or the 5 equiv of DABCO base. Almost negligible conversion was observed when the reaction was performed in the dark (entries 1-5, Supplementary Table 4). To directly compare the MOFcatalyzed reaction with the standard homogeneous conditions 18 , the same adducts and solvent (DABCO, HOAc, water, and DMPU) in equivalent amounts were added to catalyze the transformation with free catalysts, but no β-arylation product was detected. This result suggested that the interpenetrated MOF catalysts played an indispensable role in our heterogeneous system (entry 6, Supplementary Table 4). Considering the catalytic functions of H 3 NTB and PYI, 5 mol% H 3 NTB and PYI were loaded as the homogeneous catalysts to catalyze the reaction under optimal conditions. A low conversion of 11% was observed because the arms of H 3 NTB rotated quickly in the solvent and gave very poor light absorption (entry 8, Supplementary Table 4) [33][34][35] . Even when adding extra zinc salts to coordinate with the H 3 NTB and thereby hinder the rotation, only a small increase to 26% conversion could be obtained.
A further comparison of the catalytic efficiency was based on an interpenetrated achiral coordinated polymer (InP-3) with almost the same structure, except the chiral organocatalytic site was substituted by a pyridine moiety (Fig. 3c 39 . The difference in the catalytic efficiency is attributed to the absence of synergistically organocatalytic groups that used to activate the substrate. This postulation was confirmed by the conversion of the aforementioned catalytic system increasing to 58% upon the excess addition of the organocatalytic L-PYI to the reaction mixture. From a mechanistic point of view, in our heterogeneous system, the interpenetrated coordinated polymer InP-1 adsorbed Table 1 Conversion and enantiomeric excess (ee) of the photocatalytic β-arylation of saturated aldehydes with InP-1 and InP-2 as bifunctional heterogeneous catalysts a 1,4-dicyanobenzene within its channels, and this substrate quenched the excited state of the heterogeneous catalyst to afford the radical anion and oxidized InP-1 + . The aldehyde substrates readily diffuse inside the channels and are activated by the chiral amine catalyst PYI moiety to potentially form the normal chiral enamine intermediate. The close proximity between the photoredox catalytic site and the organocatalytic site increased the opportunity for the enamine intermediate to be quickly oxidized by InP-1 + through interframework electron transfer to generate the corresponding enaminyl radical cation, rather than direct coupling with the arene radical anion or other species that created bonds at the α-carbonyl position. The high-energy enaminyl radical cations were favorable to increase the acidity of the allylic C-H bonds and further promote the corresponding deprotonation at the β-position of carbonyl, eventually completing the critical 5πeactivation mode. The newly formed 5πeβ-enaminyl radical quickly coupled with the 1,4-dicyanobenzene radical anion to yield the cyclohexadienyl anion, which further underwent hydrolysis to give the β-arylation product and return the PYI (Fig. 4 and Supplementary Fig. 15). Fig. 16). These affinities implied the probability of the formation of the key enamine intermediate. Additionally, the fact that the relevant shift was not observed in IR spectrum of InP-3-adsorbed propionaldehyde confirmed the inactivity of InP-3 under the catalytic processes ( Supplementary Fig. 25).
The unique crystalline characteristic of the MOFs yields the opportunity to learn their complex internal structure by singlecrystal X-ray diffraction technique. In addition, the precise determination of the crystal structure of the frameworkencapsulated substrate molecules provides more chemical information about the host-guest interaction patterns, paths, and sites distribution, which allows us to better understand the mysterious activation and formation processes of the catalytic intermediate and has become a promising approach for catalytic studies 41,42 . Thus, the InP-1 crystals were soaked in a solution containing substrates for 12 h, and fortunately the quality of the propionaldehyde-impregnated InP-1 crystals (denoted InP-4) was suitable for X-ray crystallographic analysis (Supplementary Data 4 and Supplementary Figs. 12 and 18). The structural analysis of InP-4 revealed that the space group and unit cell sizes were almost the same as those of the original crystal. As shown in Fig. 5a, the propionaldehyde substrates were perpendicularly embedded in the channels, and the asymmetric unit of InP-4 included 1.5 propionaldehyde molecules. Each substrate molecule was disordered and divided into two parts because of molecular thermal movement. One part of the carbonyl groups was located close to the PYI moiety, and the shortest distance between the pyrrolidine nitrogen and the carbonyl oxygen atoms of propionaldehyde was 2.97 Å. Such a close distance demonstrated a substrate-framework N-H…O hydrogen bond, which may provide an additional driving force to enforce the substrate approaching the active amine catalytic sites closely and facilitating the enamine activation. Several strong O-H…O hydrogen bond interactions (2.68 and 2.35 Å) were also observed between the other part of the carbonyl groups and the free solvent molecules (O15 and O16), which was postulated to be the driving force for the capture and stable location of the substrate within the channels of the catalyst framework. When valeraldehyde and other more complex aldehydes were used as substrates (entries 2-6, Table 1), conversions between 69 and 84% were achieved in the InP-1 reaction systems, with the enantio excess reaching 52% (Supplementary Figs. 28-39 and 55-59). The insertion of the aromatic aldehyde radical coupling partners in the pores caused stronger aromatic stacking interactions and a better enantioselectivity than that of the aliphatic aldehyde product. It should be noted that these produced enantioselectivities were not found in the reported homogeneous work 18 or our control experiment (entry 1, Supplementary Table 7). These results suggested that the interpenetrated frameworks with confined chiral environments may enhance the enantioselectivity of the β-functionalization products. Interestingly, the selectivity is comparable to the best enantio excess achieved in homogeneous β-arylation reactions between 1,4-dicyanobenzene and cyclohexanone, which use chemical cocktail reaction mixtures and a larger loading (20%) of a quite complex chiral organoamine as the chiral director 18 . The simple reaction mixture in our system not only optimized the electron transfer and activation pathways to achieve efficient βarylation transformation but also provided additional spatial constraints to enhance the enantioselectivity without introducing any additional chiral sources, which demonstrates such interpenetrated MOFs to be promising catalysts for the asymmetric photocatalytic transformation. When L-PYI was replaced by Dpyrrolidin-2-yl-imidazole (D-PYI), the other enantiomorph, InP-2 (Supplementary Data 2 and Supplementary Figs. 2 and 8), was isolated with the same cell dimensions as and a mirror-image structure to InP-1, exhibiting similar catalytic activity but granting products with opposite chirality under the optimum conditions (Supplementary Table 3). Interestingly, similar substrate activation was also found in InP-2 systems. The valeraldehyde-impregnated InP-2 crystals (denoted InP-5) with good quality were fortunately obtained, and the single-crystal Xray diffraction analysis suggested the maintenance of space group and main framework (Supplementary Data 5 and Supplementary Figs. 12 and 19). As depicted in Fig. 5b, the asymmetric unit of InP-5 contained one valeraldehyde molecule, and each substrate was perpendicularly inserted in the center of the channel. Such a special spatial occupation with the minimum steric hindrance in the channel, suggested the smooth diffusion of the substrates. The exposed active amine groups of PYI (N5-H5) around the inner walls of the channels provided an affinity to the carboxyl oxygen atoms of aldehyde (O17) with the N-H…O hydrogen bond (2.85 Å) interactions, which could be the potential activation forms and were extremely beneficial to the formation of enamine intermediate. Such carbonyl activation was further supported by the redshift of C=O stretching vibration from 1736 cm −1 (free valeraldehyde) to 1710 cm −1 between the IR spectra of InP-5 and valeraldehyde (Supplementary Fig. 17).
When the bulky aromatic aldehyde (E)-3-(3-oxoprop-1-enyl) phenyl 3,5-di-tert-butylbenzoate was used as a substrate 43 , a <5% conversion under the same reaction condition was observed. The bulky aldehyde was too large to be adsorbed within the channels, and this size selectivity thus demonstrated that the cooperatively catalytic β-arylation reaction mainly occurred in the channels of the polymer, not on the external surface (entry 7, Table 1). To further test the impacts of external surface contact on the reaction, as-synthesized crystal samples (size: 50-80 μm) and finely ground particles (size: 0.1-1 μm) of InP-1 were used to catalyze the β-arylation reaction of 1,4-dicyanobenzene and benzenepropanal under the same reaction conditions (Supplementary Fig. 14). The resulting reactions gave the β-adduct with the comparable conversions of 52 and 56% after 24 h of irradiation, respectively, which demonstrated that the surface area was not the crucial factor for the transformation.
A systematic catalytic investigation was also interesting when comparing a previously reported polymer, MOF-150, involving H 3 NTB as a building block 44 . As could be expected, a 5 mol% loading of MOF-150 (per mole photosensitizer) under the same reaction conditions as the valeraldehyde system gave a negligible conversion. The excess addition of 5 mol% L-PYI to the reaction mixture led to a conversion of 52% but no ee value. Even with the addition increased to 20 mol%, only a 6% additional conversion and a negligible ee value could be detected (entries 2-4, Supplementary Table 7). The combination of NTB 3− and PYI groups in a single framework was not only advantageous to the efficient energy transfer and synergetic coupling of two catalytic cycles but also to the effective collision and constrained coupling between two activated radicals. The extension of the photocatalytic β-arylation of valeraldehyde to a previously reported non-interpenetrating Zn-PYI1 25 , which comprised both the NTB 3− and L-PYI groups, was also performed. With the shortest N···N separation of 10.9 Å between the pyrrolidine and triphenylamine nitrogen atoms, the loading of an idential amount of Zn-PYI1 under the same reaction conditions gave a conversion of 44% but negligible enantioselectivity. The modest catalytic activity and poor enantioselectivity for Zn-PYI1 may be caused by the longer distance between the catalytic centers in the photocatalyst and organocatalyst and the relatively uncontrolled pores generated by the stacked layers (Supplementary Fig. 13 and Supplementary Table 6). As the electron transfer from the enamine intermediate to the oxidized photoredox catalyst is the key step for the β-carbonyl activation, the superiority of an interpenetrated structure in heterogeneous systems is attributed to the close proximity between the two catalytic sites. This imposed vicinity encourages the direct interframework electron transfer for the β-activation of the carbonyl group to appear quickly and further promotes the formation of the β-enaminyl radicals via the critical 5πeactivation mode. The better enantioselectivity displayed by InP-1 is due to the wellmodified chirality-confined pores via framework interpenetration provided more matched and restricted chiral environments to force the substrate radicals to couple with each other in a specific direction and manner.
Photocatalytic β-functionalization of saturated ketones. The 5πe − carbonyl activation mode was also successfully applied in the photocatalytic β-coupling of cyclic ketenes with aryl ketones by MacMillan et al. under homogeneous conditions 11 . To further assess whether our MOF catalytic systems work for such reactions, InP-1 was employed to facilitate the direct β-functionalization of saturated ketones in a heterogeneous fashion. As shown in Fig. 6, the reaction was first evaluated using cyclohexanone and benzophenone as partners, along with 5 mol% of InP-1, 2 equiv of DABCO and 1 equiv of LiAsF 6 in DMF under visible-light irradiation. The resulting reaction gave the β-adduct with a conversion of 67% after 48 h without the formation of any αfunctionalization adduct, which revealed that the 5πeβ-ketone activation was indeed applied in such a heterogeneous system (Supplementary Table 8). Control experiments suggested that no desired product was detected in the absence of catalyst or light. When 5 mol% of non-interpenetrating Zn-PYI1 was used for the same catalytic system, a very low conversion (<5%) of the βadduct and fairly complicated reaction products were detected. Such a result may be ascribed to the large separation of the catalytic sites and the more active ketone substrates; the unsatisfactory separation would greatly reduce the chance of the electron being transferred from the enamine intermediate to the oxidized photosensitizer and meanwhile would create new opportunities for other species. The superiority of the interpenetrated InP-1, with its closely spaced photoredox and organocatalytic sites, was extended to the β-coupling of cyclohexanone analogs (Supplementary Figs. 40-54), which led to conversions from 38 to 62% under the same reaction conditions (6b-e in Fig. 6). The availability of the β-adduct in such ketone systems benefited from the smooth initiation of the photoredox and organocatalytic cycles, which were confirmed by the emission quenching experiments of benzophenone and IR studies of the activation of ketones ( Supplementary Figs. 26 and 27).
Discussion
We have reported a heterogeneous approach for the β-functionalization of saturated aldehydes and ketones under light irradiation and benign environments. The approach included the incorporation of photoredox and asymmetric catalytic groups in a single framework and the interpenetration of two enantiopure frameworks to modify electron transfer between the catalytic centers. The close proximity between the photoredox sites and the asymmetric organocatalytic sites via the framework interpenatration greatly increased the chance of enamine oxidation by the oxidized photosensitizer and the formation of the key 5πeenaminyl radicals. The modified environments of channels provided additional spatial constraints to enhance the regioselectivity and enantioselectivity of the β-activation products without introducing any additional chiral sources. The high selectivity, good recyclability, and clear interaction pattern render the interpenetration strategy highly promising for the regulation and optimization of reaction routes in heterogeneous photocatalysis, and allow the applied protocol to be used in the β-functionalization of saturated cyclic ketones with aryl ketones to deliver γhydroxyketone motifs.
The elemental analyses of C, H, and N were performed on a Vario EL III elemental analyzer. FT-IR spectra were recorded from KBr pellets on a JASCO FT/ IR-430. The powder XRD diffractograms were obtained on a Rigaku D/Max-2400 X-ray diffractometer with a sealed Cu tube (λ = 1.54178 Å). Thermogravimetric analyses were performed at a ramp rate of 10°C/min in a nitrogen flow with an SDTQ600 instrument. Sample morphologies were recorded via scanning electron microscopy on a Jeol JSM-6390A field emission scanning electron microscope. 1 H NMR spectra were recorded on a Varian INOVA-400M type spectrometer with chemical shifts reported as ppm (TMS as the internal standard). 13 C NMR spectra were measured on a Varian INOVA-500M spectrometer with CDCl 3 serving as an internal standard (δ = 77.23). High resolution mass spectra were obtained on a GCT-CA156 Micromass GC/TOF mass spectrometer. GC analysis was performed on an Agilent Technologies 7820 A GC system. GC-MS studies were performed on a Thermo Scientific TRACE Ultra gas chromatographic instrument.
Solid UV-vis spectra were recorded on an HP 8453 spectrometer. Liquid UV-vis spectra were recorded on a TU-1900 spectrophotometer. Fluorescent spectra and lifetime measurements were recorded on an Edinburgh FS920 fluorescence spectrometer. All confocal laser scanning microscopy micrographs were collected by an Olympus Fluoview FV1000 with λ ex = 488 nm.
HPLC analysis was performed on an Agilent 1100 using a ChIRALPAK AS-H column purchased from Daicel Chemical Industries, Ltd. Products were purified by flash column chromatography on 200-300 mesh silica gel and Kromasil Lc-80 using a SiO 2 chromatographic column. The CD spectra were measured on a JASCO J-810 by grinding the crystalline sample into powder to be recorded as KBr pellets.
Solid-state cyclic voltammograms were recorded on a Zahner PP211 instrument. The detailed experiments were performed with a commonly used three-electrode system (working electrode-an improved glassy carbon electrode; reference electrode-Ag/AgCl electrode; auxiliary electrode-platinum wire) in phosphate-buffered saline (PBS; scan rate: 50 mV s −1 ; scan range: 0.4-1.2 V).
X-ray crystallography. Single-crystal XRD data were collected on a Bruker SMART APEX diffractometer equipped with a CCD area detector and a Mo-Kα (λ = 0.71073 Å) radiation source. The data integration and reduction were processed using SAINT software 47 . An empirical absorption correction was applied to the collected reflections with SADABS 48 . The structures were solved by direct methods using SHELXTL and were refined on F 2 by the full-matrix least-squares method using the SHELXL-97 program 49,50 . All non-hydrogen atoms in the backbone of the polymers were refined anisotropically until convergence was reached. Hydrogen atoms attached to the organic ligands were located geometrically and refined in a riding model, whereas some of the disordered solvent molecules were not treated during the structural refinements. To assist in the stability of the refinement, bond distances between several disordered atoms were fixed. Crystallographic data for InP-1, InP-2, InP-3, InP-4 and InP-5 are summarized in Supplementary Table 2 General procedure for the β-functionalization of saturated aldehydes. A glass tube was filled with 1,4-dicyanobenzene (1.0 mmol), catalyst (0.025 mmol), and 1,4-diazabicyclo[2.2.2]octane (DABCO, 5.0 mmol). Then, this vial was purged with N 2 , and the corresponding aldehyde (1.4 mmol) and DMF (3.0 mL) were added via syringe. The resulting mixture was then cooled to -78°C, degassed and backfilled with N 2 three times. The vial was placed~5 cm away from a 20 W fluorescent lamp source for 48 h of irradiation. The reaction mixture was extracted with ethyl acetate, dried with anhydrous Na 2 SO 4 , concentrated in vacuo and purified by flash chromatography on silica gel using hexane/ethyl acetate as the eluent to give the βarylated aldehyde product as an oil. For the aromatic aldehyde substrates, the supernatant solution after centrifugation was diluted with 8 mL of DCM and 2 mL of MeOH. The reaction mixture was then cooled to 0°C and sodium borohydride (5.0 mmol, 5.0 equiv) was added to reduce the resulting aldehyde to the corresponding alcohol. Then the reaction mixture was treated with an identical procedure for the aliphatic aldehyde product.
General procedure for the β-functionalization of saturated ketones. A glass tube was filled with benzophenone (0.5 mmol), catalyst (12.5 μmol), LiAsF 6 (0.5 mmol), and DABCO (1.0 mmol). Then, this vial was purged with N 2 , and the corresponding cyclohexanone (2.5 mmol, 5.0 equiv), and DMF (2.0 mL) was added via syringe. The resulting mixture was then cooled to -78°C, degassed and backfilled with N 2 three times. The vial was placed~5 cm away from a 20 W fluorescent lamp source for 48 h of irradiation. The reaction mixture was diluted with water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried with anhydrous Na 2 SO 4 , concentrated in vacuo, and purified by flash chromatography on silica gel using hexane/ethyl acetate as the eluent to provide an inseparable mixture of the desired β-adduct and the corresponding hemiacetal.
General procedure for dye-uptake measurements. Before the adsorption of dye, InP-1 was first soaked in methanol solution (24 h) for guest molecule exchange and then fully dried in a vacuum oven (120°C, 12 h) to eliminate the guest molecules ( Supplementary Fig. 3). Then, the dried InP-1 (2.56 mg, 2 μmol) was soaked in a methanol solution of 2′,7′-dichlorofluorescein dye (24 mM, 2 mL) in a constant temperature oscillation incubator overnight. The resulting InP-1 was filtered and washed with methanol thoroughly until the solution became colorless and was then dried under a stream of air. The dried samples were dissociated by concentrated hydrochloric acid, and the resultant clear solution with a light olivine color was diluted to 10 mL and adjusted to a pH of 1.5. The dye concentration was determined by comparing the solution UV-vis absorption with a standard curve of the dye. | v3-fos-license |
2019-03-27T13:13:04.768Z | 2018-11-16T00:00:00.000 | 85556436 | {
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} | pes2o/s2orc | Highly-Controllable Imprinted Polymer Nanoshell on the Surface of Silica Nanoparticles for Selective Adsorption of 17 β-Estradiol
A highly-controllable core-shell silica-MIPs absorbent by anchoring a MIPs layer to the surface of SiO2 nanoparticles via a surface molecular imprinting process was prepared. The templates were covalently modified with functional monomers to form precursor EstSi. The latter together with coupling reagent KH-570, were grafted onto the surface of SiO2 nanoparticles before polymerization, to ensure the quantity and quality of imprinted sites on the surface of the covalently attached matrix. The as-synthesized core-shell nanomaterials (SiO2@MIP2) were then evaluated for selective adsorption of 17β-estradiol (E2) with Raman spectra as detection method. The results indicate that SiO2@MIP2 can fast and selectively adsorb E2 from structural analogues, with detection limit of 0.01 μmol/l.
Introduction
17β-estradiol (E2) is one of the several naturally occurring estrogens, which possesses a series of threats to wildlife and humans [1] [2].Due to its persistence and accumulation in the environment, numerous methods have been developed to decontaminate E2, such as adsorption [3], catalytic degradation [4] [5] oxidation [6] and biodegradation [7] [8] [9].Among which adsorption is a commonly adapted approach due to its relatively convenience and efficiency.
However, absorbent materials have been reported are still suffering drawbacks of slow adsorption rate, non-specific adsorption and complicated preparation process etc.
Silica nanoparticles have attracted great attention for their low toxicity, excellent chemical stability, and moderately modifiable surface [10].The large surface area and functionalized hydroxyl groups on particle surface underlie facile modification and high loading effect of various absorbents [11] [12], which allows us to tune the scales and endow them with novel properties.
Molecular imprinting has been widely used for the development of tailor-made receptor binding sites in a three-dimensional, cross-linked polymer matrix.Molecularly imprinted polymers (MIPs) are thus generated with ligand-specific recognition properties analogous to biological systems, and have gained considerable attention in applications, such as solid-phase extraction [13] [14] [15], chiral compounds isolation [16] [17], sensors [18] catalysis [19] [20] and synthetic auxiliary [21].Despite its wide applications and numerous fabrication methods of the conventional bulk MIPs, it still suffers some intrinsic limitations: the deep buried binding sites in bulk hinder subsequent template removal and the guest molecule diffusion process, thereby decreasing the imprinting efficiency; the mechanically crushing and grinding can cause the destruction of imprinted sites and cavities, resulting in the irregular shape, inhomogeneous imprinting sites and eventually poor site accessibility and low adsorption capacity to the guest molecule.In response to these intrinsic drawbacks, the surface imprinting technique has been proposed by creating the imprinted cavities with high affinity on the surface of a suitable matrix, which not only possesses high adsorption capacity but also avoids problems of limited mass transfer and template removal.
Several methodologies are adapted to prepare such MIP films include spin-coating [22] and surface-initiated atom transfer radical polymerization (ATRP) [23].
One major issue with these approaches is controlling the MIP film thickness while ensuring the quantity and quality of imprinting sites.Therefore, finding a facile way to fabricate robust and efficient nanostructured molecular imprinting systems is still greatly needed.
As is well known, various analytical methods have been applied in estrogen detection such as high-performance liquid chromatography (HPLC), gas chro- In this work, we report a facile approach to prepare a highly-controllable core-shell silica-MIPs absorbent.The covalently modified templates along with KH-570 were grafted onto the surface of SiO 2 nanoparticles and followed by polymerization.Thus, a MIPs layer was anchored on the surface of SiO 2 nanoparticles via a surface molecular imprinting process.The performance of the as-synthesized materials towards E2 and their selectivity to structural analogues were investigated, which revealed that the core-shell MIP nanomaterial coupled with Raman, could be applied to the selective adsorption and sensitive detection of estradiol.
Preparation of SiO2 Nanoparticles (SiO2 NPs)
SiO 2 NPs were prepared by the Stӧber method [24].Ethanol (44 ml), water (11 mL), and ammonia solution (1.1 mL) were mixed in a flask and vigorously stirred at 40˚C.Then a mixing solution of TEOS (4.4 mL) and ethanol (20 mL) was quickly added to the above solution and the mixture was stirred for 15 h.
The SiO 2 NPs were centrifuged and rinsed with acetone.Finally, the obtained SiO 2 NPs were dried at 120˚C for 24 h.
Anchoring of E2 and KH-570 on the Surface of SiO2 NPs
First, the template-functional monomer complex (EstSi) was prepared: E2 (1 mmol), IPTES (1.3 mmol), and TEA (8 mmol) were dissolved in THF.The mixture was stirred under N 2 at 65˚C for 24 h.The reaction solution was concentrated under reduced pressure, and the residue was purified by precipitation in n-hexane and filtration [25].EstSi was collected as a white solid (70 mg).
Preparation of 17β-Estradiol-Imprinted SiO2@MIP NPs
The above prepared SiO for 5 h, and subsequently at 60˚C for 24 h.The mixing solution was stirred at a rate of 300 rpm/min throughout the experiment.Then particles were collected by filtration and washed with acetone to remove the unreacted monomers and cross-linking agent, and dried under vacuum at SiO 2 @MIP1-E2, SiO 2 @MIP2-E2, SiO 2 @MIP3-E2, respectively.
Adsorption Experiments of E2
The equilibrium adsorption experiments were performed to evaluate the binding capability of four kinds of SiO 2 @MIP (SiO 2 @MIP1, SiO 2 @MIP2, SiO 2 @MIP3, SiO 2 @MIP4) and SiO 2 @NIP1.Standard E2 solutions were prepared in acetoni-Journal of Encapsulation and Adsorption Sciences trile and the E2 concentrations in the solutions were varied from 2 mg/L to 40 mg/L (2, 5, 10, 15, 20, 30, 40 mg/L).Then the 10 mg of SiO 2 @MIP or SiO 2 @NIP1 were dispersed into 10 mL of standard E2 solution.The obtained mixture was incubated at room temperature for 24 h, the suspension was filtered by the 0.1 µm micro-filter, and the filtered solution was analyzed by HPLC to obtain the E2 concentration.
In order to investigate the kinetics property of SiO 2 @MIP2, the adsorption kinetic experiments were performed as follows.E2 solutions were prepared in acetonitrile and the E2 concentration in the solution was 37 µmol/L.SiO 2 @MIP2 (50 mg) or SiO 2 @NIP1 (50 mg) were added into 10 mL of E2 solution.The obtained mixtures were conducted at room temperature for different contact time (5,10,15,20,30, 40, 60 min), respectively.The suspension was quickly filtered by the 0.1 µm micro-filter, and the filtrates were analyzed by HPLC to obtain the E2 concentration.
The reusability of SiO 2 @MIP2 was measured as below: standard E2 solution was prepared in acetonitrile and the E2 concentrations in the solution were 10 mg/L.The SiO 2 @MIP2 (100 mg) was dispersed into 20 mL of standard E2 solution.The mixture was incubated at room temperature for 24 h, the suspension was filtered by the 0.1 µm micro-filter.The filtrate was analyzed by HPLC.Then, the E2 of the recovered nanoparticles was removed with 10 mL of elution of methanol-acetic acid (8:2, v/v) for 24 hours to ensure complete removal of the E2 in the nanoparticles and washed with methanol, then vacuum dried at 60˚C for 24 h and reused for the adsorption of E2.The adsorption regeneration cycle was repeated five times with the same SiO 2 @MIP2.
Instruments and HPLC Analysis
Fourier transform infrared (FTIR) spectra were recorded with Perkin-Elmer system 2000 FT-IR spectrometer. 1 H and 13 C NMR spectra of EstSi were investigated by Bruker Ascend 400 MHz.Transmission electron microscopy (TEM) images were obtained on a Tecnai-G2-F20 electron microscope operating at 200 kV.Morphology of the materials were determined by scanning electron microscopy (SEM) with a JEOL 6300-F microscope.
The Raman spectra were collected on a HORIBA Lab Raman spectrometer (France) with an excitation wavelength of 532 nm, a resolution of 0.65 cm −1 and a beam diameter of 20 μm.Before Raman measurements, the SiO 2 @MIP2 (10 mg) or SiO 2 @NIP1 (10 mg) was incubated in the 2 ml of E2 acetonitrile solution (the E2 concentrations in the solution was 40 mg/L) for 24 h, then isolated by filtration, washed 3 times with acetonitrile and vacuum dried.The samples were denoted as SiO 2 @MIP2 rebinding E2 and SiO 2 @NIP1 rebinding E2, respectively.The Raman spectroscopy were recorded by focusing the 532 nm diode laser on the silica nanoparticle materials with a total exposure time of 300 s per spectrum, all spectrograms of samples were smoothed and the baselines were corrected.
Preparation
The covalently modified estradiol-functional monomer (EstSi) was synthesized according to the Yang et al.'s method (Figure 1) [25].SiO 2 nanoparticles were synthesized according to reference [24].The covalently modified templates (Est-Si) along with KH-570 were grafted onto the surface of SiO 2 nanoparticles and followed by polymerization to result in a MIPs layer on the surface of SiO 2 nanoparticles [26].The thickness of the MIPs layer was controlled by the added cross linker and the number of imprinted sites was controlled by EstSi used.The quality of the imprinted sites was controlled by both the covalently fixed template and coupling reagent (KH-570).The ratio of EstSi/KH-570 was optimized as 1:2.Finally, the template was removed by heating the SiO 2 @MIP-E2 in DMSO at 180˚C, followed by Sohlex extraction with a mixed solvent of methanol and acetic acid [25] [26].The preparation process was illustrated in Figure 2.
Binding Property Investigation of the SiO2@MIP
The adsorption capacity was calculated according to the following equation: where C 0 (mmol/L) is the initial con-centration of E2, C f (mmol/L) is the concentration of E2 in the filtrate, V (mL) is the volume of the adsorption mixture, and M (mg) is the mass of SiO 2 @MIP2.
The isotherm of imprinted materials with added acrylic acid with shell thickness of 1 nm, 2 nm and 5 nm ( Thus, the material with added acrylic acid with thickness of 2 nm was used for further investigation in the adsorption kinetics and selectivity.
The core-shell imprinted material showed fast adsorption as demonstrated in Figure 8.The adsorption capacity of SiO 2 @MIP2 increased quickly at the first 5 mins and reached equilibrium after 10 mins.For SiO 2 @NIP1, the equilibrium time was 15 mins and the binding capacity was much lower than SiO 2 @MIP2.
This phenomenon can be explained by that comparing with the SiO 2 @NIP1, SiO 2 @MIP2 have the specific binding sites for E2.The rapidly rebinding kinetics suggested SiO 2 @MIP2 has great potential in ultrafast enrichment and separation of E2 molecules.
The detection limit of the as-synthesized material was further tested with the Raman intensity changes after adding different concentrations of E2, as exhibited in Figure 9.The peaks at 829, 930 and 1450 cm −1 were selected for identifying E2.As the E2 concentration increased from 0.01 μmol/L to 1 μmol/L , the peak signals intensity slightly increased, with the lowest detectable concentration of 0.01 μmol/L , which was sufficient to meet the requirements of detection limit of E2 requirements in the environmental water samples.
The imprinting effects were further evaluated by adsorption specificity experiment for the template and its structural analogues, LU, TF and Np whose chemical structural formulae are shown in Figure10(a).The results were summarized in Figure 10(b).The adsoption ability of the non-imprinted material SiO 2 @NIP1 for E2 was much lower than the SiO 2 @MIPs, and there was no significant difference in regenerative experiments, the "n" is repeated times, Q 0 (μg/g) is the adsorption capacity of original material.
As observed in Figure 11, calculations showed that of the prepared SiO 2 @MIP2 after using for 5 times, the absorption capacity was reduced by about 10.6%.The SiO 2 @MIP2 were repeatedly used and regenerated for 5 times with no significant loss of its original adsorb ability and selectivity.SiO 2 @MIP2 as a recyclable economic material has a potential applying prospect in the extraction, separation and concentration and trace analysis of 17β-estradiol.It is because that the appropriate thickness and the high stability of the polymer layer, and monolayer recognition sites are distributed uniformly on the surface of silica nanoparticles all can enhance reusability.
Conclusion
In summary, we have developed a core-shell silica-MIPs absorbent through anchoring a MIPs layer to the surface of SiO 2 nanoparticles via a surface molecular imprinting process.The quantity and quality of imprinted sites on the surface of silica nanoparticle were ensured by covalently attached template precursors and coupling reagent KH-570 on SiO 2 before polymerized imprinting.This material, combining with Raman spectra as detection method, can fast and selectively identify E2 among structural analogues, with detection limit of 0.01 μmol/L.This method can be adapted for other environmental contaminates or biological significant molecules detection by simply switch the template in the shell imprinting process.
X.
Fu et al.DOI: 10.4236/jeas.2018.84011211 Journal of Encapsulation and Adsorption Sciences matography-mass spectrometry (GC-MS), enzyme-linked immunosorbent assay (ELISA) and some other analytical tools.Although some of these analytical techniques are widely used with good precision, their applications are seriously restricted due to the complicated and professional operation procedures, high cost and relative low sensitivity.Due to the excellent sensitivity, fast response, rich molecular information and non-destructive data acquisition, Raman is extensively used from biological analysis to environment monitoring and food safety, even clinical diagnosis and therapy.It could be a promising idea to com-X.Fu et al.Journal of Encapsulation and Adsorption Sciences bine MIPs with SERS for the selective adsorption and sensitive detection of estradiol.
SiO 2 -
(E2+KH-570) were prepared by covalently attachment of KH-570 and EstSi to the surface of SiO 2 nanoparticle.Firstly, the SiO 2 NPs were dispersed in X. Fu et al.Journal of Encapsulation and Adsorption Sciences dry toluene (75 mL per gram of particles) under inert conditions.Then, EstSi (0.5 mmol per gram of particles) and KH-570 (1 mmol per gram of particles) were added to the suspension under constant stirring.The mixture was refluxed at 90˚C for 24 h.The resultant nanoparticles were centrifuged, filtered and washed several times with acetone.The solids were vacuum dried at 60˚C for 24 h to obtain 0.95 g of SiO 2 -(E2+KH-570).The SiO 2 -(IPTES+KH-570) were prepared by the same conditions, except that IPTES (0.5 mmol per gram of particles) was used instead of EstSi (0.5 mmol per gram of particles).
2 -(E2+KH-570) (150 mg) were dispersed into 35 mL of THF under ultrasonication.MAA (0.1 mmol), EGDMA and AIBN (15 mg) were subsequently dissolved into this solution.The mixing solution was purged with nitrogen for 10 min.Polymerization was first done at 50˚C for 5 h, and subsequently at 60˚C for 24 h.The mixing solution was stirred at a rate of 300 rpm/min throughout the experiment.Then particles were collected by filtration and washed with acetone to remove the unreacted monomers and cross-linking agent, and dried under vacuum at The above prepared SiO 2 -(E2+KH-570) (150 mg) were dispersed into 35 mL of THF under ultrasonication.MAA (0.1 mmol), EGDMA and AIBN (15 mg) were dissolved into this solution.The mixing solution was purged with nitrogen for 10 min.Polymerization was first done at 50˚C
Figure 7 (
b) SiO 2 @MIP1, Figure7(c) SiO 2 @MIP2 and Figure7(d) SiO 2 @MIP3), without acrylic acid of shell thickness of 2 nm (Figure7(e) SiO 2 @MIP4), and non-imprinted material (Figure7(a) SiO 2 @NIP) are presented in Figure7, which clearly demonstrate that the material with added acrylic acid of imprinted shell thickness of 2 nm gave the highest adsorption of E2 (Figure7(c)).The one without added acrylic acid (Figure7(e)) otherwise identical with c showed a slightly lower adsorption than c.The shell thickness thicker or thinner than 2 nm showed poorer adsorption.The non-imprinted material had very low adsorption capacity for E2, due to non-specific adsorption.
Figure 10 .
Figure 10.(a) The structure of the compounds used in the selectivity adsorption; (b) Selectivity adsorption result of four kinds of molecularly imprinted materials for E2, LU, Np and TF.
Table 1 .
Raman spectral assignment for the E2 solid. | v3-fos-license |
2019-04-08T13:05:53.976Z | 2016-07-01T00:00:00.000 | 102421625 | {
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} | pes2o/s2orc | PRODUCTION AND TILLERING OF MOMBAÇA GRASS WITH DIFFERENT SOURCES AND LEVELS OF APPLIED NITROGEN
Nitrogen influences numerous physiological and morphological traits of forage grasses, which ultimately interfere directly with production and forage quality. Aimed to study was to evaluate the production of dry matter and the number of tillers of Panicum maximum cv. Mombaça grown with different sources and levels of applied nitrogen. The experiment was conducted at the Federal Institute of the Espírito Santo, Campus of Santa Teresa . The experimental design consisted of a randomised block design with three replicates in a 3 x 6 factorial design and three nitrogenous fertilizers (urea, ammonium sulphate and calcium nitrate), which were applied at six different levels (0, 120, 240, 360, 480 and 600 kg ha -1 ) during the experimental period, for a total of 54 experimental units. Nitrogen levels were divided into seven applications, and the treatments were applied every 28 days, always after the forage was cut. The results show that Mombaça grass is responsive to nitrogen fertilization, and the response in terms of dry matter production and number of tillers for the same level of nitrogen varies depending on the nitrogen source used. Of the nitrogen sources, calcium nitrate had the best performance for the variables evaluated here. For this nitrogen source, the production of dry matter achieved at the maximum level of nitrogen was 18% and 36% higher than the dry matter achieved with the use of ammonium sulphate and urea, respectively.
INTRODUCTION
Pastures are considered the most practical and economical way to feed cattle and play a key role in meat-and milk-production systems (VITOR et al., 2009). Approximately 90% of Brazilian cattle herds are grazed on pasture in extensive production systems (ANUALPEC, 2015).
To achieve high forage productivity, we should consider that forage grasses are just as demanding as many traditional cultivars. Therefore, for intensive exploitation, remediation and soil fertilization are among the factors that determine the level of production of pastures.
Pastures fall short of their potential productivity due to low levels of adopted remediation technologies (TEIXEIRA & HESPANHOL, 2014). Numerous factors contribute to these outcomes, but those factors related to soil fertility management should be noted.
Nitrogen (N) deficiency is one of the most limiting factors for achieving high productivity in forage-production systems. This nutrient is essential and can increase pasture productivity, which is reflected in more nutritious fertilizers, increased stocking rates of pastures and increased live weight gains (BARBERO et al., 2009).
According to Fagundes et al. (2005), the supply of N normally does not meet the demand of grasses, and their productivity is limited by the levels of this nutrient in the soil. Thus, providing an adequate supply of N in balanced proportions is critical in the pasture-production process (COSTA et al., 2009).
The supply of N is known to increase pasture productivity. However, this response varies among forage species. Pastures of Panicum maximum normally have a good response to N, a nutrient that has an important influence on the physiology and potential productivity of forage grasses (BRAGA et al., 2009). According to Jank et al. (1994), when supplied with high levels of N in balance with other nutrients in the soil, Panicum maximum yields can reach up to 41 tons ha -1 year -1 of dry matter.
Thus, the aim of the present study was to evaluate the effects of different sources and levels of N applications on the production of Mombaça grass (Panicum maximum Jacq.) during the rainy season in the municipality of Santa Teresa, State of Espírito Santo (ES), Brazil.
MATERIAL AND METHODS
The experiment was performed at the Midsize Animal Division of the Federal Institute of Espírito Santo (IFES), campus of Santa Teresa, which is located between 19º48'36'' south latitude and 40º40'48'' west longitude relative to the Greenwich meridian in the municipality of Santa Teresa, ES, and has an average altitude of 150 m. The regional climate is temperate humid with dry winters and hot summers; the climate is Cwa according to the Koppen classification.
The pasture area used was 1,200 m 2 , divided into 6 m 2 plots, totalling 324 m 2 of area utilised. The Mombaça grass (Panicum maximum Jacq.) pasture had been established for over four years with high production and soil fertility management techniques performed as recommended by Prezotti et al. (2007) for irrigated pastures with high nutritional requirements.
The soil was classified as a eutrophic yellow Latosol (LA) and was clayey (EMBRAPA, 1999), with 343, 170 and 487 g kg -1 of sand, silt and clay, respectively. Four months prior to the start of the experiment, a chemical analysis of the soil was performed. Based on these results, liming was performed 110 days before the start of the experiment with the application of 1,000 kg ha -1 of dolomitic limestone. Fertilization (except the N supply) was performed 90 days after liming as recommended by the Liming and Fertilization Recommendation Manual for the state of Espírito Santo (PREZOTTI et al., 2007), considering the Mombaça grass cultivar (Panicum maximum Jacq.). Fertilizer was applied in the experimental area using the broadcasting method, using 463 kg ha -1 of triple superphosphate (41% of P 2 O 5 ), 434 kg ha -1 of potassium chloride (60% of K 2 O) and 60 kg ha -1 of FTE BR 10 ® (7.0% of Zn, 4.0% of Fe, 4.0% of Mn, 2.5% of B, 1.0% of Cu, 0.1% of Mo and 0.1% of Co). Immediately prior to the start of the experiment, a new soil sampling was performed in the experimental area at depths of 0.0 -0.2 and 0.2 -0.4 m ( Table 1).
The experimental design consisted of randomised blocks. A 3 x 6 factorial design was used, consisting of three nitrogenous fertilizers 139-146p.
(urea, ammonium sulphate and calcium nitrate) and six levels of N (0, 120, 240, 360, 480 and 600 kg ha -1 ), which were divided and applied in the seven applications throughout the experimental period. In total, three replicates were used, totalling 54 experimental units.
The 3 x 2 m plots were demarcated. Uniform cuts were then performed across the entire area with the aid of a rotary mower, and the treatments were applied soon afterward. The treatments (sources and levels) were dissolved in 10 litres of water and applied to the plots with the aid of a watering can to better distribute the nitrogenous fertilizers.
The experiment was conducted during the rainy season, from 1 October 2011 to 14 April 2012. The Mombaça grass was cut every 28 days, taking into consideration the optimal grazing interval for this forage grass (SANTOS et al., 2014). After each cutting, the different sources and levels of N were applied according to each treatment, for a total of seven applications. Immediately after the application of different levels of the N sources, a water depth of 8 mm was applied throughout the experimental area using a sprinkler irrigation system with 74% application efficiency.
The forage grass was collected with the aid of a 50 x 50 cm iron square and cut with steel shears 30 cm from the soil surface, simulating the ideal cutting height for this fodder grass (SANTOS et al., 2014). After each evaluation cutting, a uniform cutting of the whole experimental area was performed, at the same cutting height as the evaluated plants. The plant material from these uniformity cuttings was then removed from the area.
The material collected in each cutting, in a 0.25 m 2 area, was placed in a plastic bucket, identified and immediately weighed to calculate the weight of the fresh mass (FM) of grass. Subsequently, an approximately 400 g representative sample of material was removed from the collected material, wrapped in paper bags, labelled and then placed in an air-circulating oven, which was maintained between 60 °C and 65 °C until a constant mass was achieved. This process was performed to determine the dry matter. After drying, the samples were ground in a Willey mill with a 1 mm sieve and packed into polyethylene bags. The ground material was placed in the oven again to remove any residual moisture. Dry matter production (DMP) per hectare was determined using the fresh mass of collected material in a 0.25 m 2 area and the dry matter content obtained after processing the sample using the following formula: DMP = [%DM x (FM x 40000)]/100, where DMP: drymatter production in kg ha -1 ; %DM: percentage of dry matter of the sample after processing, FM: fresh matter in kg, collected in 0.25 m 2 ; 40,000: factor used to convert the production into hectares.
Throughout the 196-day experimental period, the rainfall was always insufficient, and the pasture was irrigated, with an irrigation depth calculated based on the evapotranspiration of the cultivar (ETc), using the Penman-Monteith method. The meteorological data used to calculate the ETc was provided by an automatic meteorological station located 550 m from the experimental area. Some of the climate variables observed during the experimental period are presented in Figure 1. Table 1. Chemical characterisation of the soil in the experimental area at depths of 0 -20 and 20 -40 cm before and after soil liming and fertilization.
Before soil liming and fertilization The period which includes the first three cuttings, was used as pasture adaptation. Because the pasture was intensively managed and received high doses of N before the liming, we used this adaptation period to reduce the stores of N in the soil so that the response of Mombaça grass would be a function of the applied treatments and not the N stores already in soil. Thus, the results shown are for the last four cuttings.
The DMP and tiller density were evaluated for the different treatments. The data were analysed using analysis of variance, and a regression analysis was then performed using the program SAS (2002).
RESULTS AND DISCUSSION
The DMP of Mombaça grass is presented in Figure 2 as a function of the source and level of N. The DMP was influenced by the treatments evaluated. For the different sources of N, there was a linear increase up to 600 kg ha -1 of N, where the best results were obtained with the use of calcium nitrate. At levels greater than 273 kg N ha -1 of N, urea was the source with the lowest DMP response. At applications of 600 kg ha -1 of N, the production with the application of calcium nitrate was 26.6% greater than the production with the use of urea and 11.5% higher than the production with the use of ammonium sulphate. Thus, regardless of the source, there was an increase in DMP with increasing levels of N. According to Canto et al. (2013), N acts by increasing the volumetric density of the forage grass and, further, the production of the leaves in the canopy, due to the emergence and elongation of the leaves, thus increasing the DMP. Thus, balanced N fertilization is essential to the production process of pastures because the N from the mineralisation of organic matter in the soil is not sufficient to meet the demand of grasses with a high production potential (FAGUNDES et al., 2006).
In Figure 2, for the maximum level of N, the production generated with the application of ammonium sulphate was 13.6% greater than that generated with the use of urea. Similar results were reported by Costa et al. (2010). These authors reported that for the maximum dose of N (300 kg ha -1 ) for Marandu grass, there was an 18% reduction in the DMP when urea was used as the N source compared to ammonium sulphate. This fact can be explained by the changes that urea undergoes in the soil, resulting in higher losses of N. Upon application to the soil, urea [CO(NH 2 ) 2 ] is hydrolysed by the enzyme urease, resulting in formation of ammonium carbonate [CO(NH 2 ) 2 + 2H 2 O → (NH 4 ) 2 CO 3 ], which degrades rapidly into ammonium bicarbonate and a hydroxyl group
139-146p.
[(NH 4 ) 2 CO 3 + H 2 O → 2NH 4 + + OH -+ HCO 3 -], raising the pH around the fertilizer granules (ROCHETTE et al., 2009). Thus, part of the ammonia is converted into NH 3 , which can be lost into the atmosphere if the urea is not incorporated into the soil (SANGOI et al., 2003). Therefore, even with the adoption of recommended management practices of incorporating this fertilizer into the irrigation scheme after dissolving the N into the water, there may still be N losses, which can explain the lower production results for this N source. In addition, the sulphur in the ammonium sulphate might have contributed to the increase in DMP. Santos & Monteiro (1999) evaluated the response of signal grass to different levels of sulphur and concluded that forage grasses are responsive to the application of sulphur, such that as the levels increase, within certain limits, there is a proportional increase in the DMP and in the number of tillers. The significant increase seen in DMP of Mombaça grass in response to higher N levels is also due to climate changes during experimental period. High temperatures, increased solar radiation, long days and mainly water availability make forage grasses with high productive potential such as Mombaça grass reach high fodder production. According to Valente et al. (2011), Mombaça grass is sensitive to water restriction. Thus it has higher productivity in the summer. Therefore, this forage grass is usually used in intensive production systems, where irrigation is an important tool to achieve high growth in handled areas. The importance of water availability for producing Mombaça grass can be seen in the results obtained by Souza et al. (2005). In this study, the authors concluded that Mombaça grass was the crop that best responded to DMP. Thus, rain precipitation played a relevant role in the early stage of the experimental period, associated to irrigation, which supported high production of Mombaça grass in the last two cuts, due to low rain precipitation.
The different sources and levels of N also influenced the density of the profiles. In Figure 3, beginning at 430 kg N ha -1 , the tiller density was highest for the treatment with calcium nitrate, followed by urea and ammonium sulphate. According to the equations shown in Figure 3, the maximum density of the tillers was achieved at 385 kg ha -1 of N when using urea and ammonium sulphate as the N source. For calcium nitrate, there was a linear increase in the tiller density as a function of the application of N within the limits evaluated. The tiller is the basic vegetative unit of grasses (HODGSON, 1990). It presents great importance on the pastures study, due to the fact that its productivity is conditioned to population density and to the individual tillers mass. According to Barth Neto et al. (2010), N fertilization accelerates the tillering of Mombaça grass, which is reflected in a higher DMP. Costa et al. (2011) The increase in tiller density observed with the use of calcium nitrate and the results obtained in the present study for DMP using this source of N can both be explained by the contribution of calcium, which was supplied to the pasture as part of the application of N source. According to Souza et al. (2007), calcium is an essential element in the growth of meristems, especially in the growth and 139-146p. ROSADO, T. L. et al. proper functioning of the root apex. Calcium is a component of the middle lamella, where it performs a cementing function as Ca pectate. In addition, Ca plays a structural role in maintaining the integrity of the plasma membrane and acts as a modulator of the action of plant hormones, which control, for example, the senescence and abscission of leaves. According to Freitas et al. (2011), this element is essential in the development of Mombaça grass, which can have concentrations ranging from 0.55 to 0.83 g kg -1 in the plant tissues.
Also, among the N sources used, calcium nitrate is the only one that does not alter soil pH, whereas urea and especially ammonium sulphate promote soil acidity by generating ions H + during the process of ammonium nitrification. According to Malavolta (1981), calcium nitrate is a fertilizer that contains N in the nitric form, which does not undergo transformation in the soil. Therefore, its use does not promote pH reduction in the soil. Hence, this source of N, and the possible maintenance of pH close to neutral during the experimental period, contributed to development of better Mombaça grass root system, extraction of nutrients and, consequently, higher DMP. Finally, with pH close to neutral, there is an increased availability of N in the soil, as well as of other macronutrients.
There was a quadratic fit for the tiller density when using urea and ammonium sulphate as N sources. Because of its influence on morphological and physiological characteristics of forage grasses, N acts by promoting an increase in the tiller density. However, at high levels of N, there is a stabilisation or even reduction in the tiller density, as observed by Hoeschl et al. (2007) for Tanzânia grass and Eichler et al. (2008) for Mombaça grass. But this reduction in the numbers of tillers did not result in minor DMP of Mombaça grass (Figure 2). According to Santana et al. (2008), pastures submitted to elevated levels of N may present reduction on the number of tillers, due to an increase on leaf area and competition for luminosity among them. However, the productivity increase is sustained due to the higher tillers development. This way, the increase in the tillers individual weight it proportionally higher to the tiller reduction, allowing the pasture to sustain elevated productivities even with the reduction in tillers population. Similar results to the obtained in this work were presented by Carard et al. (2008), where it was observed for Brachiaria brizantha cultivars a reduction on the number of tillers and an increase on DMP with the increment on levels of N.
CONCLUSIONS
• Mombaça grass is responsive to nitrogen fertilization, and its response in terms of dry matter production and tiller density for the same levels of nitrogen varies depending on the source used; • Of the studied sources of nitrogen, calcium nitrate showed superior performance for the variables evaluated; • Regardless of the source of nitrogen, it was not possible to identify a maximum dry matter production point for Mombaça grass within the limits evaluated here. | v3-fos-license |
2018-04-03T04:06:33.336Z | 2018-03-23T00:00:00.000 | 4238081 | {
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} | pes2o/s2orc | Anti‐tumor effects of a nonsteroidal anti‐inflammatory drug zaltoprofen on chondrosarcoma via activating peroxisome proliferator‐activated receptor gamma and suppressing matrix metalloproteinase‐2 expression
Abstract Surgical resection is the only treatment for chondrosarcomas, because of their resistance to chemotherapy and radiotherapy; therefore, additional strategies are crucial to treat chondrosarcomas. Peroxisome proliferator‐activated receptor gamma (PPAR γ) is a ligand‐activated transcription factor, which has been reported as a possible therapeutic target in certain malignancies including chondrosarcomas. In this study, we demonstrated that a nonsteroidal anti‐inflammatory drug, zaltoprofen, could induce PPAR γ activation and elicit anti‐tumor effects in chondrosarcoma cells. Zaltoprofen was found to induce expressions of PPAR γ mRNA and protein in human chondrosarcoma SW1353 and OUMS27 cells, and induce PPAR γ‐responsible promoter reporter activities. Inhibitory effects of zaltoprofen were observed on cell viability, proliferation, migration, and invasion, and the activity of matrix metalloproteinase‐2 (MMP2); these effects were dependent on PPAR γ activation and evidenced by silencing PPAR γ. Moreover, we showed a case of a patient with cervical chondrosarcoma (grade 2), who was treated with zaltoprofen and has been free from disease progression for more than 2 years. Histopathological findings revealed enhanced expression of PPAR γ and reduced expression of MMP2 after administration of zaltoprofen. These findings demonstrate that zaltoprofen could be a promising drug against the malignant phenotypes in chondrosarcomas via activation of PPAR γ and inhibition of MMP2 activity.
Introduction of bone, in which complete necrosis and high expression of PPARγ appeared in the resected specimen after zaltoprofen administration [8]. Zaltoprofen is a propionic-acid derivative of nonsteroidal anti-inflammatory drugs (NSAIDs) [8]. Previous studies have shown that several NSAIDs could serve as exogenous ligands for PPARγ [9][10][11]. We consider the possibility of anti-tumorigenic effects by zaltoprofen via the activation of PPARγ in musculoskeletal neoplasms.
Matrix metalloproteinase-2 (MMP2) is a member of the MMP family and can degrade matrix collagen and basement membrane, which plays a critical role in tumor invasion and metastasis [12,13]. Overexpression of MMP2 has been detected in various types of cancer, including chondrosarcomas [14]. Therefore, the inhibition of MMP2 is a potent strategy to suppress tumor progression in chondrosarcoma [14,15]. Recent studies report that the activation of PPARγ inhibited MMP2 production and tumor cell invasion in several types of cancer, indicating that downregulation of MMP2 by PPARγ could represent an anti-tumorigenic procedure [16,17].
The purpose of this study was to assess the anti-tumor effects of zaltoprofen via regulation of MMP2 and PPARγ activity, and examine whether zaltoprofen could be a novel therapeutic agent in human chondrosarcoma.
PPARγ reporter assay
PPARγ activity was assessed using a luciferase reporter gene assay kit (INDIGO Biosciences, Inc., State College, PA), according to the manufacturer's instructions [18]. Briefly, reporter cells were dispensed into wells and then immediately treated with zaltoprofen. Following 24 h incubation, treatment media were discarded and luciferase detection reagent was added. The intensity of light emission from each sample well was quantified using a platereading luminometer (TriStar LB 941 Multimode
Cell proliferation assay
The cell proliferation assay was performed using a Cell Counting Kit-8 (Dojindo Laboratory, Mashiki-machi, Japan). Briefly, 5 × 10 3 cells/well were seeded in 96-well plates. After 24 h incubation at 37°C, indicated concentrations of zaltoprofen were added to the medium, and after further 72 h of incubation, the medium was removed and a Cell Counting Kit-8 reagent was added to the plates. After a final incubation at 37°C for 2 h, the absorbance was measured at 450 nm by a microplate reader (iMark Microplate Absorbance Reader, Bio-Rad Laboratories, Inc., Hercules, CA). In addition, 5-ethynyl-2′-deoxyuridine (EdU) proliferation assay was carried out using Click-iT ® EdU Alexa Fluor ® 555 Imaging Kit (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol [20]. Briefly, OUMS27 shPPAR , OUMS27 shNTC , SW1353 shPPAR , and SW1353 shNTC were seeded on slide chambers and incubated overnight. After treatment with zaltoprofen (400 μmol/L) or DMSO for 24 h, cells were treated with EdU (10 mmol/L) for 1 h. Then, they were fixed with 4% paraformaldehyde (Wako Pure Chemical Industries, Ltd., Osaka, Japan), washed with 3% bovine serum albumin in PBS, and permeabilized with 0.5% Triton X-100. Cells were then incubated with the Click-iT reaction cocktail, followed by Hoechst 33342 (NucBlue Live ReadyProbes Reagent, Invitrogen, Carlsbad, CA), and were observed using fluorescence microscope (BZ-9000, Keyence Co., Osaka, Japan). The number of EdU-positive cells and Hoechst 33342-positive cells was counted using Image J software according to the manufacturer's instructions [21].
Cell migration assay
Cell migration was evaluated by a monolayer denudation assay as described previously [22]. Briefly, OUMS27 shPPAR , OUMS27 shNTC , SW1353 shPPAR , and SW1353 shNTC were seeded at a density of 1 × 10 6 cells/well and grown to confluence in a 6-well plate. Cells were then wounded by denuding a strip of the monolayer approximately 1 mm in width with a 200 μL pipette tip. After washing cells with serum-free DMEM, DMSO (0.1%), zaltoprofen (200 or 400 μmol/L), a selective MMP2 inhibitor, and ARP100 (25 μmol/L) were added to each well, and then cells were incubated for 30 h. The rate of wound closure was assessed in six separate fields using Image J software [23].
Cell invasion assay
The invasive potential of OUMS27 shPPAR , OUMS27 shNTC , SW1353 shPPAR , and SW1353 shNTC was assessed using Matrigel invasion chambers (24-well, 8 μm pore size) (BD Biosciences, San Diego, CA) [24]. Cells were plated into the upper chamber at an initial density of 4 × 10 4 cell/ ml in 0.5 mL serum-free DMEM, and 0.5 mL of DMEM supplemented with 10% FBS and fibronectin 500 μg/mL was added to the bottom chambers as chemoattractant. After 24 h incubation in the presence of DMSO (0.1%), zaltoprofen (200 or 400 μmol/L) and ARP100 (25 μmol/L) into both the upper and bottom chambers, the noninvasive cells that remained at the top chambers were removed by scraping using a cotton swab, and the invasive cells at the bottom of the membranes were fixed with methanol. Cells were then washed twice with PBS and stained with 1% crystal violet for 30 min at RT. The invasive cells were counted in six separate fields using an optical microscope at 20× magnification [24].
Gelatin zymography analysis
The expression of activated MMPs in conditioned medium was assayed by gelatin zymography [25]. The cells (OUMS27 shPPAR , OUMS27 shNTC , SW1353 shPPAR , and SW1353 shNTC ) were seeded at a density of 1 × 10 6 cells/ well and grown to 70-80% confluency in a 6-well plate. Then the cells were cultured in serum-free DMEM containing DMSO (0.1%) or zaltoprofen (200 or 400 μmol/L) for 24 h. Afterward, the supernatants were collected and mixed with 5× SDS sample buffer without reducing agent or heating. The samples were loaded onto a Gelatinzymography Precast GEL (Cosmo Bio Co., Ltd., Tokyo, Japan) and subjected to electrophoresis. Following electrophoresis, the gel was washed twice with 2.5% Triton X-100, 10 mmol/L Tris-HCl (pH 8.0) for 30 min to remove SDS, and then incubated in 1 μmol/L ZnCl2, Anti-tumor effects of a NSAID zaltoprofen T. Higuchi et al.
10 mmol/L CaCl2, 0.2 mol/L NaCl, and 50 mmol/L Tris-HCl (pH 8.0) for 28 h at 37°C. Enzyme activity was visualized as negative staining with Coomassie Brilliant Blue R250 and gel was washed with destaining solution until clear bands were visible.
A clinical case of chondrosarcoma with zaltoprofen administration A 34-year-old man presented with grade 1 chondrosarcoma of cervical spine underwent tumor excision by his previous doctor. After 1 year from initial surgery, recurrent tumor was detected and excised by the same doctor, and the pathological diagnosis of the resected section was grade 2 chondrosarcoma. Although, adjuvant radiotherapy was conducted, the tumor had gradually enlarged. After 3 years from second surgery, he was referred to our hospital for severe pain and numbness of the left upper arm. There was bone destruction of the preserved vertebral arch and the tumor invaded the spinal canal, excluding the dura of cervical spine from anterolateral side of C5-6. We performed radiotherapy and tumor excision of the posterior side of C2-4. However, the tumor of C5-6 was unresectable; hence, 240 mg/day of zaltoprofen was administered to the patient against his persistent pain and numbness on the neck and the left upper arm. For approximately 2 years since zaltoprofen administration, the tumor remained within mild growth for grade 2 chondrosarcoma; however, instability and anteflexion of the cervical spine proceeded. Therefore, the patient underwent tumor excision and posterior fixation of the cervical spine. At the final follow-up, the patient was alive with disease without distant metastasis for more than 7 years after the initial diagnosis and 4 years after the administration of zaltoprofen (Fig. 5). The resection paraffin sections of two different treatment periods, the second surgery (preadministration specimen, Pre) and final surgery (postadministration specimen, Post), were used for hematoxylin-Eosin (HE) staining and PPARγ and MMP2 immunofluorescent staining with anti-PPARγ antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and anti-MMP2 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), respectively, in combination with IRDye 680 anti-mouse IgG (LI-COR Inc., Lincoln, NE) as a secondary antibody. Nuclear staining was performed with 4′, 6-diamidino-2-phenylindole (DAPI).
Consent for use of human specimens
Declaration of Helsinki protocols were followed and the patient gave their written, informed consent, which was approved by the Ethics Committee of Kanazawa University.
Statistical analysis
Each experiment was repeated independently at least three times. All data are presented as mean ± SEM. Student's t-test and ANOVA using the Tukey-Kramer post hoc test were used when mean differences were identified between the groups. All P-values were two-sided and P-values of 0.05 or less were considered statistically significant. All statistical analyses were performed with EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan) [26], which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria).
Effects of zaltoprofen on the induction and activation of PPARγ in chondrosarcoma cells
qRT-PCR demonstrated a significant upregulation of PPARγ mRNA in OUMS27 and SW1353 cells after 24 h-treatment with 400 μmol/L zaltoprofen (Fig. 1A). Moreover, zaltoprofen treatment dose-dependently increased expression levels of PPARγ protein in both cell lines as observed by western blotting (Fig. 1B), indicating that zaltoprofen induces PPARγ expression in chondrosarcoma cells. We next investigated the activity of PPARγ using a luciferase reporter assay system. The zaltoprofen treatment could significantly and dose-dependently upregulate PPARγ-responsible promoter activities, and the EC50 (effective concentration) was found to be 47.3 μmol/L (Fig. 1C); this suggests a strong induction of PPARγ activity, with a relatively mild upregulation of PPARγ expression by zaltoprofen.
Inhibitory effects of zaltoprofen on the viability and proliferation of chondrosarcoma cells
We next investigated whether zaltoprofen could reduce the viability of OUMS27 and SW1353 chondrosarcoma cell lines in vitro. When cells were treated with various concentrations (0-400 μmol/L) of zaltoprofen for 72 h, cell viability of OUMS27 and SW1353 cells was significantly and dosedependently lowered by zaltoprofen treatment (Fig. 2A). In addition, cell proliferation assay was performed to evaluate EdU incorporation into the nucleus. The number of EdUincorporated cells per Hoechst 33342-positive nuclei ratio was calculated and the involvement of PPARγ in the cell proliferation was analyzed with or without downregulation of PPARγ by a specific shRNA knockdown system (Fig. 2B). As a result, the EdU-positive cell ratio was significantly and dose-dependently decreased by the treatment of zaltoprofen in both OUMS27 and SW1353 cells, when compared to controls ( Fig. 2C and D). Moreover, the downregulation T. Higuchi et al. Anti-tumor effects of a NSAID zaltoprofen of the EdU-positive cell ratio by zaltoprofen was significantly inhibited by PPARγ silencing in chondrosarcoma cells, OUMS27 shPPAR and SW1353 shPPAR cells (Fig. 2C and D). These data suggest that inhibitory effects on cell viability and proliferation by zaltoprofen were via PPARγ.
Inhibitory effects of zaltoprofen on cell migration and invasion in chondrosarcoma cells
To examine whether zaltoprofen contributes to tumor malignancy in terms of metastatic potentials, we performed in Anti-tumor effects of a NSAID zaltoprofen T. Higuchi et al.
vitro cell migration assays and cell invasion assay using transwell inserts coated with a reconstituted basement membrane barrier (Matrigel). First, in vitro cell migration assays were performed and fielded wound area (%) was calculated (Fig. 3). Zaltoprofen at a concentration of 400 μmol/L and 200 μmol/L significantly and partially inhibited cell migration of both OUMS27 and SW1353 cells, respectively (Fig. 3). Positive control ARP100 (25 μmol/L), a selective MMP2 inhibitor, also showed a significant inhibitory effect on cell migration (Fig. 3). PPARγ silencing in chondrosarcoma cells, OUMS27 shPPAR and SW1353 shPPAR , exhibited a significant inhibition of cell migration even in the nontreated control and following cancelation of the inhibitory effect by zaltoprofen, as well as ARP100 (Fig. 3). Second, we employed a Matrigel cell invasion assay with zaltoprofen and ARP100. Zaltoprofen significantly and dose-dependently inhibited cell invasion of SW1353 cells (Fig. 4A and B). ARP100 significantly inhibited cell invasion of SW1353 cells at a concentration of 25 μmol/L (Fig. 4A and B). In the absence of zaltoprofen or ARP100, PPARγ silencing SW1353 shPPAR cells showed a significant increase in cell invasion when compared to control SW1353 shNTC cells, suggesting a PPARγ-dependent inhibition of cell invasion, which was on the contrary to obtained data on cell migration (Fig. 4A and B). However, zaltoprofen treatment significantly blocked invasion of SW1353 shPPAR cells, speculating the involvement of a PPARγindependent effect of zaltoprofen ( Fig. 4A and B).
Inhibitory effects of zaltoprofen on MMP2 expression in chondrosarcoma cells
The data on the selective MMP2 inhibitor, ARP100, led us to investigate the relationship between MMP2 and zaltoprofen. Gelatin zymography showed downregulation of MMP2 enzyme activities by zaltoprofen at 400 μmol/L in OUMS27 shNTC and SW1353 shNTC cells (Fig. 4C). However, in OUMS27 shPPAR and SW1353 shPPAR cells, the downregulation of MMP2 activities by zaltoprofen was inhibited by PPARγ silencing (Fig. 4C), thus, suggesting that a zaltoprofen-induced PPARγ activation could reduce MMP2 activities in chondrosarcoma cells.
A clinical case of chondrosarcoma treated with zaltoprofen
We experienced a case of chondrosarcoma with unintentional zaltoprofen treatment. (Fig. 5). A specimen was obtained from the patient with grade 2 chondrosarcoma before administration of zaltoprofen (preadministration, Pre), and the microscopic data showed several nuclei with irregular shape and mitoses (Fig. 6A). In contrast, in the postadministration (Post) specimens, less nuclei with irregular shape or mitoses were found, and sporadic myxoid degeneration and necrotic portions were observed (Fig. 6A). Immunofluorescent staining of PPARγ and Asterisks denote a statistically significant difference (*P < 0.05 and **P < 0.01).
T. Higuchi et al.
Anti-tumor effects of a NSAID zaltoprofen
MMP2 demonstrated that the expression of PPARγ was enhanced, but that of MMP2 was reduced, after the administration of zaltoprofen ( Fig. 6B and C).
Discussion
In the present study, we found that zaltoprofen could dose-dependently upregulate PPARγ activities and significantly, but mildly, induce PPARγ expression in human chondrosarcoma OUMS27 and SW1353 cells (Fig. 1). Zaltoprofen inhibited cell viability and proliferation as well as cell migration in a PPARγ-dependent manner in OUMS27 and SW1353 cells (Figs. 2 and 3). Although cell invasion was significantly reduced by zaltoprofen treatment, PPARγ-knockdown could not cancel the effects of zaltoprofen ( Fig. 4A and B), suggesting two possibilities including an inhibitory action of PPARγ against cell invasion and a PPARγ-independent effect of zaltoprofen on cell invasion. In addition, the downregulation of MMP2 activity by zaltoprofen was dependent on PPARγ expression (Fig. 4C).
It has been reported that PPARγ is closely associated with malignant tumors [4]. Recent studies suggest that PPARγ ligands can be useful anti-tumor agents in cancer cells [5,6]. Endogenous and exogenous ligands for PPARγ include fatty acids, eicosanoids, fibrates, thiazolidine derivatives, angiotensin receptor blockers, and NSAIDs [3,8,27]. Among them, NSAIDs such as indomethacin, diclofenac, oxaprozin, and zaltoprofen have been reported as potent ligands for PPARγ [8] and anti-tumor agents against colorectal and lung cancers [28,29]. This is the first report of anti-cancer effects of zaltoprofen against malignant musculoskeletal tumors. Activation of PPARγ is reported to drive the positive feedback loop upregulating the PPARG gene per se via direct binding of PPARγ/ RXRα heterodimers to the PPARG promoter [30]. However, the involvement of other transcriptional activation factors for PPARG, including CEBPs and KLFs, is still unclear in the PPARγ upregulation by zaltoprofen. It is also interesting to examine whether zaltoprofen can selectively activate not only PPARγ, but also PPARα or PPARδ, in terms of the anti-tumor effects of zaltoprofen. Further studies are required to elucidate the overall mechanisms of action of zaltoprofen on the anti-cancer activity.
MMP2, the 72 kDa gelatinase A/Type IV collagenase, plays an important role in the tumorigenic processes, such as tumor invasion and metastasis [12,31]. Therefore, the inhibition of MMP2 activation has been suggested to be a potent strategy for the prevention of cancer cell metastasis [31]. To date, several MMP2 inhibitors have been developed for clinical use. In chondrosarcomas, the expression of MMP2 has been confirmed and demonstrated a significant correlation with the tumor histological grade linking to the prognosis [14]. Therefore, MMP2 could be a possible [15]. Recent studies have also demonstrated that PPARγ activation could inhibit cell migration and invasion through the downregulation of MMP2 in cancer cells [16,17]. In this study, we first discovered the downregulation of MMP2 activity in a PPARγ-dependent fashion by zaltoprofen.
We experienced a case of chondrosarcoma with unintentional zaltoprofen treatment. (Fig. 5). It is known that grade 2 chondrosarcoma progresses destructively and metastasizes. Local recurrence and metastasis rates have been reported to be 27% and 50%, respectively, in grade 2 chondrosarcoma, and 70% of chondrosarcoma cases with local recurrence have developed metastasis [32]. Interestingly, for approximately 2 years during administration of zaltoprofen, the tumor remained localized without distant metastasis even though the tumor was grade 2 chondrosarcoma. Histopathological findings showed upregulation of PPARγ and downregulation of MMP2 after the administration of zaltoprofen in the patient (Fig. 6). We believe that a longterm administration of zaltoprofen could reduce the local spreading and metastatic abilities of the tumor cells.
In conclusion, this study showed that zaltoprofen activated PPARγ and subsequently decreased MMP2 activity in human chondrosarcoma cells, thereby contributing to anti-tumor effects against cell viability, proliferation, migration, and invasion. A beneficial effect of zaltoprofen was also demonstrated in a patient with grade 2 chondrosarcoma. Therefore, these findings suggest that zaltoprofen could be a novel promising remedy against chondrosarcomas via PPARγ activation and MMP2 inhibition. Future studies using orthotopic xenograft mouse models will be required to show the in vivo effect of zaltoprofen on chondrosarcomas, based on previous reports of osteosarcoma [33,34], rhabdomyosarcoma [35], and undifferentiated pleomorphic sarcoma [36][37][38] with various anti-cancer drugs. In addition, large prospective cohort studies will be required to draw a final conclusion about the efficacy of zaltoprofen and to evaluate the effect of clinical applications in patients with chondrosarcoma. | v3-fos-license |
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} | pes2o/s2orc | New uracil analog U-332 is an inhibitor of NF-κB in 5-fluorouracil-resistant human leukemia HL-60 cell line
Background 5-Fluorouracil (5-FU) is an antimetabolite that interferes with DNA synthesis and has been widely used as a chemotherapeutic drug in various types of cancers. However, the development of drug resistance greatly limits its application. Overexpression of ATP-binding cassette (ABC) transporters in many types of cancer is responsible for the reduction of the cellular uptake of various anticancer drugs causing multidrug resistance (MDR), the major obstacle in cancer chemotherapy. Recently, we have obtained a novel synthetic 5-FU analog, U-332 [(R)-3-(4-bromophenyl)-1-ethyl-5-methylidene-6-phenyldihydrouracil], combining a uracil skeleton with an exo-cyclic methylidene group. U-332 was highly cytotoxic for HL-60 cells and showed similar cytotoxicity in the 5-FU resistant subclone (HL-60/5FU), in which this analog almost completely abolished expression of the ATP-binding cassette (ABC) transporter, multidrug resistance associate protein 1 (ABCC1). The expression of ABC transporters is usually correlated with NF-κB activation. The aim of this study was to determine the level of NF-κB subunits in the resistant HL-60/5-FU cells and to evaluate the potential of U-332 to inhibit activation of NF-κB family members in this cell line. Methods Anti-proliferative activity of compound U-332 was assessed by the MTT assay. In order to disclose the mechanism of U-332 cytotoxicity, quantitative real-time PCR analysis of the NF-κB family genes, c-Rel, RelA, RelB, NF-κB1, and NF-κB2, was investigated. The ability of U-332 to reduce the activity of NF-κB members was studied by ELISA test. Results In this report it was demonstrated, using RT-PCR and ELISA assay, that members of the NF-κB family c-Rel, RelA, RelB, NF-κB1, and NF-κB2 were all overexpressed in the 5-FU-resistant HL-60/5FU cells and that U-332 potently reduced the activity of c-Rel, RelA and NF-κB1 subunits in this cell line. Conclusions This finding indicates that c-Rel, RelA and NF-κB1 subunits are responsible for the resistance of HL-60/5FU cells to 5-FU and that U-332 is able to reverse this resistance. U-332 can be viewed as an important lead compound in the search for novel drug candidates that would not cause multidrug resistance in cancer cells.
Background
The incidence of acute myeloid leukemia (AML) and heterogeneous clonal disorders of hematopoietic progenitor cells are increasing worldwide [1]. There are several therapies which are offered for patients with AML but survival after relapse remains poor, necessitating the search for novel chemotherapeutic candidates. At present, the toxic effect of chemotherapy and the occurrence of secondary malignancies associated with AML are the major drawbacks in the pharmacotherapy of AML. Moreover, the frequent acquisition of multidrug resistance (MDR) phenotype is the additional serious problem in the treatment of AML patients. Neoplastic cells are able to develop many different mechanisms of MDR, such as DNA mutations, cell metabolic changes and very often, overexpression of ATP-binding cassette (ABC) transporters and activation of the nuclear factor κB (NF-κB) [2][3][4]. 5-Fluorouracil (5-FU) was the first synthetic fluoropyrimidine analog that showed pharmacological activity. At the molecular level, 5-FU is an antineoplastic antimetabolite that interferes with DNA synthesis by blocking the thymidylate synthase-catalyzed conversion of deoxyuridylic acid to thymidylic acid [5]. In numerous cancer cells, 5-FU inducesh67hu apoptosis and cell cycle arrest and inhibits proliferation [6][7][8].
Since its first synthesis in 1957 [9] 5-FU has been widely used as a chemotherapeutic drug in various types of cancer [10]. However, its application is now limited due to the serious side-effects and the development of resistance [11]. Numerous modifications of 5-FU have been proposed so far, and new analogs modified at position 1, such as tegafur, carmofur and floxuridine ( Fig. 1) are now replacing 5-FU in clinical practice [12].
Though new anticancer agents continue to be discovered, the problem of resistance is still a major obstacle in obtaining efficient drug candidates. Among various mechanisms that can be developed by neoplastic cells in order to escape medical intervention, overexpression of ATP-binding cassette (ABC) transporters is the one well documented [3,4]. The ABC transporter family are transmembrane proteins involved in the normal physiological process but also in the mechanism of drug resistance in cancer cells. The ABC proteins function as efflux pumps that can transport drugs out of the cells in the ATP energy-dependent mechanism, reducing their intracellular concentration [13]. The best known transporters that were shown to reduce the cellular uptake of anticancer drugs, including 5-FU, are Pglycoprotein (ABCB1), multidrug resistance associate protein 1 (ABCC1) and breast cancer resistance protein (ABCG2) [14][15][16][17][18][19][20].
The mechanism of ABC transporter regulation is still not fully understood. Recently, many transcription factorbinding sequences, such as those for p53, AP-1 and, very often, for NF-κB have been identified in the promoter region of the ABCB1 gene [21][22][23].
NF-κB consists of a family of five proteins, p65 (RelA), RelB, c-Rel, p105/p50 (NF-κB1), and p100/52 (NF-κB2) that may form different transcriptionally active homo-and heterodimeric complexes [24] (Fig. 2). The most important subunit of the NF-κB family is RelA/p65, which is phosphorylated in the posttranslational activation mechanism [24,25]. RelB is the only NF-κB subunit that does not form homodimers and can trigger a potent transcriptional activation only when coupled to p50 or p52. The c-Rel plays an essential role in the regulation of T-cell-mediated immunity [26]. NF-κB1 and NF-κB2 are the precursor forms of p50 and p52, respectively [27]. Fig. 2 The members of NF-κB family. The Rel homology domain is characteristic for all memb ers the NF-κB family, while the ankyrin repeats only for the NF-κB inhibitors and the NF-κB precursors of p50 and p52 subunits (p100, p105). The transactivation domain is a C-terminal nonhomologous domain that is important for NF-κB-mediated gene transactivation. For activation of transactivation domain, RelB requires an aminoterminal leucine zipper region Various chemotherapeutic drugs, including 5-FU, were shown to activate NF-κB. The members of the NF-κB family can be activated in several ways. In the classical NF-κB pathway, various cellular receptors, such as tumor necrosis factor receptors (TNFRs), are activated. Then, the inhibitory IκB proteins, that form dimmers with NF-κB subunits, are phosphorylated at two specific serine residues. The IκB activation leads to disintegration of the NF-κB complexes. RelA-and c-Rel-containing dimers translocate to the nucleus where they regulate over 100 transcription targets. The genes whose expression is regulated by NF-κB play an important role in immune/stress responses, apoptosis, proliferation, differentiation and development [28,29].
Continuing the search for novel compounds with anticancer properties, we have recently described a series of uracil analogs, combining uracil skeleton with an exo-cyclic methylidene group conjugated with a carbonyl function [30]. These analogs were all highly cytotoxic against leukemia HL-60 cell line. The most potent analog of the series, (R)-3-(4-bromophenyl)-1ethyl-5-methylidene-6-phenyldihydrouracil, designated U-332 ( Fig. 1), caused in HL-60 cells excessive DNA damage which led to the cell cycle arrest in G2/M phase and apoptosis. To determine the activity of U-332 in the resistant cells, we recently selected a 5-FU resistant subclone (HL-60/5FU) of HL-60 cell line by the conventional method of the continuous exposure of the cells to 5-FU up to 0.08 mmol/L concentration [31]. HL-60/5FU cells exhibited a 6-fold enhanced resistance to 5-FU as compared with HL-60 cells. RT-PCR and ELISA assay showed significant overexpression of MDR-related ABC transporters, ABCB1, ABCG2 but especially ABCC1 in the HL-60/5FU, as compared with the parental cell line. U-332 almost completely abolished ABCC1 expression in the resistant HL-60/5FU cells disturbing therefore drug efflux [31].
The aim of this study was to determine the level of NF-κB subunits in the resistant HL-60/5-FU cell line and to evaluate the potential of a novel uracil analog U-332 to inhibit activation of NF-κB family members in this cell line. For comparison, bengamide (BGD) [32], a potent inhibitor of NF-κB activation was included in the research.
Materials
BGD was obtained from Tocris Bioscience (Bristol, UK). U-332 and BGD were dissolved in DMSO and diluted in culture medium to obtain less than 0.1% DMSO v/v concentration.
Cell culture
The human leukemia cell line, HL-60 was purchased from the European Collection of Authenticated Cell Cultures (ECACC) and was cultured in RPMI 1640 plus GlutaMax I medium (Invitrogen, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS) and antibiotics (100 μg/mL streptomycin and 100 U/mL penicillin).
5-FU-resistant HL-60 cell line was obtained by a longtime exposure of HL-60 cells to increasing 5-FU concentrations (0.01-10 mg/L). The process was repeated until the cells were able to tolerate up to 10 mg/L of 5-FU. The detailed description of this procedure was given elsewhere [31].
Metabolic activity -MTT assay
The determination of anti-proliferative activity of U-332 and BGD was performed by the MTT assay, according to the Mosmann method, as described elsewhere [34].
Quantitative real-time PCR assay
The expression of NF-κB genes was analyzed by realtime PCR (RT-PCR). Briefly, the HL-60 and HL-60/5FU cells were seeded on the 6-well plates at the optimal cell density (3.0 × 10 5 cells/well in 3 mL of culture media), treated with the tested compounds at IC 50 concentration each and left to grow for 24 h. Total RNA was directly extracted from cultured cells, using the Total RNA Mini Kit (A&A Biotechnology, Gdynia, Poland) while Transcriba Kit (A&A Biotechnology, Gdynia, Poland) was used for cDNA synthesis, according to the manufacturer's procedure.
Amplification of cDNA was performed using Real-Time 2x-PCR SYBR Master Mix (A&A Biotechnology, Gdynia, Poland) and gene specific primers (RelA, RelB, NF-κB1, NF-κB2 and c-Rel) ( Table 1) in Stratagene MX3005P QPCR System (Agilent Technologies, Inc. Santa Clara, CA, USA) according to the manufacturer's protocol. The housekeeping gene, GAPDH, was used as an internal reference gene for normalization of qPCR results. The gene expression levels were determined by the 2 -ΔΔCT method [35].
Determination of NF-κB subunit activity by ELISA-based method
The activity of NF-κB family members (p50, p52, p65, c-Rel, RelB) was analyzed in the cellular protein extracts (10 μg) by the ELISA-based method using NF-κB (p50, p52, p65, c-Rel, RelB) Transcription Factor Assay Kit (ABCAM). Briefly, HL-60 and HL-60/ 5FUres cells were seeded in triplicate into 6-well plates at the optimal cell density (4.5 × 10 5 cells/well in 3 mL of culture media). Then, U-332 and BGD at IC 50 concentration each were added and cells were left to grow for 24 h. After incubation, cells were washed 3x in PBS and immediately collected by centrifugation (200×g, 5 min).
The Nuclear Extraction Kit was used in the preparation of nuclear extracts which were then analyzed using NF-κB (p50, p52, p65, c-Rel, RelB) Transcription Factor Assay Kit containing a 96-well plate with immobilized oligonucleotides for NF-κB subunit binding site (5-GGGACTTTCC-3́). Active NF-κB heterodimers present in the whole-cell extracts appropriately bind to this oligonucleotide.
For detection of p50, p52, p65, c-Rel or RelB, the primary antibodies recognizing an epitope on these NF-κB subunits were used. The secondary antibodies conjugated to horseradish peroxidase (HRP) provided sensitive colorimetric readout at OD 450 nm. The data were visualized using Flexstation 3.
Statistical analysis
Statistical analyses and all graphs were prepared using Prism 6.0 (GraphPad Software Inc., San Diego, CA, USA). All data are presented as mean ± SEM. Statistical significance was assessed using one-way ANOVA followed by a post-hoc multiple comparison Student-Newman-Keuls test (for comparisons of three or more groups) or Student's t-test (for comparisons of two groups).*p < 0.05, **p < 0.01, and ***p < 0.001, ****p < 0.0001 were considered significant.
Then, both types of cells were treated for 24 h with U-332 or BGD (at IC 50 concentration each). In HL-60 cells, U-332 down-regulated the expression level of c-Rel, RelA, NF-κB1 and NF-κB2 genes, but the most pronounced effect (3.7-fold) was observed for NF-κB2 (Fig. 4a). A potent NF-κB inhibitor, BGD did not influence NF-κB gene expression in this cell line (Fig. 4b).
Activity of NF-κB subunits
To determine the activity of NF-κB family members in HL-60 and HL60/5FU cell lines, the ELISA-based method was used. Cancer cell lysates were prepared after 24 h exposure of cells to analog U-332 or BGD (at IC 50 concentration each). Consistent with the enhanced gene expression, the activity of cRel, RelA and NF-κB1 subunits was significantly up-regulated in HL-60/5FU cells in comparison with the effect observed in HL-60 cells (Fig. 6a). Incubation of HL-60 cells with U-332 drastically reduced the activity of c-Rel, while BGD did not influence any of NF-κB subunits (Fig. 6b). In the resistant cell line, U-332 potently reduced activity of c-Rel, RelA and NF-κB1, while BGD exerted the strongest effect on c-Rel, RelB and NF-κB1 subunits (Fig. 6c).
Discussion
Chemotherapeutic drugs are meant to kill disseminated cancer cells and prevent metastasis but many cancers develop resistance during treatment [36]. Activation of NF-κB or/and overexpression of ABC transmembrane proteins play a major role in the resistance of tumor cells to chemotherapy [2][3][4]. Many chemotherapeutic agents, including 5-FU, doxorubicin, etoposide or cisplatin have been reported to activate NF-κB and to upregulate expression of ABC transporters [37][38][39][40]. In several cancer cell lines the inhibition of either NF-κB or ABC transporter activity increased intracellular accumulation of chemotherapeutic drugs [41][42][43]. For example imatinib, a known NF-κB inhibitor, reversed the acquired resistance to doxorubicin by down-regulating the level of ABCB1 through the inhibition of RelA (p65) activity [44]. Therefore, the combination of anticancer drugs with NF-κB and ABC transporter inhibitors can be considered an efficient approach to sensitize cancer cells to chemotherapy. Various members of the NF-κB family are constitutively activated in many cancers via one of the two pathways: the canonical pathway involving RelA, NF-κB1 p50 and c-Rel and the non-canonical pathway engaging RelB and NF-κB2 p52. Generally, the canonical NF-κB pathway is known to mediate mostly inflammatory responses, while the non-canonical NF-κB and its components have been shown to have pro-tumorigenic effects in many cancer types. However, there is significant cross-regulation between the components of these pathways, emphasizing the importance of the NF-κB as a single, highly complex system with disease relevance in many types of cancer [45,46].
In this report, we have shown that in HL-60/5FU resistant cells all 5 NF-κB subunit genes (RelA, RelB, NF-kB1, NF-kB2 and cRel) were significantly upregulated. By monitoring activation of both, the non-canonical and the canonical NF-κB pathway members we have demonstrated that in HL-60/5FU resistant cells U-332 was involved in the down-regulation of the canonical pathway (RelA, c-Rel and NF-κB1), while BGD inactivated some subunits of both pathways (c-Rel, RelB and NF-κB2). BGD is usually considered a universal inhibitor of both NF-κB pathways. Here, we have shown that in leukemic HL-60/5FU cells the levels of only three out of 5 NF-κB subunits were affected by BGD.
Conclusions
The important finding of the research presented here and in our previous papers [31,33] was the identification of the novel uracil analog as a potent inhibitor of NF-κB and ABCC1 transporter expression. The inactivation of NF-κB subunits was shown to correlate with the inhibition of ABC transporter activity which may lead to the increased accumulation of a drug in cancer cells. Fig. 4 Real-time PCR analysis of c-Rel, RelA, RelB, NF-κB1 (p100/p50), and NF-κB2 (p100/p52) mRNA levels in HL-60 cells treated with U-332 (a) and BGD (b). Data are expressed as mean ± SEM Statistical significance was assessed by test t. *p < 0.05, ** p < 0.01, *** p < 0.001 Fig. 5 Real-time PCR analysis of c-Rel, RelA, RelB, NF-κB1 (p100/p50), and NF-κB2 (p100/p52) mRNA levels in HL-60/5FU cells treated with U-332 (a) and BGD (b). Data are expressed as mean ± SEM. Statistical significance was assessed by the test t. ** p < 0.01, *** p < 0.001 Fig. 6 The activity of c-Rel, RelA, RelB, NF-κB1 (p100/p50), and NF-κB2 (p100/p52) in untreated HL-60 and HL-60/5FU (a); in HL-60 treated with U-332 or BGD (b); in HL-60/5FU treated with U-332 or BGD (c). Human ABC ELISA kits and extracts from cancer cells treated with U-332 or BGD (at IC 50 concentration each) were used. As a positive control Raji nuclear extract was added. Data are expressed as mean ± SEM. Statistical significance was assessed using test t and oneway ANOVA and a post-hoc multiple comparison Student-Newman-Keuls test. **p < 0.01; ***p < 0.001 in comparison with control | v3-fos-license |
2016-05-04T20:20:58.661Z | 2014-10-03T00:00:00.000 | 10606456 | {
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} | pes2o/s2orc | The Involvement of Nek2 and Notch in the Proliferation of Rat Adrenal Cortex Triggered by POMC-Derived Peptides
The adrenal gland is a dynamic organ that undergoes constant cell turnover. This allows for rapid organ remodeling in response to the physiological demands of the HPA axis, which is controlled by proopiomelanocortin (POMC)-derived peptides, such as adrenocorticotropic hormone (ACTH) and N-Terminal peptides (N-POMC). In the rat adrenal cortex, POMC-derived peptides trigger a mitogenic effect, and this process increases cyclins D and E, while inhibiting p27Kip1. The goal of the present study was to further explore the mitogenic effect of ACTH and synthetic N-POMC1–28 peptides by investigating the differences in the expression of key genes involved in the cell cycle of the rat adrenal cortex, following inhibition of the HPA axis. Moreover, we evaluated the differences between the inner and outer fractions of the adrenal cortex (ZF-fraction and ZG-fraction) in terms of their response patterns to different stimuli. In the current study, the inhibition of the HPA axis repressed the expression of Ccnb2, Camk2a, and Nek2 genes throughout the adrenal cortex, while treatments with POMC-derived peptides stimulated Nek2, gene and protein expression, and Notch2 gene expression. Furthermore, Notch1 protein expression was restricted to the subcapsular region of the cortex, an area of the adrenal cortex that is well-known for proliferation. We also showed that different regions of the adrenal cortex respond to HPA-axis inhibition and to induction with POMC-derived peptides at different times. These results suggest that cells in the ZG and ZF fractions could be at different phases of the cell cycle. Our results contribute to the understanding of the mechanisms involved in cell cycle regulation in adrenocortical cells triggered by N-POMC peptides and ACTH, and highlight the involvement of genes such as Nek2 and Notch.
Introduction
The adrenal gland is composed of two distinct regions: the cortex and the medulla. The cortex consists of three concentric zones: the zona glomerulosa (ZG), the zona fasciculata (ZF), and the zona reticularis (ZR). The adrenal cortex requires stimuli from the adrenocorticotropic hormone (ACTH) precursor pro-opiomelanocortin (POMC) for adrenocortical maintenance and remodeling. Several other peptides are secreted from the anterior pituitary, including pro-gamma-MSH. Pro-gamma-MSH potentiates the steroidogenic actions of ACTH, triggering an increase of cholesterol and cholesterol ester hydroxylase activity [1]- [3]. Moreover, the N-terminal sequences of pro-gamma-MSH that do not contain the MSH-gamma sequence (known as the N-terminal portion of POMC, or N-POMC) are able to induce cell proliferation in the adrenal cortex [4] [5]. Previous studies have shown that N-POMC and N-POMC 2-54 peptides are potent mitogens, both in vitro [4] and in vivo [6]. Recent work from our group has shown that synthetic N-POMC 1-28 induced in vivo Sphase entry in the entire rat adrenal cortex by up-regulating cyclins D and E [7] [8]. Moreover, N-POMC 1-28 induced proliferation of rat adrenal cells in primary culture via the ERK1/2 pathway [9].
It is well-known that inhibition of the HPA axis by dexamethasone (DEX) or hypophysectomy can cause atrophy only in the innermost portion of the adrenal cortex [10]- [13]. However, the mechanisms involved in the regulation of cell cycle genes in adrenal cells after the absence of POMC-derived peptides remain poorly understood. This study aimed to evaluate the expression pattern of 86 genes associated with cell cycle regulation in the adrenal cortex of dexamethasone-treated rats after administration of ACTH or synthetic N-POMC 1-28 peptides (these N-POMC peptides contain correctly aligned disulfide bonds in cysteines [N-POMC Cys ] or a linear structure with methionines [N-POMC Met ]). Moreover, we evaluated the differences between the inner and outer fractions of the adrenal cortex in terms of their response patterns to different stimuli, as well as the differences and similarities between the proliferative effects triggered in the adrenal cortex by ACTH and N-POMC peptides. Our results highlight the involvement of some key genes in adrenocortical cellular proliferation, such as Nek2 and Notch.
Animals
Male Sprague-Dawley rats with an average weight of 250630 g were obtained from the Institute of Biomedical Sciences of the University of São Paulo and maintained in a temperaturecontrolled environment and 12-h light/dark cycle. The study was approved by the Animal Experimentation Ethics Committee at the University's Institute of Biomedical Sciences. The animals were fed with standard rat food and received water ad libitum. All experimental procedures were conducted between 9 and 11 am; the rats were euthanized by decapitation and adrenal glands were harvested.
N-POMC 1-28 peptide synthesis N-POMC 1-28 peptides were synthesized with cysteine (N-POMC Cys ) or methionine (N-POMC Met ). The N-POMC Cys peptide was synthesized and characterized by Bachem America Inc. (Torrance, CA, USA) and contained correctly aligned disulfide bridges in the cysteine 2-24 and 8-20 positions. In order to avoid undefined disulfide bonds, the N-POMC Met peptide was synthesized and characterized in the Department of Biochemistry and Biophysics at the Federal University of São Paulo (UNIFESP) by replacing cysteine with methionine.
Dexamethasone, ACTH, and N-POMC 1-28 treatments
The animals were treated as previously described in [8]. Briefly, a concentration of 50 mg/100 g of body weight (BW) of Dexamethasone (DEX) (Aché Laboratórios Farmacêuticos, Campinas, SP, Brazil) was administered intraperitoneally, once a day at 9 am for 2 days, with the purpose of inhibiting the HPA axis. The control animals received saline injections and followed the same treatment regimen as the experimental animals. Subsequently, DEX-treated rats were divided into four groups (n = 4-5) and received a single injection of ACTH, N-POMC Cys , N-POMC Met , or saline. The ACTH, N-POMC Cys , and N-POMC Met treatments were administered at a concentration of 10 27 M in the dose of 100 ml/100 g BW.
Preparation of protein lysates
The preparation of protein lysates was performed as described in [7]. Briefly, at 6 hours post treatment, the adrenal glands were removed and gently decapsulated to separate the capsule/zona glomerulosa (ZG fraction, composed mainly of ZG cells) from the zona fasciculata/reticularis and medulla (ZF fraction, composed mainly of ZF cells). The medulla was removed from the ZF fraction and the samples were lysed in ice-cold RIPA and protease inhibitors. Protein concentrations were quantified with the Figure 1. Clustergram showing the expression profile of significantly (p,0.05) altered genes related to the cell cycle in adrenal ZF fraction or ZG fraction after inhibition of the HPA axis with DEX for two days (50 mg/100 g BW) (A) Correlation of up-or downregulated genes in ZF fraction and ZG fraction; genes regulated in both DEX and control treatments are presented in the figure intersection (B). Analysis of Nek2b protein expression in adrenal ZF fraction and ZG fraction after inhibition of HPA axis with DEX (C). Adrenal glands were separated in two fractions (ZG and ZF), and total protein or total RNA, respectively, was extracted for the immunoblotting or PCR array analysis. Control animals received saline only (Control). *p,0.05, n = 3. doi:10.1371/journal.pone.0108657.g001 Bradford assay. Previously published data concerning the quality of preparation and separation of the adrenal fractions, showed that the contamination between different fractions was minimal and acceptable [7].
Nek2 protein expression
Total lysate protein samples from the ZG and ZF fractions (30 mg from each) were sorted in 10% SDS-PAGE. To test the Nek2 protein isoforms, we used a commercial HeLa nuclear extract (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for positive control of the antibody. We also obtained Y1 and primary rat adrenal cell culture total protein extracts, as described in [9]. After electrophoresis, the gel was electroblotted onto Hybond-C nitrocellulose membranes using a semi-dry Bio-Rad apparatus. Non-specific sites were blocked overnight at 4uC with 5% dried non-fat milk dissolved in TBS (150 mM NaCl, 10 mM Tris, 1% Tween 20, pH 7.5). The membranes were subsequently incubated with anti-Nek2 (1:1000; Santa Cruz) or b-actin (1:2000, Santa Cruz) for two hours at room temperature. Proteins were detected using chemiluminescent secondary peroxidase-conjugated antirabbit or anti-mouse polyclonal antibodies (ECL-Amersham-Pharmacia, Piscataway, NJ, USA). The immunoblot results were quantified by densitometry using the Gel-Pro Imager and Gel-Pro Imager kit Version 1.0 quantification program for Windows. The average and standard error were calculated for the data obtained from each fraction. Ponceau staining was used to monitor protein transfer amounts and total protein loaded.
Immunohistochemistry of Nek2 and Notch expression
The rats were euthanized 6 h after the final treatments; adrenal glands were removed, fixed in 4% paraformaldehyde in a solution of 0.1 M phosphate buffered saline (PBS; pH 7.4) for 8 h at room temperature, and subsequently immersed in 6% sucrose PBS for 12 h. The fixed and deparaffinized sections were rehydrated through a graded ethanol series, washed with sterile Milli-Q water, PBS, and PBS with 0.5% hydrogen peroxide. Sections were incubated for 10 minutes in boiling citrate buffer (pH 6.0) for antigen retrieval, and the background staining was blocked using a commercial blocker solution (Background Blocker; Diagnostic BioSystems, Pleasanton, CA, USA). These sections were washed and incubated in PBS with anti-Nek2 1:200 (Santa Cruz), or anti-Notch 1 1:100 (Cell Signaling Technology, Inc. Danvers, MA, USA) or anti-Notch 2 1:100 (Cell Signaling) or even Notch 3 1:500 (Cell Signaling). The immune complexes were detected by immunoperoxidase staining using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA, USA) and visualized with diaminobenzidine (Sigma Fast; Sigma Aldrich, St. Louis, MO, USA) followed by counterstaining with Harris' hematoxylin and a saturated solution of lithium carbonate. The adrenal sections incubated in non-immune primary sera tested negative (data not shown).
Total RNA isolation and PCR array
Two hours after final treatments with Saline, ACTH, or N-POMC peptides, the rats were decapitated and their adrenal glands were removed and separated into ZG and ZF fractions. The total RNA was isolated using Trizol extraction according to the manufacturer's instructions (Life Technologies, USA). The cDNA synthesis was prepared according to the PCR array manufacturer's instructions (Sabiosciences -Qiagen, Germantown, MD USA). cDNA was mixed with SYBER Green MIX provided by the manufacturer and applied in the PCR array plates (Rat Cell Cycle PARN-020). The assays were performed on a standard real-time PCR device ABI7500 (Applied Biosystems -Life Technology). Data were analyzed using online software provided by the PCR array manufacturer.
Statistical data analyses
For the Western-Blotting assay, statistical comparisons of three independent immunoblot results were tested using analysis of variance (ANOVA) followed by the Tukey-Kramer Multiple Comparisons Test. For the PCR array experiments, the p values were calculated based on a Student's t-test of the replicate 2 ' (2 Delta Ct) values for each gene in the control and treatment groups. Values of p#0.05 were considered statistically significant. The results corresponded to three independent experiments.
Ethics Statement
This study was carried out in full accordance with the ethical principles of animal experimentation adopted by the Brazilian Society of Laboratory Animals in Science and was approved by the ethics committee on animal use of the Institute of Biomedical Sciences under protocol number 156, page 112, book 02.
Results
HPA-axis inhibition reduced Nek2, Ccnb2, and Camk2b expression in the entire cortex To evaluate the effects of HPA-axis inhibition by dexamethasone (DEX) in the expression of genes involved in the cell cycle of the adrenal cortex, we used a real-time RT-PCR-based plate assay. The ZG and ZF fractions showed distinct profiles of gene Table 2. Genes significantly regulated in the ZG fraction of the adrenal cortex after inhibition of the HPA axis followed by treatments with ACTH, N-POMC Met , or N-POMC Cys . expression after HPA-axis inhibition with Dex treatment (Figure 1A). Among all significantly regulated genes, three genes were significantly (p,0.05) down-regulated in both fractions of the cortex: Nek2, Ccnb2, and Camk2b ( Figure 1B). Nek2 was the most repressed gene, irrespective of the adrenal fraction (ZG: 217.8 fold and ZF: 212.4 fold). All significantly regulated genes are listed in Table 1. In order to validate the expression of Nek2 on a protein level, we first evaluated the protein isoform found in the rat adrenal cortex. Our results show that the isoform 2 (Nek2b ,38 kDa) was expressed in the rat adrenal gland ( Figure S1). The expression of Nek2b was reduced in both fractions after HPA-axis inhibition: 0.57-fold and 0.36-fold in the ZG and ZF fractions, respectively ( Figure 1C). Immunohistochemistry revealed no Table 3. Genes significantly regulated in the ZF fraction of the adrenal cortex after inhibition of the HPA axis followed by treatments with ACTH, N-POMC Met , or N-POMC Cys . expression of Nek2b in the capsule, and a decreased expression in the entire cortex following HPA-axis inhibition (Figure 2).
ACTH and N-POMC peptides induce distinct gene expression profiles in the outer and inner zones of the adrenal cortex
We report results for treatments with ACTH and N-POMC peptides on genes that were significantly (p,0.05) up-or downregulated. In the ZG fraction, treatment with ACTH promoted a distinct profile of gene expression compared to treatment with N-POMC peptides ( Figure 3A). These distinct responses could be partially due to the larger number of repressed genes in common between treatments with the two N-POMC peptides (14) compared to those repressed in common between treatment with ACTH and either N-POMC peptide (4). Only three genes were down-regulated in all treatments: Ccnc, Cdkn1a, and Pkd1. Our data also reveal that ACTH up-regulated the expression of Camk2 (as did N-POMC Cys ) and of Dnajc2 (as did N-POMC Met ). Moreover, Inha expression was up-regulated after stimulation with both N-POMC peptides ( Figure 3B). When we analyzed cell cycle-related genes, we observed that ACTH up-regulated four genes related to the G1 phase (Camk2a, Ccne1, Cdk4, and Gpr132) and two related to the G2 phase (Chek1 and Dnajc2). Treatment with N-POMC Met up-regulated the expression of genes related to the S phase (Mcm3 and Mre11a), the G2 phase (Dnajc2), and the M phase (Ran, Psmg2, and Pes1), whereas N-POMC Cys up-regulated the expression of only one gene (Camk2a), which is related to the G1 phase ( Figure 3C). Furthermore, N-POMC Cys down-regulated more genes (7) related to a late phase of the cell cycle (M-phase) than ACTH (2) or N-POMC Met (3). The list of all significantly altered genes in the ZG fraction, including their fold regulation data, is available in Table 2. In the ZF fraction, the clustergram based only on significantly altered genes reveals that N-POMC Cys treatment promoted a distinct pattern of gene expression when compared to the N-POMC Met and ACTH treatments ( Figure 4A). Also, when we analyzed the genes that were regulated in common by the different treatments, we observed that Nek2 was up-regulated in all three treatments. Stag1, Mdm2, Casp3, Dnajc2, and Nfatc1 were up-regulated after treatment with both ACTH and N-POMC Met , whereas Ppm1d was up-regulated after treatment with either ACTH or N-POMC Cys . Among the genes down-regulated in common between the treatments with POMC-derived peptides, only Camk2a was repressed after treatment with ACTH or N-POMC Cys . Interestingly, N-POMC Met did not repress the expression of any gene ( Figure 4B). When we analyzed genes related to specific phases of the cell cycle, we observed that ACTH up-regulated genes related to the G1 phase (Nfatc1), the G2 phase (Ppm1d and Dnajc2), and the M phase (Nek2 and Stag1). N-POMC Met up-regulated genes related to the G1 phase (Nfatc1), the G2 phase (Dnajc2), and the M phase (Nek2, Rad21, Shc1, and Stag1). On the other hand, N-POMC Cys treatment up-regulated the expression of genes related to the S phase (Msh2 and Sumo1) and the M phase (Ccna1 and Nek2). Concomitantly with these up-regulations, ACTH treatment repressed genes related to the G1 phase (Camk2a, Ccnd1, and Ppp3ca), the S phase (Rad51), the G2 phase (Ccnf), and the M phase (Prm1 and Wee1). Also, N-POMC Cys treatment downregulated genes related to the G1 phase (Camk2a and Slfn1) and the M phase (Ccna2 and Ccnb1), as shown in Figure 4C. All significantly altered genes in the ZF fraction, including their fold regulation data, are listed in Table 3.
ACTH and N-POMC Met increase Nek2b expression in the entire adrenal cortex
Based on previous data with PCR array, we chose Nek2 to evaluate the variation in the expression of some candidate genes on a protein level. This gene appeared down-regulated in the entire adrenal cortex after inhibition of the HPA axis and all three treatments up-regulated its expression in the ZF fraction. After treatments with either ACTH or N-POMC Met , there was an increase in Nek2b expression in the ZG fraction (2.35-fold and 2.31-fold, respectively) and also in the ZF fraction (1.93-fold and 1.85-fold, respectively) ( Figure 5A). Immunohistochemistry showed increased Nek2b expression in the entire cortex following treatments with ACTH and N-POMC Met (Figure 5B and C). On the other hand, N-POMC Cys treatment did not alter Nek2b expression, irrespective of the adrenal fraction analyzed (Figure 5D).
The expression of Notch proteins in the adrenal gland
Our PCR array data showed that Notch2 was up-regulated in the ZG and ZF fractions after ACTH or N-POMC Met , respectively. In order to determine the expression of the Notch2 protein, we performed an immunohistochemistry assay. Immunohistochemistry labeling for Notch2 showed no positive cells in the adrenal cortex or medulla, regardless of treatment. Positive Notch2 cells were present only in the adrenal capsule, and there was no modulation in their expression, irrespectively of treatment ( Figure 6A). This result led us to evaluate the expression of two other Notch proteins in the adrenal gland, Notch1 and Notch3, even though we did not previously analyze their gene expression in the PCR array. The immunohistochemistry results for Notch1 revealed that this protein is expressed in both adrenal cortex and medulla ( Figure 7A). Also, the expression of Notch1 is slightly reduced in the ZG of the cortex after HPA-axis inhibition with DEX ( Figure 7B and C). ACTH and N-POMC Met treatments promoted an increase in Notch1 expression in sub-capsular cells ( Figure 7D and E), whereas N-POMC Cys treatment did not alter its expression anywhere in the cortex ( Figure 7F). Notch3 protein expression analyses revealed that the adrenal medulla has remarkable positive staining for this protein, when compared to the cortex ( Figure 8A). Further, the inhibition of the HPA axis by DEX promoted a decrease in the levels of Notch3 protein expression in the entire cortex ( Figure 8B and C). After ACTH treatment, there was no significant modification in the expression of this protein ( Figure 8D). However, after N-POMC Met or N-POMC Cys treatments, we measured an increase in Notch3 expression ( Figure 8E and F). The induction of Notch3 by N-POMC Met or N-POMC Cys did not reestablish the Notch3 expression observed in the control with saline only.
Discussion
In this study we found that three genes (Ccnb2, Camk2a, and Nek2) were negatively regulated throughout the adrenal cortex when the HPA axis was inhibited. Ccnb2 and Camk2a are related to the G2/M transition and Nek2 is associated with key processes of spindle checkpoint. Cyclin B2/CDK1 is a key kinase complex involved in the transition of phase G2/M and its inhibition. Cyclins B1 and B2 act at the same CDK complex, promoting the progression of the cell cycle, and cyclin B2 is able to compensate for the down-regulation of cyclin B1 during mitosis in human cells [14]. Cyclin B2 has been shown to be overexpressed in adrenocortical carcinoma and is thus a good candidate for distinguishing benign from malignant adrenocortical tumors [15]. Calcium/Calmodulin-Dependent Protein Kinase II Alpha (Camk2a) is essential for regulating G2/M and the metaphaseanaphase transition in many cell types [16]. It is a member of the family of serine/threonine protein kinases and is a downstream effector of the angiotensin II-triggered signaling cascade that synthesizes and releases aldosterone in the ZG-cells [17]. Moreover, aldosterone production stimulated by ACTH in ZG cells is triggered by an increase in Ca 2+ influx, which activates Camk signaling [18]. Therefore, the repression of the HPA axis could, at least in part, compromise the production of aldosterone in ZG by down-regulating the expression of Camk2a.
Equally important is NEK gene expression, which encodes a serine/threonine enzyme involved in key processes such as the centrosome cycle, kinetochore-microtubule attachment, spindle checkpoint/assembly, chromosome condensation, and cytokinesis (for a review, see [19]). Furthermore, overexpression of NEK2 has been associated with breast tumorigenesis [20]. In 2009, Giordano et al. published a microarray dataset for adrenocortical tumors (carcinomas and adenomas) and normal adrenals [21] (freely available on the NCBI website under Gene Expression Omnibus Series accession number GSE10927, www.ncbi.nlm.nih.gov/geo/). When we analyzed these data, we observed that NEK2 is overexpressed in carcinomas compared to adenomas and normal adrenals ( Figure S4). To our knowledge, this is the first report showing that Nek2 expression is associated with cell cycle regulation in adrenal glands. Also, studies that screened the gene expression profile of serumstimulated human fibroblasts showed that Nek2 mRNA levels are highest in S and G2 [22]. In the current study, we showed that Nek2b gene and protein are induced in both adrenal fractions after treatments with ACTH or N-POMC Met , suggesting that Nek2b could be associated with the control of adrenocortical cell proliferation, since these treatments promoted an increase in the number of cells in the S-phase [7]. To the best of our knowledge, this is the first report showing that Nek2 expression is associated with cell cycle regulation in adrenal glands.
Following HPA-axis inhibition, there was an increase in the expression of Tumor protein p53 (Tp53) and Cyclin-dependent kinase 1A (Cdkn1a) in the ZG fraction. Both of these genes are associated with cell cycle arrest. In the ZG fraction, the absence of POMC-derived peptides does not promote a decrease in the number of cells in the S-phase, even with the increase of these two genes associated with cell cycle arrest. On the other hand, the Wee1 gene was up-regulated in the ZF fraction. Wee1 kinase is able to phosphorylate and inhibit the cyclin B/CDK complex, which causes cell cycle arrest [23]. These data are in agreement with our own results showing the down-regulation of the cyclin B gene at the same time point and in the same adrenal fraction. Curiously, no gene was up-regulated in either fraction, reflecting the distinct peptide responses among adrenal cortex cells. The analysis of gene expression data plotted in the rat cell cycle pathway from the Kyoto Encyclopedia of Genes and Genomes (KEGG) shows that most cells in the ZG and ZF fractions seem to be in a different phase of the cell cycle, when analyzed at the same time point (Figures S2 and S3). Indeed, in the ZG fraction, HPAaxis inhibition down-regulates genes that are mainly related to the G1-S phases of the cell cycle, whereas in the ZF fraction, most repressed genes are in the G2 phase. Therefore, these data suggest that the zones comprising the adrenal cortex respond to inhibition of the HPA axis at different time points. This reinforces the notion of the independence of HPA axis control over the different zones of the adrenal cortex [24].
Regarding the effects of POMC-derived peptides in the adrenal cortex, we previously demonstrated that N-POMC Met peptide and ACTH promoted an increase in the number of cells in S-phase in all adrenal zones, whereas N-POMC Cys was effective only in ZG and ZR [7]. These data are in contrast with previous observations in POMC-null mice in which peripheral administration of N-POMC peptide did not change adrenal growth, but caused a reduction in food intake and body weight [25]. Moreover, results published previously by the same group [26] showed that the synthetic peptide N-POMC Cys is able to stimulate cell proliferation of tumor adrenocortical cells and primary culture of bovine adrenal glands. In addition, results from our group, which used primary cultures of rat adrenal cells, detected the involvement of MAPK-ERK signaling after stimulation with N-POMC peptides [9]. Consistent with these results, we showed that only N-POMC Met and ACTH up-regulated the expression of Nek2b in ZG and ZF, which highlights the differences between the two N-POMC peptides. Moreover, we observed that these mitotic treatments are able to increase the expression of genes related to the cohesin protein complex ( Figure S3), which is associated with a late phase of cell division (for review see [27]). Interestingly, in N-POMC Cys treatments, which do not stimulate ZF to enter the Sphase [7], the expression of the cohesin protein complex was not modulated.
The amino acid sequence of the N-terminal region of POMC is highly conserved between mammals [28], suggesting an important biological role for this peptide. The disulfide bridges in the Nterminal region of the native molecule induce the formation of a tertiary hairpin structure, which appears to be essential for accurate targeting of the peptide to the secretory region of the cell [29]. Here, the comparative analysis of the peptides' effects on the expression of cell cycle genes in the ZG fraction, suggest that ZG cells are driven to different stages of the cell cycle, depending on the treatment (2h-ACTH or 2h-N-POMC). In fact, while 2h-ACTH treatment induced up-regulation of genes related to the G1-S transition, both 2h-N-POMC peptides down-regulated the genes associated with this phase. On the other hand, both 2h-N-POMC peptides up-regulated genes associated with the S-phase (Pcna and Gadd45; Figure S2).
The differences in gene expression profiles between the ZF and ZG fractions might be explained by the composition of the extracellular matrix (ECM) and the microenvironment of the adrenal zones [30]. There is some evidence that the ECM and integrins are differentially expressed throughout the rat adrenal cortex, and that the proliferative effect in vitro of ACTH in ZG and ZF cells depends on the composition of the substrate that these cells are exposed to [30]. Also, the vascularization of the adrenal gland, which begins at the periphery of the organ and reaches the marrow centripetally, promotes a diffusion gradient between the adrenal zones [31]. This diffusion gradient might be directly reflected in the concentration of POMC-derived peptides that each zone is influenced by, resulting in the observed differences in gene expression profiles.
Regarding Notch protein expression, we observed higher Notch1 expression in the subcapsular cells than in the rest of the cortex after ACTH and N-POMC Met treatments, but not after N-POMC Cys treatment. Moreover, Notch2 appears to be expressed only in the capsular cells, irrespective of the treatment. The subcapsular and capsular cells of the adrenal cortex have been considered to be the main site of undifferentiated cells, involved in adrenocortical zonation, remodeling, and homeostasis (for a review, see [32]). It has recently been shown that Delta-like homologue 1 (Dlk1), which physically interacts with Notch1 [33], is expressed in the subcapsular region of the cortex and stimulates Gli1 expression in the mesenchymal cells of the adrenal capsule [34]. Therefore, after proliferative treatments, Notch1 is expressed in the same subcapsular region as Dlk1 and Sonic-Hedgehog (Shh), which suggests that this protein might be involved in the regulation of adrenocortical remodeling and homeostasis. Further studies must be conducted to elucidate the role of Notch1 in subcapsular cells. The Notch signaling pathway is one of the most altered pathways regarding adrenal tumors, suggesting that this pathway is also crucial for the proliferation of adrenocortical cells [35]. Interestingly, Notch3 is highly expressed in the adrenal medulla, where the presence of chromaffin progenitor cells has already been reported [36].
In conclusion, our study shows that the inhibition of the HPA axis represses the expression of Ccnb2, Camk2a, and Nek2 genes throughout the adrenal cortex, and that Nek2 is up-regulated after treatments with proliferative POMC-derived peptides. We also demonstrated that the zones of the adrenal cortex respond to HPA-axis inhibition and POMC-derived peptides at different times, reinforcing the notion that cells from the ZG and ZF fractions are in different phases of the cell cycle. Moreover, our results contribute to the understanding of the mechanisms triggered by N-POMC peptides and ACTH in genes associated with cell cycle regulation in adrenocortical cells. Figure S1 Isoforms of Nek2 protein in commercial HeLa nuclear extracts, Y1 total extracts, rat adrenal gland extracts, and primary rat adrenal cell culture. Total proteins from Y1 cells, rat adrenal gland, and primary rat adrenal cell culture were extracted by using RIPA buffer. Encyclopedia of Genes and Genomes (KEGG). The genes highlighted in green are down-regulated and those in red are upregulated. (TIF) Figure S4 The expression of NEK2 comparing the microarray dataset from adrenocortical tumors, carcinomas and adenomas, and normal adrenals. The microarray dataset is published in [21] and is freely available on the website www.ncbi.nlm.nih.gov/geo/through Gene Expression Omnibus Series accession number GSE10927. (TIF) | v3-fos-license |
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} | pes2o/s2orc | Increased ionization supports growth of aerosols into cloud condensation nuclei
Ions produced by cosmic rays have been thought to influence aerosols and clouds. In this study, the effect of ionization on the growth of aerosols into cloud condensation nuclei is investigated theoretically and experimentally. We show that the mass-flux of small ions can constitute an important addition to the growth caused by condensation of neutral molecules. Under atmospheric conditions the growth from ions can constitute several percent of the neutral growth. We performed experimental studies which quantify the effect of ions on the growth of aerosols between nucleation and sizes >20 nm and find good agreement with theory. Ion-induced condensation should be of importance not just in Earth’s present day atmosphere for the growth of aerosols into cloud condensation nuclei under pristine marine conditions, but also under elevated atmospheric ionization caused by increased supernova activity.
C louds are a fundamental part of the terrestrial energy budget, and any process that can cause systematic changes in cloud micro-physics is of general interest. To form a cloud droplet, water vapor needs to condense to aerosols acting as cloud condensation nuclei (CCN) of sizes of at least 50-100 nm 1 , and changes in the number of CCN will influence the cloud microphysics 2, 3 . One process that has been pursued is driven by ionization caused by cosmic rays, which has been suggested to be of importance by influencing the density of CCN in the atmosphere and thereby Earth's cloud cover [4][5][6][7] . Support for this idea came from experiments, which demonstrated that ions significantly amplify the nucleation rate of small aerosols (≈1.7 nm) 8,9 . However, to affect cloud properties, any change in small aerosols needs to propagate to CCN sizes 50-100 nm, but such changes were subsequently found by numerical modeling to be too small to affect clouds 3,10,11 . The proposed explanation for this deficit is that additional aerosols reduce the concentration of the gases from which the particles grow, and a slower growth increases the probability of smaller aerosols being lost to preexisting aerosols. This has lead to the conclusion that no significant link between cosmic rays and clouds exists in Earth's atmosphere.
This conclusion stands in stark contrast to a recent experiment demonstrating that when excess ions are present in the experimental volume, all extra nucleated aerosols can grow to CCN sizes 12 . But without excess ions in the experimental volume, any extra small aerosols (3 nm) are lost before reaching CCN sizes, in accordance with the above mentioned model results. The conjecture was that an unknown mechanism is operating, whereby ions facilitate the growth and formation of CCN. Additional evidence comes from atmospheric observations of sudden decreases in cosmic rays during solar eruptions in which a subsequent response is observed in aerosols and clouds 6,7 . Again, this is in agreement with a mechanism by which a change in ionization translates into a change in CCN number density. However, the nature of this micro-physical link has been elusive.
In this work we demonstrate, theoretically and experimentally, the presence of an ion mechanism, relevant under atmospheric conditions, where variations in the ion density enhance the growth rate from condensation nuclei (≈1.7 nm) to CCN. It is found that an increase in ionization results in a faster aerosol growth, which lowers the probability for the growing aerosol to be lost to existing particles, and more aerosols can survive to CCN sizes. It is argued that the mechanism is significant under present atmospheric conditions and even more so during prehistoric elevated ionization caused by a nearby supernova. The mechanism could therefore be a natural explanation for the observed correlations between past climate variations and cosmic rays, modulated by either solar activity [13][14][15][16][17] or caused by supernova activity in the solar neighborhood on very long time scales where the mechanism will be of profound importance [18][19][20] .
Results
Theoretical model and predictions. Cosmic rays are the main producers of ions in Earth's lower atmosphere 21 . These ions interact with the existing aerosols, and charge a fraction of them. However, this fraction of charged aerosols is independent of the ionization rate in steady state-even though the electrostatic interactions enhance the interactions among the charged aerosols and between these aerosols and neutral molecules, the increased recombination ensures that the equilibrium aerosol charged fraction remains the same 22 . Ion-induced nucleation will cause the small nucleated aerosols to be more frequently charged relative to an equilibrium charge distribution, but ion recombination will move the distribution towards charge equlibrium, typically before the aerosols reach~4 nm 23 . Changing the ionization is therefore not expected to have an influence on the number of CCN through Coulomb interactions between aerosols.
However, this argument disregards that the frequency of interactions between ions and aerosols is a function of the ion density, and that each time an ion condenses onto an aerosol, a small mass (m ion ) is added to the aerosol. As a result, a change in ion density has a small but important effect on the aerosol growth rate, since the mass flux from the ions to the aerosols increases with the ion density. This mass flux is normally neglected when compared to the mass flux of neutral molecules (for example sulfuric acid, SA) to the aerosols by condensation growth, as can be seen from the following simple estimate: the typical ion concentration in the atmosphere is on the order of ≈10 3 ions cm −3 , however, the condensing vapor concentration (SA) is typically on the order of ≈10 6 molecules cm −3 . The ratio between them is 10 −3 , from which one might conclude that the effect of ions on the aerosol growth is negligible. Why this is not always the case will now be shown.
The mass flux to neutral aerosols consists not only of the condensation of neutral molecules, but also of two terms which add mass due to recombination of a positive (negative) ion and a negative (positive) aerosol. Furthermore, as an ion charges a neutral aerosol, the ion adds m ion to its mass. Explicitly, taking the above mentioned flux of ion mass into account, the growth of aerosols by condensation of a neutral gas and singly charged ions becomes, ∂N i ðr;tÞ ∂t ¼ À P j ∂ ∂r I i;j ðr; tÞN j ðr; tÞ; with i and j = (0, +, −) referring to neutral, positively, and negatively charged particles. Here r and t are the radius of the aerosol and the time. N i = (N 0 , N + , N − ) is the number density of neutral, positive, and negative aerosols. n 0 is the concentration of condensible gas, n + , n − are the concentration of positive and negative ions, while A i = (m i /4πr 2 ρ), with m i being the mass of the neutral gas molecule (i = 0), and the average mass of positive/ negative ions, i = (+, −), ρ is the mass density of condensed gas, and β is the interaction coefficient between the molecules (or ions) and neutral and/or charged aerosols (See Methods for details on derivation of the equations, the interaction coefficients, details of the experiment, and the (m ion /m 0 ) of 2.25). β 00 , β +0 , and β −0 correspond to the interaction coefficients describing the interaction between neutral aerosols of radius r and neutral molecules, positive ions and negative ions respectively, whereas β 0+ , and β 0− are the interaction coefficients between neutral molecules and positively/negatively charged aerosols. Finally β +− corresponds to the recombination between a positive ion and a negative aerosol of radius r, and vice versa for β −+24 . If no ions are present, the above equations simplify to the well known condensation equation 25 , where is the growth rate of the aerosol radius due to the condensation of molecules onto the aerosols. It is the change in growth rate caused by ions that is of interest here.
By assuming a steady state for the interactions between ions and aerosols, we find 22 which using N tot = N 0 + N + + N − gives N 0 ðr; tÞ N tot ðr; tÞ Equations (3) and (4) can be inserted into the components of Eq.
(1) (for i = (0, +, −)). Assuming symmetry between the positive and negative charges, i.e., m ion ≡ m + = m − , β ±0 ≡ β −0 = β +0 , β ±∓ ≡ β +− = β −+ , and n ion ≡ n + = n − , finally leads to (See Methods for details on derivation of the equations, the interaction coefficients, details of the experiment, and the (m ion /m 0 ) of 2.25): where The 1 term appearing in Eq. (5) is the result of the approximation (1 + 2(β 0± β ±0 )/(β ±∓ β 00 ))/(1 + 2β ±0 /β ±∓ ) ≈ 1, good to 3 × 10 −4 for a 10 nm aerosol and decreasing for d > 10 nm. The bracketed term in Eq. (5) is related to the rate of change in the aerosol radius This growth rate is one of the characteristic equations describing aerosol evolution, and it is valid independent of any losses 26 . It is Γ, in Eq. (6), which quantifies the net effect of ion condensation. The term 4(β ±0 /β 00 )(N 0 /N tot ) depends on electrostatic interactions, and where (n ion /n 0 ) and (m ion /m 0 ) depend on the specific concentrations and parameters. Figure 1a portrays this part together with (β ±0 /β 00 ) and (N 0 /N tot ). Figure 1b depicts the size of Γ in % of the neutral condensation, as a function of the ionization rate q and diameter d of the aerosols for an average atmospheric sulfuric acid concentration of n 0 ≈ 1 × 10 6 molecules cm −3 and m 0 = 100 AMU and a mass ratio (m ion /m 0 ) of 2.25 (See Methods for details on derivation of the equations, the interaction coefficients, details of the experiment, and the (m ion /m 0 ) of 2.25.). It should be noted that the terms β ±0 and β 00 also depend on the mass and diameter of the ions and neutral molecules, which may vary depending on composition. Both exact masses and the mass asymmetry between ions can vary-observationally positive ions tend to be heavier than negative ions 27 . There are additional caveats to the theory, which will be examined in Discussion section.
Experimental results. We now proceed to show that the predictions of the theory of ion-induced condensation outlined above can be measured in experiments. The latter were done in an 8 m 3 stainless steel reaction chamber 12 . Due to wall losses, the growth rate of the aerosols could not be too slow, therefore the sulfuric acid concentration needed to be larger than n 0 ≈ 2 × 10 7 molecules cm −3 . This decreases the effect that ionization has on the aerosol growth by more than an order of magnitude when compared to typical atmospheric values. It is however a necessary constraint given the finite size of the chamber. The number of nucleated particles had to be low enough that coagulation was unimportant, thus keeping the growth fronts in size-space relatively sharp, allowing accurate growth rate measurements.
The ionization in the chamber could be varied from 16 to 212 ion pairs cm −3 s −1 using two γ-sources. At maximum ionization, the nucleation rate of aerosols was increased by~30% over the minimum ionization.
The experiments were performed with a constant UV photolytic production of sulfuric acid, and every 4 h (in some cases 2) the ionization was changed from one extreme to the next, giving a cycle period P of 8 h (or 4) (See Methods for details on derivation of the equations, the interaction coefficients, details of the experiment, and the (m ion /m 0 ) of 2.25.). The effect of ioninduced nucleation during the part of the cycle with maximum ionization results in an increased formation of new aerosols (Fig. 2a). To improve the statistics, the cycle P was repeated up to 99 times. A total of 11 experimental runs were performed, representing 3100 h. Each data set was subsequently superposed over the period P resulting in a statistically averaged cycle. An example of a superposed cycle can be seen in Fig. 2b), where locations of the transition regions between the low and high aerosol density data can be used to extract the effect of ions on aerosols growth. The two transitions determine two trajectories, profile 1 and profile 2, in the (d, t)-plane, from which it is possible to estimate the difference in the growth time to a particular size d (See Methods for details on derivation of the equations, the interaction coefficients, details of the experiment, and the (m ion / m 0 ) of 2.25.). A CI API-ToF mass spectrometer was used to measure the sulfuric acid concentration during some of the experiments and to estimate the average ion mass 28 .
The above theory predicts a difference in the time it takes the two profiles to reach a size r due to a growth velocity difference caused by ion condensation. The time it takes for aerosols to grow 6)) in %, in an atmosphere with a condensible gas concentration of 1 × 10 6 molecules cm −3 as a function of aerosol diameter d and ionization rate q (left hand axis) or ion density (right hand axis). The contour lines show the relative size of the growth due to ion condensation in % of the usual condensation growth. The mass ratio (m ion /m 0 ) is set to 2.25, and the mass of the neutral molecule is set to 100 AMU to size r along the two possible profiles is expressed as where t 1 and t 2 refers to the time it takes profiles 1 and 2 to reach size r. The integrand is given by Eq. (7) and it considers that after half the period, the γ-sources are switched off (or on). The above equations can be integrated numerically to find ΔT = t 2 (r) − t 1 (r) and allow comparison with the experiments. During the first~12 nm of growth, profile 1 grows with the γsources on and it thus grows faster than profile 2 in the γ-off region, consequently, t 1 (r) < t 2 (r) and ΔT is increasing (Fig. 2b). This increase is due to the (nearly) constant difference in growth rate between the two profiles. But when profile 1 enters the second part of the cycle, when the γ-sources are off, profile 2 enters the high ion state and is now growing faster than profile 1. Therefore, it is now profile 2 that grows faster and ΔT starts to decrease. Figure 3 depicts three examples of ΔT as a function of the diameter d. It is seen that the data scatter around the theoretical curves (red (γ-on) and blue (γ-off)) obtained from Eqs. (7) and (8). The gray curves were produced by performing a LOESS (locally weighted smoothing) smoothing of the experimental data. It also indicates that the enhanced growth is continuing up to at least 20 nm, and in good agreement with theory. Note that although some of the experiments contain size distribution data above 20 nm, the profiles at those sizes become poorly defined at which point we stop the analysis.
All 11 experimental runs are summarized in Fig. 4, where ΔT is averaged between 6 and 12 nm, and shown as a function of the SA concentration, which is obtained from either CI-API-ToF measurements and/or slopes of the growth profiles. The red curve is the theoretical expectation for the γ-sources at maximum, and the blue curve is obtained with a 45% reduction in the ion density. Both are found by numerically solving Eqs. (7) and (8). The relative importance of ion condensation increases as the SA concentration is lowered, as predicted and in good agreement with theory.
Discussion
The most common effect of ions considered in aerosol models is aerosol charging which increases the interaction between the charged aerosols and neutral aerosols/molecules, thereby increasing aerosol growth. However, as mentioned previously, the ion density does not affect the steady state fraction of aerosols that are charged such that the ion-induced interactions remain nearly constant, implying that no effect on the aerosol growth is expected by changing the background ionization. Nonetheless, experiments and observations do suggest that ions have an effect on the formation of CCN, the question has therefore been, how is this possible?
The present work demonstrates that the mass flux associated with the aerosol charging by ions and ion-aerosol recombination is important and should not be neglected. Γ in Eq. (7) contains the effect of the mass-flux of ions to aerosols and demonstrates the inherent amplifications by the interaction between the ions and aerosols. This function Γ shows that the initial estimate of the mass-flux, (n ion /n 0 ) = 10 −3 , made in the introduction, gets multiplied by the size-dependent function 4 β ± 0 =β 00 À Á mion m0 N 0 =N tot ð Þ which at maximum is about 60 m ion =m 0 $ 2:25 ð Þ , and therefore nearly two orders of magnitude larger, than the naive estimate. The simple expression for the growth rate, Eq. (7), can conveniently be used as a parametrization in global aerosol models.
As a test of the theoretical model, extensive experiments were performed to study the effect on growth of the flux of ion-mass to the aerosols. One complication in the experiments was that aerosols were lost to the walls of the chamber. This meant that the concentration of SA could not be as low as the typical values in the atmosphere~10 6 molecules cm −3 , but had to be higher thañ 2 × 10 7 molecules cm −3 . Therefore, the relative effect on the growth caused by the ions was more than an order of magnitude smaller, as can be seen from Eq. (7). The experimental challenge was therefore to measure a <1% change in growth rate, which was done by cyclic repeating the experiments up to 99 times and average the results in order to minimize the fluctuations, with a total of 3100 h of experiments. Figures 3 and 4 demonstrate both the importance of varying the neutral SA gas concentration and the effect of changing the ion density, and show excellent agreement with the theoretical expectations. One important feature is that the effect on the growth rate continues up to~20 nm, as can be seen in Fig. 3, which is larger sizes than predicted for charged aerosols interacting with neutral molecules [29][30][31] , and is expected to increase for atmospherically relevant concentrations of SA. It should be noted that the early stages of growth are very important since the smallest aerosols are the most vulnerable to scavenging by large pre-existing aerosols, and by reaching larger sizes~20 nm faster, the survivability increases fast.
The presented theory is an approximation to a complex problem, and a number of simplifications have been made which gives rise to some questions. We will now discuss the most ). Note that profile 1 (profile 2) is initially growing with γ-on (γ-off) until d ≈ 13 nm. However when d > 13 nm profile 1 (profile 2) grows with γoff (γ-on). It is the difference in timing of profile 1 and 2 that contain information about the effect of ions on the growth rate pertinent: Will the material that constitute the ions condense onto the aerosols in any case as neutral molecules? This will certainly be the case for the negative HSO À 4 ions. Assuming that all negative ions, n − , are HSO À 4 , then the number of neutral SA molecules would be n 0 − n − , where n − is the total negative ion density. Inserting values in the right hand side of Eq. (7), for example for the present experiment n 0~1 0 7 molecules cm −3 , and n −~1 0 4 ions cm −3 the correction to the growth rate from the decrease in neutral molecules is, Δðdr=dtÞ=ðdr=dtÞ j j n 0 À n À ð ÞÀn 0 ð Þ =n 0 j j <10 À3 , but the ion condensation impact on the growth rate is of the order 10 −2 (Fig. 4) and therefore an order of magnitude smaller. So even if the neutral molecules would condense eventually, it does not change the estimated growth rate by ion condensation significantly. This would also be the case under atmospheric conditions, where n 0 is of the order 10 6 cm −3 and n ion~1 0 3 ions cm −3 , again a correction an order of magnitude lower than the ion condensation effect. Also note that the mass-flux from ions is larger than from the neutral molecules, which is part of the faster growth rate. In fact, even if the larger particles grow slightly slower due to a decrease in neutral molecules, the growth rate of the smaller particles is enhanced due to the ion interactions, which make the cross-section of the small particles larger (Fig. 5). This leads to the second question: Will the ion-mass that condenses onto the small aerosols stay in the aerosol and not evaporate after the aerosol is neutralized? This is slightly more difficult to answer, since the composition of all the . a Experimental run V9 (Fig. 4) Interaction coefficients. The interaction coefficients between a small neutral particle of mass 100 AMU and a small ion of mass 225 AMU interacting with aerosols of diameter d. The interaction between neutral particles, β 00 , is given by the blue curve, the interaction between small neutral particles and charged aerosols, β 0± , is given by the red curve. The interaction between a positive or negative ion and neutral aerosols, β ±0 , is described with the yellow curve. Finally, the recombination coefficient between two oppositely charged particles is given by the brown curve. The coefficients were calculated assuming Brownian diffusion while including Van der Waals-forces, Coulomb-forces (including image charges) and viscous forces 24 . Symmetry between positive and negative ions has been assumed, see text ions are not known. The abundant terminal negative HSO À 4 ions are not more likely to evaporate than the neutral SA molecules. With respect to unknown positive or negative ions the possibility of evaporation is more uncertain. If the material of some of the ions are prone to evaporate more readily, it would of course diminish the ion effect. The present experimental conditions did not indicate that this was a serious problem, but in an atmosphere of e.g. more volatile organics it could be. Another issue is that sulfate ions typically carry more water than their neutral counterparts 32 , and it is uncertain what happens with this excess water after neutralization of the aerosol. It was also assumed that the ion density was in steady state with the aerosol density at all times. This is of course an approximation, but from measurements of the ion density with a Gerdien tube 33 the typical time scale for reaching steady state is minutes and the assumption of an ion density in steady state is thus a reasonable approximation 12 . It is worth noting that in the experiments two types of losses for ions are present, in addition to recombination: Wall losses and condensation sink to aerosols. Based on the loss rate of sulfuric acid the wall loss rate is about 7 × 10 −4 s −1 , while the condensation sink for experiment V2 was 1.2 × 10 −4 s −1 . This means that the wall losses were dominant and changes in the aerosol population will thus have a minimal influence on the ion concentration. Furthermore recombination is by far the dominant loss mechanism for ions. For an ion production rate of 16 cm −3 s −1 , the actual ion concentration is 92% of what a calculation based only on recombination gives-for larger ion production the recombination becomes more dominant and vice versa. Under atmospheric conditions of high condensation sink and low ion production this may constitute a significant decrease to the effect due to the reduced ion concentration, but under clean conditions and in the experiment the condensation sink has an minor effect. In order to calculate the interaction coefficients between ions and aerosols it is necessary to know the mass of the ions and mass of the aerosols. This is complex due to the many ion species and their water content, and as a simplification an average ion mass was chosen to be 225 AMU. The sensitivity of the theory to changes in ion mass in the range (130-300 AMU) and mass of a neutral SA molecule in the range (100-130) could change the important ratio (β ±0 /β 00 ) by up to 20%.
The possible relevance of the presented theory in Earth's atmosphere will now be discussed. From Eq. (6), the factor (n ion / n 0 ) indicates that the relative importance of ion condensation will be largest when the concentration of condensing gas n 0 is small and the ion density is large. Secondly, the number density of aerosols should also be small so the majority of ions are not located on aerosols. This points to pristine marine settings over the oceans, away from continental and polluted areas. Results based on airborne measurements suggest that the free troposphere is a major source of CCN for the Pacific boundary layer, where nucleation of new aerosols in clean cloud processed air in the Inter-Tropical Convergence Zone are carried aloft with the Hadley circulation and via long tele-connections distributed over ± 30°latitude 34,35 . In these flight measurements, the typical growth rate of aerosols was estimated to be of the order~0.4 nm h −135 , which implies an average low gas concentration of condensing gas of n 0~4 × 10 6 molecules cm −3 . Measurements and simulations of SA concentration in the free troposphere annually averaged over day and night is of the order n 0~1 0 6 molecules cm −336 . This may well be consistent with the above slightly larger estimate, since the aerosol cross-section for scavenging smaller aerosols increases with size, which adds to the growth rate. Secondly, the observations suggest that as the aerosols enters the marine boundary layer, some of the aerosols are further grown to CCN sizes 35 . Since the effect of ion condensation scales inversely with n 0 , a concentration of n 0~4 × 10 6 molecules cm −3 would diminish the effect by a factor of four. As can be seen in Fig. 1b, the effect of ion condensation for an ionization rate of q = 10 ion pairs cm −3 s −1 would change from 10 to 2.5% which may still be important. Note that other gases than sulfuric acid can contribute to n 0 in the atmosphere. As aerosols are transported in the Hadley circulation, they are moved in to the higher part of the troposphere, where the intensity and variation in cosmic rays ionization are the largest 37 . This suggests that there are vast regions where conditions are such that the proposed mechanism could be important, i.e., where aerosols are nucleated in Inter-Tropical Convergence Zone and moved to regions where relative large variations ionization can be found. Here the aerosols could grow faster under the influence of ion condensation, and the perturbed growth rate will influence the survivability of the aerosols and thereby the resulting CCN density. Finally the aerosols are brought down and entrained into the marine boundary layer, where clouds properties are sensitive to the CCN density 2 .
Although the above is on its own speculative, there are observations to further support the idea. On rare occasions the Sun ejects solar plasma (coronal mass ejections) that may pass Earth, with the effect that the cosmic ray flux decreases suddenly and stays low for a week or two. Such events, with a significant reduction in the cosmic rays flux, are called Forbush decreases, and can be used to test the link between cosmic ray ionization and clouds. A recent comprehensive study identified the strongest Forbush decreases, ranked them according to strength, and disussed some of the controversies that have surrounded this subject 7 . Atmospheric data consisted of three independent cloud satellite data sets and one data set for aerosols. A clear response to the five strongest Forbush decreases was seen in both aerosols and all low cloud data 7 . The global average response time from the change in ionization to the change in clouds was~7 days 7 , consistent with the above growth rate of~0.4 nm h −1 . The five strongest Forbush decreases (with ionization changes comparable to those observed over a solar cycle) exhibited inferred aerosol changes and cloud micro-physics changes of the order~2% 7 . The range of ion production in the atmosphere varies between 2 and 35 ions pairs s −1 cm −337 and from Fig. 1b it can be inferred from that a 20% variation in the ion production can impact the growth rate in the range 1-4% (under the pristine conditions). It is suggested that such changes in the growth rate can explain thẽ 2% changes in clouds and aerosol change observed during Forbush decreases 7 . It should be stressed that there is not just one effect of CCN on clouds, but that the impact will depend on regional differences and cloud types. In regions with a relative high number of CCN the presented effect will be small, in addition the effect on convective clouds and on ice clouds is expected to be negligible. Additional CCNs can even result in fewer clouds 38 . Since the ion condensation effect is largest for low SA concentrations and aerosol densities, the impact is believed to be largest in marine stratus clouds.
On astronomical timescales, as the solar system moves through spiral-arms and inter-arm regions of the Galaxy, changes in the cosmic ray flux can be much larger [18][19][20] . Inter-arm regions can have half the present day cosmic ray flux, whereas spiral arm regions should have at least 1.5 times the present day flux. This should correspond to a~10% change in aerosol growth rate, between arm and inter-arm regions. Finally, if a near-Earth supernova occurs, as may have happened between 2 and 3 million years ago 39 , the ionization can increase 100 to 1000 fold depending on its distance to Earth and time since event. Figure 1b shows that the aerosol growth rate in this case increases by more than 50%. Such large changes should have profound impact on CCN concentrations, the formation of clouds and ultimately climate.
In conclusion, a mechanism by which ions condense their mass onto small aerosols and thereby increase the growth rate of the aerosols, has been formulated theoretically and shown to be in good agreement with extensive experiments. The mechanism of ion-induced condensation may be relevant in the Earth's atmosphere under pristine conditions, and able to influence the formation of CCN. It is conjectured that this mechanism could be the explanation for the observed correlations between past climate variations and cosmic rays, modulated by either solar activity [13][14][15][16][17] or supernova activity in the solar neighborhood on very long time scales [18][19][20] . The theory of ion-induced condensation should be incorporated into global aerosol models, to fully test the atmospheric implications.
Methods
Correction to condensation due to ions. Expanding Eq. (1) gives where the indexes 0, +, and − refer to neutral, positively, and negatively charged particles. Here r and t are the radius of the aerosol and the time. N 0 , N + , and N − is the number density of neutral, positive, and negative aerosols. n 0 is the concentration of the condensible gas (usually sulfuric acid in the gas phase), n + and n − are the concentration of positive and negative ions, A 0 = (m 0 /4πr 2 ρ), A + = (m + /4πr 2 ρ), and A − = (m − /4πr 2 ρ), where m 0 is the mass of the neutral gas molecule, m + and m − are the average mass of positive/negative ions, ρ is the mass density of condensing gas, and β the interaction coefficient between the monomers and the neutral and/or charged aerosols. The parameters of the above model are shown in Fig. 5.
Using equilibrium between aerosols and ions we have while defining N tot = N 0 + N + + N − gives N 0 ðr; tÞ N tot ðr; tÞ If we further assume symmetry between the positive and negative charges, i.e., that m ion ≡ m + = m − , β ±0 ≡ β −0 = β +0 , β ±∓ ≡ β +− = β −+ as well as n ion ≡ n + = n − , such that and for N tot = N 0 + N + + N − , we obtain N 0 ðr; tÞ N tot ðr; tÞ Using Eq. (12) in Eq. (9) and using the charge symmetry gives Adding the three equations then results in Using N tot as a common factor, we then have Taking β 00 as a common factor and plugging Eq. (13) into the first term gives the expression The above function is equal to 1 + O(10 −2 ), and F is therefore replaced with 1. A simple rearrangement provides the final form where Detailed description of the experimental setup. The experiments were conducted in a cubic 8 m 3 stainless steel reaction chamber used in Svensmark et al. 12 , and shown schematically in Fig. 6. One side of the chamber is made of Teflon foil to allow the transmission of collimated UV light (253.7 nm), that was used for photolysis of ozone to generate sulfuric acid that initiates aerosol nucleation. The chamber was continuously flushed with 20 L min −1 of purified air passing through a humidifier, 5 L min −1 of purified air passing through an ozone generator, and 3.5 mL min −1 of SO 2 (5 ppm in air, AGA). The purified air was supplied by a compressor with a drying unit and a filter with active charcoal and citric acid. The chamber was equipped with gas analyzers for ozone and sulfur dioxide (a Teledyne 400 and Thermo 43 CTL, respectively) and sensors for temperature and Fig. 6 The experimental setup NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02082-2 ARTICLE relative humidity. For aerosol measurements, a scanning mobility particle sizing (SMPS) system was used. The system consisted of an electrostatic classifier (TSI model 3080 with a model 3077A Kr-85 neutralizer) using a nano-DMA (TSI model 3085) along with either one of two condensation particle counters (TSI model 3775 or 3776). For some of the experiments, a CI API-ToF 28 using HNO 3 as the ionizing agent was used to measure the sulfuric acid in the chamber. The ionization in the chamber could be increased by two 27 MBq Cs-137 gamma sources placed 0.6 m from opposing sides of the chamber, with the option of putting attenuating lead plates of 0.5, 1.0, and 2.0 cm thickness in front of each source. At full strength the sources increase the ionization in the chamber to 212 ion pairs cm −3 s −1 .
Details of the data analysis. A total of 11 experimental runs totaling 3100 h of measurements were made with varying settings. The settings for each of the experiments are shown in Table 1.
To detect an eventual difference in growth rate the following method was employed. For each experimental run each size-bin was normalized and then the individual periods were superposed to reduce the noise in the data, as shown in Fig. 2 of the main paper. The superposed data was then used for further analysis. For each size-bin recorded by the SMPS, the number of aerosols relative to the mean number NðdÞ h i¼ 1=T R T 0 N tot d; t′ ð Þdt′ was then plotted-as exemplified in the top curve of Fig. 7. The derivative of this curve, is the rate of change of aerosol density of a given size, is used to determine the temporal position of the profiles 1 and 2. This can be achieved by first calculating the derivative d N tot = NðdÞ h i ð Þ =dt ð Þ 2 À Á , then normalizing with this function's maximum value at diameter d, (the square was used to get a positive definite and sharply defined profile), and then smoothed using a boxcar filter with a width of typically 7-16 min -shown as the lower black curve in Fig. 7. The width of the boxcar filter was typically determined from the requirement that the Gaussian fit converged-for instance, in some cases with low sulfuric acid concentration a longer boxcar filter was used, due to the relatively higher noise.
On top of the black curve in Fig. 7, a dashed red and a dashed blue curve are superimposed. These are Gaussian fits to the two maxima. The position of the center of each of the Gaussian profiles gives the growth time relative to the time the γ sources were opened (profile 1) or closed (profile 2). The difference between these growth times then gives the ΔT for each bin size, as shown in Fig. 3. The ΔT values can then be compared with the theoretical expectations. Averaging the individual ΔT values for sizes between 6 and 12 nm finally results in the ΔT shown in Fig. 4.
The m ion /m 0 ratio. Table 2 summarizes the average masses (m/q) of a series of runs using the API-ToF without the CI-unit to measure negative ions in order to determine the ratio m ion /m 0 . Note that water evaporates in the API-ToF so the masses measured are lower than the actual masses of the clusters. The ratio of 2.25 for m ion /m 0 used in the calculations would imply that for a dry (0 water) neutral sulfuric acid molecule (98 AMU) m ion should be 220 m/q. The amount of water on a sulfuric acid molecule varies according to relative humidity-for 50% RH it is typically 1-2 water molecules. Assuming 1.5 waters and m ion /m 0 = 2.25 this would a Shows the name of the experiment, used for reference. An asterisk (*) next to the name indicates that sulfuric acid was measured during the experiment b Length of the period (P) where a P of 4 h means that the experiment had 2 h of γ-rays on and 2 h of γ-rays off c Number of repetitions (periods) of the experiment d Scan range of the DMA, which was narrowed in later runs without changing the scan-time to improve counting statistics e Setting of the UV light used to produce sulfuric acid, in percentage of maximum power. On 175 212 Each line shows the conditions and average m/q for a 4-h API-ToF mass spectrum without the CI. Column 1 shows the UV level as percentage of maximum power. Column 2 shows whether the γ-ray sources were on or off. Column 3 is the average m/q of the spectrum. Column 4 is the average mass of the spectrum, when 1 water (m/q 18) has been added to all masses except the first four sulfuric acid peaks (m/q 97, 195, 293, 391) which has 1.5 water per sulfuric acid give a wet mass of 281 AMU. However, the experiments were performed at lower RH than 50% and also note that hydrogen sulfate ions attract more water than the neutral sulfuric acid molecule 32 . Last, the positive ions were not measured and these are typically heavier than the negative ions 27 .
Data availability. The data generated during the current study are available from the corresponding author on reasonable request. | v3-fos-license |
2020-10-19T18:08:03.374Z | 2020-10-01T00:00:00.000 | 224914507 | {
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} | pes2o/s2orc | An ultrasensitive electrochemical sensing platform for the detection of cTnI based on aptamer recognition and signal amplification assisted by TdT
We have developed an ultrasensitive and highly specific electrochemical sensing platform for the detection of cardiac troponin I (cTnI), a recognized biomarker for the diagnosis of acute myocardial infarction (AMI) and related cardiovascular diseases (CVDs). This strategy is based on the assists of terminal deoxynucleotidyl transferase (TdT)-mediated signal amplification and the specific recognition between cTnI and the aptamer of cTnI. In this experiment, we prepared a gold electrode that modified with probe 2 (P2), in the presence of cTnI, the aptamer of cTnI that in probe 1 (P1)/aptamer complexes bond with cTnI specifically and release the free P1. P1 would bind with P2, resulting in the formation of 3′-OH of DNA. In the presence of terminal deoxynucleotidyl transferase (TdT) and dTTP, TdT mediated P1 to extend and formed the structure of poly T. Methylene blue (MB)-poly A hybridized with the extended poly T and generated an electrochemical signal. The detection limit can be as low as 40 pg mL−1. This sensor was also successfully applied to the detection of cTnI in numerous spiked biological samples, and it can be a great reference for the clinical diagnosis, prognosis, and treatment of CVDs and AMI.
Introduction
Cardiovascular diseases (CVDs) are major causes of death in the world, causing serious problems to human health. 1,2 The rates of morbidity and mortality due to CVDs are expected to continue to rise rapidly with the aging of the population in the future. 3 Acute myocardial infarction (AMI) is a common cardiovascular disease; it is also one of the most serious adverse cardiac events, which may lead to irreversible myocardial injury or necrosis. 4 At present, the diagnosis of AMI is still based on electrocardiography (ECG), but only a small proportion of patients with AMI show changes in ECG and can be diagnosed. 5,6 Therefore, we need a timely way that can assist the early diagnosis and treatment of the patients with AMI and improve their survival rate. Clinicians oen use biomarkers to assist in the assessment of AMI. 7 Since the redenition of the diagnostic standard of AMI in 2000, cardiac troponins (cTns) have been regarded as better biomarkers in the diagnosis of AMI. 8 The recent related literature reports on CVDs and AMI strongly prefer cardiac troponin I (cTnI) as the biomarker 9 of choice due to its sensitivity and specicity in the diagnosis of CVDs and AMI. 8,10,11 cTnI is a polypeptide involved in myocardial contraction, 12 which is released only during cardiomyocyte necrosis, 13 and it is not found in smooth muscle nor is it found in substantial concentrations in striated muscle, showing little or no changes in patients with a skeletal muscle disease or trauma. 14 Thus, at present, cTnI is widely used as one of the reliable biomarkers of AMI due to its high specicity and high sensitivity, 12,[15][16][17] and it is gradually becoming the "gold standard" in clinical practice for the diagnosis of AMI. 13,18,19 cTnI may also be used to monitor the state of patients with AMI and predict the prognosis. 20 In the previously related literature, several techniques have been reported to monitor cTnI, including the enzyme-linked immunosorbent assay (ELISA), 21 uoro-microbead guiding chip, 14 surface plasmon resonance (SPR) biosensors, 18 silicon nanowire biosensors, 21 electrochemiluminescence immunosensors, 22 nanoelectrode arrays, 23 uorescence probes, 19 radioimmunoassay (RIA), 24 and optomagnetic biosensors. 25 However, all of the above methods have their own disadvantages: ELISA requires a long time to operate and a well-trained operator; 21 the other biosensor methods for cTnI detection are usually complex in process or operation, labor-intensive, time-consuming, and involve expensive reagents. 14,18-25 Therefore, we need to develop a convenient and highly specic method for the detection of cTnI, which can assist the diagnosis and treatment of CVDs and AMI.
Nowadays, electrochemical biosensors have attracted particular attention as a powerful diagnostic platform due to their remarkable advantages of being ultrasensitive, highly selective, simple, fast, portable, real-time, and inexpensive for sensing applications. [26][27][28] In this study, we have designed a novel, highly specic, and ultrasensitive electrochemical biosensor for the signal amplication and detection of cTnI, which can assist the clinical diagnosis of CVDs and AMI. In this study, rst, we achieved high selectivity through the specic binding of aptamers to the target cTnI. An aptamer is a small oligonucleotide sequence screened in vitro, which can bind with corresponding ligands with high affinity and strong specicity. 29 Compared with antibodies, an aptamer has higher stability and can be easily synthesized. Its emergence provides a new research platform for biochemical and biomedical elds with high efficiency and fast recognition, and shows good application prospects in many aspects. 30 Accordingly, the aptamer of cTnI can bind with cTnI specically in our sensing process. Second, we used terminal deoxynucleotidyl transferase (TdT) to complete the amplication of the electrical signal and thus achieved the aim of ultrasensitive detection of cTnI in this electrochemical sensing platform. Terminal deoxynucleotidyl transferase (TdT), also known as terminal transferase, 31 is a kind of DNA polymerase that can catalyze the sequential addition of deoxy-ribonucleoside triphosphates (dNTPs) at the 3 0 hydroxyl (3 0 -OH group) terminus of the target DNA [32][33][34][35] and extend the strand of DNA to form a polynucleotide tail (such as poly T) to advocate signal amplication. 36,37 Deoxythymidine triphosphate (dTTP) is one of the dNTPs and is also one of the direct precursors of DNA biosynthesis. 35,[38][39][40] Finally, we used methylene blue (MB), an aromatic heterocycle 41,42 that was modied with poly A, as an effective electroactive hybridization indicator [43][44][45] in this electrochemical sensing platform for the detection of cTnI.
Hence, in the present research, we have developed an ultrasensitive and specic electrochemical biosensor that combined the signal amplication strategy based on TdT with specic recognition between the aptamer of cTnI and cTnI. The aptamer of cTnI specically bonds with cTnI in the presence of cTnI, resulting in the release of free P1. The free P1 hybridized with P2 modied on the surface of the gold electrode and formed the strand of P1/P2. With the addition of TdT and dTTP, TdT extended the strand of P1 and formed the structure of poly T due to the presence of free -OH at the 3 0 end of P1. The extended poly T hybridized with MB-poly A and thus introduced MB to the surface of the gold electrode, resulting in the formation of the electrochemical signal. One extended poly T can hybridize with multiple MB-poly A and lead to signal amplication. By detecting the amplied electrochemical signal, we can achieve the detection of low content cTnI. In addition, this highly specic and ultrasensitive electrochemical biosensor for the detection of cTnI is simple, novel, inexpensive, and fast. This study can accelerate the development of myocardial markers' determination, which provides a potential reference for the clinical diagnosis, prognosis, and treatment of CVDs and AMI.
Materials
The ve proteins (cTnI, myoglobin, EpCAM, CRP, and CEA) used in this study were purchased from Cusabio Biotechnology Co. Ltd (Wuhan, China). TdT and dTTP were purchased from Takara Biotech Co. Ltd (Dalian, China). Gold electrodes were obtained from Gaoss Union Technology Co. Ltd (Wuhan, China), and 6-mercapto-1-hexanol (MCH) was obtained from Sigma-Aldrich (St. Louis, MO, USA). All synthetic DNA sequences (P1, P2, MB-poly A, and the aptamer of cTnI) used in the experiments were obtained from Sangon Biotechnology Co. Ltd (Shanghai, China); their sequences are listed in Table 1. The buffer solution used in the experiments is 10 mM Tris (10 mM MgCl 2 , 50 mM NaCl). All other reagents used in the experiments are of analytical grade, and ultrapure water was obtained from the Millipore water purication system (18.2 MU cm resistivity, Milli-Q Direct 8). The biological samples, including the serum, saliva and urine, of healthy normal human subjects were all from Dongfeng General Hospital affiliated to Hubei University of Medicine. All experiments in this study were conducted in strict accordance with the institutional guidelines and relevant laws (National Health Commission of the People's Republic of China, ethical review of biomedical research involving human beings), and it has been approved by the Ethics Committee of Dongfeng General Hospital (Shiyan, China). Informed consent has been obtained from human subjects involved in any experiments.
Apparatus
In this study, square wave voltammetry (SWV) and chronoamperometry were performed on a CHI660D workstation (CH Instruments Inc., Shanghai, China). SWV was carried out in a 10 mM Tris buffer by scanning the potential from À0.4 to 0 V at an amplitude of 25 mV, a frequency of 25 Hz, and a step potential of 4 mV. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) experiments were performed in 10 mM phosphate-buffered saline (PBS) solution containing 0.1 M KCl and 5 mM [Fe(CN) 6 ] 3À/4À in the potential window range of À0.2 to 0.6 V and the frequency range of 0.1 Hz to 10 kHz at a scanning rate of 0.1 V s À1 and amplitude of 5 mV.
Sensing process of cTnI
First, the aptamer of cTnI and P1 were added in equal proportion and hybridized, and then a certain concentration of cTnI antigen was added. 30 min later, 10 mL of the above solution was dropped onto the surface of the gold electrode that was modi-ed with P2. Aer 15 min of reaction, the obtained gold electrode was immersed in the solution containing TdT and dTTP (2 mM). Then, the gold electrode was immersed in the MB-poly A (MB-AAA AAA AAA) solution, and 10 mM Tris buffer was used to wash aer each step of the reaction. Finally, detect the electrochemical signal produced in the above process.
Electrochemical characterization of the sensing platform
Prior to the experiments of this study, we conducted electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) to analyze the feasibility and construction of the electrochemical sensing platform; the results are shown in Fig. 1. EIS is usually used to investigate the modication process of the electrochemical sensors, and an increase in the semicircle diameter indicates an increase in the electron transfer resistance (R et ) on the electrode surface. Additionally, CV is used to further characterize the construction process. As shown in Fig. 1a and b, the bare gold electrode presents a small semicircle area with an electron transfer R et of 150 U (Fig. 1a, curve a) and shows the maximum redox peak current (Fig. 1b, curve a). Aer modifying the surface of gold electrode with P2 and sealing with 6-mercapto-1-hexanol (MCH), the semicircle diameter increased and the electron transfer R et was 1500 U (Fig. 1a, curve b). The peak current decreased signicantly, while the separation of the peak increased (Fig. 1b, curve b) in comparison with the bare gold electrode (Fig. 1b, curve a). It shows that P2 was modied on the surface of the bare gold electrode successfully, thus forming an Au-S bond via self-assembly. Aer adding cTnI aptamer/P1 and cTnI, P1 was released and hybridized with P2 (there was no signicant change in the electrode). Aer adding TdT, TdT mediated P1 (because the 3 0 end of P1 chain is free OH) to extend and obtain the structure of poly T, resulting in further increase in the semicircle with the R et value of 2300 U (Fig. 1a, curve c) and further decrease in the peak current (Fig. 1b, curve c), which indicates the formation of P1/P2 and poly T. When the modied electrode was further hybridized with MB-poly A in the presence of P1/P2 and poly T, the semicircular diameter (R et ¼ 2900 U) further increased signicantly (Fig. 1a, curve d), and the redox peak current further decreased (Fig. 1b, curve d). The above results show that the hybridization and extension of MB-poly A and poly-T triggered the signal amplication reaction that is based on the catalysis of TdT, and more MB bind to the surface of the electrode to form a stronger electrical signal. amplication can be observed. By detecting the amplied electrochemical signal in the aforementioned process, a highly sensitive and specic detection of cTnI can be achieved.
Optimization of experimental parameters
In order to obtain the best experimental performance of this electrochemical sensing platform, we used square wave voltammetry (SWV) to optimize the following experimental parameters: the concentration of P2, the concentration of cTnI aptamer, the amount of TdT and the reaction time. In each optimization experiment, the concentration of cTnI used was 10 ng mL À1 , and each experiment was repeated three times. The electrochemical signal of this sensor depends on the concentration of P2 xed on the surface of the gold electrode, and too high or too low assembly concentration of P2 will make the electrode crowding and probe sinking, thus reducing the molecular recognition ability and the hybridization efficiency, and weakening the signal amplication performance. Therefore, rst, we studied the effects of the concentration of P2 in the range of 0.1 to 1.4 mM on the current response of SWV. As shown in Fig. 3a, the current response increases gradually with the increase in the concentration of P2 from 0.1 mM to 1.0 mM, and then decreases gradually when the concentration of P2 exceeds 1.0 mM. The above results show that the optimal concentration of P2 is 1.0 mM, and it can be xed on the electrode surface in a relatively vertical direction to obtain the best molecular recognition and hybridization efficiency. With the increase in the concentration of P2, the current response decreases gradually, which is due to the increase in the steric resistance and electrostatic repulsion, and it is unfavorable for Fig. 2 The schematic of this electrochemical sensing platform for the detection of cTnI. This journal is © The Royal Society of Chemistry 2020 RSC Adv., 2020, 10, 36396-36403 | 36399 surface hybridization. Consequently, the concentration of 1.0 mM for P2 was considered as the optimal concentration. The concentration of the cTnI aptamer also has an important effect on signal amplication, which may affect the efficiency of P1 to form the structure of poly T and trigger the electrochemical signal amplication in the presence of cTnI and TdT. As shown in Fig. 3b, the current response increases gradually with the increase in the concentration of the cTnI aptamer, and then it tends to be stable. When the concentration of the cTnI aptamer reaches 40 nM, the value of the current response also reaches a plateau. When the concentration of the cTnI aptamer is more than 40 nM, the corresponding change in the current response is negligible. Thus, we chose 40 nM as the optimal concentration of the cTnI aptamer.
Moreover, the amount of TdT is also one of the important factors that affected the efficiency of this biosensor, and TdT can mediate the extension of P1 to obtain the structure of poly T, which is the key part of the signal amplication of this sensing platform. The effect of the amount of TdT in this experiment is shown in Fig. 3c. From Fig. 3c, we can see that the value of the current response gradually increases with the increase in the amount of TdT (from 5 U mL À1 to 40 U mL À1 ); when the amount of TdT reaches 40 U mL À1 , the value of the current response tends to be stable. When the amount of TdT is 60 U mL À1 or 80 U mL À1 , the corresponding change in the current response can be ignored, so we selected 40 U mL À1 as the optimal amount of TdT that can obtain the maximum experimental signal. Finally, we also studied the effects of the reaction time, and the results are shown in Fig. 3d. It can be seen from Fig. 3d that the value of the current response increases gradually with the increase in the reaction time. When the reaction time is 80 min, the corresponding value of the current response reaches a relatively stable state. When the reaction time continues to increase to 100 min, the change in the current response value can be ignored. Therefore, 80 min is set as the optimal reaction time.
Sensitivity towards cTnI detection
Dynamic response range and sensitivity are both signicant indexes to evaluate the analysis performance of the biosensor.
Under the above optimization experimental parameters, we have also investigated the sensitivity of this sensor. The sensitivity and sensing performance of this sensing platform were studied with the concentration of cTnI from 0 to 500 ng mL À1 . As shown in Fig. 4a, it can be clearly seen that with the increase in cTnI, the corresponding current response also shows a gradual increase, which indicates that the concentration of cTnI has a strong correlation with the current response.
Simultaneously, Fig. 4b shows that there is a good linear correlation between the value of the current response and the logarithm of the concentration of cTnI in the linear range from 0.5 ng mL À1 to 100 ng mL À1 . The regression equation of linear correlation was calculated as i ¼ 2.1838 lg C À 4.3281 (R 2 ¼ 0.982), where i is the value of the SWV current response and lg C is the logarithm of the concentration of cTnI (ng mL À1 ). Also, the detection limit of this sensor platform can be as low as 40 pg mL À1 aer calculation, which shows that the sensor has high sensitivity. In addition, we also compared this method with the other related literature reports listed in Table 2.
Specicity and regeneration
Due to the specic recognition between cTnI and the aptamers of cTnI, the electrochemical sensing platform shows high specicity. There are numerous proteins in real biological samples, so we tested whether these proteins will interfere with the determination of cTnI. The specicity of this strategy for the determination of cTnI was also reviewed by adding ve different kinds of proteins, which included cTnI and four other types of proteins, namely myoglobin, EpCAM, CRP, and CEA. As shown in Fig. 5a, we used this sensor to detect the other four kinds of proteins, namely myoglobin (20 ng mL À1 ), EpCAM (20 ng mL À1 ), CRP (20 ng mL À1 ), and CEA (20 ng mL À1 ); by comparing with the blank control group, we can see that there is no signicant difference between the current response values of these four groups of proteins and the blank control group (the difference can be ignored). However, when we use this sensor to detect the value of the current response generated by cTnI (10 ng mL À1 ), we can see that the current response of cTnI is signicantly stronger than any of the above four groups of proteins. In other words, the aforementioned results show that the sensing platform has an excellent specicity towards the detection of cTnI. Moreover, we have further studied the regeneration ability of the sensor; the experiment of regeneration was conducted by washing the sensor with a 5 M urea solution at 37 C for 10 min. It can be seen from Fig. 5b that aer ve cycles of regeneration the value of the current response is almost at the same level as the rst current response signal, indicating that the sensor has a satisfactory ability of regeneration.
Application in biological samples
In order to further study the selectivity and applicability of the sensing platform in various biological samples, we prepared three biological samples, namely human serum (10%), urine (10%) and saliva (10%), by diluting them. cTnI with the same volume and same concentration of 10 ng mL À1 was diluted to the above three biological samples and Tris buffer respectively. Also, each group was set with a blank control, and then the SWV current response values of each group were detected. The results in Fig. 6 show that in the presence of 10 ng mL À1 cTnI, the SWV current response of each group in different biological samples signicantly enhanced in comparison with the blank control. The above results show that the sensing platform has excellent applicability and selectivity in biological samples, which is due to the specic recognition and high affinity of cTnI aptamer and cTnI. In addition, due to the excellent applicability and feasibility in biological samples of this sensing platform, this method also shows potential clinical practicability for the detection of cTnI.
Conclusion
In conclusion, we have successfully developed an electrochemical aptasensing platform for the detection of cTnI, which is based on the combination of highly specic recognition of aptamers with the TdT-mediated signal amplication strategy. The designed sensor has satisfactory specicity, sensitivity and Optomagnetic biosensors 0.03 ng mL À1 0.03-6.5 ng mL À1 25 Nanoelectrode arrays 0.2 ng mL À1 0.1-100 ng mL À1 23 Electrochemical biosensors 0.04 ng mL À1 0.5-100 ng mL À1 This sensor Fig. 5 The specificity (a) and regeneration (b) of this electrochemical sensing platform for the detection of cTnI. Error bars: SD, n ¼ 3. Fig. 6 The SWV currents of electrochemical sensing platform for the detection of cTnI in several spiked biological samples, including urine, saliva and serum. Error bar: SD, n ¼ 3.
This journal is © The Royal Society of Chemistry 2020 RSC Adv., 2020, 10, 36396-36403 | 36401 regeneration, and the detection limit of this sensor can be as low as 40 pg mL À1 . In addition, this method has been also applied to detect cTnI in various biological samples successfully, which shows that this method has a good potential for the clinical diagnosis, prognosis, and treatment of CVDs and AMI. Also, this method has universal applicability for the detection of other markers of disease. We only need to adjust the sequence of nucleic acid accordingly if the target to be detected is replaced by another marker. Compared with the antibody, the aptamer used in this study has good stability and low cost. Finally, the electrode used in this research can be regenerated, further reducing the cost. We believe that this sensing strategy will be a useful method to directly monitor other biomarkers by selecting appropriate aptamers, and will also provide potential references for the early detection of clinical biomarkers in the future.
Conflicts of interest
There are no conicts to declare. | v3-fos-license |
2019-01-02T16:31:13.963Z | 1999-10-30T00:00:00.000 | 86816282 | {
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} | pes2o/s2orc | Antioxidant activity of rosemary { Rosmarinus officinaiis L . ) extracts on natural olive and sesame oils
Se determinaron las actividades antioxidantes de extractos de cloroformo y de metanol de romero (Rosmarinus officinalis L.) en aceites de sésamo y de oliva almacenados a 55 °C mediante medidas de los índices de peróxidos a intervalos regulares. Se utilizaron concentraciones al 1% y 2% de extractos y ácido cítrico. Los extractos (excepto para los días 12,16 y 20 de almacenamiento de aceite de oliva) mostraron una alta actividad antioxidante comparados con la muestra control en ambos aceites. Los efectos antioxidantes de ambos extractos y los niveles de ácido cítrico en el aceite de oliva tuvieron diferencias significativas después de 4 días de almacenamiento (p < 0.01). La concentración más efectiva en aceite de sésamo durante el almacenamiento fue del 2% en extracto de cloroformo. Además, especialmente concentraciones del 2% de ambos extractos de disolventes de romero mostraron una actividad antioxidante significativa en comparación con el ácido cítrico en aceite de sésamo.
INTRODUCTION
Synthetic antioxidants are widely used to retard undesirable changes as a result of oxidation in many foods.Excessively oxidized fats and oils are not suitable for nutritive purposes.Because, the oxidation products of oils have toxic effects.Many synthetic substances such as butylated hydroxyanisol (BHA), propyl gállate and citric acid are comnnonly used in lipids to prevent oxidation.Recently, these synthetic substances have been shown to cause such as enlarge the liver size and increase microsomal enzyme activity.Therefore, there is need for other compounds to effect as antioxidants and to render food products safer for mankind (Farag et a/.,1989, Farag eia/.,1990, Brookman, 1991).
Plant originated antioxidants have been used in oils or lipid containing foods in order to prevent oxidative deterioration (Gur and Gulden, 1997).The antioxidant activity displayed by spices or other antioxidants depends on several factors such as the concentration, the temperature, the hydrophobic, hydrophylic or amphiphatic character, the presence of synergists and the chemical nature of the food or medium to which they are added (Logouri and Boskou, 1995).Chang et al. (1977) obtained similar results while investigating the antioxidative effect of rosemary and sage due to the peroxide value.Essential oils such as rosemary and sage oils lack any antioxidant activity although the herbs are known antioxidants and find many applications in food preparations (Chipault et a!., 1952;Chipault et a!., 1956;Bishou eta!., 1977;Hermann eta!., 1981;Barbut efa/., 1985;Pizzocaro eta!., 1985;Inatani et al., 1984;Houlihan et al., 1984;Ózcan and Akgül, 1995).Naturally occurring compounds in rosemary extracts (Wu etal., 1982;Ho etal., 1983) have been reported to exhibit antioxidant properties greater than BHA and equal BHT The extracts rosemary (Chang et al., 1977;Economou et al., 1991;Banlas et al., 1992), were examined in order to determine their antioxidative activity against autoxidation in different substrates, mostly in lard.Their antioxidative activity depends on the solvent used, but the structure activity relationship of them have not been completely investigated (Chang et al., 1977;Economou et al., 1991;Banlas eí a/., 1992).
Antioxidant effects of 35 methanol extracts and 20 essential oils from Turkish spices were tested in sunflower oil stored at 70 °C (Ózcan and Akgül, 1995).Thus, the objective of this study was to evaluate to efficacy of adding a natural rosemary oleoresin to sesame and olive oils and to compare it with a commercial citric acid used as an antioxidant.
Plant Material and Preparation of Extracts:
Rosemary was purchased from market.Ground material was extracted with pure methanol and chloroform for 3 hr in a stirred vessel, at a liquid-to-solid ratio 4:1 and a temperature of 60 °C.The mixture was filtered and concentrated in rotary evaporator and solvent was completely removed.Extracts were kept in sealed bottles under refrigerated using.
Olive and Sesame Oils: Naturel olive and sesame oils without adding any antioxidant were kindly supplied by Kristal and Salur company in Yzmir and Kenya, respectively.Their peroxide numbers were 15 and 18.1 meq/kg, the same respectively.The sesame oil was selected for their high degree unsaturation levels and for being the most widely used as edible and tehina (sesame paste) oil in Turkey.
Citric Acid: Citric acid (E.Merck, Darmstadt) was preferred because of using commonly as prevent to the deterioration at oil company.
Antioxidant Activity Measurement:
The rate of oxidation was followed by periodic determination of peroxide values of the oil stored at 55 °C by using chloroform and methanol as extraction solvents which are at different polarities.A calculated quantity of the extract and citric acid was added at the 1 and 2% concetrations into olive and sesame oil, and the mixture was stirred.A control sample was prepared under the same conditions without adding any antioxidant.All samples of 20 g each were storaged in 10 X 100 mm open beakers at 55 °C in the dark.For the peroxide number, a known weight of olive and sesame oils (2 g) was dissolved in a mixture of CH3COOH: CHCI3 (3:2, v/v), and saturated solution of Kl (1ml) was then added.The liberated iodine was titrated with sodium thiosulfate solution (0.01 N) in the presence of starch as an indicator (A.O.C.S., 1989).The codes of the samples are shown in Table I.It should be mentioned that in experiments, with the plant extracts at various concentrations, raw olive and sesame oils were used.
Statistical Analyses: Findings of*the research were analysed for statistical significance by analyses of variance, and differences among groups were established according to Düzgünes et al. (1987).Experiments and analysis were replicated and duplicated.
Table I The codes of the extract and citric acid added samples
Olive
RESULTS AND DISCUSSION
Table II represents the antioxidant effect of the chloroform and methanol extracts of rosemary, and citric acid in olive and sesame oils as determined by the peroxide value.
After 4 days, the extract concentrations showed antioxidant effect in varying degrees on olive and sesame oil compaered with the control test (p < 0.01).Peroxide values of all the samples partly increased during storage.After 12 days, peroxide values of sesame oil at all the concentrations had higher than those of olive oil.At both extract concentrations of chloroform and methanol peroxide values were high according to control on 16 and 20 days.But, antioxidant effects of both solvent extract concentrations (1%) in olive oil are higher than that of the same citric acid concentration as from 4,8 and 12 days.But, the same concentrations (1%) of extracts in sesame oil showed antioxidant effect according to citric acid (1%) concentration.
The 2% concentration of chloroform and methanol in sesame oil had more effect than those of only 2% concentration of chloroform and methanol extracts and both concentrations of citric acid (p < 0.01 ).The most effective concentration on sesame oil during storage had 2% chloroform (Fig. 1).Furthermore, especially 2% levels of both solvent extracts of rosemary exhibited remarkable antioxidative activity in comparison with citric acid on sesame oil (p < 0.01).
Overall strongest activity of rosemary was not surprising, because of various findings reported on its stabilizing effect (Chang etal., 1977;Braceo et al., 1981), and related active components such as carnosol, rosmanol, rosmariquinone, carnosic and ursolic acids etc. (Wu et al., 1982;Inatani et al., 1983;Houlihan étal., 1985;Chen étal., 1992).The results indicated that rosemary extract obtained by different polarities solvents an antioxidant activity on olive and sesame oils at 55 °C.Antioxidant effects of both extracts and citric acid levels in olive oil had found significantly different after fronn 4 days of storage.However, antioxidant effects of both extract concentrations of solvents were silightly weaker than that of control on 16 and 20 days of storage.Peroxide values of sesame oil, especially 12 days, had higher than those of olive oil as from 12 days of storage.The reason for this is probably that initial peroxide value of sesame oil was higher than that of other oil.However, it was seen that the antioxidant activity of the extract depends on the polarity of the solvents used for extraction.
Table II Antioxidant effect of rosemary extract added to olive and sesame oils* stored in thie darl< at 55 °C (meq/kg)
Initial peroxide value of the olive and sesame oil were 15.0 and 18.1 meq/kg, respectively.* Differences among means indicated with majuscules are significant in p < 0.01. | v3-fos-license |
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} | pes2o/s2orc | Optogenetic manipulation of calcium signals in single T cells in vivo
By offering the possibility to manipulate cellular functions with spatiotemporal control, optogenetics represents an attractive tool for dissecting immune responses. However, applying these approaches to single cells in vivo remains particularly challenging for immune cells that are typically located in scattering tissues. Here, we introduce an improved calcium actuator with sensitivity allowing for two-photon photoactivation. Furthermore, we identify an actuator/reporter combination that permits the simultaneous manipulation and visualization of calcium signals in individual T cells in vivo. With this strategy, we document the consequences of defined patterns of calcium signals on T cell migration, adhesion, and chemokine release. Manipulation of individual immune cells in vivo should open new avenues for establishing the functional contribution of single immune cells engaged in complex reactions.
I mmune responses rely on highly coordinated sequences of cellular processes. This coordination is enforced by various mechanisms of cellular communication allowing immune cells to sense the information from their environment and respond by altering their behavior. The introduction of intravital imaging for the study of immune responses in vivo has provided new insights into the cellular orchestration of immune reactions during infectious processes, autoimmunity, or cancer at the single-cell resolution 1,2 . While these approaches have turned out to be very informative, it remains difficult to fully understand the functional contribution of single immune cells involved in these complex reactions.
Ideally, such an understanding would require the ability to manipulate the function of individual immune cells in real-time in vivo and follow the consequences of such activity. The development of optogenetics offers the possibility to manipulate cellular function with exquisite spatiotemporal control 3,4 . By exploiting photosensitive proteins that bind, aggregate, dissociate, or change conformation upon light illumination, optogenetic tools (referred to as 'actuators') can control a variety of cellular functions and pathways. Optogenetics using wide-field illumination strategies typically activate many cells simultaneously. Manipulation of individual cells using optogenetic actuators has been possible using light-targeting strategies with one photon or multiphoton excitation and has been applied primarily on cell cultures or brain tissue [5][6][7][8][9] . In particular, manipulation of neural circuit activity using two-photon optogenetics has been achieved in vivo 6,[9][10][11] . Applying these approaches to immune cells in their native environment faces several technical challenges, including the ability to efficiently express actuators in primary immune cells and, most importantly, the capacity to photoactivate selected cells located in densely packed tissue environments.
To achieve these goals, we sought to improve the sensitivity of optogenetic actuators in order to increase their responsiveness to two-photon excitation and to design efficient strategies for in vivo photoactivation. As a proof of concept, we focused on calcium manipulation, as calcium is a key signaling pathway for many immune cells, including T and B lymphocytes, and several optogenetics strategies based on calcium release-activated channel (CRAC) have been proposed [12][13][14][15][16] . In T cells, calcium signals are in particular elicited upon interactions between the T cell receptor (TCR) and antigenic complexes present at the surface of antigenpresenting cells and regulate multiple aspects of T cell motility, activation, and function. It has been shown for example that chemical induction of calcium influx induces T cell arrest in vitro 17,18 . Calcium signals are usually correlated with low T cell motility in vivo [17][18][19][20][21][22][23] . Furthermore, inhibition of Orai-1 channel activity in T cells delays Ag-mediated arrest 24 and reduced the frequency of spontaneous pauses during basal T cell motility 22 . Whether elevation of intracellular calcium concentration is sufficient to induce T cell deceleration in vivo remains to be formally established. In addition, how specific patterns of calcium signals regulate T cell dynamics in vivo is not completely understood.
Recent studies have demonstrated the possibility to optogenetically activate bulk immune cells, including T cells and dendritic cells using wide-field illumination and calcium actuators 23,[25][26][27] . However, simultaneous imaging and control of a spatially defined population of immune cells in vivo has yet to be achieved. In the present report, we have improved the OptoSTIM1 actuator (hereafter abbreviated OS1) 16 that uses light-induced Cry2-based aggregation of stromal interaction molecule 1 (STIM-1) to trigger calcium entry and showed that the modified actuator could efficiently respond to two-photon excitation even in densely packed tissues, such as lymph nodes. We used both in vitro and in vivo experiments to investigate the consequences of spatiotemporally controlled calcium signals on T cell migration and behavior. The possibility to simultaneously visualize and manipulate single immune cells in vivo should provide new opportunities to decode the complexity of immune cellular networks.
Results
Optogenetic calcium actuators with enhanced sensitivity. To manipulate individual T cells with light, we relied on the OS1 actuator that uses Cry2-mediated clustering of the STIM-1 calcium sensor to trigger CRAC channel activation and extracellular calcium influx 16 . We reasoned that increasing the sensitivity of the actuator and reducing light exposure needed for photoactivation should be of importance to manipulate individual cells in vivo. We therefore introduced a mutation in the Cry2 domain that increases its oligomerization properties 28 ( Supplementary Fig. 1). To compare the response of the original and the new actuator (abbreviated eOS1 for enhanced OptoSTIM1), we transduced the B3Z T cell hybridoma 29 and analyzed calcium responses upon light exposure by flow cytometry. As shown in Fig. 1a and Supplementary Fig. 2a, b, both OS1-and eOS1expressing B3Z cells displayed calcium elevation in response to the photoactivation using a 470 nm light-emitting diode (LED) but eOS1 triggered a more robust response. In fact, only a subset of cells with strong overexpression of the original OS1 actuator responded to light exposure ( Supplementary Fig. 2c). As expected, B3Z cells expressing a mutant version of OS1 (OS1-mut), known to be non-responsive to light, did not show calcium elevation (Fig. 1a). Similar results were obtained using individual B3Z clones with a homogenous expression of either OS1 or eOS1, and selected to express identical levels of the actuator (Fig. 1b, Supplementary Fig. 2a, b). No calcium signals were detected in eOS1-expressing cells in the absence of light exposure (Supplementary Fig. 2d) strongly suggesting that the increase response of the eOS1 actuator is not associated with a higher background activity at steady state. Finally, photoactivation by itself did not lead to detectable toxicity ( Supplementary Fig. 3). As expected with this reversible optogenetic system, the calcium elevation was transient, with intracellular calcium concentration returning to basal levels after~10 min (Fig. 1c). Of note, the original OS1 is fused to enhanced green fluorescent protein (eGFP). To provide additional possibility to combine eOS1 with other reporters, we generated a new version by replacing the eGFP with the mScarlet fluorescent protein. Photoactivation of mScarlet-eOS1-expressing B3Z cells resulted in a robust calcium elevation confirming that this actuator is also suitable for calcium manipulation (Fig. 1d). To test the functional consequence of light-induced calcium signaling, we took advantage of the expression of a NFAT-LacZ reporter gene in B3Z cells 29 . In good agreement with their stronger calcium responses, eOS1-expressing B3Z cells displayed higher NFAT activity than their OS1-expressing counterparts (Fig. 1e). Together, these results establish that the eOS1 actuator exhibits enhanced cellular responses to photoactivation. To confirm that these results obtained with a T cell hybridoma also pertained to primary T cells, we introduced the eOS1 actuator in primary effector CD8 + T cells by retroviral transduction (Supplementary Fig. 4a, b). Photoactivation led to a rapid calcium influx that was maximal with the eOS1 actuator ( Supplementary Fig. 4c).
Manipulating T cell dynamics in vitro using the eOS1 actuator. Previous manipulations of intracellular calcium in T cells using either ionomycin or thapsigargin have shown that calcium influx can trigger T cell arrest 17,18 . These supraphysiological stimuli are nevertheless poorly suited to assess the consequence of specific patterns of calcium signals with distinct durations or frequencies.
By contrast, optogenetic manipulations with eOS1 offer the possibility to shape the duration and repetition of Ca 2+ signals, but also to study cell behavior when Ca 2+ progressively returns to baseline levels. These possibilities may provide a better mimic of calcium signals seen during distinct types of TCR stimulation (seen for example during synapse and kinapse formation). We first characterized the impact of triggering calcium signals in eOS1-expressing B3Z T cell hybridoma in vitro. B3Z cells exhibited typical ameboid migration on Poly-L-Lysine (PLL) + ICAM-1-coated surfaces. Upon photoactivation, most B3Z cells immediately stopped and rounded up (Fig. 2a). After~10 min, B3Z cells spread (as measured by an increased cellular area) and adhered to the surface, exhibiting either no motility or a slow, mesenchymal-like, adhesive migration (Fig. 2a, Supplementary Movie 1). These results suggest that Ca 2+ influx contribute to B3Z T cell arrest through a two-step process, including a transient phase of rounding followed by increased adhesion. To confirm that T cell arrest upon photoactivation was dependent on extracellular calcium influx, we photoactivated B3Z cells after chelating calcium with ethylene glycol tetraacetic acid (EGTA). B3Z cells did not alter their motile behavior upon photoactivation in the absence of extracellular calcium. To test how the intensity of calcium signals may impact B3Z cell dynamics, we partly chelated extracellular calcium with EGTA in order to reduce the intracellular calcium elevation upon photoactivation (Fig. 2b, c). In these settings, B3Z cells briefly stopped after photoactivation but rapidly regained motility without evidence of cell spreading (Fig. 2c). This observation suggests that spreading requires a higher calcium response than that required for the initial T cell arrest, although it remains possible that the presence of EGTA directly impacts LFA-1/ICAM-1 interactions. Next, we tested the role of LFA-1/ICAM-1 interaction during the adhesion phase. We compared the behavior of photoactivated B3Z cells on PLL versus Representative of four independent experiments. e eOS1 actuators elicit enhanced NFAT activity in B3Z clones after photoactivation as measured by the production of β-galactosidase 3 h post photoactivation. Photoactivation was performed by LED illumination (12 photoactivations of 5 s every 5 min). The βgalactosidase activity observed in the absence of photoactivation was set to 1; NT, non-transduced B3Z. Results are compiled from four independent experiments (each dot represents one experiment, bars show mean ±SEM). Clones were compared using a two-tailed unpaired Student's t-test (ns p = 0.8263, **p < 0.01, *p < 0.05). Source data are provided as a Source Data File.
PLL + ICAM-1-coated surfaces (Fig. 2d). While B3Z cells arrested and rounded up on both type of surfaces, spreading and adhesion was only evident on ICAM-1-coated plates. In the absence of ICAM-1, B3Z cells regained ameboid motility after the initial rounding phase (Fig. 2d, Supplementary Movie 2). These results suggest that B3Z cell spreading is ICAM-1 dependent. Upon photoactivation, we noted that the onset of cell spreading coincided with the end of the calcium response. Thus, we asked whether the return to basal Ca 2+ level after photoactivation was a prerequisite for the acquisition of increased adhesiveness. When we repeated the photoactivation every 5 min, B3Z cells remained rounded with little evidence for spreading ( Fig. 2e), suggesting that adhesion is indeed initiated upon termination of Ca 2+ signals. Finally, we assessed the effect of eOS1 photoactivation in primary CD8 + T cells. As shown in Supplementary Figs. 4d and 2f, CD8 + T cells stopped their migration upon photoactivation and full arrest was maintained by repeated photoactivations. Changes in T cell dynamics after photoactivation were due to the actuator activity and not to unspecific effects of light exposure since T cells that did not express eOS1 maintained their motile behavior when subjected to the photoactivation procedure ( Supplementary Fig. 4e). Overall, our in vitro results suggest that Ca 2+ influx in migrating T cells is responsible for a sequence of events that includes stopping and rounding followed by a phase of increased adhesion as intracellular Ca 2+ concentration returns to basal levels.
Manipulation of chemokine release using the eOS1 actuator. We next ask whether the eOS1 actuator could be used to manipulate T cell effector functions. To delineate the early effector response of activated CD8 + T cells promoted by calcium elevation or TCR signaling, we analyzed cytokine and chemokine release after ionomycin treatment or anti-CD3/CD28 ligation. Both types of stimulation lead to the early release of the chemokines CCL3, CCL4, and CLL5 (Fig. 3a). Focusing on CCL3, we photoactivated primary activated eOS1-expressing CD8 + T cells and measured rapid release of the chemokine as early as 1 h after photoactivation (Fig. 3b). Release of CCL3 was not detected with control activated CD8 + T cells (that did not express eOS1), confirming that CCL3 release was a specific consequence of eOS1 photoactivation (Fig. 3b). By collecting the supernatant at different time points after the photoactivation (Fig. 3c), we found that calcium elevation led to a transient pulse of chemokine release that lasted~1-2 h (Fig. 3d). Our results indicate that rapid release of CCL3 is a function of stimulated effector T cells and that the eOS1 actuator can be used to study the properties and consequences of early effector functions in T cells.
eOS1 actuator allows for two-photon photoactivation. Optogenetic manipulation of single cells in deep tissue environments, Fig. 3 The eOS1 actuator allows to control the rapid chemokine release in T cells. a Effector primary CD8 + T cells were stimulated for 1 h at 37°C with anti-CD3 + anti-CD28-coated antibodies or with ionomycin or were left unstimulated. Supernatants were recovered and the secretion of the chemokines CCL3, CCL4, and CCL5 were measured using a mouse cytokine multiplex assay. Representative of two independent experiments. Results are shown as mean ± SEM. Dots correspond to experimental replicates. Statistical significance was assessed using a two-tailed unpaired t-test (*p < 0.05, **p < 0.01, ***p < 0.001). b Primary CD8 + T cells were transduced with eOS1 (or with eGFP as a control) and the calcium pathway was stimulated for 1 h either by photoactivation or thapsigargin. such as lymphoid organs could potentially benefit from the use of two-photon microscopy, a technique of choice to image immune cells in vivo. Two-photon optogenetics remains challenging in part due to the small two-photon excitation volume and might be facilitated by optimizing actuator sensitivity. We therefore assessed whether the robust responses to light seen with eOS1 might allow for two-photon photoactivation. To test this idea, we initially conducted a set of in vitro experiments. First, we subjected OS1-and eOS1-expressing B3Z cells (loaded with the calcium-sensitive dye Indo-1) to two-photon photoactivation.
While we failed to efficiently photoactivate cells expressing the original OS1 actuator, we observed a robust calcium response in eOS1-expressing cells (Fig. 4a, Supplementary Movie 3). Even by substantially increasing the laser power and duration of photoactivation ( Supplementary Fig. 5), we did not reach optimal photoactivation with OS1 and instead promoted phototoxicity. Of note, the increase in intracellular Ca 2+ induced by two-photon photoactivation of eOS1-expressing cells was associated with nuclear translocation of a NFAT1-mCherry reporter (Fig. 4b). As shown in Fig. 4c, d, only cells located in the region delineated for the photoactivation exhibited calcium influx, indicating that this approach can be used to manipulate calcium levels in individual cells with high spatial precision (Fig. 4d). Overall, two-photon photoactivation led to an elevation of intracellular calcium that lasted~5-15 min (Fig. 4c, d) and could be used repeatedly to generate specific temporal patterns of calcium signal (Fig. 4c, Supplementary Movie 4). Primary CD8 + T cells expressing the eOS1 actuator also responded to two-photon photoactivation with rapid calcium elevation, cell rounding, and motility arrest ( Supplementary Fig. 4f). A second key step toward simultaneous in vivo two-photon imaging and photoactivation is to establish imaging conditions to visualize cells without causing undesired photoactivation. In our aforementioned in vitro experiments, two-photon imaging was performed at 720 nm to visualize Indo-1 fluorescence, a wavelength that did not cause photoactivation. However, Indo-1 staining is typically lost within a couple of hours in vivo, so this strategy is not appropriate for in situ imaging. It is also difficult to rely on the eGFP expressed by the eOS1 actuator for cell imaging, as excitation wavelength used to visualize eGFP also causes photoactivation. To circumvent these difficulties, we tested whether the eOS1 actuator expressing the mScarlet fluorescent protein as a fusion molecule could be used for tracking cells without causing photoactivation. To validate these settings, we imaged cells using a dual excitation at 720 and 1040 nm to visualize Indo-1 signals and mScarlet fluorescence, respectively (Fig. 4e, Supplementary Movie 5). Visualization of mScarlet fluorescence using 1040 nm excitation did not cause any detectable photoactivation as measured by the lack of calcium signals prior to photoactivation. After photoactivation (performed using a 940 nm excitation wavelength), we detected both calcium signals and changes in mScarlet-eOS1 cellular localization consistent with membrane recruitment expected following STIM-1-Orai-1 interactions (Fig. 4e, Supplementary Movie 5). Thus, the mScarlet-eOS1 actuator provides the opportunity to visualize cells using 1040 nm excitation wavelength without triggering unwanted photoactivation.
We sought to establish a second imaging strategy to visualize cell response using a genetically encoded calcium indicator. To this end, we co-expressed the mScarlet-eOS1 or GFP-eOS1 actuator and the Fluorescence Resonance Energy Transfer (FRET)-based Twitch2B calcium reporter in B3Z cells 30 . This strategy was possible since the expression of mScarlet fluorescent protein was not interfering with the FRET efficiency (Supplementary Fig. 6). Twitch2B fluorescence (and FRET signals) could be visualized using 830 nm two-photon excitation without causing photoactivation (Fig. 4f, g). After photoactivation (940 nm), calcium signals could be readily detected in B3Z cells, with an efficacy correlated to the intensity of photoactivation (Fig. 4f, g). Thus, the combination of Twitch2B and eOS1 represents a fully genetically encoded strategy for the simultaneous manipulation and visualization of calcium signals in individual cells. In sum, we have established distinct strategies (summarized in Supplementary Table 1) compatible with twophoton-based imaging and activation of eOS1-expressing cells.
Optogenetic manipulation of calcium signals in T cells in vivo.
We next investigated the possibility to optogenetically manipulate T cells in lymph nodes of live animals. Effector CD8 + T cells transduced and isolated to express both eOS1, and the Twitch2B calcium reporter were adoptively transferred and recipient mice were subjected to two-photon imaging of the popliteal lymph node either as an explant or intravitally (Fig. 5a). As shown in Fig. 5b, c, two-photon-based photoactivation triggered rapid calcium elevation in T cells. Next, we co-transferred CD8 + T cells transduced and isolated to express both eOS1 and the Twitch2B with untransduced CFP-expressing control CD8 + T cells. Photoactivation was associated with cell arrest and rounding of eOS1expressing T cells (Fig. 5d, Supplementary Movie 6) while control T cells maintained high motility after photoactivation. These observations confirmed that changes in T cell behavior were indeed mediated by the eOS1 actuator and not due to unspecific effects originating from the photoactivation procedure (such as toxicity or temperature increase; Fig. 5d, Supplementary Movie 6). In a second set of experiments, we only transferred CD8 + T cells coexpressing eOS1 and Twitch2B, and followed the fate of T cells after photoactivation. A few minutes after the initial phase of calcium elevation associated with T cell arrest and rounding (Fig. 5e, f), intracellular calcium returned to baseline levels in most T cells (Fig. 5e, g, Supplementary Movie 7). While some T cells regained motility as calcium levels went back to normal, a substantial fraction of T cells remained arrested (Fig. 5e, g, Supplementary Movie 7). This result is reminiscent of our in vitro observations and suggest that calcium influx can lead to a prolonged cellular arrest, possibly by modulating cell adhesiveness.
We report here the possibility to manipulate individual T cells within deep tissue environments, such as lymph nodes using a sensitive optogenetic actuator while imaging them in real time. As a proof of concept, we combined both in vitro and in vivo experiments to dissect the impact of calcium signals on T cell migration and behavior. Extending previous in vitro studies, we provide in vitro and in vivo evidence that calcium elevation leads to cell rounding and arrest followed by increased adhesion when calcium levels return to baseline and to the release of chemokines such as CCL3. It will be interesting to assess how calcium signals also regulate other T cell effector functions in vivo or impact the dynamics of other immune cell types. In addition, the capacity to manipulate cell arrest and adhesion offer new possibilities to alter and study intercellular communication. Finally, the ability to pattern calcium stimulation with repeated photoactivations provides a valuable approach to study how a specific sequence of signals is integrated into T cell transcriptional program.
By offering the ability to visualize and perturb cellular behavior and activity at will, the combination of optogenetics and intravital imaging represents an attractive strategy to unlock new avenues for the study of immune responses in vivo.
Methods
Mice and cell lines. Six to 12-week-old C57BL/6 (B6) mice were obtained from Charles River France. Rag1 −/− OT-I TCR transgenic mice were bred in our animal facility. All experiments were carried out in agreement with relevant guidelines and regulations and approved by the Institut Pasteur committee on Animal Welfare (CETEA) under the protocol code of CETEA 2013-0089. B3Z T cell hybridoma contains the LacZ reporter gene driven by the transcriptional control of NFAT elements 29 . The β-galactosidase activity can be measured by the hydrolysis of the chromogenic substrate CPRG.
Plasmids. The nucleotide sequence coding for the fusion protein eGFP-Cry2PHR (1-498)-humanStim1(238-465) (OS1) 16 , the D387A inactive mutant (OS1-mut), and the E490G mutant (eOS1) were synthetized by Genewiz (Merck group) and subcloned into pMSCV. The version of the eOS1 actuator fused to mScarlet (mScarlet-eOS1) and the fusion protein NFAT1-mCherry were also synthetized by Genewiz and cloned in pMSCV. pMSCV-Twitch2B was generated by subcloning the coding sequence for Twitch2B 30 Measurement of chemokine production. Effector CD8 + T cells were stimulated for 1 h at 37°C with anti-CD3 + anti-CD28-coated antibodies (2.5 μg/mL) or with ionomycin (1 μg/mL) or were left unstimulated. Supernatants were recovered and the secretion of the cytokines/chemokines were measured using a mouse cytokine multiplex assay (Invitrogen). For experiments using photoactivation, CD8 + T cells were transduced with eOS1 (or with eGFP as a control) stimulated for 1 h by LED photoactivation. Secretion of CCL3 was measured in the supernatants by enzymelinked immunosorbent assay (ELISA; R&D Systems). For kinetic analysis of chemokine secretion, the supernatants were collected every 20 min and replaced by warm medium. CCL3 concentration in the samples collected over time was analyzed by ELISA (R&D Systems).
β-galactosidase assay. The indicated B3Z clones were photoactivated using 470 nm LEDs for 10 s every 5 min for a total period of 1 h. After three additional hours of culture, cells were washed twice in phosphate-buffered saline (PBS) and lysed in 100 μL per well of CPRG buffer (PBS + 9 mM MgCl2 + 0.125% NP40 + 100 μm β-mercaptoethanol + 0.15 mM chlorophenol red-β-D-galactopyranoside (Roche, #10884308001)). Plates were incubated in the dark at room temperature for 30 min to 1 h and the optical density was read at 570 nm (reading at 620 nm was used as reference and subtracted).
In vitro cell migration assays. Coverslips (Fluorodish 10 mm, World Precision Instruments) were coated with PLL (Sigma, 0.01% diluted in H2O) for 10 min at room temperature then with recombinant mouse ICAM-1 (R&D systems #796-IC-050, at 5 μg/mL) for 1 h at 37°C. Cells were incubated in the culture dishes for 30 min at 37°C. Phase-contrast images were recorded using a DMI-6000B automated microscope (Leica) with a motorized stage (Pecon), an HQ2 Roper camera, 20×/0.45 NA dry objective (Olympus) and an environmental chamber (Pecon). Images were acquired every 30-40 s for 20-30 min using Metamorph software (Molecular Devices). Photoactivation was performed using a 100 ms pulse of blue light using an EL6000 mercury lamp (Leica) and a 470/40 excitation filter, and image acquisition was continued for 1 h thereafter. Images were analyzed using Fiji software (ImageJ 1.52f). Cell spreading was evaluated by measuring the cell surface area manually. Cell roundness was determined by calculating the ratio of the length of the minor to the major axes of the cells. For motility analysis, phase-contrast images were treated using ITrack4U and then subjected to cell tracking using Imaris software (Bitplane). No cells were excluded from the analysis. When graphed over time, cell velocities were smoothed (average ten consecutive time points). For in vitro migration assay using two-photon imaging, plastic dishes were coated with recombinant mouse ICAM-1 (R&D systems #796-IC-050, at 5 μg/mL) for 1 h at 37°C. Cells were stained with Indo-1 as described above. Two-photon imaging was performed on an upright microscope (FVMPE-RS, Olympus) with a 25×/1.05 NA dipping objective (Olympus) and using FV31S-SW software (Olympus). Photoactivation within a region of interest was performed at 940 nm (laser power set at 20%) using galvano scanner at 10 µs/pixel for a total of 20 s. Photoactivation of single cells was performed at 940 nm (laser power set to 12%) using tornado scanning at 100 µs/pixel for a total of 20 s. Excitation was provided by an Insight DS+ Dual laser (Spectra-Physics) tuned at either 720, 830, 940, or 1040 nm. The following filter sets were used for imaging Indo-1 and mScarlet-eOS1 (Indo-1: 483/32 and 390/40, and mScarlet: 607/70), Twitch2B (CFP: 483/32 and YFP: 542/27), and NFAT-mCherry (624/40). For quantification of NFAT translocation, B3Z T cells expressing the eOS1 actuator and a NFAT-mCherry reporter were imaged before and after photoactivation. The concentration of mCherry fluorescence upon NFAT translocation in the nucleus was quantified in each cell by surface tracking using Imaris software (and referred to as translocation score).
Intravital two-photon imaging. CD8 + T cells were retrovirally transduced to express eOS1 and Twitch2B. Double-expressing T cells were isolated on a MoFlo Astrios (Beckman Coulter) using AQUIOS Designer Software 2.0 (ADS 2.0, Beckman Coulter), and expanded for 24 h in presence of recombinant human IL-2. T cells (1-2 × 10 7 cells) were injected intravenously into naive recipient mice. One to 5 days after transfer, recipient mice were anesthetized with a mixture of xylazine (Rompun, 10 mg/kg) and ketamine (Imalgene, 100 mg/kg), and the popliteal lymph nodes prepared for intravital imaging 31 . Briefly, a cover slip, onto which was glued a heated metal ring, was placed on a surgically exposed popliteal lymph node. The ring was filled with sufficient water to immerge a 25×/1.05 NA dipping objective (Olympus) and temperature was maintained at 37°C. Alternatively, explanted lymph nodes were imaged as explants 32 . Imaging was performed at 820-830 nm and photoactivation at 900 nm. Photoactivation in lymph nodes was performed using a resonant scanner (0.067 µs/pixel, laser power set at 30%, zoom 3), with ten scans per z-plane and repeating this sequence 15 times. The following filter set was Fig. 4 Two-photon optogenetic manipulation of calcium signals using eOS1 actuator. a B3Z T cell clones expressing either the OS1 or eOS1 calcium actuators were stained with Indo-1 and deposited on ICAM-1-coated surface. Cells were visualized using a two-photon laser tuned at 720 nm. PA: 940 nm; laser power 20%; galvano scanner at 10 µs/pixel. Scale bar: 30 μm. Representative of two independent experiments. b Nuclear translocation of NFAT following two-photon photoactivation of eOS1. A B3Z T cell clone expressing the eOS1 actuator and a NFAT-mCherry reporter was visualized using a twophoton laser tuned at 1040 nm. PA: 940 nm; laser power 20%; galvano scanner at 100 µs/pixel. Fig. 5 Simultaneous imaging and manipulation of calcium signals in individual T cells in lymph nodes. a Experimental setup for optogenetic manipulation of T cells in lymph nodes. b-g CD8 + T cells expressing the eOS1 actuator and the Twitch2B calcium indicator were adoptively transferred. After 1-5 days, intravital imaging of the popliteal lymph node or explanted lymph node imaging was performed using two-photon microscopy. b, c Time-lapse images b and quantification c showing calcium levels (Twitch2B signals, YFP:CFP ratio) in T cells before and after photoactivation during intravital imaging. Photoactivation was performed by tuning the laser at 900 nm with the laser power set at 25%, using the resonant scanner (0.067 µs/pixel). Scale bar: 10 µm. Each dot represents one cell. Bars represent mean values. Statistical significance was assessed using a two-tailed Mann-Whitney test (***p < 0.001). d eOS1-expressing T cells but not control T cells react to two-photon photoactivation. CD8 + T cells expressing the eOS1 actuator and the Twitch2B calcium indicator were adoptively transferred together with CFP-expressing control T cells. After photoactivation in explanted lymph nodes, eOS1-expressing T cells (yellow cells) arrested while control T cells (green cells) maintained their motile behavior. Photoactivation was performed at 900 nm with the laser power set at 30% using the resonant scanner (0.067 µs/pixel). Scale bar: 10 µm. Times are in min:sec. White arrows show an eOS1expressing T cell. Representative of five independent experiments. e Calcium signals can promote prolonged arrest in T cells. Time-lapse images show that two-photon photoactivation elicits a transient calcium elevation and T cell arrest in explanted lymph nodes. Dotted circles highlight one T cell over time.
Photoactivation was performed at 900 nm with the laser power set at 30% using the resonant scanner (0.067 µs/pixel). Scale bar: 10 µm. Times are in min:sec. Representative of five independent experiments. f Quantification of T cell velocities before or just after photoactivation. Each dot represents one cell. Bars represent mean values. Statistical significance was assessed using a two-tailed Mann-Whitney test (***p < 0.001). g Calcium signals can promote prolonged T cell arrest. The figure compiles the fate of individual T cells after photoactivation. Each line represents a cell. Note that after a few minutes, calcium levels return to baseline levels but many T cells remain arrested. Source data are provided as a Source Data File.
Statistical analyses. All statistical tests were performed with Prism v.6.0 g (GraphPad) and data are represented as mean ± SEM (or SD when specified). Unless mentioned otherwise, we used Mann-Whitney U test for two-group comparison. All p-values were calculated with two-tailed statistical tests and 95% confidence intervals. *p < 0.05, **p < 0.01, ***p < 0.001; ns, non-significant.
Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article.
Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request. The Source data underlying Figs. 1a-e, 2a-f, 3a, b, d, 4b, c, d, g, 5c, f, Supplementary Figs. 2b, c, d, 3, 4c-f, 5 and 6 can be found in a Source Data file. | v3-fos-license |
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} | pes2o/s2orc | Influence of bark on fuel ethanol production from steam-pretreated spruce
Background Bark and bark-containing forest residues have the potential for utilization as raw material for lignocellulosic ethanol production due to their abundance and low cost. However, the different physical properties and chemical composition of bark compared to the conventionally used wood chips may influence the spruce-to-ethanol bioconversion process. This study assesses the impact of bark on the overall bioconversion in two process configurations, separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF), utilizing steam-pretreated spruce bark and wood mixtures. Results Mixtures of different proportions of spruce bark and wood chips were subjected to SO2-catalyzed steam pretreatment at 210°C for five minutes, which has been shown to be effective for the pretreatment of spruce wood chips. The final ethanol concentration was the highest without bark and decreased significantly with increasing proportions of bark in both process configurations. However, this decrease cannot be attributed solely to the lower availability of the carbohydrates in mixtures containing bark, as the ethanol yield also decreased, from 85 to 59% in SSF and from 84 to 51% in SHF, as the mass fraction of bark was increased from 0 to 100%. Conclusions The results show that it was significantly more difficult to hydrolyse spruce bark to monomeric sugars than wood chips. Bark had an adverse effect on the whole bioconversion process due to its lower enzymatic hydrolyzability. On the other hand, bark inclusion had no detrimental effect on the fermentability of steam-pretreated spruce wood and bark mixtures. It was also observed that lower amounts of inhibitory degradation products were formed during the steam pretreatment of spruce bark than during the steam pretreatment of wood chips.
Background
The driving force behind the exploitation of renewable energy sources is the necessity to shift from a fossil-fuel dependent economy to one based on renewable resources. Biomass can be used to efficiently produce renewable liquid or gaseous fuels, providing alternatives to fossil fuels [1]. Ethanol, for instance, is already being produced from sugar and starch crops, and is used worldwide, as a consequence of policies promoting ethanol production [2][3][4][5]. However, the controversy of ethanol production from sugar and starch crops (first-generation ethanol) has led to the development of technologies employing lignocellulosic biomass as raw material [6][7][8].
The utilization of lignocellulosic biomass to produce ethanol provides an alternative to sugar and starch crops.
However, the additional cost of the lignocellulosic ethanol production process, resulting from the necessity of pretreatment and enzymes for lignocellulosic biomass refining, has led to limited profitability in comparison with sugar-and starch-based ethanol production [9]. Thus, there is a need to further decrease the production cost of lignocellulosic ethanol in order for it to become competitive with the first-generation ethanol [10].
One possible means of cost reduction is to utilize abundant low-cost lignocellulosic raw materials such as bark [11]. Bark and bark-containing forest residues could serve as a potential feedstock for ethanol production, although bark is considered to be an inferior raw material to highervalue wood chips due to its composition. Compared with spruce wood chips, spruce bark has a lower content of carbohydrates, and contains significantly more extractives and ash [12]. The lower content of carbohydrates results in decreased sugar concentration after hydrolysis, and thus a lower ethanol concentration after fermentation. The prehydrolyzates obtained from pretreated bark can contain elevated amounts of water-soluble extractives and polyphenols, which may have inhibitory effects on fermenting microorganisms and cellulolytic enzymes [13][14][15]. Therefore, the combined utilization of bark and wood chips for ethanol production might pose an even greater challenge than the use of softwood chips, which is already demanding. In this case, not only the inherent recalcitrance of the material must be overcome, but also the problems resulting from the significant difference in the physical properties and chemical composition of bark and wood chips. However, if these limitations could be overcome, then abundantly available, low-value forestry residues could be exploited, and existing spruce-to-ethanol production processes could be simplified by not having to debark the material. Ultimately, it is likely that forestry residues available for bioethanol production will contain varying amounts of bark, and it is therefore important to investigate the effects of bark on production processes previously optimized for wood chips only.
Previous studies have mainly focused on the effects of bark on fermentability. Boussaid et al. found that including bark led to decreased fermentability of pre-hydrolyzates prepared under low-severity pretreatment conditions, while pre-hydrolyzates prepared under higher severity conditions could be fermented comparably well to ethanol when 9% bark was included [13]. Similar results were obtained by Robinson et al., who found that up to 30% bark, on a dry basis, had negligible effects on the fermentability of pre-hydrolyzates obtained from SO 2 -catalyzed steamexploded Douglas fir whitewood [16]. Although the enzymatic hydrolyzability of bark has been investigated previously [17], there are few reports on the influence of bark on enzymatic hydrolysis and the overall ethanol yield of the ethanol bioconversion process when performed at higher water-insoluble solids (WIS) content [18]. Enzymatic hydrolysis and fermentation must be performed at a higher WIS loading in order to increase the ethanol concentration after fermentation, which is essential to reduce the cost of distillation and thus the marginal production cost production [19].
The aim of the present study was to assess the feasibility of utilizing bark together with spruce wood chips for the fermentative conversion of biomass to ethanol at 10% WIS content, using a commercial enzyme cocktail to hydrolyse the steam-pretreated material, and an industrial strain of Saccharomyces cerevisiae as the fermenting microorganism. Spruce bark mixed with wood chips at different ratios ranging from 0 to 100% were subjected to SO 2 -catalyzed steam pretreatment at 210°C for five minutes, which has previously been shown to be effective for spruce wood chips [20]. The effects of bark inclusion on the spruce-to-ethanol bioconversion process were investigated by performing separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) of the steam-pretreated wood and bark mixtures.
Steam pretreatment of wood and bark mixtures
The composition of the spruce wood chips and bark is given in Table 1. The content of carbohydrates, with the exception of arabinan, was lower in the bark than in the wood chips. The amount of hexose sugars available in the bark feedstock was only about half of that in the wood feedstock. Even though only neutral carbohydrates were analyzed, spruce bark also contains a significant amount of other polysaccharides, such as pectin [21,22]. The bark contained significantly more extractives and ash than were found in the wood chips. The content of acid-soluble lignin may be slightly overestimated for both feedstocks due to possible interference from other non-lignin components [23].
The compositions of the water-insoluble solid fractions of the steam-pretreated materials were determined, and the results are presented in Table 2. As a result of the lower glucan content of the bark feedstock, the glucan content of the steam-pretreated mixtures decreased with increasing proportions of bark (Table 2). Detectable amounts of sugars originating from the hemicellulose were observed in the solid fraction after pretreatment of 100% bark. Steam pretreatment dissolved most of the hemicelluloses in all other steam-pretreated materials. This could be due to the higher recalcitrance of bark or the possible neutralization of the SO 2 added to the raw material by the higher ash and extractives content of the bark feedstock. This indicates that bark requires more SO 2 or higher severity steam pretreatment conditions to dissolve hemicellulose to the same extent as in wood chips. Interestingly, the acid-insoluble lignin content of the water-insoluble fractions increased as the bark content increased in the feedstock (Table 2), although the acid-insoluble lignin content was found to be higher in the wood chips than in the bark (Table 1). This has been found in previous studies reporting that bark contains water-soluble phenolic compounds that can condense with lignin during acid-catalyzed steam pretreatment and appear as acid-insoluble lignin in the subsequent compositional analysis [18,24]. This phenomenon could play a significant part in the structural changes of the bark during the acid-catalyzed steam pretreatment carried out at the optimal condition for the wood chips. The composition of liquid fractions obtained from the steam-pretreated materials is presented in Table 3. As a consequence of the lower carbohydrate content of bark, the concentration of total sugars (expressed in monomeric form) in the liquid fraction decreased with increasing proportions of bark, with the exception of arabinose due to the higher arabinan content of bark. As it can be seen in Figure 1, the addition of bark seemed to have a negligible effect on the overall recovery of glucose (94 to 96% for all steam-pretreated materials) and mannose (80 to 82% for all steam-pretreated materials) in the steam pretreatment step, although a lower proportion of sugars was dissolved in monomeric form in the liquid fraction as bark was added. This confirms the hypothesis that the pretreatment conditions previously found to be optimal for spruce wood chips may be too mild to overcome the recalcitrance of bark.
The amount of degradation products generated during steam pretreatment is a function of the severity of the pretreatment and the concentration of carbohydrates present in the feedstock. Therefore, the most likely explanations of the decreasing concentrations (Table 3), and also the amount of all measured inhibitors expressed as grams of inhibitors formed per 100 g dry raw material ( Figure 2) with increasing bark inclusion, are the lower carbohydrate content and the higher recalcitrance of the bark feedstock.
Fermentability of pre-hydrolyzates
In order to evaluate the effect of the inhibitory compounds present in the liquid fractions obtained from the steam-pretreated materials, the pre-hydrolyzates were subjected to a fermentation test. As shown in Figure 3, all the pre-hydrolyzates showed similar high degrees of fermentability to ethanol, and the ethanol yields were in the same range as in the control solution, which contained only pure monomeric glucose and mannose.
Although bark contained more extractives than wood chips (Table 1), these had no detrimental effect on the fermentability of the pre-hydrolyzates. Boussaid et al. reported decreased fermentability of pre-hydrolyzates following lowseverity steam pretreatment of bark-containing softwood, and attributed it to extractives, which were recovered in the water-soluble fraction [13]. However, additional acid hydrolysis of the pre-hydrolyzates increased the ethanol yield significantly, and they believed this to be due to the condensation and polymerization of water-soluble phenolic compounds. Moreover, they also showed that pre-hydrolyzates obtained from steam pretreatment at a severity factor above three (as defined by Overend et al. [25]) fermented well to ethanol despite the inclusion of 9% bark. This indicates that the relatively high severity of the steam pretreatment applied in our study also made the condensation and polymerization of water-soluble phenolic compounds possible, hence possibly eliminating their inhibitory effect. These results support previous findings that lower amounts of inhibitory compounds are generally formed during steam pretreatment of softwood bark than in the case of wood chips, and as a consequence, prehydrolyzates of steam-pretreated spruce bark can be fermented comparably well into ethanol [12,16,18].
Separate hydrolysis and fermentation of wood and bark mixtures
Previous studies have mostly been devoted to the investigation of the fermentability of steam-pretreated softwood bark [12,13,16,17], while little has been reported on the effect of including bark on the enzymatic hydrolyzability of steam-pretreated softwoods. Furthermore, most previous studies have been performed at lower WIS contents [17,18]. The implementation of enzymatic hydrolysis and fermentation, either separately or simultaneously, at a higher WIS content is driven by the possible energy savings in the distillation step [26]. In order to investigate the effects of bark on enzymatic hydrolyzability and fermentability of the hydrolyzed pretreated materials separately, SHF experiments were performed at 10% WIS content. The major advantage of SHF is that hydrolysis and fermentation can be carried out under their optimal conditions. However, SHF in general requires longer overall process time in comparison with SSF [27], and the endproduct inhibition of enzymes by glucose and cellobiose results in a reduced rate of saccharification [28]. Figure 4 shows the concentration profiles for glucose during the enzymatic hydrolysis of steam-pretreated wood and bark mixtures and final glucose yields. The highest final glucose concentration (80.6 g/L) was achieved with Figure 2 The amounts of inhibitory compounds formed during steam pretreatment of wood and bark mixtures. HMF, 5-hydroxymethylfurfural. no bark addition, and decreased to 34 g/L with 100% bark. Furthermore, the highest glucose yield (90% based on all available glucose) and the highest rate of hydrolysis were obtained when no bark was added. The significant extractives content of bark can be a possible explanation for the lower glucose yields with bark addition. Phenolic compounds, either in monomeric or oligomeric form, deriving from bark can have inhibitory effect on the enzymes [14,15,29], which makes the enzymatic hydrolysis of bark more challenging than in the case of spruce wood chips. As can be seen in Figure 4, the glucose yield decreased significantly with increasing proportions of bark, and reached 53% at 100% bark. However, the same trend of decreasing hydrolyzability was also observed with increasing proportions of bark at 5% WIS loading, both on whole slurry and on washed fibre (data not shown). This suggests that the underlying reason for the lower enzymatic hydrolyzability is not the inhibitory effect of phenolic compounds in the liquid phase, but rather the higher recalcitrance or the structural changes caused by the relocation of bark extractives during acid-catalyzed steam pretreatment.
Previous studies have reported higher sugar yields from bark-containing softwood or bark as the only raw material. However, these enzymatic hydrolysis experiments were performed at a lower WIS content. For instance, Kemppainen et al. reported between 70 and 80% yields, depending on the steam pretreatment conditions, after 48 hours of enzymatic hydrolysis of spruce bark at 1% dry matter content [18]. A glucose yield of 79.6% was obtained by Robinson et al. in the enzymatic hydrolysis of softwood containing bark pretreated with steam and an additional alkaline peroxide treatment [17]. Another possible way can be to use additional accessory enzymes in order to increase the yield of the enzymatic hydrolysis of bark. For instance, 24% improvement was observed in hydrolysis of spruce bark after 48 hours when pectinase enzymes were used as a supplementation to the cellulolytic enzymes [18]. Due to the presence of pectin in bark, the pectinase activity of the applied enzyme cocktail may also significantly affect the hydrolysis yields.
After the removal of the solid fraction of enzymatically hydrolyzed mixtures by filtration, the liquid fractions were subjected to fermentation. Figure 5 shows the concentration profiles for total hexose sugars and the ethanol produced during fermentation, together with the final ethanol yields (percentage of the theoretical maximum stoichiometric yield based on all available hexose sugars). The ethanol concentrations reached their maximum values after 24 hours in all cases. However, the final ethanol concentrations were considerably lower as the amount of bark was increased, due to the lower concentration of hexose sugars available for ethanol production in the enzymatically hydrolyzed bark-containing mixtures. As can be seen in Figure 5, almost all hexose sugars were consumed by the yeast and fermented into ethanol. The ethanol yields were largely unaffected by the addition of bark, and were in the same range; above 90%. Table 4A shows the final concentrations of the substrates and products, together with the ethanol yield and the initial volumetric ethanol productivity in the SHF experiments. The volumetric ethanol productivity during the first two hours increased with bark inclusion, from 1.7 g/L⋅h to 3.2 g/L⋅h as the bark content was increased from 0 to 100%. A possible explanation for the higher initial volumetric ethanol productivity could be the lower concentration of 5-hydroxymethylfurfural (HMF) and furfural in the mixtures with higher bark content (Table 3). HMF and furfural are known to be inhibitory to yeast [30], and the fermentability of pre-hydrolyzates is significantly decreased with increasing concentrations of HMF and furfural. Taherzadeh et al. found that the rate of fermentation decreased considerably when the combined amount of HMF and furfural exceeded approximately 2 g/L [12]. In the present study, the HMF and furfural concentrations were highest when no bark was added to the spruce chips (1.6 g/L and 1.0 g/L, respectively), which were completely detoxified by the yeast in the first four hours of fermentation. As a consequence of the inhibitory effect of these degradation products, lower initial volumetric ethanol productivity was observed with the pretreated mixtures containing wood chips, than that for 100% bark, which contained the lowest concentrations of HMF and furfural.
The results of the SHF experiments showed that bark had no detrimental effects on the fermentability, which is in agreement with the results of the pre-hydrolyzates fermentation. However, the addition of bark has an adverse effect on enzymatic hydrolyzability, which limits the amount of ethanol that can be produced by the yeast in the bioconversion process.
Simultaneous saccharification and fermentation of wood and bark mixtures
In the SSF process configuration, enzymatic hydrolysis and fermentation are performed simultaneously in the same vessel, and the end-product inhibition during hydrolysis is minimized by the continuous conversion of glucose to ethanol by the fermenting microorganism [31]. However, enzymatic hydrolysis and fermentation are performed under sub-optimal conditions. In the present study, SSF was performed at 10% WIS content to assess the effect of including bark on both fermentability and enzymatic hydrolyzability. Figure 6 shows the concentration profiles for total hexose sugars and the ethanol produced during SSF, together with the ethanol yields obtained (percentage of the theoretical based on all available hexose sugars).
As can be seen in Figure 6, the highest final ethanol concentration (46 g/L) was obtained when no bark was added, and it decreased significantly with increasing additions of bark. However, this decrease cannot be attributed solely to the lower amount of carbohydrates available in the bark-containing mixtures, as the ethanol yield also decreased, from 85 to 59%, as the proportion of bark was increased from 0 to 100%.
As can be seen in Table 4B, the volumetric ethanol productivity during the first two hours of SSF increased from 1.7 to 4.0 g/L⋅h as the amount of bark was increased from 0 to 100%. Furthermore, accumulation of the hexose sugars was also observed during the same time period in all SSF experiments, with the exception of 100% bark, where no accumulation of hexose sugars occurred during SSF (Figure 6). Similarly to the SHF experiments, the lower concentrations of inhibitory compounds with increasing amounts of bark contributed to higher initial volumetric ethanol productivities than without bark. This indicates that the fermentability was not affected negatively by the inclusion of bark. This is also confirmed by that the addition of bark had no noticeable negative effects on the sugar utilization of the yeast in the SSF experiments ( Figure 6). All the available glucose and mannose were consumed by the yeast, and only galactose was detected at low concentrations after 96 hours. Glycerol was the main by-product produced by the yeast in all cases (Table 4), and there was no significant difference in the yield of glycerol based on all available hexose sugars (approximately 4% in all SSF experiments). The ethanol yield obtained in the SSF experiments with no bark was in the same range as reported for spruce chips in previous studies [32,33], while Kemppainen et al.
reported an ethanol yield of 66.4% of the theoretical in SSF of sequentially hot-water extracted and steampretreated spruce bark [18]. This higher yield might be explained by the removal of the extractives from the bark prior steam pretreatment. Moreover, the six-hour prehydrolysis applied before SSF, and the possible structural differences between industrial bark and the freshly processed bark used in the present study, should not be neglected.
Comparing the two process configurations, it is apparent that SSF was superior to SHF in all cases, since SSF resulted in higher overall yields regardless of the bark content (Table 4). It is also evident that the decreased enzymatic hydrolyzability of bark is a decisive factor behind the declining ethanol yields, with increasing amounts of bark in both process configurations. Bark was found to be significantly more difficult to hydrolyse to monomeric sugars than wood chips. Lower amounts of monomeric sugars were recovered in the liquid fraction after steam pretreatment, and lower yields were observed in the enzymatic hydrolysis step with increasing bark content. Although, it appears that bark might require more severe steam pretreatment to overcome its inherent recalcitrance, the possible unfavorable structural changes in the steam-pretreated bark could also hamper the enzymatic hydrolysis. The relocation of extractives and bark lignin during the acid-catalyzed steam pretreatment might reduce the accessibility for the enzymes to cellulose, which can result in lower enzymatic hydrolyzability. Delignification methods might also be an alternative to achieve higher yields in enzymatic hydrolysis, and thus provide more sugars for ethanol fermentation; however, chemical delignification operations are expensive and would constitute an additional burden on the already sensitive economics of second-generation ethanol production [34]. Thus, further research is needed to improve the enzymatic hydrolyzability of bark in order to achieve higher yields at lower enzyme dosages.
Conclusions
The effect of including bark in the spruce-to-ethanol production process has been assessed. The results showed that adding bark had no detrimental effects on the fermentability of steam-pretreated spruce bark and wood mixtures, and it was observed that lower amounts of degradation products were formed during the steam pretreatment of spruce bark than spruce wood chips. However, the addition of bark had an adverse effect on the whole bioconversion process due to the low hydrolyzability of bark. This was reflected by the decreasing overall ethanol yield with increasing proportions of bark in both process configurations. SSF proved to be more efficient than SHF for all wood and bark mixtures, since this process configuration resulted in higher overall yields, regardless of bark content.
Materials
Fresh spruce, Picea abies, was debarked and kindly provided by a local sawmill (ATA Timber Widtskövle AB, Everöd, Sweden), together with the bark fraction. The bark and the bark-free chipped wood were further chipped using a knife mill (Retsch GmbH, Haan, Germany) and sieved in order to obtain the fraction with a size range between 2 and 10 mm. The spruce had a dry matter content of 40%, while the bark had a somewhat lower dry matter content of 35%. The raw materials were stored in plastic bags at 4°C until used.
The enzyme preparation used was Cellic CTec3, kindly provided by Novozymes A/S (Bagsvaerd, Denmark). The yeast used in both the SSF and SHF experiments was Ethanol Red, kindly provided by Leaf Technologies (Marcq-en-Baroeul Cedex, France). The yeast used to determine the fermentability of the pre-hydrolyzates was prepared on an agar plate from ordinary baker's yeast, Saccharomyces cerevisiae, produced by Jästbolaget (Rotebo, Sweden). Vitahop, kindly provided by BetaTec (Schwabach, Germany), was used in the SSF and SHF experiments to avoid bacterial contamination. All chemicals used were of reagent grade quality.
Feedstock preparation and steam pretreatment
The bark and wood fractions were mixed to obtain batches containing 0, 10, 30, 50 and 100% bark on a dry weight basis. Each batch had a total dry weight of 700 g. The mixtures were impregnated with gaseous SO 2 (2.5% w/w, based on the water content of the mixtures) in tightly sealed plastic bags for 20 minutes at room temperature, and then subjected to steam pretreatment.
Steam pretreatment was performed at 210°C for five minutes in a 10 L reactor (Process-& Industriteknik AB, Kristianstad, Sweden), as described previously by Palmqvist et al. [35]. The pretreated materials were stored at 4°C before subsequent analysis and experiments.
Fermentation of pre-hydrolyzates
Yeast that was used to evaluate the fermentability of pre-hydrolyzates was aerobically cultivated. The inoculum culture was prepared by adding yeast cells, previously grown on a YPG agar plate (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose and 15 g/L agar) for three days at 30°C, to two 250 mL Erlenmeyer flasks, together with 70 mL of an aqueous solution containing 23.8 g/L glucose, 10. The aeration rate was 1 L/min, corresponding to 1 vvm (gas volume flow per unit working volume per minute). The stirrer speed was 700 rpm and the pH was maintained at pH 5 with 2.5 M NaOH solution. The dissolved oxygen concentration was monitored continuously with an O 2 -sensor (Mettler-Toledo GmbH, Urdorf, Switzerland). When all the sugars had been consumed as indicated by the O 2 -sensor, cultivation was stopped and the cells were harvested by centrifugation in 700 mL bottles at 3,600 × g for 10 minutes. The supernatant was discarded and the dry matter content of the harvested cells was determined. The time between cell harvesting and the initialization of the fermentation tests was less than two hours.
Fermentation tests were carried out on the prehydrolyzates to assess their fermentability and the extent of inhibition by the compounds formed during steam pretreatment. Pre-hydrolyzates were obtained from the steam-pretreated materials by vacuum filtration using grade five filter paper (Munktell Filter AB, Falun, Sweden). The pre-hydrolyzates were then diluted with deionized water to obtain an equivalent solids concentration (the concentration of inhibitors corresponding to an SSF with a certain WIS load) corresponding to a WIS load of 10% mass fraction. The initial concentrations of fermentable sugars were adjusted to 30 g/L glucose and 20 g/L mannose in order to obtain comparable fermentation results. A reference solution was prepared with the same sugar concentrations to serve as a control. Fermentation was performed anaerobically on a rotary shaker in shake flasks with a working volume of 100 mL, containing 0.5 g/L (NH 4 ) 2 HPO 4 , 0.025 g/L MgSO 4 •7 H 2 O and 0.2 mL/L Vitahop. Fermentation tests were conducted at 30°C and pH 5.5 for 24 hours with a yeast concentration of 5 g/L. The fermentation experiments were performed in duplicate.
Separate hydrolysis and fermentation
Enzymatic hydrolysis of the whole pretreated slurry was performed in 2 L LABFORS bioreactors with a working weight of 1.2 kg. A WIS load of 10% mass fraction and Cellic CTec3 enzyme cocktail at a load of 20 FPU/g WIS based on the final weight, were applied. The hydrolysis experiments were performed at 45°C, with a stirring rate of 400 rpm, at pH 5 maintained with 2.5 M NaOH solution. After 96 hours of enzymatic hydrolysis the supernatants were separated by vacuum filtration using grade five filter paper (Munktell Filter AB). The supernatants obtained from the duplicates were mixed and stored at −20°C prior to fermentation.
Fermentation of the supernatant was performed in 2 L LABFORS bioreactors with a working weight of 0.55 kg. Ethanol Red yeast was added at a dry weight concentration of 5 g/L based on the final weight. The supernatant was supplemented with (NH 4 ) 2 HPO 4 solution at a concentration of 0.5 g/L and 0.125 mL/L Vitahop. Fermentation was carried out at 30°C, with a stirring rate of 250 rpm for 96 hours, at pH 5 maintained with 2.5 M NaOH solution. All experiments were performed in duplicate.
Simultaneous saccharification and fermentation
The SSF experiments using the whole pretreated slurry were performed in sterilized 2 L LABFORS bioreactors with a working weight of 1 kg. A WIS load of 10% mass fraction, the Cellic CTec3 enzyme cocktail at a load of 20 FPU/g WIS and Ethanol Red yeast at a dry weight concentration of 5 g/L based on the final amount, were applied. The experiments were carried out at 35°C, with a stirring rate of 400 rpm for 96 hours, at pH 5 maintained with 2.5 M NaOH solution. The SSF media were supplemented with (NH 4 ) 2 HPO 4 solution at a concentration of 0.5 g/L and 0.125 mL/L Vitahop. All experiments were performed in duplicate.
Analysis
The total solids content of biomass materials and total dissolved solids content of liquid samples were determined according to the National Renewable Energy Laboratory (NREL) standardized laboratory analytical procedure [37].
The structural carbohydrates, lignin, extractives and ash content of the solid fractions and the composition of the liquid fractions were determined according to NREL standardized laboratory analytical procedures [38][39][40][41].
All samples obtained from experiments or compositional analysis were centrifuged in 2 mL Eppendorf tubes at 16,000 × g for 10 minutes. The supernatant was filtered using 0.2 μm syringe filters (GVS Filter Technology Inc., Indiana, United States), and filtered samples were stored at −20°C prior to high-performance liquid chromatography (HPLC) analysis. Sugars, ethanol, organic acids and other by-products were analyzed using a Shimadzu LC-20 AD HPLC system equipped with a Shimadzu RID 10A refractive index detector (Shimadzu Corporation, Kyoto, Japan). Monomeric sugars were quantified with isocratic ion-exchange chromatography using an Aminex HPX-87P column with a De-Ashing Bio-Rad micro-guard column at 85°C (both from Bio-Rad Laboratories, Hercules, California, United States) using reagent grade water as the mobile phase at a flow rate of 0.5 mL/min. Ethanol, organic acids and other by-products were determined using an Aminex HPX-87H chromatography column with a Cation-H Bio-Rad micro-guard column at 50°C (Bio-Rad Laboratories, Hercules, California, United States), with a mobile phase of 5 mM sulfuric acid at a flow rate of 0.5 mL/min.
Yield calculations
The glucose yield in the enzymatic hydrolysis experiments was calculated on the basis of total available glucose in the liquid and the solid fraction of the steam-pretreated materials. The theoretical amount of glucose released during enzymatic hydrolysis is 1.11 times the amount of glucan in the solid fraction of the steam-pretreated materials (due to the addition of water in hydrolysis). The ethanol yield is expressed as a percentage of the theoretical stoichiometric ethanol yield (0.51 g/g), based on total available hexose sugars, namely glucose, mannose and galactose, in the solid and/or the liquid fraction of the steam-pretreated materials. | v3-fos-license |
2020-12-10T09:06:53.118Z | 2020-12-01T00:00:00.000 | 228081166 | {
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} | pes2o/s2orc | Silencing of VEGFR2 by RGD-Modified Lipid Nanoparticles Enhanced the Efficacy of Anti-PD-1 Antibody by Accelerating Vascular Normalization and Infiltration of T Cells in Tumors
Simple Summary siRNA delivery to tumor endothelial cells was achieved using arginyl-glycyl-aspartic acid (RGD)-modified lipid nanoparticles containing a novel pH-sensitive and biodegradable lipid. The anti-tumor efficacy of an immune checkpoint inhibitor was improved by the silencing of VEGFR2 using the delivery system, because the combination therapy induced vascular normalization and increased CD8+ T cell infiltration into tumors. The efficient delivery of nucleic acids is a promising strategy to improve therapeutic outcomes in immune checkpoint inhibitor-resistant cancers. Abstract Despite the promising anticancer effects of immune checkpoint inhibitors, their low objective response rate remains to be resolved; thus, combination therapies have been investigated. We investigated the combination of an anti-programmed cell death 1 (aPD-1) monoclonal antibody with the knockdown of vascular endothelial factor receptor 2 (VEGFR2) on tumor endothelial cells to overcome resistance to immune checkpoint inhibitors and improve the objective response rate. The successful delivery of small interfering RNA to tumor endothelial cells was achieved by RGD peptide-modified lipid nanoparticles composed of a novel, pH-sensitive, and biodegradable ssPalmO-Phe. RGD-modified lipid nanoparticles efficiently induced the knockdown of VEGFR2 in tumor endothelial cells (TECs), which induced vascular normalization. The combination of a PD-1 monoclonal antibody with Vegfr2 knockdown enhanced CD8+ T cell infiltration into tumors and successfully suppressed tumor growth and improved response rate compared with monotherapy. Our combination approach provides a promising strategy to improve therapeutic outcomes in immune checkpoint inhibitor-resistant cancers.
Introduction
Immune checkpoint inhibitors (ICIs) that target programmed cell death-1 (PD-1) or programmed cell death-ligand 1 (PD-L1) proteins are being used extensively, for the treatment of many types of Cancers 2020, 12 cancers. Anti-PD-1/PD-L1 monoclonal antibodies (mAbs) have been used as monotherapies and are associated with low objective response rates (ORRs) and acquired tumor resistance [1,2]. Thus, there is a need for combination therapies. Clinical trials have been conducted to investigate the synergistic effect of ICIs and anti-angiogenesis in patients [3,4]. Angiogenesis, the generation of new blood vessels from preexisting vessels, occurs in many physiological processes, including tumorigenesis by pro-angiogenic factors [5]. The vessel maturation process is impeded because of the persistent hypersecretion of pro-angiogenic factors in the tumor microenvironment [6]. Abnormal angiogenesis leads to a lack of pericyte coverage and leaky blood vessels, resulting in increased vascular permeability and higher interstitial fluid pressure, which inhibits the infiltration of cytotoxic lymphocytes into tumors [7,8]. Immature blood vessels cannot supply enough oxygen to compensate for consumption, thus resulting in hypoxia, which directly impairs the functions of tumor infiltrating lymphocytes (TILs).
Vascular endothelial growth factor (VEGF) plays a pivotal role in angiogenesis in tumor microenvironments [9,10]. Bevacizumab, an anti-VEGF mAb, is the first anti-angiogenic agent approved for multiple cancers [11]. However, the neutralization of VEGF results in systemic side effects, including hemorrhage, hypertension, proteinuria, impaired wound healing, and thrombosis [12]. Therefore, angiogenesis factors must be selectively targeted in the tumor microenvironment. The angiogenic signal of VEGF is mainly transmitted by its receptor, VEGFR2, which is expressed on tumor endothelial cells (TECs) [10]. We previously developed arginyl-glycyl-aspartic acid (RGD)-modified liposomal delivery systems targeting TECs [13][14][15][16][17]. RGD-modified liposomes were used to deliver doxorubicin to TECs, killing these cells and destroying the tumor vessel structure in doxorubicin-resistant renal cell carcinoma, while the blood vessels in normal organs such as the liver and spleen were not affected by this drug delivery system [13]. This research showed that RGD-modified liposomes selectively deliver doxorubicin to TECs. We also demonstrated that small interfering RNAs (siRNA) against Vegfr2 delivered to TECs in murine tumors by RGD-modified lipid nanoparticles (LNPs) resulted in vascular normalization [15,17].
We reported the development of a series of ionizable lipids, an SS-cleavable and pH-activated lipid-like material (ssPalm), as a material for use as a nucleic acid carrier [18][19][20]. Recently, we synthesized a novel ssPalmO-Phenyl-P4C2 (ssPalmO-Phe) that was characterized by self-degradability driven by redox-responsive concentrations of the thiol group and subsequent nucleophilic attacks of thiol groups against phenyl esters [21]. This spontaneous degradation inside the cells allows its cargo to be effectively released into the cytosol, thus achieving efficient delivery of mRNA. In the study reported here, we demonstrated the improved effect of an anti-programmed cell death 1 (aPD-1) mAb with vascular normalization caused by the knockdown of Vegfr2, by delivering siRNA using RGD-modified LNP composed of ssPalmO-Phe.
Characterization of LNPs
LNPs were prepared using ethanol dilution, and the PEG-lipid or RGD-PEG-lipid was modified using the post-insertion method, as described in the Materials and Methods section. The average diameters of the prepared PEG-LNP and RGD-LNP comprising ssPalmO-Phe were approximately 160 nm, and they possessed a slightly negative charge (Table 1); this indicates that RGD modification did not affect the physicochemical characteristics of LNPs. The polydispersity index (PDI) of the two LNPs, as an indicator of homogeneity of particle diameter distribution, was approximately 0.2. The value meant that PEG-LNP and RGD-LNP were homogeneous nanoparticles. siRNA in LNPs had an encapsulation efficiency of more than 90%, comparable to that of LNPs composed of other types of ssPalm [18]. The recovery rate of each LNP suggests that approximately half of the starting amount of siRNA was encapsulated in LNP, which is also consistent with our previous data [18]. The siRNAs were successfully encapsulated in LNPs prepared with ssPalmO-Phe.
In Vitro Cellular Uptake and Knockdown by LNPs in Endothelial Cells
To evaluate the expression of the integrin receptors on the cell surface, lymphatic endothelial cells (LEC) and Human umbilical vein endothelial cells (HUVEC) were analyzed using flow cytometry. HUVEC cells expressed substantial levels of integrin; however, no expression of integrin was detected in the LEC cells ( Figure 1A). We next examined the cellular uptake of PEG-LNP and RGD-LNP by LEC and HUVEC cells. Although no difference in uptake of LNPs was detected in LEC cells, a significant increase in cellular uptake was observed for RGD-LNP in HUVEC cells ( Figure 1B,C). These results demonstrated that RGD modification selectively enhanced the cellular uptake of LNPs in integrin-positive cells such as HUVEC cells. The knockdown efficacy of RGD-LNP and RGD-LNP in HUVEC cells was examined using RT-PCR, which revealed that PEG-LNP showed negligible knockdown of PLK1 in HUVEC cells at a dose of 100 nM of siRNA, compared with untreated HUVEC. However, RGD-LNP decreased the expression of PLK1 in a dose-dependent manner ( Figure 1D). This finding clearly demonstrated that RGD-LNP could induce the knockdown of target genes by delivering siRNA selectively to integrin-positive endothelial cells. of siRNA was encapsulated in LNP, which is also consistent with our previous data [18]. The siRNAs were successfully encapsulated in LNPs prepared with ssPalmO-Phe.
In Vitro Cellular Uptake and Knockdown by LNPs in Endothelial Cells
To evaluate the expression of the integrin receptors on the cell surface, lymphatic endothelial cells (LEC) and Human umbilical vein endothelial cells (HUVEC) were analyzed using flow cytometry. HUVEC cells expressed substantial levels of integrin; however, no expression of integrin was detected in the LEC cells ( Figure 1A). We next examined the cellular uptake of PEG-LNP and RGD-LNP by LEC and HUVEC cells. Although no difference in uptake of LNPs was detected in LEC cells, a significant increase in cellular uptake was observed for RGD-LNP in HUVEC cells ( Figure 1B,C). These results demonstrated that RGD modification selectively enhanced the cellular uptake of LNPs in integrin-positive cells such as HUVEC cells. The knockdown efficacy of RGD-LNP and RGD-LNP in HUVEC cells was examined using RT-PCR, which revealed that PEG-LNP showed negligible knockdown of PLK1 in HUVEC cells at a dose of 100 nM of siRNA, compared with untreated HUVEC. However, RGD-LNP decreased the expression of PLK1 in a dose-dependent manner ( Figure 1D). This finding clearly demonstrated that RGD-LNP could induce the knockdown of target genes by delivering siRNA selectively to integrin-positive endothelial cells.
In Vivo Knockdown of VEGFR2 on TECs and Vascular Normalization
To evaluate whether RGD-LNP could induce the knockdown of a target gene in murine TECs, RGD-LNP encapsulating siVegfr2 was intravenously administered to MC38 tumor-bearing mice.
To distinguish endothelial cells from other cells, CD31, a marker of endothelial cells, was also stained as described in the Materials and Methods section. VEGFR2 expression on CD31-positive cells was significantly decreased after treatment with RGD-LNP encapsulating siVegfr2 ( Figure 2). Vascular normalization was initiated in tumors treated with RGD-LNP encapsulating siVegfr2. The marge ratio of αSMA, a marker of pericytes, in positive TECs with CD31 in tumors treated with control siRNA was comparable to that in untreated tumors ( Figure 3). An increase in the ratio of αSMA to CD31 was, however, observed in tumors treated with siVegfr2. The results clearly showed that knockdown of Vegfr2 promoted coverage of tumor vasculature with pericytes, i.e., vascular normalization. When used in combination, RGD-LNP successfully delivered siVegfr2 to TECs in MC38 tumors.
In Vivo Knockdown of VEGFR2 on TECs and Vascular Normalization
To evaluate whether RGD-LNP could induce the knockdown of a target gene in murine TECs, RGD-LNP encapsulating siVegfr2 was intravenously administered to MC38 tumor-bearing mice. To distinguish endothelial cells from other cells, CD31, a marker of endothelial cells, was also stained as described in the Materials and Methods section. VEGFR2 expression on CD31-positive cells was significantly decreased after treatment with RGD-LNP encapsulating siVegfr2 ( Figure 2). Vascular normalization was initiated in tumors treated with RGD-LNP encapsulating siVegfr2. The marge ratio of αSMA, a marker of pericytes, in positive TECs with CD31 in tumors treated with control siRNA was comparable to that in untreated tumors ( Figure 3). An increase in the ratio of αSMA to CD31 was, however, observed in tumors treated with siVegfr2. The results clearly showed that knockdown of Vegfr2 promoted coverage of tumor vasculature with pericytes, i.e., vascular normalization. When used in combination, RGD-LNP successfully delivered siVegfr2 to TECs in MC38 tumors. In vivo Vegfr2 knockdown in MC38 tumors by delivery of siVegfr2 using RGD-LNP. MC38 tumor-bearing mice were administered with RGD-LNP encapsulating either siPLK1 as a control, or siVegfr2, at a dose of 40 μg siRNA. Tumors were harvested 24 h after administration, sectioned, and stained as described in the Materials and Methods section. Four tumors from each group were imaged, and CD31-positive cells stained with VEGFR2 or not stained were quantified in each image. Red: VEGFR2, green: CD31 (marker of endothelial cells), blue: nuclei. * p < 0.05 determined using oneway ANOVA followed by Tukey's test. Bars: 50 μm. In vivo Vegfr2 knockdown in MC38 tumors by delivery of siVegfr2 using RGD-LNP. MC38 tumor-bearing mice were administered with RGD-LNP encapsulating either siPLK1 as a control, or siVegfr2, at a dose of 40 µg siRNA. Tumors were harvested 24 h after administration, sectioned, and stained as described in the Materials and Methods section. Four tumors from each group were imaged, and CD31-positive cells stained with VEGFR2 or not stained were quantified in each image. Red: VEGFR2, green: CD31 (marker of endothelial cells), blue: nuclei. * p < 0.05 determined using one-way ANOVA followed by Tukey's test. Bars: 50 µm. MC38 tumor-bearing mice were administered with RGD-LNP encapsulating either siPLK1 as a control, or siVegfr2, at a dose of 40 μg siRNA twice, three days apart. Tumors were harvested 24 h after the final administration, sectioned, and stained as described in the Materials and Methods section. Four tumors from each group were imaged, and CD31-positive cells that were stained with αSAM or not stained were quantified in each image. Red: αSMA (marker of pericytes), green: CD31 (marker of endothelial cells), blue: nuclei. * p < 0.05, ** p <0.01 determined using one-way ANOVA followed by Tukey's test. Bars: 50 μm.
In Vivo Anti-Tumor Efficacy
We examined the anti-tumor efficacy of aPD-1 mAb combined with Vegfr2 knockdown ( Figure 4A). Monotherapy with either aPD-1 mAb or RGD-LNP had a minor effect on MC38 tumor growth ( Figure 4B). Combination therapy significantly suppressed tumor growth compared with controls and monotherapies by day 12. We also assessed the interaction index of the combination therapy versus monotherapy [22]. The interaction indexes of the combination therapy at days 12 and 15 were −0.90 (95% confidence interval (CI), −1.55 to −0.26) and −0.64 (95% CI, −1.56 to 0.29), respectively, which indicates that the combination of aPD-1 and siVegfr2 was additive to supra-additive as compared with the monotherapies. The combination therapy also induced strong responses in seven of ten tumor-bearing mice ( Figure 4C). Significant differences were observed in the response rate of the combination therapy compared with those of other groups. Anti-angiogenesis caused by the silencing of Vegfr2 improved the response to an immune checkpoint inhibitor. Thereafter, we investigated the mechanisms by which the combination successfully suppressed tumor growth by analyzing CD8+ T cells that infiltrated the tumors. When compared with control IgG treatment, aPD-1 mAb or siVegfr2 treatment resulted in a 2.7-and 1.9-fold increase in infiltrated CD8+ T cells in tumors ( Figure 5A,B). The infiltration of CD8+ T cells increased 5.5-fold after the combination therapy. We further analyzed the exhausted markers such as PD-1 and T-cell immunoglobulin mucin-3 (Tim-3) on CD8+ T cells by isolating CD8+ T cells from tumors. No changes in the expression of these markers were found due to the treatments ( Figure 5C,D), which suggested that the unresponsiveness of CD8+ T cells to aPD-1 mAb in MC38 was not caused by the exhaustion state. The improved anti-tumor efficacy of aPD-1 mAb combined with the knockdown of Vegfr2 resulted MC38 tumor-bearing mice were administered with RGD-LNP encapsulating either siPLK1 as a control, or siVegfr2, at a dose of 40 µg siRNA twice, three days apart. Tumors were harvested 24 h after the final administration, sectioned, and stained as described in the Materials and Methods section. Four tumors from each group were imaged, and CD31-positive cells that were stained with αSAM or not stained were quantified in each image. Red: αSMA (marker of pericytes), green: CD31 (marker of endothelial cells), blue: nuclei. * p < 0.05, ** p < 0.01 determined using one-way ANOVA followed by Tukey's test. Bars: 50 µm.
In Vivo Anti-Tumor Efficacy
We examined the anti-tumor efficacy of aPD-1 mAb combined with Vegfr2 knockdown ( Figure 4A). Monotherapy with either aPD-1 mAb or RGD-LNP had a minor effect on MC38 tumor growth ( Figure 4B). Combination therapy significantly suppressed tumor growth compared with controls and monotherapies by day 12. We also assessed the interaction index of the combination therapy versus monotherapy [22]. The interaction indexes of the combination therapy at days 12 and 15 were −0.90 (95% confidence interval (CI), −1.55 to −0.26) and −0.64 (95% CI, −1.56 to 0.29), respectively, which indicates that the combination of aPD-1 and siVegfr2 was additive to supra-additive as compared with the monotherapies. The combination therapy also induced strong responses in seven of ten tumor-bearing mice ( Figure 4C). Significant differences were observed in the response rate of the combination therapy compared with those of other groups. Anti-angiogenesis caused by the silencing of Vegfr2 improved the response to an immune checkpoint inhibitor. Thereafter, we investigated the mechanisms by which the combination successfully suppressed tumor growth by analyzing CD8+ T cells that infiltrated the tumors. When compared with control IgG treatment, aPD-1 mAb or siVegfr2 treatment resulted in a 2.7-and 1.9-fold increase in infiltrated CD8+ T cells in tumors ( Figure 5A,B). The infiltration of CD8+ T cells increased 5.5-fold after the combination therapy. We further analyzed the exhausted markers such as PD-1 and T-cell immunoglobulin mucin-3 (Tim-3) on CD8+ T cells by isolating CD8+ T cells from tumors. No changes in the expression of these markers were found due to the treatments ( Figure 5C,D), which suggested that the unresponsiveness of CD8+ T cells to aPD-1 mAb in MC38 was not caused by the exhaustion state. The improved anti-tumor efficacy of aPD-1 mAb combined with the knockdown of Vegfr2 resulted from the increase in infiltration of CD8+ T cells in tumors, but not from the prevention of CD8+ T cell exhaustion.
Discussion
The objective response rate (ORR) to aPD-1 mAbs such as nivolumab and pembrolizumab has been reported to be limited to around 20% [2]. Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and PD-1 blockade have proven successful in improving survival rates, resulting in the approval of the ipilimumab and nivolumab combination for the treatment of metastatic melanoma and renal cell carcinoma [23]. However, severe side effects have been observed with combination therapy. The development of safer alternative means is necessary.
Tumors with high levels of TIL respond better to ICIs than tumors with low TIL [1]. Therefore, strategies that can convert "cold" tumors into "hot" tumors have attracted attention, as they improve the ORR of ICIs. One promising approach involves combining ICIs with anti-angiogenic agents, because pro-angiogenic factors can play both direct and indirect roles in the immunosuppressive tumor microenvironment [4]. Several studies have shown that treatment of xenograft cancer models with inhibitors of VEGF or VEGFR2, such as anti-VEGFR2 receptor antibodies and apatinib, can exert a synergistic effect with aPD-1 or aPD-L1 mAbs [24][25][26][27][28][29][30]. Anti-PD-L1 atezolizumab in combination with bevacizumab has been shown to enhance the migration of TILs in patients with metastatic renal cell carcinoma [31]. Clinical trials combining immunotherapy and anti-angiogenic factors such as apatinib, lenvatinib, and ramucirumab are currently in progress for several types of cancer, suggesting that combining a strategy with anti-angiogenesis is beneficial for immunotherapy [32][33][34][35].
We found that the knockdown of Vegfr2 in TECs enhanced the anti-tumor efficacy of aPD-1 mAb. Selective targeting of TECs was achieved by RGD-modified LNP, because RGD modification alters the biodistribution of nanocarrier systems, facilitating their delivery to integrin αvβ3-expresisng TECs [36][37][38]. The size of LNPs which target cancer cells in tumors is generally less than 100 nm in diameter, because LNPs have to pass through the gap between the TECs of the neovasculature, based on the enhanced permeability and retention (EPR) effect [39]. In this study, the diameter of the prepared LNPs was 160 nm, which is appropriate for targeting TECs of the neovasculature due to a reduction in the EPR effect [13,40]. LNPs composed of ssPalmO-Phe showed lower toxicity because of their biodegradability [21]. Biodegradable LNPs deliver nucleic acids in vivo without any apparent toxicity. In a preclinical safety study, no significant changes in the biochemical parameters of blood,
Discussion
The objective response rate (ORR) to aPD-1 mAbs such as nivolumab and pembrolizumab has been reported to be limited to around 20% [2]. Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and PD-1 blockade have proven successful in improving survival rates, resulting in the approval of the ipilimumab and nivolumab combination for the treatment of metastatic melanoma and renal cell carcinoma [23]. However, severe side effects have been observed with combination therapy. The development of safer alternative means is necessary.
Tumors with high levels of TIL respond better to ICIs than tumors with low TIL [1]. Therefore, strategies that can convert "cold" tumors into "hot" tumors have attracted attention, as they improve the ORR of ICIs. One promising approach involves combining ICIs with anti-angiogenic agents, because pro-angiogenic factors can play both direct and indirect roles in the immunosuppressive tumor microenvironment [4]. Several studies have shown that treatment of xenograft cancer models with inhibitors of VEGF or VEGFR2, such as anti-VEGFR2 receptor antibodies and apatinib, can exert a synergistic effect with aPD-1 or aPD-L1 mAbs [24][25][26][27][28][29][30]. Anti-PD-L1 atezolizumab in combination with bevacizumab has been shown to enhance the migration of TILs in patients with metastatic renal cell carcinoma [31]. Clinical trials combining immunotherapy and anti-angiogenic factors such as apatinib, lenvatinib, and ramucirumab are currently in progress for several types of cancer, suggesting that combining a strategy with anti-angiogenesis is beneficial for immunotherapy [32][33][34][35].
We found that the knockdown of Vegfr2 in TECs enhanced the anti-tumor efficacy of aPD-1 mAb. Selective targeting of TECs was achieved by RGD-modified LNP, because RGD modification alters the biodistribution of nanocarrier systems, facilitating their delivery to integrin αvβ3-expresisng TECs [36][37][38]. The size of LNPs which target cancer cells in tumors is generally less than 100 nm in diameter, because LNPs have to pass through the gap between the TECs of the neovasculature, based on the enhanced permeability and retention (EPR) effect [39]. In this study, the diameter of the prepared LNPs was 160 nm, which is appropriate for targeting TECs of the neovasculature due to a reduction in the EPR effect [13,40]. LNPs composed of ssPalmO-Phe showed lower toxicity because of their biodegradability [21]. Biodegradable LNPs deliver nucleic acids in vivo without any apparent toxicity. In a preclinical safety study, no significant changes in the biochemical parameters of blood, such as the number of red blood cells, hematocrit, mean corpuscular volume, number of platelets, or the number of white blood cells, was observed, even when LNPs composed of ssPalmO-Phe were administered to rats at a dose of 175 mg nucleic acids/kg body weight [21]. This delivery system can be used for delivering not only siRNA but also other nucleic acids such as mRNA to TECs, to efficiently manipulate undruggable angiogenesis factors that cannot be inhibited by small compounds or antibodies.
It was demonstrated that the efficacy of ICIs could be improved in combination with the knockdown of Vegfr2 in TECs. Although treatment with either aPD-1 mAb or siVegfr2 enhanced tumor infiltrating CD8+ T cells in tumors by two to three-fold compared with control treatment, the combination further increased the infiltration of CD8+ T cells into tumors by 5.5-fold. Type 1 T helper (Th1) cells, such as CD8+ T cells in tumors, promote vascular normalization by secreting interferon-γ (IFN-γ) [41]. T cells that infiltrate tumors after vascular normalization were activated by aPD-1 mAbs and could secrete IFN-γ to promote further vascular normalization, which could explain the increase in CD8+ T cells after the combination treatment. Vascular normalization in tumors also enhanced the delivery of antibody and protein therapeutics to tumors [42,43]. Therefore, vascular normalization induced by the knockdown of Vegfr2 could contribute to an increase in the amount of aPD-1 mAb delivered in tumors. When used in combination, a blockade of both pathways efficiently converts the cold tumor microenvironment into a hot microenvironment. As previously reported, the enhanced anti-tumor effect of ICIs combined with anti-angiogenesis was observed in subcutaneous xenograft models [24,25,28]. However, subcutaneous xenograft tumors do not necessarily reflect the actual tumor microenvironment. The synergistic effect between ICIs and anti-angiogenesis has been also observed in orthotopic models [26,29,30]. Even though further study is required to evaluate the delivery of LNPs in orthotopic models of colon cancer in terms of the delivery mechanisms of LNPs in the peritoneal compartment and the efficacy of their cargos, the combination of PD-1/PD-L1 blockade with siVegfr2 delivery by the LNP system could be applicable for the treatment of peritoneal disseminations.
In some cases, T cells are ineffective against cancer because the T cells enter exhaustion, a state of T cell dysfunction [44]. It was reported that anti-PD-1/PD-L1 upregulates the Ras-Raf-MEK-ERK and PI3K-AKT signaling pathways in immune cells by blocking the PD-1/PD-L1 axis [45]. Anti-PD-1/PD-L1 therapy therefore restores T cells from an exhausted state and enhances their tumor-killing activity [3]. In the present study, a reduction in the expression of the markers of exhaustion, PD-1 and Tim-3, was not observed, even after the combination therapy. Even though further studies are required to elucidate the precise mechanisms of action, T cell exhaustion in tumors is not a dominant factor for the immune surveillance of MC38 tumors.
Preparation and Characterization of LNPs
cRGD was conjugated to NHS-PEG-DSPE (RGD-PEG-lipid) via amide bonds by incubation in dimethylformamide with 1.2 equivalent of triethylamine as previously described [16,47]. siRNA-loaded LNPs were prepared by the alcohol dilution method [16,17]. ssPalmO-Phe and cholesterol in ethanol were mixed at a molar ratio of 50/50 (molar ratio) at 1500 nmol total lipid. PEG-DMG was added to the solution at 3.0 mol% to control the particle size distribution. To the lipid solution, 20 µg of siRNA in 124 µL of 20 mM malic buffer (pH 3.0) was gradually added under vigorous mixing. The mixture was sequentially diluted with 1 mL of malic buffer and then 3 mL of PBS. The resulting mixture was subjected to ultrafiltration with Amicon Ultra-14 (MWCO 100,000) twice. To modify LNP with RGD-PEG-lipid, LNP was incubated with 3.0 mol% of RGD-PEG-lipid in 10% ethanol/20 mM malic buffer solution at 45 • C for 45 min. Ethanol was removed by ultrafiltration. As a control, MeO-PEG-DSPE was incorporated into LNP instead of RGD-PEG-DSPE. The mean size and zeta potential of the prepared LNPs were determined using a Zetasizer Nano ZS ZEN3600 instrument (Malvern Instruments, Westborough, MA, USA). The encapsulation efficiency and recovery ratio of siRNA were measured by RiboGreen assay as previously described [48].
Flow Cytometry
LEC and HUVEC cells were washed with PBS and detached using Accutase (Innovative Cell Technologies, San Diego, CA, USA). Detached cells were incubated with anti-PE-conjugated human integrin αvβ3 antibody (Table S1) for 30 min at 4 • C. Cells were then washed with 0.5% BSA/0.1% sodium azide in PBS (FACS buffer). Ten thousand cells per sample were analyzed using a Novocyte flow cytometer (ACEA Biosciences, San Diego, CA, USA). Tumors were dissociated into single cells using mouse Tumor Dissociation Kits, and a gentleMACS™ Octo Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). Single cells were incubated with 10 µg/mL of anti-mouse CD16/32 antibody (clone 93, Biolegend, San Diego, CA, USA) in FACS buffer for 10 min at 4 • C to block Fc receptors. After washing the cells with FACS buffer, cells were stained with fluorophore-labeled antibodies (listed in Table S1) for 30 min at 4 • C. After washing, cells were stained with 7-AAD (5 µg/mL, Biolegend, #42040) for 5 min at 25 • C to determine cell viability. Cells were analyzed using a Novocyte flow cytometer. CD8+ T cells were further isolated from single cell suspension using mouse CD8a+ T Cell Isolation Kits (Miltenyi Biotec). Isolated CD8+ T cells were stained and analyzed as described above.
Cellular Uptake of PEG-LNP and RGD-LNP
To investigate the cellular uptake of LNPs, HUVEC and LEC cells (1.5 × 10 5 cells/well) were seeded on a six-well plate 24 h before LNP was added. Cells were incubated with DiO-labeled PEG-LNP or RGD-LNP at 20 µM of lipid for 2 h and then washed twice with PBS. Cells were trypsinized and centrifuged at 4 • C at 500× g for 5 min. The supernatant was removed with an aspirator, and FACS buffer was added to the cell pellet. The cell suspension was then analyzed on a Novocyte flow cytometer.
In Vitro Knockdown Studies
HUVEC cells (1.5 × 10 5 cells/well) were seeded in 24-well plates 24 h before LNP addition. Cells were incubated with PEG-LNP at 100 nM of siPLK1 or RGD-LNPs at indicated concentrations of siPLK1 for 24 h. Anti-human PLK1 siRNA is used as negative control siRNA because this sequence does not have any effect on mouse Plk1 and physiology or on immune system via RNA sensors [49]. To isolate RNA from the cells, 250 µL of TRIreagent (Molecular Research Center, Cincinnati, OH, USA) was added after washing with PBS. Total RNA was purified according to the manufacturer's protocol. Complementary DNA was synthesized using High Capacity RNA-to-cDNA kit (ThermoFisher, Wilmington, DE, USA) at 0.5 µg RNA. The obtained cDNA was diluted 10-fold and then evaluated by quantitative PCR with THUNDERBIRD Master Mix (TOYOBO, Osaka, Japan). The expression of PLK1 mRNA was calculated by the ∆∆Ct-value method. GAPDH was regarded as an internal control. The primers used were PLK1 forward; CTCCTTGATGAAGAAGATCACC, reverse; GAAGAAGTTGATCTGCACGC, GAPDH forward; CCTCTGACTTCAACAGCGAC, reverse; CGTTGTCATACCAGGAAATGAG as previously reported [47,50].
Tumor Inoculation
C57BL/6JJ mice (six weeks old, female) were purchased from Japan SLC (Shizuoka, Japan). Cancer cells were subcutaneously (s.c.) transplanted into syngeneic mice using 1 × 10 6 cells in 100 µL Hanks' balanced salt solution. All animal procedures were approved by the Chiba University Institutional Animal Care and Use Committee (A1-219).
Immunofluorescence Analysis for In Vivo Knockdown of Vegfr2
When tumor volumes reached 100-200 mm 3 , tumor-bearing mice were intravenously (i.v.) treated with RGD-LNP encapsulating either siPLK1 as a control or siVegfr2, at a dose of 40 µg siRNA/100 µL PBS/mouse. At 24 h after the administration, tumors were harvested to examine the knockdown of Vegfr2 in MC38 tumors. The RGD-LNPs at a dose of 40 µg siRNA/100 µL PBS/mouse were i.v. administrated twice, three days apart, to tumor-bearing mice. At 24 h after the final administration, tumors were harvested to examine vascular coverage. Tumors were fixed with 4% PFA in PBS and embedded in OCT compound (Sakura Finetek, Tokyo, Japan). Tissues were sectioned (10 µm) onto glass slides (MAS coated glass, Matsunami, Osaka, Japan). The slices were then immersed in 100-fold diluted anti-mouse VEGFR2, anti-mouse CD31, and anti-mouse actin, α-Smooth muscle (αSMA) antibodies (Table S1) at room temperature for 30 min. The secondary antibodies used were goat anti-Rat IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 and Goat anti-Hamster IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488, respectively. Images were taken using an LSM780 system (Carl Zeiss, Oberkochen, Germany). Images were then quantified using Image J software [51].
Tumor Inoculation
Tumor-bearing mice were randomized and treated i.p. with control IgG or aPD-1 mAb at 200 µg/mouse in 100 µL PBS and treated intravenously with RGD-LNP encapsulating siPLK1 or siVegfr2 at 40 µg siRNA/mouse in 100 µL PBS on days 5, 8, and 12 (post-tumor inoculation). Tumor volume was calculated using the formula 1/2 × a × b 2 , where a and b represent the largest and smallest tumor diameters, respectively. Tumor volumes of 30% or less than that of tumors treated with control IgG were regarded as efficiently suppressed.
Statistical Analysis
All data are presented as the mean value ± S.E. Pair-wise comparisons of subgroups were made using Student's t-tests with Welch's correction. Comparisons between multiple treatments were made using one-way analysis of variance (ANOVA), followed by an appropriate post-hoc test, and chi-squared tests followed by adjusted standardized residual analysis. P values (both sides) were considered significant if p was less than 0.05. Statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). An interaction index of combination therapy versus monotherapy of the fixed-dose drug combination was assessed according to the methods described in a previous report [22]. If the 95% CI was less than zero, the drug combination was considered to be supra-additive; if it contained zero, the drug combination was considered to be additive; otherwise, the drug combination was considered to be sub-additive.
Conclusions
In this study, we demonstrated successful siRNA delivery to TECs in xenograft tumors using RGD-modified LNP composed of ssPalmO-Phe. The combination of aPD-1 mAb and VEGFR2 knockdown decreased the number of TILs in tumors via vascular normalization and suppressed tumor growth. Further studies are required to test whether this combination strategy could be applied to other types of cancer therapies, but these results indicate that this delivery system implements a combination strategy which can improve the therapeutic outcome of ICIs. | v3-fos-license |
2020-12-02T14:11:18.404Z | 2020-11-25T00:00:00.000 | 227242900 | {
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} | pes2o/s2orc | Pharmacokinetic Characterization of (Poly)phenolic Metabolites in Human Plasma and Urine after Acute and Short-Term Daily Consumption of Mango Pulp
Pharmacokinetic (PK) evaluation of polyphenolic metabolites over 24 h was conducted in human subjects (n = 13, BMI = 22.7 ± 0.4 kg/m2) after acute mango pulp (MP), vitamin C (VC) or MP + VC test beverage intake and after 14 days of MP beverage intake. Plasma and urine samples were collected at different time intervals and analyzed using targeted and non-targeted mass spectrometry. The maximum concentrations (Cmax) of gallotannin metabolites were significantly increased (p < 0.05) after acute MP beverage intake compared to VC beverage alone. MP + VC beverage non-significantly enhanced the Cmax of gallic acid metabolites compared to MP beverage alone. Pyrogallol (microbial-derived metabolite) derivatives increased (3.6%) after the 14 days of MP beverage intake compared to 24 h acute MP beverage intake (p < 0.05). These results indicate extensive absorption and breakdown of gallotannins to galloyl and other (poly)phenolic metabolites after MP consumption, suggesting modulation and/or acclimation of gut microbiota to daily MP intake.
Introduction
Mango (Mangifera indica L.) is one of the most economically important fruits in the world due to its appealing taste and high nutritional value [1]. Mango has a unique and diverse phytochemical profile [2] consisting of carotenoids and a number of polyphenol compounds including mangiferin, gallotannins, catechin, quercetin, kaempferol, gallic acid, ellagic acid and other phenolic acids [3]. The polyphenolic profile of mango is associated with reducing the risk of developing a number of chronic diseases and their related complications [4,5]. Several in vitro and in vivo animal studies support anti-diabetic [6,7], anti-cancer [8,9], anti-inflammatory [10,11], anti-oxidant [12,13] and anti-bacterial [14] activities linked with the intake of mango pulp, peel, seed, juice, extracts and other mango products. Additionally, bioavailability and bioaccessibility analyses of isolated compounds (mangiferin) or extracts from mango leaf and mango seed kernel have been conducted in pre-clinical and in vitro models [15][16][17][18][19]. However, mango pulp (MP) consumed by humans involves delivery/absorption of bioactive components from a complex food matrix potentially influencing bioavailability. Only a few studies have assessed the absorption and metabolism of polyphenols from mango fruit in humans [4,20,21].
A previous study identified and quantified five compounds in plasma and seven compounds in urine after "Ataulfo" mango consumption (500 g of flesh or 721 g of juice) [21]. Barnes et al. reported seven galloyl metabolites in urine after a 10-day consumption of 400 g of MP from the Keitt variety [20]. The same research group reported five galloyl-derivatives in plasma after 6 weeks of mango supplementation [4]. Collectively, only a few metabolites have been reported in plasma after MP intake and mangiferin has never been detected in previous studies.
Therefore, the first objective of this study was to characterize and investigate the absorption/kinetic profile of mango (poly)phenols and their metabolites in plasma over 24 h after MP consumption.
Black pepper (piperine), vitamin C (VC) and vitamin E have all been documented to enhance the absorption of polyphenol compounds when added to foods and beverages [22,23]. Vitamin C enhanced the absorption of green tea polyphenols in humans [24] and worked synergistically with oat (poly)phenolics and almond skin flavonoids to protect low density lipoprotein (LDL) against oxidation in a hamster and human model [25,26]. Vitamin C is inherent to the mango fruit; however, amounts may not be sufficient to stabilize polyphenols during digestion and transit through the gastrointestinal tract to enhance absorption in the small intestine or help maintain structural integrity for microbial metabolism in the large intestine. So, the second objective of this study was to test the hypothesis that concomitant intake of VC with MP would enhance absorption of mango (poly)phenols by increasing the number and/or concentration of mango (poly)phenolic metabolites.
Our previous work with berries indicates that regular intake or exposure to fruit polyphenols increases concentration of some metabolites in blood [27][28][29][30]. Likewise, increased excretion of certain mango metabolites was observed in urine after a 10-day intake of MP, of which some were microbial-derived [20]. Therefore, the third objective of this study was to assess the influence of daily intake of MP for 14 days on (poly)phenolic metabolite profiles.
The potential health benefits of various mango polyphenols are well documented in previous studies. For example, gallotannins and generated metabolites from intestinal microbial catabolism have been shown to possess anti-obesogenic effects in human and mouse models [4,33] and antibacterial effects in vitro [34,35]. Therefore, understanding the metabolic fate of mango polyphenols and their PK (pharmacokinetic) profiles after consumption will facilitate interpreting their roles in providing health benefits in humans.
Acute Study
The objective of the acute 24 h study was to characterize and determine PK parameters of mango polyphenols and the generated metabolites in human plasma after a single, one-time intake of a MP beverage. The acute study also aimed to evaluate the potential of VC to enhance absorption of MP polyphenols. The (poly)phenolic metabolites were identified using UHPLC-Q-TOF-MS in the pooled plasma (n = 13) at time points 0, 2, 8 and 24 h. A total of 18 (poly)phenolic metabolites were characterized in the pooled plasma samples at 2, 8 and 24 h after MP beverage intake (Table 1). For the acute part of the study the quantitative analysis was conducted for 11 (poly)phenolic compounds and their metabolites in plasma. The mean plasma concentration vs. time profiles (0−24 h) are reported for 10 metabolites that were detected above the limit of quantitation (LOQ): gallic acid (C1), galloyl glucose (C2), methylgallic acid (C3), methylgallic acid sulfate (C4), ferulic acid hexoside (C5), methylpyrogallol sulfate isomer 1 (C6), methylpyrogallol sulfate isomer 2 (C7), pyrogallol sulfate (C8), methylpyrogallol glucuronide (C9) and catechol glucuronide (C10), after ingestion of MP, MP + VC and VC test beverages ( Figure 1). The C max and AUC 0-24h values of these 10 metabolites were significantly higher in plasma after MP and MP + VC beverages intake compared to VC beverage intake only except for methylpyrogallol glucuronide (C9) which failed the normality test for C max during statistical analysis and had no significant differences in AUC 0-24h among treatments (Table 2). Mangiferin (C11) was detected but was below the LOQ. The pharmacokinetic curve of mangiferin is reported as the area vs. time profile (0−24 h) instead of mean plasma concentration vs. time profile ( Figure 2). Mangiferin had significantly higher content in plasma (as observed from areas) after MP and MP + VC beverages intake compared to VC beverage intake alone ( Table 2). The presence of gallic acid and mangiferin in plasma after MP beverage intake was confirmed by matching the MRM (multiple-reaction monitoring) ion transitions of the gallic acid and mangiferin standard with the compounds detected in the plasma. The identification of plasma metabolites including galloyl glucose, methylgallic acid, methylgallic acid sulfate, ferulic acid hexoside and methylpyrogallol glucuronide was confirmed by UHPLC-Q-TOF-MS and previous literature reports [2,20]. In general, (poly)phenolic compounds undergo structural modification before being absorbed into the blood. Compounds that escape absorption from the small intestine proceed to the colon where they are converted to various small molecules (phenolic acids, valerolactones, urolithins, etc.) by micro-organisms present in the lower bowel [36,37]. The metabolic products from the colon and the deconjugated (poly)phenols and aglycone structures from the upper digestive tract undergo phase II metabolism in the small intestine, liver and/or kidney resulting in methylated, glucuronidated and sulfoconjugated metabolites [38,39], while phase I metabolism (oxidation/reduction reactions) occurs to a lesser extent [40]. The resulting metabolites circulate in the blood and are transported to various body tissues and organs. Finally, the majority of metabolites are excreted in the urine by the kidneys [41]. Similar to mango pulp composition, most of the quantified compounds in plasma after acute supplementation of mango were gallic acid derivatives ( Table 2, Figures 1 and 2). Free gallic acid, galloyl glucose and gallic acid released from gallotannins were absorbed within 1-2 h and formed methyl, sulfate and glucuronide metabolites. Compounds such as gallic acid and its derivatives (C1-C4), ferulic acid hexoside (C5) and mangiferin (C11) reached their maximum concentration/area in plasma within 1-2 h after mango intake, and were cleared from the bloodstream within 6-8 h, suggesting absorption from the small intestine. On the other hand, several other metabolites (C6-C10) peaked at a much later time (8-10 h), suggesting microbial metabolism and absorption from the lower bowel. Pyrogallol is a major microbial metabolite of gallotannins [42] and gallic acid [43]. Several studies support a colonic origin of pyrogallol and its derivatives [21,33,[44][45][46][47]. The conjugates of pyrogallol (C6-C9) achieved maximum concentrations in plasma between 6-10 h after mango intake. Catechol glucuronide (C10) showed a similar absorption pattern as pyrogallol derivatives. Inter-individual variability (high standard error) was observed in some metabolites especially those derived from gut microbial action possibly due to differences in the intestinal flora of individuals.
The enhancement of the absorption of mango polyphenols in the presence of VC was also explored in this acute study. There was an increase in the concentrations/areas of five metabolites, including gallic acid (C1), galloyl glucose (C2), methylgallic acid (C3), methylgallic acid sulfate (C4) and mangiferin (C11) after intake of MP + VC beverage compared to MP beverage alone (Figures 1 and 2); however, the differences were not statistically significant ( Table 2 and Figure 2). The polyphenols are prone to autoxidation when exposed to slightly basic environments such as the small intestine [48]. Vitamin C as an antioxidant may prevent the oxidation of mango polyphenols to some extent, such that simultaneous intake of mango with VC may reduce the gastrointestinal degradation of mango polyphenols. Previous studies have reported enhanced absorption of green tea polyphenols with VC. An increased absorption of epigallocatechin (EGC) and epigallocatechin gallate (EGCG) were observed in vitro [49], in vivo [50] and in humans [51] when green tea or its extracts were combined with VC-rich mixtures. In this study, even though higher concentrations/areas were observed for some metabolites in the MP + VC group compared to the MP group, the differences were not statistically significant. Further research with a higher dose of VC or combination with other bioactive compounds such as piperine [22] is needed to determine if dose or compound type could be the factors affecting absorption of (poly)phenolic compounds.
Short-Term 14 Day Mango Feeding Trial
In this part of the study, metabolite pool changes after daily intake of MP for 14 days were assessed. As noted in the acute study, several MP polyphenols are subjected to gut microbial metabolism. We hypothesized that with consistent daily intake/exposure to these polyphenols, an increase in generated metabolites would be observed due to an increase in microbial population or upregulation of the microbial mechanisms metabolizing the consistently available substrate.
A total of 166 (poly)phenolic metabolites were quantified in urine (Appendix A, Table A2). The compound which showed the highest urinary excretion when compared to fasting baseline urine was pyrogallol sulfate isomer (∆ = 1415.1 ± 955.2 nmol/L for 24 h fasting sample, ∆ = 5298.0 ± 56.6 nmol/L for 15th day fasting sample). Similarly, Barnes et al. (2016) observed significantly increased excretion of pyrogallol sulfate in human urine after 10 days of MP intake (cv. Keitt, 400 g/day) [20]. Most of the compounds quantified in urine were detected in the plasma. However, we were not able to quantify them in the plasma due to their low concentrations (<LOQ).
To get a better understanding of the changes in the polyphenolic metabolite pools after 14 days consumption of MP, the (poly)phenolic compounds quantified in urine samples were divided into 10 classes: benzoic acid derivatives, phenylacetic acid derivatives, phenylpropanoic acid derivatives, benzaldehyde derivatives, pyrogallol derivatives, catechol derivatives, hippuric acid derivatives, cinnamic acid derivatives, valerolactone derivatives and others (Appendix A, Table A2). The concentrations of polyphenol classes in urine were compared at baseline (0 h, before MP beverage intake), 24 h fasting (24 h after single-time MP beverage intake) and 15th day fasting (24 h after 14 days of MP beverage intake) ( Figure 3, Appendix A, Table A2). The concentration increase of pyrogallol derivatives was highest in the 15th day fasting urine samples (3.6% increase) compared to 24 h fasting urine (p < 0.05). Minor increases (<1.0%) were also observed in other classes of compounds ( Figure 3).
These results are in agreement with previous studies on mango conducted by the Talcott group where they observed significant (p < 0.05) increases of pyrogallol and benzoic acid derivatives in urine including methylpyrogallol sulfate, pyrogallol sulfate and methylgallic acid sulfate after consumption of MP [20,52,53]. The increased excretion of mango (poly)phenol (mainly gallotannins) metabolites could be due to the adaptive increase in microbial metabolism after 14 days of daily intake of mango polyphenols [4,33,54]. Generated metabolites support the growth of specific bacterial species, which in turn enhance the production of certain metabolites suggesting reciprocal interaction between them. Several gut microbial species have been associated with gallotannins catabolism. For example, colon microorganisms such as Lactobacillus plantarum, Streptococcus galloylitcus, Aspergillus oryzae and Lactococcus lactis can utilize gallotannins and produce gallic acid, pyrogallol and catechol [47,55]. Previous studies in humans demonstrated that repetitive mango supplementation can increase the abundance of pyrogallol-producing microbiota, e.g., Aspergillus oryzae and Lactococcus lactis, and decrease the abundance of Bacteroides thetaiotaomicron and Clostridium leptum [47,53]. Additionally, gallotannins and their metabolites may inhibit growth of other bacteria such as Bacteroides fragilis, Escherichia coli, Enterobacter cloacae, Salmonella typhimurium, Salmonella aureus, etc. [34,35,56], possibly increasing community space for blooming microbiota that can utilize gallotannins and increasing net metabolite concentrations. Enhanced microbial efficiency to metabolize polyphenols may also influence concentration of metabolites [57] although this may not be only true for mango polyphenols, but also for non mango-specific metabolites (i.e., hippuric acid).
Inter-Individual Variability
The inter-individual variability in (poly)phenolic compounds and their metabolites was calculated for baseline (0 h), 24 h fasting and 15th day fasting urine samples after MP beverage intake ( Table 3). The percentage of the coefficient of variation (%CV) can show the extent of variability in relation to mean of the population. The highest inter-individual variability in terms of total polyphenols excreted in urine was observed at baseline (172%CV) which decreased tremendously after acute (64%CV) and 14 days (72%CV) of mango intake. The inter-individual variability was also determined in terms of classes of (poly)phenolic metabolites. Benzaldehyde derivatives (217%CV), pyrogallol derivatives (179%CV) and catechol derivatives (182%CV) had the highest %CV in urine at baseline (0 h) which decreased to 57%CV, 81%CV and 73%CV, respectively in 15th day fasting urine samples. On the other hand, the %CV of cinnamic acid derivatives, one of the major phenolic acid groups in mango pulp [58], increased from 68%CV to 136%CV after 14 days of daily intake of mango compared to baseline (Table 3). Inter-individual variability has been documented previously in metabolite studies and may be attributed to a number of factors including but not limited to the differences in body weight/health status, the composition of individual gut microbiota [59,60], habitual dietary intake as these were free living individuals, host genetics including genetic polymorphism of cytochrome P450 enzymes resulting in an altered expression and function of individual enzymes, and work and living/lifestyle environments, among other influences [61,62].
In this study, we identified and quantified (poly)phenolic compounds in MP, as well as their metabolites after MP consumption in human plasma and urine with advanced instruments such as UHPLC-Q-TOF-MS and UHPLC-QQQ-MS. With our crossover design, we reduced the influence of confounding covariates because each subject served as their own control. However, the study has some limitations. The standards of most metabolites (glucuronides, sulfates etc.) were not commercially available at the time when the study was conducted, so those metabolites were quantified either with the respective parent compounds or with the standards that share similar structures or molecular weight with them. This may lead to differences between their actual concentrations and the reported values.
In summary, this study provides comprehensive characterization of (poly)phenolic metabolites generated after mango consumption in humans both in acute and repetitive intake settings. In addition, this is the first study to explore the effectiveness of the addition of VC for enhancing the absorption/metabolism of MP polyphenols. Understanding the metabolic fate and PK parameters of polyphenols from mangoes will aid in developing future research for assessing potential health benefits associated with mango consumption.
Ethics, Study Design and Study Subjects
The research was conducted at the Clinical Nutrition Research Center (CNRC), Illinois Institute of Technology (IIT) (Chicago, IL, USA). The study was approved by the Institutional Review Board (IRB) and the trial was registered at clinicaltrials.gov (registration number: NCT03365739). All subjects signed the IRB approved informed consent form prior to the start of any study-related procedures. This was a two-part human clinical trial including an acute (24 h) and short-term (14 days) evaluation of MP intake. Thirteen healthy subjects were enrolled and completed both parts of the study protocol (Table 4, Figure 4). Subjects were nonsmokers and were not taking any supplements or medications (i.e., laxatives, proton pump inhibitors etc.) that would interfere with study outcomes. Subjects did not have documented atherosclerotic disease, digestive disorders, inflammatory disease, diabetes mellitus, or other systemic diseases. They did not have any allergy or intolerance to test beverages and study foods. Females of reproductive age were monitored, avoiding the menstruation phase of their menstrual cycle for study day visits.
Acute 24 h Trial
The acute trial was a randomized, 3 arm, within-subject crossover study design ( Figure 5A). The trial initiated with 3 days of food record collection to assess background (pre-study) dietary intake followed by counseling to adhere to a diet devoid of mango or its parts and relatively low in polyphenol-rich beverages/foods, which was maintained throughout the duration of the trial. Subjects were also provided a list of "Foods to Avoid" and alternate options to avoid high polyphenol foods and beverages, particularly for the 24 h before each study day, such as avoidance of coffee, tea, caffeinated products and alcohol. In general, subjects were encouraged to follow their "usual" diet with the exception of foods/beverages that may interfere with the outcomes of the study. Food intake diaries and, phone call and email reminders helped subjects maintain compliance throughout the study period. After an initial 7-day run-in period on the limited polyphenol diet, subjects were assigned to a randomization sequence providing 1 of 3 study test beverages (recipes provided in Appendix A, Table A3) on three separate days in a random order: MP (500 g), MP (500 g) + VC (100 mg) or VC (100 mg) beverages. On each study day subjects arrived fasted and well hydrated. After standard admission procedures were completed (e.g., review of compliance with dietary requirements for 3 days prior to study day, dinner meal intake, usual sleep patterns), a catheter was placed by a registered nurse in subjects' non-dominant arm and a fasting/baseline blood sample was collected (0 h). Subjects were provided with a standard breakfast meal 2 h after the study beverages. The breakfast meal was comprised of a buttermilk biscuit with butter and jelly, and scrambled egg white with shredded white cheddar cheese. After the 6 h blood collection, subjects ate a low polyphenol lunch consisting of dry roasted peanuts and fresh peeled cucumber with ranch dressing (Appendix A, Table A3). Each visit lasted~10.5 h and subjects were required to remain at the CNRC on the IIT Campus the entire time. After catheters were removed, subjects were given a controlled low polyphenolic dinner meal (provided at every study visit) to eat at home, with reminder instructions for overnight fasting and their scheduled time to report back to the CNRC the next morning for the 24 h blood and urine collection. Blood samples were collected at 0, 0.5, 2, 4, 6, 8, 10 and 24 h and urine samples were collected at 0 and 24 h ( Figure 5A). Blood samples were collected in vacutainers containing ethylenediaminetetraacetic acid (EDTA) and were centrifuged at 425 × g for 15 min at 4 • C to separate plasma. Spot urine samples were collected in urine collection cups, immediately placed on ice and then aliquoted into individual cryovials. All the samples were stored at −80 • C until analysis.
Short-Term 14-Day Trial
A 14-day feeding trial was conducted with the MP only to study the effect of repetitive intake of MP on (poly)phenolic metabolite profiles. Upon completion of the acute trial, subjects were instructed to continue on the low polyphenolic diet for 2 weeks while consuming MP beverages (provided by CNRC) daily in the morning (250 g MP + 50 g water) and evening (250 g MP + 50 g water). On day 14, subjects consumed both beverages (500 g of MP in total) in the morning and reported to the CNRC on the 15th day after an overnight fast to provide a fasting blood and urine sample ( Figure 5B). Blood and urine samples were collected as described above and stored at −80 • C until analysis.
Extraction of (Poly)phenolic Compounds from MP
MP (5 g) was extracted with 20 mL of extraction solution (acetone: water: acetic acid = 70:29.7:0.3 v/v) followed by two subsequent extractions with 10 mL extraction solution. The samples were vortexed for 30 s, followed by 10 mins sonication in iced water. The samples were kept in the dark for 10 mins before centrifugation at 8228 g for 10 min at 4 • C. The supernatants were pooled together after three extractions and final volume was made up to 45 mL. Extract (1 mL) was dried under nitrogen gas and reconstituted in 2 mL of acidified water (0.1% formic acid) for SPE procedure. The elution of the compounds was done with 1 mL of acidified methanol (0.1% formic acid). The eluent was collected and dried under nitrogen gas at room temperature. The dried samples were reconstituted in 200 µL of starting mobile phase (SMP) (0.1% formic acid, 5% acetonitrile in water) and centrifuged at 18,514 × g for 10 min at 4 • C. Supernatant was collected in amber HPLC vials before analysis.
Extraction of (Poly)phenolic Compounds from Plasma
SPE procedure was conducted to extract and concentrate the (poly)phenolic compounds and their metabolites from the plasma samples. Briefly, plasma samples were thawed on ice and 500 µL plasma was diluted with 1.5 mL of 0.1% formic acid in water with addition of internal standards (2-methylhippuric acid (2 ng/mL), phloridizin (1 ng/mL)) before being loaded on preconditioned SPE cartridges. The cartridges were washed with 0.1% formic acid in water (1 mL) after loading with plasma samples. The elution of the compounds was achieved by 0.1% formic acid in methanol (1 mL) and the eluent was dried under nitrogen gas at room temperature. The dried samples were reconstituted in 100 µL of SMP and centrifuged at 18,514× g for 10 mins at 4 • C. Supernatant was collected in amber HPLC vials before analysis.
Extraction of (Poly)phenolic Compounds from Urine
Urine (500 µL) was diluted and centrifuged at 18,514 × g for 10 mins at 4 • C. The supernatant was collected and filtered through 0.2 µm polypropylene syringe filter before HPLC analysis.
Identification and Quantification of (Poly)phenolic Compounds
The (poly)phenolic compounds were identified using an Agilent 1290 Infinity ultrahigh-performance liquid chromatography (UHPLC) system coupled with Agilent 6550 electrospray ionization (ESI) quadrupole time of flight (Q-TOF) mass spectrometer (MS) (Agilent Technologies, Santa Clara, CA, USA). The system was equipped with a binary pump with an integrated vacuum degasser, autosampler with a thermostat and column compartment with a thermostat. Spectra were recorded in negative mode with the following parameters: gas temperature 250 • C, gas flow 10 L/min, nebulizer pressure 35 psi, sheath gas temperature 300 • C, sheath gas flow 11 L/min and capillary 3500 V. MS scan method and Auto MS preferred list of the compounds was created based on personal compound database library (PCDL) and literature reports. Spectra were acquired in MS scan with the m/z range of 100-1200 and an acquisition rate of 2 spectra per s, and in MS/MS mode with the m/z range of 50-1200 and an acquisition rate of 4 spectra per s. The compound identification was based on the MS/MS fragmentation pattern, exact mass (PCDL), retention time match with available standards and previous literature reports. Compounds with mass error less than 5 ppm and/or retention time match with the standards were considered for identification. The data were analyzed using the Mass Hunter Qualitative Analysis software (version B.06.00, Agilent Technologies, Santa Clara, CA, USA). Pooled plasma (n = 13) samples were analyzed at 0, 2, 8 and 24 h for identification of (poly)phenolic metabolites.
A UHPLC system coupled with a 6460 Series Triple Quadrupole (QQQ) (Agilent Technologies, Santa Clara, CA, USA) was used for quantitative analysis. The ESI conditions were the same as those used in the UHPLC-Q-TOF analysis. The quantification of the compounds was conducted by multiple-reaction monitoring (MRM) transitions. Spectra were recorded in both positive-and negative-ion mode with capillary voltage of 4500 V and drying gas flow rate of 9 L/min at 200 • C. The sheath gas temperature and flow rate were 300 • C and 11 L/min, respectively. Standards were optimized for collision energies, fragmentor voltages and MRM transitions using Mass Hunter Optimizer. The MRM transition for metabolites were created based on the results from UHPLC-Q-TOF-MS. Standards for plasma analysis were prepared in blank plasma (charcoal-stripped human plasma obtained from Bioreclamation IVT) for matrix match and for urine analysis were prepared in SMP.
(Poly)phenolic compounds and their metabolites in plasma and urine were separated on a Pursuit 3 PFP column (150 × 2.0 mm, 3.0 µm, Agilent Technologies) equipped with a Pursuit MetaGuard column (10 × 2.0 mm, 3.0 µm, Agilent Technologies) at a constant temperature of 35 • C using our previously established method [29]. The flow rate was maintained at 0.4 mL/min and the injection volume was 5 µL. The mobile phase consisted of acidified water (0.1% formic acid) (A) and acidified acetonitrile (0.1% formic acid) (B). The gradient for the separation of compounds was as follows: 5 to 5% B from 0 to 1 min; 10% B at 3 min; 15% B at 7 min; 15% B at 9 min; 20% B at 10 min; 20% B at 11 min; 25% B at 12 min; 30% B at 13 min; 30% B at 14 min; 95% B at 15 min; and back to 5% B at 16 min. The column was re-equilibrated to the initial mobile phase conditions for 4 min before the next injection. The data were analyzed using the Mass Hunter Quantitative Analysis software (version B.07.00, Agilent Technologies, Santa Clara, CA, USA).
Pharmacokinetic and Statistical Analysis
The absorption and elimination of 11 major mango metabolites over 24 h was determined after MP, MP + VC, and VC intake. The plasma concentration/area at each time point was determined by the mean of 13 subjects. The time vs. concentration curves and the time vs. area curves were created using GraphPad Prism (version 8.2.1). C max (nmol/L) is defined as the maximum concentration of (poly)phenolic metabolites in plasma between 0−24 h after test beverage intake. T max is the time (h) when C max was achieved. Wilcoxon signed-rank test was used for T max analysis due to non-normal distribution. Area under the plasma concentration time curve (AUC 0−24h , nmol·h/L) was calculated by the linear trapezoidal method using Microsoft Excel 2013, version 15 [63].
Subject characteristics were analyzed from data collected at the screening visit and tabulated using descriptive statistics. Results are presented as numbers and percentages, as appropriate. For continuous variables, normality was assessed using Shapiro-Wilk tests, skewness and kurtosis. Data not conforming to normal distribution patterns were log transformed prior to analysis and noted accordingly. Mixed-model repeated measure analysis of covariance (ANCOVA) was performed on each quantitative outcome variable to test main effects of test beverages and time (hour) using PROC MIXED via Window PC-SAS (version 9.4; SAS Institute Inc, Cary, NC). Multiple comparisons within and among test beverages (MP, MP + VC and VC) over 24 h were performed by mixed model statistical significance (p < 0.05). A two-tailed distribution, paired t-test (Microsoft Excel 2013, version 15) was conducted for a short-term 14-day feeding trial to compare the (poly)phenolic metabolite concentrations in fasting urine collected after acute (24 h) and 14-day MP intake (15th day). The pharmacokinetic (PK) curves of the (poly)phenolic metabolites with plasma concentrations below LOQ are presented as area vs. time instead of area vs. concentration curves.
The results of the statistical analysis are presented as mean ± standard error unless indicated otherwise. Statistical significance was based on 2-sided test beverage comparison at the 5% significance level under a null hypothesis of no difference between test beverages.
Funding:
We are grateful to the National Mango Board for funding this study.
Conflicts of Interest:
The authors have declared no conflict of interest. All these compounds were tentatively identified based on the Agilent's personal compound database library (mass error < 5 ppm). * Identity of compounds confirmed by standards. | v3-fos-license |
2017-04-14T21:30:29.834Z | 2012-09-11T00:00:00.000 | 1464420 | {
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} | pes2o/s2orc | Food Sources and Intake of Calcium in a Representative Sample of Spanish Adults
The present study aimed to assess calcium intake, dietary sources of this nutrient and its adequacy with respect to the dietary reference intake (DRI) in a representative sample of Spanish adults. In this study 418 adults (18 to 60 years) from 15 Spanish provinces were studied. Energy and nutrient intake were determined using a 24-hour recall questionnaire for two days. Adequacy of calcium intake was assessed using the established DRI for calcium. Anthropometric data (weight and height) were measured and the body mass index was calculated. Seventy eight percent of the participants in the study did not meet the DRI for calcium. Additionally, 33.7% of the participants did not meet the 67% of the DRI. The daily intake of calcium was 916.6 ± 288.1 mg/day, which represented the 81.3% of the DRI of calcium. Interestingly , subjects who had higher intake of calcium were taller. Additionally, it has been observed that individuals with normal body weight (BMI < 25 kg/m 2) had higher intakes of dairy products in comparison with overweight and obese individuals (BMI ≥ 25 kg/m 2). The main food sources of calcium were dairy products (58.7% of calcium), cereals (13.6%) and vegetables (6.5%). Less than 1% (0.5%) of the calcium intake came from dietary supplements. It was observed that individuals who met the DRI for calcium had a significantly higher intake of dairy products (551.3 ± 240.4 g/day) than individuals who did not meet the DRI of calcium (305.0 ± 150.3 g/day). Calcium intake was inadequate in this sample of the adult Spanish population. Therefore, an increase in the consumption of dairy products, as well as cereals , vegetables and food items fortified with calcium seems to be necessary to achieve an adequate intake of calcium and to prevent diseases caused by calcium deficiency.
Introduction
Calcium plays an important role strengthening the bones and preventing bone related diseases such as osteoporosis [1].An adequate intake of calcium has been associated with the prevention of obesity, hypertension [2][3][4], type 2 diabetes [2,4,5] and dyslipidemia [2].Several studies performed in Spain and Europe, have observed that calcium intake was generally below the dietary reference intake (DRI) due principally to a low consumption of dairy products [6][7][8][9].Although dairy foods are valuable from a nutritional standpoint and epidemiologic studies have been suggested that they may have favorable effects on body weight in adults [10], recently tendencies indicate that the dairy consumption is only justified during infancy [10][11][12].These tendencies have been led to reduce or avoid the dairy products consumption in some groups of adults, which may negatively affects the intake of different nutrients and particularly the calcium intake [10,12].There are few studies that have assessed calcium intake in a representative sample of the Spanish population.Therefore, the main aim of the present study was to assess calcium intake and its adequacy with respect to the DRI.In addition, the main dietary sources of calcium were identified.
Material
A group of 418 adults (196 men and 222 women) with ages ranging from 18 to 60 years were selected as a representative sample of the Spanish adult population as whole.All data were collected from January to September 2009.
The sample size was calculated, taking into account data provided by the Spanish Intersalt study [13], to be representative for each gender, assuming a dropout rate of 25%.The initial sample size required was set at 406 participants.Sampling was performed in fifteen randomly selected provinces (selected with the proviso that the great majority of Spain's autonomous regions were represented), including the capital city of each province and a semiurban/rural town (randomly chosen).The total number of cities/towns included was 30.The participants on each sampling point were divided into six subgroups, taking into account their gender (male/female) and age (18 -30, 31 -44 and 45 -60 years).
All selected participants were healthy and lived in their own homes; neither hospitalized people nor those living in institutions were included in the present study.Individuals with a diagnosis of diabetes, hypertension or renal disease, or who had been prescribed diuretics, were excluded.
The present study was conducted according to the guidelines laid down in the Declaration of Helsinki and all procedures were approved by the Ethics Committee of the Faculty of Pharmacy (Universidad Complutense de Madrid, Madrid, Spain).Written informed consent was obtained from all subjects.
Participants were randomly selected among the residents of each population and were invited to take part in the study via telephone (or in person in some of the rural areas).In each of the 30 sampling areas, when a participant was excluded at any site, or when participation was declined, another person of the same gender and age group was recruited.A group of 1835 individuals were initially recruited, where only 492 (26.8%) accepted to take part in the study.From these, 74 individuals were excluded.The final study sample size was 418 participants (53.1% women) (22.8% of the originally contacted sample).
Methods
All participants received the same instructions to complete the questionnaires.All the measurements were carried out by trained personal following the same protocol.
Dietary Information
Food intake was determined using the 24-hour recall method on two consecutive days [14].Participants were asked to write all foods consumed on the preceding days (where appropriate the brand of these foods were also registered).They indicated the size of the servings consumed (approximate weights or household measures [cups, bowls, plates, etc.]), whether these weights or sizes corresponded to raw or cooked food, with or without bone or skin, etc.In addition, participants completed a questionnaire where they indicated the consumption of dietary supplements and manufactured dietary foods.
The energy and nutrient intake were then calculated using the DIAL software [15], which uses the Food Composition Tables of the Department of Nutrition, Complutense University of Madrid [16].The participants' calcium intake was compared with the DRI for calcium [17].The number of servings of the different food groups consumed by participants was calculated by dividing the grams of the food consumed by the size of the standard serving [16,18], and these were compared with the recommended servings described in the Dietary Guidelines for the Spanish population [19].
The dietary data have been validated considering the discrepancy between each individual energy intake and the estimated energy expenditure.The estimated energy expenditure was determined taking into account the participants' weight, sex and age (for the calculation of the basal metabolic rate) and the physical activity coefficient.The discrepancy was determined with the following equation:
Theoretical energy expenditure energy intake 100 Theoretical energy exp enditure
In individuals with stable body weight, energy intake should be similar to the estimated energy expenditure and the gap between these values can be used as an indicator of undervaluation/overvaluation of intake.In some cases, individuals that take part in a nutritional study modify their nutritional intakes.Although the recorded intake is real, the discrepancy in energy intake and expenditure may suggest a departure from the usual intake [20].
Once calcium intake has been established, the food sources of calcium were assessed to identify the most important sources of the mineral.The contribution of each food to total calcium intake has been calculated by adding the amount of calcium provided by each specific food and dividing by the total intake of calcium, all multiplied by 100 [21].
Anthropometric Information
Weight and height were determined using a digital electronic scale (Seca Alpha, GmbH & Company, Igni, France; range 0.1 -150 kg, precision 100 g) and a Harpenden digital stadiometer (Pfifter, Carlstadt, NJ, USA; range 70 -205 cm, precision 1 mm).For both measurements, participants were barefoot and wore minimal clothes, in agreement with the norms set up by the World Health Organization (WHO) [22].Body mass index (BMI) was calculated and used to classify the individuals following the WHO classification (normal weight: BMI < 25 kg/m 2 , overweight: BMI ≥ 25 kg/m 2 and BMI < 30 kg/m 2 and obesity: BMI ≥ 30 kg/m 2 [22,23].
Physical Activity Study
Participants completed two physical activity questionnaires, describing the time spent on the different daily activities, such as sleeping, eating, walking, resting, exercising, one during the weekdays and other during the weekend [24].An activity coefficient was established for each participant by multiplying the time spent in each activity by coefficients related with the activity type and dividing the result by 24 (hours) [24,25].A coefficient of 1 for sleeping and resting, 1.5 for very light activities (activities that can be done sitting or standing), 2.5 for light activities (e.g.walking), 5 for moderate activities (e.g.playing tennis, skiing and dancing) and 7 for intensive activities (e.g.cutting down trees and playing basketball).The weekday coefficient was multiplied by 6, the Sunday coefficient was then added to the weekday result and the total was divided by 7.This provided a final activity coefficient for each participant, which was multiplied by the basal energy expenditure estimated with the WHO formulae [25,26] to provide the estimated daily energy expenditure for each participant.
Statistical Analysis
All data were presented as the mean ± standard deviation.The differences between genders were assessed using Student t-test with quantitative data where the data was normally distributed, using the Mann-Whitney test otherwise.Chi-square test was used to assess the differences between genders in the qualitative variables.Two-way ANOVA test was used to assess the differences within the studied variables using gender and the achievement of the DRI of calcium.Due to the inter-correlation of energy intake with nutrients, an adjustment of the nutrients using the residual method of Willet [27] was performed.The statistical significance was set at P < 0.05.All calculations were made using the statistical software Rsigma Babel (RSIGMA 2.0 BABEL, 1992, Horus Hardware; Madrid, Spain)
Results
Table 1 shows the participants' characteristics.There were no differences in age between gender groups.However, males' height, weight and BMI were significantly higher compared with the female participants.Interestingly, 48.9% of the study sample presented weight excess (35.3% overweight and 13.6% obesity).Male participants had higher percentages of weight excess problems (overweight: 43.9% and obesity: 16.4%) compared with the female participants (overweight: 25.7% and obesity: 11.3%).The mean daily intake of calcium was 916.6 ± 288.1 mg/day), which represented the 81.3% of the DRI for calcium.Seventy-eight percent of the participants did not meet the DRI for calcium, with intakes lower than the 67% of the DRI's in 33.7% of the participants.Protein intake was 89.4 ± 16.4 g/day, which met in excess the DRI for this nutrient.However, the calcium:protein ratio (10.4 ± 3.2 mg/g) and the calcium:phosphorus ratio (0.62 ± 0.15 mg/mg) did not meet the established optimal values of 20 mg/g for calcium:protein ratio and 1 -2 mg/mg for calcium:phosphorus ratio in 98.5%, 98.8% of the participants, respectively.
Participants were classified whether they met the DRI for calcium intake or not.Participants who met the DRI for calcium intake and those who did not meet the DRI had mean calcium intakes of 1300.7 ± 231.0 mg/day and 813.5 ± 203.7 mg/day, respectively.The calcium:protein and calcium:phosphorus ratios were also higher in the participants who met the DRI for calcium intake compared to the participants who did not meet the DRI.Interestingly, the study revealed that adults with an adequate intake of calcium were taller than the ones with calcium intakes lower than the DRI (Table 3).Values are mean and standard deviations; *** P < 0.001 (differences by gender).
There were also differences between the participants who met the DRI for calcium intake and those who did not meet the DRI in relation with the dietary sources of calcium and the intake of other nutrients.Participants who met the DRI for calcium consumed more servings of dairy products (3.9 ± 1.3 servings/day) than participants who did not meet the DRI (2.1 ± 1.1 servings/day).Additionally, participants who met the DRI for calcium intake consumed more fish and less meat and pulses than individuals who did not meet the DRI (Table 4).
Discussion
In the present study, mean calcium intake of the participants was below the DRI in 70% of the males and 86% of females.Calcium intake (917 ± 288 mg/day) was similar to those observed in the Spanish Encat study in 2002 [28] (830 ± 200 mg/day in males and 778 ± 170 mg/day in females).Additionally, other studies in Europe, such that performed by Vriese, et al. [29] in Belgian adults or the study carried out by Hulshof, et al. [30] in Dutch young adults had similar calcium intakes.
Calcium uptake is affected by the intake or the bioavailability of other nutrients such as phosphorus and protein.
In any growth situation or to prevent disease, both calcium and phosphorus are needed to support an optimal increase in bone mass.
Lower calcium:phosphorus ratios are associated with reductions in the bioavailability of calcium [31].
However, an excess intake of calcium in relation with phosphorus (1.6 -1.8) may reduce the absorption of calcium and other minerals (magnesium, manganese, zinc), resulting in reduced bone mineral densities [32].The calcium:phosphorus ratio should be 1:1 [33].
The present study indicated that 98.8% of the sample had a calcium:phosphorus ratio lower than this value.Low calcium:phosphorus ratios may interfere with calcium metabolism, decreasing the absorption of calcium [34].A study carried out by Teegarden, et al. [35] showed that higher intake of calcium (1200 -1400 mg), resulting in calcium:phosphorus ratios of 1.2 to 1.4, could increase bone mineral density and reduced the risk of bone fractures later in life.
Therefore, an increase of calcium intake combined with phosphorus intakes of 900 -1000 mg would provide the adequate protection against bone disease maintaining an optimal bone metabolism in adult individuals.
The high intake of proteins has been associated with urinary calcium losses, affecting calcium absorption and possibly bone mineral content and density [35].However, protein intakes below the DRI can be detrimental for bone formation and its conservation through adult life.
Several studies have associated high protein intakes with increased bone mineral content and density and reductions of hip fractures incidence [35,36].The present study indicated that the 98.5% of the sample had a calcium:protein ratio lower than 20 mg/g (10.4 ± 3.2 mg/g) [36], which reveals again a low intake of calcium associated with a high intake of proteins, which may have an adverse effect on the utilization of calcium and maintenance of bone mass.
Table 3. Dietary and anthropometric differences between adults who did not meet the dietary references intakes for calcium (<DRI) and those who met (≥DRI).
Interestingly, subjects who had higher intake of calcium were taller (Table 3).Although there are other variables influencing the anthropometry of humans, calcium intake and the intake of dairy products have been associated with higher bone mineral densities and content, and taller individuals [34,37,38].
Additionally, it has been observed that individuals with normal body weight (BMI < 25 kg/m 2 ) had higher intakes of dairy products when compared with overweight and obese individuals (BMI ≥ 25 kg/m 2 ).This may be caused by the restriction of dairy products consumption in overweight and obese individuals.However, some studies have observed that dairy product intake did not have a negative effect on body weight [39], and even helped overweight individuals to maintain a stable body weight and an adequate calcium intake [4].
The main sources of dietary calcium are dairy products, which was similar to other studies [8,40] noting the importance of calcium from dairy products in bone mineralization, which is why the contribution must be adequate.The intake of calcium is important to maintain an adequate bone mineral composition.The low intakes of calcium observed in the present study could be associated with low intakes of dairy products (below the recommended 2 -3 servings/day in 42.3% of the sample), as these provided almost 60% (58.7%) of the calcium within the diet (Table 2).It was observed that individuals who met the DRI for calcium had a significantly higher consumption of dairy products (551.3 ± 240.4 g/day) than individuals who did not meet the DRI of calcium (305.0 ± 150.3 g/day).In conclusion, calcium intake was inadequate in this sample of the adult Spanish population.Therefore, an increase in the consumption of dairy products, as well as cereals, vegetables and food items fortified with calcium seems to be necessary to achieve an adequate intake of calcium and to prevent diseases caused by calcium deficiency.
Table 4 . Food consumption differences between adults who did not meet the dietary references intakes for calcium (<DRI) and those who met (≥DRI).
Values are mean and standard deviations; Two-way ANOVA was performed, taking into account gender influence (G) and the meeting of DRI for calcium (DRI); * P<0.05; ** P<0.01; *** P<0.001. | v3-fos-license |
2018-04-03T00:22:48.045Z | 2004-05-28T00:00:00.000 | 11140671 | {
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} | pes2o/s2orc | Coupling of Folding and Binding of Thymosin (cid:1) 4 upon Interaction with Monomeric Actin Monitored by Nuclear Magnetic Resonance*
Thymosin (cid:1) 4 is a major actin-sequestering protein, yet the structural basis for its biological function is still unknown. This study provides insight regarding the way this 43-amino acid peptide, mostly unstructured in solution, binds to monomeric actin and prevents its assembly in filaments. We show here that the whole backbone of thymosin (cid:1) 4 is highly affected upon binding to G-actin. The assignment of all amide protons and nitrogens of thymosin in the bound state, obtained using a combination of NMR experiments and selective labelings, shows that thymosin folds completely upon binding and displays a central extended region flanked by two N- and C-terminal helices. The cleavage of actin by subtilisin in the DNase I binding loop does not modify the structure of thymosin (cid:1) 4 in the complex, showing that the backbone of the peptide is not in close proximity
Cell locomotion or changes in cell shape are mediated by the rapid polymerization of actin in response to signaling. The source for this massive increase in F-actin is provided by a large reservoir of unassembled, "sequestered" actin, which is kept monomeric by interaction with G-actin-binding proteins (1). Thymosin 4 (T4), 1 first identified as the most abundant (300 M) actin-sequestering protein in human blood platelets (2,3) and neutrophils (4), was eventually shown, together with variants of the -thymosin family, to bind G-actin in all vertebrates (5)(6)(7)(8) and some invertebrates (9). In contrast to profilin, another G-actin-binding protein, which can either sequester actin when filament barbed ends are blocked by capping proteins or participate in barbed end assembly when barbed ends are uncapped (10), T4 acts as a simple sequestering protein that prevents G-actin association to both ends of actin filaments. It reacts passively to the changes in critical concentration for actin assembly while amplifying them. T4 essentially binds G-actin with a high specificity for the ATP-bound form (11), an affinity in the 10 6 M Ϫ1 range (3,11), and relatively slow association-dissociation kinetics that are indicative of a structural change of the complex after rapid equilibrium binding (12). Finally, in binding to G-actin, T4 slows down metal ion/nucleotide dissociation (13,14). This protein is composed of a single WH2 domain, which has recently been identified in 37 other proteins belonging to various organisms, and was shown to be an evolutionarily conserved actin-monomer binding motif (15). Interestingly, different WH2 domains were found to perform different functions in the regulation of actin monomeric concentration and actin assembly processes. Whereas -thymosins inhibit the assembly of actin filaments, the other WH2 domains characterized thus far display a profiling-like activity, promoting the assembly of actin filaments at the barbed end (16 -20). A fine structural and dynamic characterization of WH2 domain interfaces with actin is therefore essential to understand the functional variability of WH2 domains.
Whereas the structure and interface with actin of other G-actin binding proteins such as DNase I (21), gelsolin segment-1 (22), and profilin (23) are well known from crystallographic studies, T4 and, more generally, WH2 domains remain elusive. NMR studies have shown that T4 was mostly unstructured in aqueous solution, except for a short helical region (residues 5-16) at low temperature (24). Mutagenesis studies indicated that the ␣-helical structure of this region was required for binding to actin (25)(26)(27). Biochemical results based mainly on chemical cross-linking indicated that T4 bound G-actin in an extended conformation, with the N-terminal helical region (residues 1-19) making contacts with residues in subdomain 1 of actin, whereas the C-terminal region could be cross-linked to His 40 and Gln 41 of actin in subdomain 2 (12,28,29). According to these results, the ability of T4 to interfere with actin-actin contacts involved in filament assembly at both the barbed and pointed ends of the actin monomer was thought to account for its inhibition of polymerization via a simple steric mechanism and also for its competition with profilin and DNase I for binding to G-actin. Recent biochemical and thermodynamic studies of the binding of T4 to CaATP-actin and MgATP-actin, based mainly on fluorescence quenching of 5-(2acetylaminoethylamino)naphtalene-5-sulfonate conjugated to Cys 374 of actin upon T4 binding and on tritium exchange, also suggested that this small protein changed the conformation and structural dynamics of actin monomers (12).
Our study constitutes the first essential step for the under-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and structure factors ( standing of the molecular basis of G-actin sequestration by thymosin 4. A number of specificities of the biological system under study render its structural analysis especially challenging for both crystallography and NMR. The tendency of G-actin to self-assemble, even at low ionic strength, at concentrations greater than 50 -70 M, the high flexibility of T4, and the limited stability of the T4-G-actin complex at high concentrations have hampered crystallization of the complex thus far. These difficulties, combined with the large size of the complex (47 kDa), preclude 1 H, 15 N, 13 C NMR spectroscopy studies of the T4-actin complex that require long acquisition periods. Here we show that the assignment of the whole 15 N-1 H N correlation spectra of T4 bound to monomeric CaATP-actin and MgATP-actin is successfully performed using an alternative strategy based on appropriately chosen specific labels. Our results demonstrate that the whole T4 backbone is tightly bound to monomeric actin and that the structures of the peptide bound to CaATP-actin, MgATP-actin, and subtilin-cleaved actin are identical. The secondary structure of actin-bound thymosin 4 is obtained, and novel information is also provided about its fast dynamics using 1 H-15 N heteronuclear NOE experiments. A hypothetical model of the complex is proposed, based on biochemical data and our NMR results.
Thymosin 4 and Thymosin 10 -Recombinant thymosins 4 and 10 were bacterially expressed in Escherichia coli using pET3d vectors kindly provided by Dr. Helen Lu Yin (University of Texas Southwestern Medical Center). Bacteria (BL21(DE3) strain) were grown at 28°C in diverse media depending on the labeling: (a) [U- 15 N]AA T4 (AA was alternatively Thr, Lys, Leu, and Ser), M9 medium implemented with amino acids (40 mg/liter), except the one to be labeled. 50 mg/liter of the labeled amino acid was added when the optical density was equal to 0.2.
The expression of all T4 variants was induced by addition of isopropyl-1-thio--D-galactopyranoside when the optical density of the culture was 0.4. Bacteria were collected 2 h and 30 min later. The bacterial extract was treated with HClO 4 (up to 4%/v). The acid-soluble T4 solution was brought to pH 4, loaded on a 4 ϫ 25-cm RP 18 (Merck) column, eluted by 33% n-propanol in H 2 O, and further purified by high pressure liquid chromatography using two consecutive preparative columns (2.5 ϫ 25 cm, Hibar Lichrospher 100 RP-18E; Merck) and a 5-28% acetonitrile gradient.
Selective Labeling of Thymosin 4 -The selective labeling strategy was chosen to minimally perturb the culture conditions already used for the previous production of recombinant T4. In particular, the same bacteria strain was used. Because "pseudo-rich" conditions were used for bacteria growth, the protein yield was better than for uniform labeling, around 7 mg protein/culture liter.
Two main criteria were important for the choice of the labeled amino acid: (a) first, the frequency and position of the amino acid along the sequence; and (b) second, the possibility to specifically label the amino acid using the BL21(DE3) bacteria strain. This second criteria can be quite restrictive because a number of metabolic leakages and transaminase activities can lead to partial or complete dilution of the labeling for a number of amino acids (33,34). Considering the first criteria, the four amino acids Lys, Leu, Thr, and Ser were particularly interesting.
Lysine is the most frequent amino acid in the sequence (Lys 3 , Lys 11 , Lys 14 , Lys 16 , Lys 18 , Lys 19 , Lys 25 , Lys 31 , and Lys 38 ); it is nicely spread along the whole sequence and is part of the LKKTET fragment (residues 17-22 in T4), which is known to be a consensus recognition motif for actin-binding proteins. Threonine and leucine were also selected for their presence in the consensus motif (Thr 20 , Thr 22 , and Thr 33 ; Leu 17 and Leu 28 ). Moreover, the assignment of Leu 28 is particularly difficult due to its sandwiched position between two prolines. Finally, serine was initially selected due to its distribution along the sequence (Ser 1 , Ser 15 , Ser 30 , and Ser 43 ). However, the specific labeling of this residue could not be obtained using the BL21 bacteria strain, and a partial uniform labeling was obtained instead. This is probably due to a significant metabolic leakage from serine to glutamate, which itself leads to a metabolic leakage toward all other amino acids (33). In the case of leucines, leakages were also observed, but to a much smaller extent, and two main peaks corresponding to Leu 17 and Leu 28 were observed in the HSQC spectra of free [ 15 N]Leu T4. The most intense secondary peaks correspond to isoleucines Ile 9 and Ile 34 . These were, however, beyond detection in the complex.
NMR Samples and Experiments
NMR Samples-Thymosin NMR samples consisted of 15 N-labeled thymosins at a concentration of 1-2 mM in a 4 mM phosphate buffer (pH 6.9) containing 10 M ATP, 20 M Ca 2ϩ , 10% D 2 O, and 0.01% sodium azide. NMR samples of complexes between actin and thymosins were prepared by mixing 20 ml of 47 M actin with thymosins taken from a 2 mM stock solution up to a 1:1 molar ratio, occasionally with a small excess of thymosins. The proteins were then concentrated up to 350 -500 M complex using a Centriprep 30 device (Amicon) and centrifuged at 3000 rpm for 45 min. The complexes were stored in G buffer containing 5 mM Tris-Cl, 0.1 mM CaCl 2 , 0.2 mM ATP, 0.2 mM dithiothreitol, 5% D 2 O, and 0.01% sodium azide. The final pH value was 6.9.
NMR Experiments-NMR experiments were performed on Bruker Avance 600 MHz and 800 MHz spectrometers equipped with 5-mm triple resonance gradient probes. Quadrature phase detection in the indirectly detected dimension was obtained via States-TPPI mode (35). A Watergate sequence was used in all experiments to achieve water signal suppression (36).
Assignment of 1 H, 15 N, and 13 C resonances of T4 free in solution was obtained using standard procedures from two-dimensional 1 H-1 H NOESY and total correlation spectroscopy experiments, three-dimensional 1 H-15 N NOESY-HSQC, and three-dimensional 1 H-15 N total correlation spectroscopy-HSQC experiments and HNCA, HN(CO)CA, HNCO, HN(CA)CO, CBCANH, and CBCA(CO)NH experiments. Mixing time for NOESY experiments was set to 120 ms. Two mixing times of 50 and 80 ms were used for total correlation spectroscopy experiments.
Pulse sequences used to determine 15 N R 1 (N Z ), R 2 (N XY ), and heteronuclear 1 H-15 N NOE values were similar to those described previously (41)(42)(43). R 1 (N Z ) experiments were performed with 10 relaxation delays (0.012, 0.048, 0.072, 0.144, 0.300, 0.420, 0.600, 0.840, 1.200, and 2.400 s). For R 2 (N XY ) experiments, a set of 10 experiments was acquired, with relaxation delays of 0.008, 0.024, 0.040, 0.080, 0.136, 0.200, 0.304, 0.480, 0.640, and 0.800 s. In the two sets of experiments, the points corresponding to different relaxation delays were acquired in an interleaved manner to avoid any bias that could arise from progressive shim degradation. For all experiments, carrier frequencies were set at 117 ppm in the F1 dimension ( 15 N) and 7.6 ppm in the F2 dimension. Spectral widths were 23 ppm in the F1 ( 15 N) dimension and 3.5 ppm in the F2 ( 1 H N ) dimension. Cross-peak intensities were determined from peak heights using the SPARKY peak picking routine (Goddard, T. D., and Kneller, D. G. SPARKY 3 Software, University of California, San Francisco, CA). The relaxation rate constants R 1 (N Z ) and R 2 (N XY ) were obtained from nonlinear fits to mono-exponential functions using the Levenburg-Marquardt algorithm (45). The quality of the fits and error estimations were obtained using established Monte Carlo procedures, with an experimental gaussian error set to 4 times the rms base plane noise level and using 500 synthetic data for each NH vector. 15 N NOE enhancements were obtained as the ratio of the peak heights in the spectra recorded with and without saturation of protons during the relaxation delay.
For 15 N-1 H HSQC experiments carried on T4-actin complex, carrier frequencies were set at 117 ppm in the F1 dimension ( 15 N) and 7.6 ppm in the F2 dimension. Spectral widths were 28 ppm in the F1 ( 15 N) dimension and 5 ppm in the F2 ( 1 H N ) dimension. The three-dimensional 1 H-15 N NOESY-HSQC experiment used for the assignment of T4 bound to actin was performed with a mixing time equal to 40 ms.
Measurements of the Binding of T4 to G-actin and Subtilisin-cleaved Actin
The affinity of T4 for actin or subtilisin-cleaved actin (subactin) was derived from the increase in actin tryptophane fluorescence ( exc , 295 nm; em , 350 nm) linked to the binding of T4 (46). Experiments were carried out in a Spex spectrofluorimeter at 20°C, using 2 M CaATP-G-actin or subtilisin-cleaved actin in G buffer containing only 20 M ATP to minimize the screen effect.
The relative increase in fluorescence, (F Ϫ F 0 )/(F max Ϫ F 0 ), was expressed as function of the molar fraction of actin in complex with T4, (TA)/(A 0 ), according to the following equation.
Alternatively, the affinity of T4 for subtilisin-cleaved actin could be derived from its sequestering activity, by measuring the amount of F-actin at steady state in the presence of different concentrations of T4 (25,47). The concentration of F-actin, C w , was derived from the fluorescence of pyrenyl-actin. As the T4-actin complex formed, C w decreased linearly with the total concentration [T 0 ] of T4, according to the following equation: in which [TA] is the concentration of T4-actin complex; C w 0 and C w are the mass concentrations of F-actin in the absence and presence of T4, respectively; C C is the critical concentration for F-actin assembly; and K T is the equilibrium dissociation constant of the T4-actin complex. The values of C C for actin and subtilisin-cleaved actin were independently derived from critical concentration plots. The value of K T was derived from the slope C C /(C C ϩ K T ) of the lines describing the linear decrease of C w with [T 0 ].
Finally, the affinity of T4 for actin and subtilisin-cleaved actin could also be derived from the inhibition of nucleotide exchange linked to T4 binding. The CaATP-subtilisin-cleaved actin 1:1 complex was prepared by eliminating free ATP from the solution by Dowex-1 treatment (48) and diluted to 2.2 M in G buffer containing no nucleotide, 10 M CaCl 2 (final concentration), and variable amounts of T4. At time 0, 5.8 M ⑀ATP was added to the solution, and the rate of ⑀ATP exchange for bound ATP was derived from the increase in fluorescence of ⑀ATP associated to its binding to actin ( exc , 350 nm; em , 410 nm). The process was mono-exponential, and the observed first order rate constant varied with the concentration of T4, reflecting the binding of T4 to subtilisin-cleaved actin in rapid equilibrium, as follows where k Ϫ and k Ϫ Ј are the rate constants for dissociation of CaATP from subtilisin-cleaved actin and T4-subtilisin-cleaved actin complex, respectively; [T] is the concentration of free T4; and K T Ј is the equilibrium dissociation constant of the T4-subtilisin-cleaved actin complex.
Building of a Model of the Thymosin 4-actin G Complex
Software and Hardware-The X-PLOR (49) software was run on a Macintosh G4 PowerBook. The graphical analyses were performed using either MOLMOL (50) run on a G4 PowerBook or Insight (Accelerys) run on an O2 Silicon Graphics work station.
Model Building-The initial actin structure was extracted from the Protein Data Bank entry 1atn.pdb (51). A thymosin initial structure was built manually using the Builder module of Insight software. According to NMR results, a helical geometry was imposed to the Met 6 -Asp 13 and Ile 34 -Ala 40 segments, whereas the Lys 18 -Ser 30 region was constrained to adopt an extended conformation. The and angles of residues 5, 18, 31, and 32 were randomized, and the structure was submitted to a short torsion dynamic to eliminate eventual bad van der Waals interactions. Distances of 30 Ϯ 10, 50 Ϯ 10, and 55 Ϯ 10 Å between residues 3 and 18, 18 and 38, and 3 and 38 were imposed during this first dynamic to obtain an initial structure already compatible with the previously identified cross-links between the peptide and G-actin (12,29). The two files were merged, and the thymosin was aligned and translated such that its main axis lies parallel to the actin plane at a distance of 30 Å above it.
Three sets of constraints were designed to drive the formation of the complex. The first set was introduced to maintain the helical geometry of the thymosin 6 -13 and 34 -40 segments. It was composed of 12 H N -O (1.8 -2.2Å) and N-O (1.8 -3.2Å) distance restraints, enforcing the ␣-helices hydrogen bonds, and of 12 (47 Ϯ 5°) and (57 Ϯ 5°) dihedral angle restraints. The second set was derived from cross-linking experiments. The N⑀ atoms of the thymosin Lys 3 , Lys 18 , and Lys 38 were constrained to be in close vicinity (Ͻ5 Å) of the carboxylic functions of the actin Glu 167 (Lys 3 ), Asp 1 , or Glu 4 (Lys 18 ) and Glu 41 (Lys 38 ). In the case of Lys 18 of thymosin, an ambiguous restraint was used to allow both possibilities. Finally, the last set was designed to impose close packing between the thymosin and the actin. More precisely, each C␣ of the thymosin Thr 20 -Ser 30 segment was constrained to be in proximity (Ͻ9 Å) of an actin C␣ atom through the application of an ambiguous restraint. Similarly, the side chains of Met 6 , Ile 9 , and Phe 12 were constrained to be in contact with the protein by introducing a restraint between their C atoms and the actin C␣ atoms (Ͻ10 Å). These last constraints allowed the positioning of hydrophobic side chains of the N-terminal amphiphilic helix of thymosin toward actin, as shown by previously published mutagenesis data (27).
The system was optimized in three steps. First, thymosin was simply rotated above actin to minimize the distance between the thymosin Lys 3 and Lys 18 and actin Asp 1 , Glu 4 , and Glu 167 . Second, the system was submitted to a torsion dynamic, in presence of the cross-linking-derived and C-C␣ restraints, followed by a cartesian dynamic in presence of all restraints. The two dynamics (5000 steps of 1 fs) were realized at a temperature of 5000 K. The structure was finally minimized to rectify the geometry and optimize the van der Waals interactions. The topallhsa and parallhsa parameter and topology files of the X-PLOR software were used in conjunction with an energy function in which the electrostatic term was turned off and the van der Waals interaction was modeled by a simple quadratic repulsion term (except for the final minimization, which was realized with a true van der Waals potential). A square NOE potential was used for the distance restraints of the first set, and a soft potential was used for the others. In both cases, the R-6 mean was used to allow the application of the ambiguous restraints.
RESULTS
Thymosin 4 Is Mainly Unfolded in Solution at 25°C, Except for a Small Helical Content in Its N-terminal Region-The assignment of the proton, nitrogen, and carbon resonances of T4 free in solution were obtained using standard procedures. The assignments of the proton resonances of T4 in solution at 2°C are in agreement with those reported previously (24). The HSQC spectrum of T4 in solution is typical of an unfolded protein, with a very narrow range of amide proton resonances (Fig. 1A). However, structural and dynamic data clearly show the partial formation of secondary structures in the N-terminal and C-terminal segments of the peptide. At 2°C, the presence of NH-NH(i, iϩ1), H␣-NH(i, iϩ2), H␣-NH(i, iϩ3), H␣-NH(i, iϩ4), and H␣-H(i, iϩ3) NOE correlations for all residues along segment 5-16 indicates that this part of the protein folds as a ␣-helix. This conclusion is corroborated by the CSI of H␣ and C␣ nuclei (Fig. 1B) (37)(38)(39). The concomitant presence of strong H␣-NH(i, iϩ1) correlations in this region, however, shows that the helix form is in fast exchange with an extended strand conformation. No NOEs characteristic of the existence of secondary structures were found in any other region of the protein. However, the small negative H␣ and positive C␣ shifts relative to the random coil shifts observed for residues 31-37 suggest that this segment has a weak tendency to fold into an ␣-helix. The tendency of segment 31-37 to form a helix at 2°C in water is also in agreement with the folding of this segment into an ␣-helix in trifluoroethanol (52). The ␣-helical fold of the N-terminal part of T4 is anticipated from the analysis of primary sequence. The N-terminal sequence Pro 4 -Asp 5 -Met 6 -Ala 7 -Glu 8 -Ile 9 indeed corresponds to an N-capping box motif: h-(D,N,T,S)-N1-N2-(E,Q)-h, where N stands for any amino acid, and h is a hydrophobic residue (53)(54)(55). This motif is known to initiate the first turns and the propagation of ␣-helices in the C-terminal direction and to strongly stabilize their N-terminal extremity. In contrast with data obtained at 2°C, no NOE correlation indicative of any secondary structure was found at 25°C. The 15 N relaxation data obtained on T4 free in solution at 2°C and 25°C reinforces the conclusion obtained from structural data. The low values of heteronuclear NOEs, which are all below 0.5 at 2°C and below 0 at 25°C, provide clear evidence of the high flexibility of T4 in aqueous solution (Fig. 2). In a fully structured, globular protein of this size, these values are expected to lie around 0.7 at 2°C and 0.4 at 25°C. Heteronuclear NOEs found here are thus compatible with a globally unfolded protein. The spectral densities for the N-H vectors of the backbone at the three frequencies 0, N (60 MHz), and around H -N (540 MHz) were calculated from R 1 (N z ) and R 2 (N x,y ) relaxation time constants and heteronuclear 1 H-15 N NOEs measured at 2°C and 25°C using a reduced matrix approach (56, 57) (data not shown). Both absolute and relative values of these spectral densities at the three frequencies again emphasize the internal flexibility along the whole polypeptide chain. However, their profiles along the sequence are not completely homogeneous. At both temperatures, higher values of J(0) compensated by lower values of (J H ) in the segment 10 -16 demonstrate a restriction of internal flexibility in this fragment typical of a nascent helix. This restriction of motions in the hundred of picoseconds time scale can be also seen in the C-terminal segment 31-36 of the protein, although to a smaller degree.
Thymosin 4 Forms a Tight 1:1 Complex with Monomeric CaATP-actin at 25°C- Fig. 3A shows the HSQC spectra recorded at 25°C for a sample containing equimolar amounts of [U- 15 residues. Use of TROSY versions of these experiments was unsuccessful. The three-dimensional 1 H-15 N NOESY-HSQC was the only experiment that provided reliable results. Assuming inter-residual and sequential amide/side chain protons proximities, the analysis of H N -H N and H N -H␣ connectivities allowed a possible assignment of the N-terminal segment Met 6 -Ser 15 and the C-terminal fragment Glu 35 -Ser 43 . However, no correlation could be found allowing the assignment of amino acids in the central fragment 16 -34. A method based on selective labeling was then considered.
The strategy for selecting Leu, Thr, and Lys as 15 N-labeled amino acids is described under "Experimental Procedures." All the results obtained for selective labeling of T4 were in good agreement with amino acid evolution in the nitrogen cycle of E. coli (33,34). 15 N HSQC spectra of [ 15 N]AA T4 (where AA is Leu, Thr, or Lys) bound to G-actin are shown in Fig. 3B. Resonances obtained for the selectively labeled T4 perfectly superimposed those obtained for the uniformly labeled protein.
Finally, the three successful selective labelings, in conjunction with the 15 N NOESY-HSQC experiment, allowed the complete assignment of nitrogen and amide protons of T4 bound to G-actin. All amide protons strips could be assigned by researching similarity between strips, with the additional knowledge of the type of the amino acids that had been selectively labeled. The amino side chain undergoing the larger shift upon binding to G-actin could be assigned to Asn 26 , suggesting its location at the interface of the complex. Assignment of H␣ protons can also be suggested from the cross peaks between amide and H␣ protons in the 15 N NOESY-HSQC. When H N -H N correlations indicative of a helical folding were present, the most intense H N -H␣ correlation was considered to be intraresidual. In the other cases, the most intense H N -H␣ correlation was considered to be sequential (H N (i)-H␣(i-1)).
Besides this strategy, an HSQC spectrum of the complex between T10 and monomeric CaATP-actin was performed. A large spreading of the resonances upon binding was observed, as for T4, demonstrating the formation of an analogous tight complex of T10 with actin. The central segment 16 KLKK-TETQEKN is strictly conserved between T4 and T10. The absence of chemical shift variations in the HSQC spectra of the two thymosins for the peaks previously assigned to 18 LKK-TETQEK in the T4-CaATP-actin complex confirmed the assignment of these 9 residues (data not shown).
T4 Bound to G-actin Is Composed of an Extended Central Segment Flanked by Two ␣-Helices, and Its Dynamic Behavior
Demonstrates the Binding of the Whole Peptide's Backbone to G-actin-The very large variations of chemical shifts observed for amide nitrogens and protons along the whole backbone of free and actin-bound T4 demonstrate that the polypeptide chain undergoes a complete reorganization upon binding (Fig. 3C). The CSI calculated on H N, 15 converges on the existence of two ␣-helices, Asp 5 -Leu 17 and Lys 31 -Ala 40 , linked by an extended fragment, Lys 18 -Asn 26 (Fig. 3D).
Heteronuclear 1 H-15 N NOEs could be measured on T4 bound to G-actin at 25°C. The histogram of resulting NOEs is shifted from a value centered around Ϫ0.2 for free T4 to 0.7 for bound T4 (Fig. 2B). Due to the low concentration of the complex and the restricted recording time imposed by the limited stability of the sample, a low signal to noise ratio and therefore imprecise values of heteronuclear NOEs were obtained. However, the large positive shift of NOE values upon T4 binding to actin clearly indicates that T4 adopts the correlation time of a fully bound peptide. The smaller values of heteronuclear NOEs in the center of the sequence indicate that the corresponding N-H bonds of T4 in the complex retain some internal flexibility in the hundred of picoseconds range ( Fig. 2A).
The Structures of T4 Bound to CaATP-actin and MgATPactin Are Identical-De La Cruz et al. (12) recently observed significant differences in the binding characteristics of T4 to CaATP-actin and MgATP-actin. 15 N-1 H HSQC spectra of T4 bound to CaATP-actin or MgATP-actin are identical (data not shown). Amide nitrogen and proton chemical shifts are very sensitive to local electronic environment, and the identical 1 H N -15 N chemical shifts found for the bound state of T4 bound to either CaATP-actin or MgATP-actin demonstrate that the fold of T4 is identical in both cases. The only difference was the systematic presence of a higher amount of free T4 when MgATP-actin was used. This is likely due to the higher efficiency of polymerization of MgATP-actin as compared with CaATP-actin, leading to a higher excess of free T4 in the NMR sample.
Cleavage of G-actin by Subtilisin Does Not Alter the T4actin Complex-T4 binding to subtilisin-cleaved actin (subactin) was linked to a 9% increase in the actin tryptophane fluorescence, as compared with an 11.6% increase upon binding to unmodified G-actin (Fig. 4A). Binding constants of 1.5 Ϯ 0.2 and 2.7 Ϯ 0.4 M were found for the complexes of T4 with CaATP-bound actin and subactin, respectively. The actin-and subactin-sequestering activities of T4 were compared under different ionic conditions. Within low ionic strength polymerization buffer (1 mM MgCl 2 and no KCl), the critical concentrations for filament assembly were 0.35 M for actin and 1.7 M for subactin (Fig. 4B, inset). The slopes of the linear decrease in F-actin versus the total concentration of T4 yielded values of 0.4 M for K T and 1 M for K T Ј (Fig. 4B, a). These values refer to binding of T4 to the MgATP-bound form of G-actin. At physiological ionic strength (1 mM MgCl 2 and 0.1 M KCl), the critical concentrations were 0.1 and 0.5 ⌴ for actin and subactin, respectively (Fig. 4B, a, inset). When increasing amounts of T4 were added to a 16 M unmodified F-actin solution in Mg/KCl polymerization buffer, the decrease in F-actin was not a linear function of T4 concentration, as observed previously (11), which reflected the interaction of T4 with F-actin at high concentration (Fig. 4B, b). From the linear portion of the curve at low T4 concentration, a K T value of 1.2 M was derived. In contrast, the concentration of subtilisin-cleaved F-actin decreased perfectly linearly with increasing T4, demonstrating that when loop 38 -52 is cleaved, T4 no longer interacts with F-actin at high ionic strength (Fig. 4B, b). A K T Ј value of 2.7 M was derived from these experiments.
Nucleotide exchange is known to be strongly inhibited on G-actin upon T4 binding (13). We find that this property is conserved for subtilisin-cleaved actin. The rate of nucleotide exchange was 1.6-fold higher on subtilisin-cleaved actin than on unmodified actin. Binding of T4 to subactin lowered the exchange rate by 100-fold, as for unmodified actin. The T4 concentration dependence of the observed first order rate constant was consistent with a K T Ј value of 1.5 M for binding of T4 to subactin (data not shown).
In conclusion, cleavage of loop 38 -52 on G-actin, which leads to higher critical concentrations for actin filament assembly, causes only a 2-fold decrease in the affinity of T4 and does not affect the functional properties of T 4 , except for the loss in ability of T4 to weakly interact with subtilisin-cleaved F-actin.
The 15 N-1 H HSQC spectrum of T4-subactin at 0.5 mM at 25°C was fully superimposable over the one obtained with unmodified actin (data not shown), except for slightly narrower linewidths in the case of subactin, due to the lower concentration of polymerized actin. NMR data provide information on the localization of T4 on actin. The structural integrity of actin is known to be conserved after subtilisin cleavage of segment Val 43 -Met 47 in the DNase I binding loop, which is locked up in the three-dimensional structure of G-actin by a -sheet itself stabilized by an ␣-helix (Ref. 58 and this study). The lack of difference in the chemical shifts between T4 bound to actin and subactin demonstrates that all amide nitrogens and protons of T4 are in the same environment in both complexes and that they are thus not likely to be in the vicinity of the cleaved segment Val 43 -Met 47 in subactin.
DISCUSSION
Most structural studies of protein-protein complexes address the interaction of two folded partners with defined three-dimensional structures (59 -63). However, a number of proteins are physiologically unstructured and fold only upon binding to their biological target (64). Many of these proteins are involved in cell cycle regulation processes in which their unstructured state provides significant advantages such as a fast turnover, relatively low binding constants, and the ability to bind several targets (64,65). The unfavorable binding entropy linked to the induced folding process requires a precise control over the thermodynamics of the binding. A structural characterization of the proteins, both alone and bound to their target, is crucial to understand how these polypeptides selectively recognize their targets and perform specific functions. Nuclear magnetic resonance is uniquely well suited to provide detailed structural and dynamic information about unstructured proteins and the level of structure of each partner upon binding and thus to describe the local and global thermodynamics governing the binding process of unfolded states (43,66,67).
The present work constitutes the first structural study of the interaction between a major actin-sequestering protein, thymosin 4, and unmodified G-actin at concentrations (0.3-0.5 mM) that are physiological.
Strategies for the Assignment of Thymosin 4 Bound to Gactin-Despite the small size of T4, its assignment when bound to G-actin presents major difficulties due to the low stability and large size of the complex. The impossibility of growing recombinant actin at a significant level precludes the opportunity to obtain large quantities of deuterated actin that could drastically reduce the relaxation of the system and allow the use of classical three-dimensional NMR experiments or their TROSY counterparts for the assignment. We show here that this drawback can be partly bypassed by using a selective labeling strategy. In the case of a relatively small polypeptide such as thymosin, the combined use of the three selective labelings of threonines, lysines, and leucines and a three-dimensional 1 H-15 N NOESY-HSQC experiment was sufficient to obtain the complete assignment of amide protons and nitrogen resonances.
T4 Undergoes a Complete Folding Process upon Binding to G-actin-The large spreading of all resonances in the HSQC of T4 upon binding to G-actin clearly demonstrates that all amide protons and nitrogens of T4 are in a structured environment in the complex. When two folded proteins interact, differences of chemical shifts between the free and bound state can be exploited to delineate the interface of the complex using the chemical shift mapping technique. When the folding is induced by the binding, the resonance shifts are due to the organization of both intra-and inter-molecular protein interfaces, and it is not possible to differentiate between them. However, because the physical principles of protein folding and protein-protein association are similar, the way chemical shifts vary between the bound, folded state and the free, unfolded state is directly connected to the CSI and can be related to the type of local folding in the bound state. Its analysis demonstrates that the binding of T4 to actin is linked to the stabilization of the nascent N-terminal helix (residues 6 -13), the formation of a C-terminal helix (residues [31][32][33][34][35][36][37][38][39][40], and the organization of an extended interface with actin of the central segment (residues [17][18][19][20][21][22][23][24][25][26]. Implications for the Binding Site of Thymosin 4 on Gactin-Although the assignment and secondary structure characterization of the bound form of T4 do not allow us to characterize the organization of these secondary structures on G-actin, our results, combined with those of previous studies, give clear indications about the T4 binding site on actin. Mutagenesis and cross-linking studies have shown that the correct formation of the N-terminal ␣-helix (residues 6 -16) was crucial for the binding of T4 to G-actin and that this amphiphilic helix very likely made hydrophobic contacts with domain 1 of G-actin via the side chains of Met 6 , Ile 9 , Phe 12 , and Lys 16 (25,(27)(28)(29). Cross-links were obtained between Lys 38 of T4 and Gln 41 of G-actin and between Cys 43 of the mutant S43C-T4 and His 40 of G-actin, suggesting that the C-terminal segment of T4 was located near domain 2 of actin (12,29). Additional cross-links between Lys 3 /Lys 18 of T4 and Glu 167 / Asp 1 or Glu 4 of G-actin located the N-terminal segment of T4 near the pointed end of actin (29). Safer et al. (29) proposed from these cross-links that the central segment containing the consensus sequence 17 LKKTET adopts an extended conformation allowing T4 to run from the pointed end to the barbed end of G-actin.
Our results confirm the stabilization of the N-terminal helix upon binding to actin and are compatible with the hypothesis of an extended central segment, 17 LKKTET, possibly extended to 23 QEKNPLP. The very large variations of chemical shifts in this region are indeed indicative of the formation of an ordered, extended structure and suggest the formation of hydrogen bonds with residues of actin. The large chemical shift variations of the amino nitrogen and protons of Asn 26 is a strong indication for the direct involvement of this side chain in the binding. In an extended structure, a maximal translation of 3.4 Å/residue is allowed; hence an extended segment of 14 residues extends over 47 Å, allowing thymosin 4 to extend from the pointed end to the barbed end of actin. The fact that the modified F-actin (q) or subtilisin-cleaved actin (E). The fluorescence of pyrenyl-actin was measured after a 16-h incubation at room temperature. a, measurements in low ionic strength assembly buffer (1 mM MgCl 2 added to G buffer). Actin concentration, 1.5 M; subtilisincleaved actin concentration, 3 M. Inset, critical concentration plots for actin (f and q) and subtilisin-cleaved actin (Ⅺ and E) in low ionic strength (f and Ⅺ) and physiological ionic strength (q and E) buffers. Note that the specific fluorescence of subtilisin-cleaved F-actin is 28% lower than that of unmodified F-actin. b, measurements at physiological ionic strength (1 mM MgCl 2 , 0.1 M KCl). Actin and subtilisin-cleaved actin concentrations ϭ 16 M . FIG. 4. Cleavage of G-actin by subtilisin does not alter the thymosin 4-actin complex. A, fluorescence titration curves of unmodified and subtilisin-cleaved actin by thymosin 4. T4 at the indicated concentrations was added to 2 M CaATP-G-actin (q) or subtilisin-cleaved actin (E). The percentage increase in tryptophane fluorescence was measured. Solid lines are binding curves calculated according to Eq. 1 and using maximal relative increases in fluorescence of 11.6% and 9% for actin and subtilisin-cleaved actin, respectively, and values of 1.5 and 2.7 M for K T and K T Ј . B, thymosin 4 sequesters subtilisin-cleaved actin as well as unmodified actin. T4 at the indicated concentrations was added to solutions of 2% pyrenyl-labeled un-cleavage of actin by subtilisin does not modify the electronic environment of T4 amide protons demonstrates that the backbone of the C-terminal end of the peptide does not make direct contact with segment 42-47 of actin, in agreement with the accessibility of Gly 46 of actin in T4-actin complex to proteolytic enzymes (12). However, it seems at first sight less compatible with the formation of cross-links between C-terminal residues of T4 and His 40 -Gln 41 of G-actin. All results, however, can be accommodated by the high flexibility of the DNase I binding loop that allows the cross-link of Lys 38 and Ser 43 of T4 with actin. Finally, the results are in favor of an induced fit between the C-terminal end of T4 and actin, resulting in a locked conformation of the beginning of the DNase I binding loop that is not perturbed by the cleavage of peptide 43-47 by subtilisin.
Models of the complex were built that are compatible with our NMR results and with the previously mentioned biochemical data (see "Experimental Procedures"). The problem of the docking of two proteins is particularly arduous when one of the proteins undergoes large conformational changes upon binding, as is the case for thymosin here. The use of experimentally derived constraints allowed a delimiting of the interaction surface (from the cross-links and mutagenesis results) and a restriction of thymosin 4-bound conformations (from our NMR results) but was not sufficient to converge toward a unique cluster of complexes. In particular, the lack of orientational restraints for the two helices of T4 toward actin leads to a large uncertainty in the position of the extended central segment. However, the models clearly show that thymosin 4 can cross the whole surface of G-actin, with the N-terminal and C-terminal helices making contacts with the pointed and barbed end of G-actin, respectively (Fig. 5).
HSQC spectra unambiguously demonstrate that the structure of T4 is the same when bound to CaATP-actin or MgATPactin. De La Cruz et al. (12) reported significant differences in the thermodynamic and kinetics parameters for the binding of T4 to CaATP-actin and MgATP-actin. This suggests that the peptide is able to drive the two different initial states of CaATP-actin and MgATP-actin toward a unique Ca/MgATPactin-T4 bound state.
Implication for the Sequestering Activity of Thymosin 4 -Thymosin 4 belongs to the class of WH2 domain proteins that contain an evolutionarily conserved actin monomer-binding mo-tif originally found in -thymosins. They are all thought to play a role in the regulation of actin dynamics through their binding to actin monomers. The few structural studies of WH2 domains show that they are mostly unfolded alone under physiological conditions. Remarkably, they perform different functions in the regulation of actin polymerization, with some preventing and others promoting actin filament assembly, and the structural basis of these differences has not yet been elucidated.
The extended conformation of T4 on G-actin leads to a broad interface, which is compatible with the wide conformational changes undergone by the protein upon binding. The large entropy loss generated by the folding of intrinsically unfolded proteins upon binding to their target is usually compensated by broad interfaces, allowing the formation of numerous hydrogen bonds and the expulsion of a large number of water molecules. In the case of thymosin, the helical propensity of the N-terminal segment also leads to a favored entropy of binding and most probably constitutes the driving force for binding actin. Moreover, it is known that protein-protein binding sites often contain hydrophobic patches. The hydrophobic face of the N-terminal amphiphilic helix of T4, consisting of Met 6 , Ile 9 , Phe 12 , and the side chain of Lys 16 , constitutes a unique favorable recognition hydrophobic patch along the T4 primary sequence. This hydrophobic N-terminal surface, which is present in most WH2 domains (15), probably forms a standard interface with actin that suffices to bring a stable association, but not to bring the function specificity of these proteins. The unique sequestering activity of  thymosins among the family of WH2 domains characterized thus far is then very likely due to a higher specificity of the C-terminal segment for actin or to a specific orientation of this C-terminal segment that prevents the nucleotide exchange and impedes actin-actin interaction at the pointed end of the filament. To investigate the specificity of the sequestering function of thymosins, some mutations that could modify this function can be proposed from our NMR results. Two regions can be probed first. The central region containing the so-called "actin binding motif" LKKTET is highly specific of the thymosin family. Residues of this region undergo the largest chemical shift variations upon binding to actin, indicative of a tight implication in the interaction with actin, most probably through the formation of intermolecular hydrogen bounds. Some of these residues are not strictly conserved in other WH2 domains, especially Thr 20 and Asn 26 , which are replaced by hydrophobic residues (Ala or Val for Thr 20 and Ile for Asn 26 ) in a number of other WH2 domains such as the domain 1 of ciboulot, which was recently shown to exhibit a profilin-like function (19). Subtle variations of the interactions in this region could lead to a variation of the orientation or the dynamics of the C-terminal segment of the WH2 domain. The location and stability of this C-terminal segment, which extends to the pointed end of actin in the case of T4, are likely to be crucial for the sequestering activity (29). In this segment, the acidic residue Glu 35 , which also undergoes a large chemical shift variation upon actin binding, is replaced by non-acidic, hydrophobic residues in a number of other WH2 domains. The analysis of the activity and structural features of mutants suggested from this study should bring additional information on the key interactions that govern the specificity of function of T4 and, more generally, actin-binding WH2 domains.
Acknowledgments-We thank Nicolas Birlirakis for help in the recording of NMR experiments. The 800 MHz spectra were run on the spectrometer installed at Institut de Chimie des Substances Naturelles-Centre National de la Recherche Scientifique, procured with the help of Région Ile de France and the Association pour la Recherche contre le Cancer. (49) using structural constraints derived from our NMR results and previously published biochemical data. The two different orientations shown were generated using MOLMOL software (50). G-actin is depicted in gray, whereas thymosin is colored in blue and red (helices). Side chains of residues Lys 3 , Lys 18 , Lys 38 , and Ser 43 of thymosin and Asp 1 , Glu 4 , His 40 , Gln 41 , and Gln 167 of actin were shown to form cross-links and are shown in yellow. The cross-link constraints are all satisfied and are visualized as green dotted lines. | v3-fos-license |
2018-11-16T19:36:54.693Z | 2018-11-01T00:00:00.000 | 53302164 | {
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} | pes2o/s2orc | Structural Studies of the 3′,3′-cGAMP Riboswitch Induced by Cognate and Noncognate Ligands Using Molecular Dynamics Simulation
Riboswtich RNAs can control gene expression through the structural change induced by the corresponding small-molecule ligands. Molecular dynamics simulations and free energy calculations on the aptamer domain of the 3′,3′-cGAMP riboswitch in the ligand-free, cognate-bound and noncognate-bound states were performed to investigate the structural features of the 3′,3′-cGAMP riboswitch induced by the 3′,3′-cGAMP ligand and the specificity of ligand recognition. The results revealed that the aptamer of the 3′,3′-cGAMP riboswitch in the ligand-free state has a smaller binding pocket and a relatively compact structure versus that in the 3′,3′-cGAMP-bound state. The binding of the 3′,3′-cGAMP molecule to the 3′,3′-cGAMP riboswitch induces the rotation of P1 helix through the allosteric communication from the binding sites pocket containing the J1/2, J1/3 and J2/3 junction to the P1 helix. Simultaneously, these simulations also revealed that the preferential binding of the 3′,3′-cGAMP riboswitch to its cognate ligand, 3′,3′-cGAMP, over its noncognate ligand, c-di-GMP and c-di-AMP. The J1/2 junction in the 3′,3′-cGAMP riboswitch contributing to the specificity of ligand recognition have also been found.
Introduction
Riboswitch, located at the 5 untranslated region of mRNAs, are the gene regulatory elements, which can control gene expression induced by small-molecule ligand binding [1][2][3][4][5][6]. So far, more than 20 riboswitch families have been found, due to different types of small-molecule ligands [5,. Riboswitches have been proposed as antibiotic drug targets [30], which have attracted structural biologists and biophysicists great attention.
To get a better understanding of the conformational change for the 3 ,3 -cGAMP riboswitch induced by the cognate and noncognate ligands binding, molecular dynamics simulations were employed on the apo 3 ,3 -cGAMP riboswitch, the 3 ,3 -cGAMP riboswitch bound by the 3 ,3 -cGAMP molecule and by the c-di-GMP and c-di-AMP molecules. Our main goals were (1) to address the apo 3 ,3 -cGAMP riboswitch structural feature at the nucleotide level; (2) to explain specificity of ligand recognition of the 3 ,3 -cGAMP riboswitch.
Results
We monitored the root-mean-square deviation (RMSD) values with respect to the average structure for the apo riboswitch, riboswitch + 3 ,3 -cGAMP, riboswitch + c-di-GMP, riboswitch + c-di-AMP and G20A-GEMM-I + 3 ,3 -cGAMP systems ( Figure S1). The small RMSD fluctuations of one simulation indicate that the system has attained equilibrium. It can be seen that the five systems had the small fluctuations of RMSD values after 20 ns, which indicate that all the studied systems have reached equilibrium after 20 ns, and the energies of these systems were also stable during the equilibrated simulation. The nucleotides that have atoms within 5 Å of 3 ,3 -cGAMP (or c-di-GMP) in the riboswitch + 3 ,3 -cGAMP model (or the riboswitch + c-di-GMP model) were defined as the binding pocket. The RMSD values, solvent accessible surface area (SASA) and radius of gyration (Rg) of the binding pocket for the apo riboswitch, riboswitch + 3 ,3 -cGAMP, riboswitch + c-di-GMP and riboswitch + c-di-AMP systems are measured (Figure 2A-C). The RMSD values of the binding pocket in the riboswitch + 3 ,3 -cGAMP model are relatively stable throughout the trajectory. The increase of the RMSD values of the binding pocket in the riboswitch + c-di-GMP, riboswitch + c-di-AMP and apo riboswitch systems predict the binding pocket has a conformational change occurring. The SASA and Rg values of binding pockets in these four systems verify the occurrence of the conformational changes, which indicate that the binding pocket in the apo riboswitch has a smaller binding pocket compare to that in the ligand-bound state. Computational techniques, and especially molecular dynamics (MD) simulations, play an important role in investigating structure and dynamics of macromelecules at the atomistic level [41][42][43][44]. MD simulations have been carried on the adenine riboswitch [45,46], the SAM-I, SAM-II riboswitches [47][48][49][50][51][52][53], the Glms riboswitch [54,55], the preQ1 riboswitch [56][57][58][59][60][61][62], the guanine riboswitch [63,64], and the c-di-GMP class I GEMM riboswitch studied in our team [65], which are useful for providing interpretations for existing experimental results and producing predictions for novel experiments on these riboswitches [66]. However, the computational investigation on the 3′,3′-cGAMP riboswitch are very limited so far.
Results
We monitored the root-mean-square deviation (RMSD) values with respect to the average structure for the apo riboswitch, riboswitch + 3′,3′-cGAMP, riboswitch + c-di-GMP, riboswitch + c-di-AMP and G20A-GEMM-I + 3′,3′-cGAMP systems ( Figure S1). The small RMSD fluctuations of one simulation indicate that the system has attained equilibrium. It can be seen that the five systems had the small fluctuations of RMSD values after 20 ns, which indicate that all the studied systems have reached equilibrium after 20 ns, and the energies of these systems were also stable during the equilibrated simulation. The nucleotides that have atoms within 5 Å of 3′,3′-cGAMP (or c-di-GMP) in the riboswitch + 3′,3′-cGAMP model (or the riboswitch + c-di-GMP model) were defined as the binding pocket. The RMSD values, solvent accessible surface area (SASA) and radius of gyration (Rg) of the binding pocket for the apo riboswitch, riboswitch + 3′,3′-cGAMP, riboswitch + c-di-GMP and riboswitch + c-di-AMP systems are measured (Figure 2A-C). The RMSD values of the binding pocket in the riboswitch + 3′,3′-cGAMP model are relatively stable throughout the trajectory. The increase of the RMSD values of the binding pocket in the riboswitch + c-di-GMP, riboswitch + c-di-AMP and apo riboswitch systems predict the binding pocket has a conformational change occurring. The SASA and Rg values of binding pockets in these four systems verify the occurrence of the conformational changes, which indicate that the binding pocket in the apo riboswitch has a smaller binding pocket compare to that in the ligand-bound state.
Energy Calculations Revealed the Preferential Binding of the 3 ,3 -cGAMP Riboswitch to 3 ,3 -cGAMP over c-di-GMP and c-di-AMP
To compare the competitive binding characteristics of the 3 ,3 -cGAMP riboswitch to the cognate ligand, the 3 ,3 -cGAMP molecule, and to the noncognate ligand, the c-di-GMP and c-di-AMP molecules, MM-GBSA methods were used to calculate the binding free energies for the riboswitch + 3 ,3 -cGAMP, riboswitch + c-di-GMP and riboswitch + c-di-AMP systems ( Table 1). The binding of the 3 ,3 -cGAMP riboswitch to the 3 ,3 -cGAMP molecule with the binding free energy of −11.62 kcal/mol in the riboswitch + 3 ,3 -cGAMP system is more energetically favorable over the binding of the 3 ,3 -cGAMP riboswitch to the c-di-GMP molecule with that of −8.74 kcal/mol, and the binding of the 3 ,3 -cGAMP riboswitch to the c-di-AMP molecule with the binding free energy of 13.31 kcal/mol in the riboswitch + c-di-AMP system indicate the c-di-AMP molecule can't bind to the 3 ,3 -cGAMP riboswitch, which agrees with the experiment result [39]. The ranking of binding energy predicts that the 3 ,3 -cGAMP riboswitch binds preferentially to its cognate ligand, the 3 ,3 -cGAMP molecule, over its noncognate ligand, the c-di-GMP and c-di-AMP molecules. In addition, the binding of the 3 ,3 -cGAMP molecule to the class I GEMM riboswitch with G20A mutation with the binding free energy of −7.64 kcal/mol, which agrees with experiment result analyzed by the dissociation constants K d [39] (Table S1). Table 1. MM-GBSA free energy (kcal·mol −1 ) components for the riboswitch + 3 ,3 -cGAMP and riboswitch + c-di-GMP models.
Interaction Analysis between the 3 ,3 -cGAMP Riboswitch and Ligands
To address the interactions of the 3 ,3 -cGAMP riboswitch with the cognate ligand (i.e., the 3 ,3 -cGAMP molecule) and with the noncognate liand (i.e., the c-di-GMP molecule), the percentages of all possible hydrogen bonds between the 3 ,3 -cGAMP riboswitch and the 3 ,3 -cGAMP molecule for the riboswitch + 3 ,3 -cGAMP system, and between the 3 ,3 -cGAMP riboswitch and the c-di-GMP molecule for the riboswitch + c-di-GMP system were analyzed from the equilibrated MD simulations, with the error bar analyzed by the block averaging [67,68] (Table 2). The hydrogen bond interaction was defined as follow: The distance between donor and acceptor was shorter than 3.5 Å, and the angle among donor, proton and acceptor was greater than 120 • [69,70]. The are some varied hydrogen bonds between the 3 ,3 -cGAMP riboswitch and the 3 ,3 -cGAMP molecule for the riboswitch + 3 ,3 -cGAMP model, and between the 3 ,3 -cGAMP riboswitch and the c-di-GMP molecule for the riboswitch + c-di-GMP model, namely, the nucleotides G8-C15 around the J1/2 junction and G40-A42 in the J2/3 junction ( Table 2). The hydrogen bond occupancies between nucleotides G8-C15 around the J1/2 junction of the 3 ,3 -cGAMP riboswitch and the 3 ,3 -cGAMP molecule increased 316.58%, compared to those between the 3 ,3 -cGAMP riboswitch and the c-di-GMP molecule in the riboswitch + c-di-GMP model, which might cause the stronger interaction of the 3 ,3 -cGAMP riboswitch with the 3 ,3 -cGAMP molecule. At the same time, the occupancies of hydrogen bond between nucleotide G40-A42 in the J2/3 junction of the 3 ,3 -cGAMP riboswitch and the 3 ,3 -cGAMP molecule in the riboswitch + 3 ,3 -cGAMP model were reduced by 79.08% compared to those between the 3 ,3 -cGAMP riboswitch and the c-di-GMP molecule. Overall, however, the hydrogen bond occupancies have an increase in the riboswitch + 3 ,3 -cGAMP model, which suggest the stronger interaction of the 3 ,3 -cGAMP riboswitch with the 3 ,3 -cGAMP molecule compare with the c-di-GMP molecule, which is in good agreement with the binding energy analysis discussed above. Table 2. The occupancies (%) of hydrogen bonds with the error bars between the 3 ,3 -cGAMP riboswitch and the 3 ,3 -cGAMP molecule for the riboswitch + 3 ,3 -cGAMP model, and between the 3 ,3 -cGAMP riboswitch and the c-di-GMP molecule for the riboswitch + c-di-GMP model.
In addition to hydrogen bond interaction, the base-stacking interactions between A α , G β of the 3 ,3 -cGAMP molecule and the 3 ,3 -cGAMP riboswitch in the riboswitch + 3 ,3 -cGAMP model, and between G α , G β of the c-di-GMP molecule and the 3 ,3 -cGAMP riboswitch in the riboswitch + c-di-GMP model have been detected (Figure 4 and Figure S2). The criteria for the base-stacking was defined as follow: The base-base distance ≤4.0 Å, the base-base angle ≤ 30 • and overlap with each other [71]. As shown in Figure 4 and Figure S2, the base of G40, A α , base of A41, G β , base of G8 in the riboswitch + 3 ,3 -cGAMP model and the base of G40, G α , base of A41, G β , base of G8 in the riboswitch + c-di-GMP model are plane-parallel approximation and overlap between the bases, which meet the angle criteria for base stacking. The percentage of distance ≤4.0 Å between base of G8 and G β of 3 ,3 -cGAMP, base of A41 and A α of 3 ,3 -cGAMP increased 45.54%, 30.58% compared to that between base of G8 and G β of c-di-GMP, A41 and G α of c-di-GMP, which also suggest the stronger interaction of the 3 ,3 -cGAMP riboswitch with the 3 ,3 -cGAMP molecule compare with the c-di-GMP molecule. In addition to hydrogen bond interaction, the base-stacking interactions between Aα, Gβ of the 3′,3′-cGAMP molecule and the 3′,3′-cGAMP riboswitch in the riboswitch + 3′,3′-cGAMP model, and between Gα, Gβ of the c-di-GMP molecule and the 3′,3′-cGAMP riboswitch in the riboswitch + c-di-GMP model have been detected (Figures 4 and S2). The criteria for the base-stacking was defined as follow: The base-base distance ≤4.0 Å, the base-base angle ≤ 30° and overlap with each other [71]. As shown in Figures 4 and S2, the base of G40, Aα, base of A41, Gβ, base of G8 in the riboswitch + 3′,3′-cGAMP model and the base of G40, Gα, base of A41, Gβ, base of G8 in the riboswitch + c-di-GMP model are plane-parallel approximation and overlap between the bases, which meet the angle criteria for base stacking. The percentage of distance ≤4.0 Å between base of G8 and Gβ of 3′,3′-cGAMP, base of A41 and Aα of 3′,3′-cGAMP increased 45.54%, 30.58% compared to that between base of G8 and Gβ of c-di-GMP, A41 and Gα of c-di-GMP, which also suggest the stronger interaction of the 3′,3′-cGAMP riboswitch with the 3′,3′-cGAMP molecule compare with the c-di-GMP molecule. Additionally, all possible hydrogen bond interaction sites of the 3′,3′-cGAMP riboswitch in the riboswitch + 3′,3′-cGAMP system occurred at the nucleotides G8, A9, A11, A12, A14, C15 around the J1/2 junction, G40, A41, A42 around the J2/3 junction and C75 of the J1/3 junction (Table 2), which are in good agreement with energy decomposition discussed above [39]. Such hydrogen bond and base-stacking interactions bridge the P1, P2 and P3 through the J1/2, J2/3 and J1/3 junctions, which may be necessary for the orientation of the P1, P2 and P3 helices.
Structural Characteristics of the 3 ,3 -cGAMP Riboswitch in the 3 ,3 -cGAMP-Bound and c-di-GMP-Bound State
To address the structural change of the 3 ,3 -cGAMP riboswitch in the 3 ,3 -cGAMP-bound and c-di-GMP-bound state, the average structures from the equilibrated simulations of the riboswitch + To investigate the differences of interaction of the 3′,3′-cGAMP riboswitch in the 3′,3′-cGAMPbound and c-di-GMP-bound state, the distances between the J1/2 junction and 3′,3′-cGAMP or c-di-GMP, between the J2/3 junction and 3′,3′-cGAMP or c-di-GMP for the riboswitch + 3′,3′-cGAMP and the riboswitch + c-di-GMP systems have been measured ( Figure 6). The average distance between the J1/2 junction and the 3′,3′-cGAMP molecule for the riboswitch + 3′,3′-cGAMP model is 5.17 Å, while the average distance between the J1/2 junction and the c-di-GMP molecule for the riboswitch + c-di-GMP model is 6.08 Å ( Figure 6). This distance with a decrease in the riboswitch + 3′,3′-cGAMP system indicates that the J1/2 junction move toward the 3′,3′-cGAMP molecule, which result in the increase of the hydrogen bond occupancies of the J1/2 junction discussed above. As shown in Figure 6, the average distance between the J2/3 junction and the 3′,3′-cGAMP molecule for the riboswitch + 3′,3′-cGAMP model is 5.93 Å, while the average distance between the J2/3 junction and the c-di-GMP molecule for the riboswitch + c-di-GMP model is 4.84 Å. This distance with an increase in the riboswitch + 3′,3′-cGAMP system indicates that the J2/3 move away to the 3′,3′-cGAMP molecule, which result in the decrease of the hydrogen bond occupancies of the J2/3 junction and have no influence on the ranking of binding energy discussed above.
In addition, the distances between the J1/2 junction and c-di-AMP, between the J2/3 junction and c-di-AMP, between the J1/3 junction and c-di-AMP for the riboswitch + c-di-AMP system, and between the J1/3 junction and 3′,3′-cGAMP in the riboswitch + 3′,3′-cGAMP system have also been measured ( Figure S4). It can be seen that the distances between J1/2, J2/3 and J1/3 junctions and c-di-AMP in the riboswitch + c-di-AMP system are larger than that in the riboswitch + 3′,3′-cGAMP system, which indicate that the J1/2, J2/3 and J1/3 junctions move away to the ligand in the riboswitch + c-di-AMP system. The movements of the J1/2, J2/3 and J1/3 junctions may result in the unbinding of the c-di-AMP molecule to the 3′,3′-cGAMP riboswitch, which is consistent with the binding energy discussed above. To investigate the differences of interaction of the 3 ,3 -cGAMP riboswitch in the 3 ,3 -cGAMP-bound and c-di-GMP-bound state, the distances between the J1/2 junction and 3 ,3 -cGAMP or c-di-GMP, between the J2/3 junction and 3 ,3 -cGAMP or c-di-GMP for the riboswitch + 3 ,3 -cGAMP and the riboswitch + c-di-GMP systems have been measured ( Figure 6). The average distance between the J1/2 junction and the 3 ,3 -cGAMP molecule for the riboswitch + 3 ,3 -cGAMP model is 5.17 Å, while the average distance between the J1/2 junction and the c-di-GMP molecule for the riboswitch + c-di-GMP model is 6.08 Å ( Figure 6). This distance with a decrease in the riboswitch + 3 ,3 -cGAMP system indicates that the J1/2 junction move toward the 3 ,3 -cGAMP molecule, which result in the increase of the hydrogen bond occupancies of the J1/2 junction discussed above. As shown in Figure 6, the average distance between the J2/3 junction and the 3 ,3 -cGAMP molecule for the riboswitch + 3 ,3 -cGAMP model is 5.93 Å, while the average distance between the J2/3 junction and the c-di-GMP molecule for the riboswitch + c-di-GMP model is 4.84 Å. This distance with an increase in the riboswitch + 3 ,3 -cGAMP system indicates that the J2/3 move away to the 3 ,3 -cGAMP molecule, which result in the decrease of the hydrogen bond occupancies of the J2/3 junction and have no influence on the ranking of binding energy discussed above.
In addition, the distances between the J1/2 junction and c-di-AMP, between the J2/3 junction and c-di-AMP, between the J1/3 junction and c-di-AMP for the riboswitch + c-di-AMP system, and between the J1/3 junction and 3 ,3 -cGAMP in the riboswitch + 3 ,3 -cGAMP system have also been measured ( Figure S4). It can be seen that the distances between J1/2, J2/3 and J1/3 junctions and c-di-AMP in the riboswitch + c-di-AMP system are larger than that in the riboswitch + 3 ,3 -cGAMP system, which indicate that the J1/2, J2/3 and J1/3 junctions move away to the ligand in the riboswitch + c-di-AMP system. The movements of the J1/2, J2/3 and J1/3 junctions may result in the unbinding of the c-di-AMP molecule to the 3 ,3 -cGAMP riboswitch, which is consistent with the binding energy discussed above. Figure 6. Time-dependences of distances between the J1/2 junction and ligand, between the J2/3 junction and ligand of the 3′,3′-cGAMP riboswitch for the riboswitch + 3′,3′-cGAMP (magentas) and riboswitch + c-di-GMP (blue) models. The bottom panel highlights these distance differences between the J1/2 junction and ligand, between the J2/3 junction and ligand for the riboswitch + 3′,3′-cGAMP (magentas dotted line) and riboswitch + c-di-GMP (blue dotted line) models in the two models. The yellow circles indicate the centroid of J1/2, J2/3 and ligand for the two models.
Structural Characteristics of the 3′,3′-cGAMP Riboswitch in the Ligand-Free and 3′,3′-cGAMP-Bound State
To address the structural change of the 3′,3′-cGAMP riboswitch in the ligand-free and 3′,3′-cGAMPbound state, the angle changes among the centroid of P2, P3 and P1 of 3′,3′-cGAMP riboswitch of the apo riboswitch and riboswitch + 3′,3′-cGAMP systems have been analyzed and are shown in Figure 7A, and the average structures are shown in Figure 7B. It can be seen that the average angle among the centriod of P2, P3 and P1 decrease from 92.0° in the riboswitch + 3′,3′-cGAMP system to 80.8° in the apo riboswitch system, which indicates that the 3′,3′-cGAMP riboswitch leads to the rotation of P1 helix with 11.2° induced by the 3′,3′-cGAMP molecule unbinding ( Figure 7A,B). Such rotation results in the tighter packing of riboswitch in its free state that is validated by the radius of gyration (Rg) analysis of the 3′,3′-cGAMP riboswitch in the apo riboswitch and riboswitch + 3′,3′-cGAMP systems (Figure 8). The Rg of the apo riboswitch system was lower than that of the riboswitch + 3′,3′-cGAMP system (Figure 8). The P1 helix rotation in the apo riboswitch system may be because of the destruction of hydrogen bonds and base stacking interactions compared with the riboswitch + 3′,3′-cGAMP system. The destruction of these interactions also disturbs the 3′,3′-cGAMP binding pocket conformation. Figure 6. Time-dependences of distances between the J1/2 junction and ligand, between the J2/3 junction and ligand of the 3 ,3 -cGAMP riboswitch for the riboswitch + 3 ,3 -cGAMP (magentas) and riboswitch + c-di-GMP (blue) models. The bottom panel highlights these distance differences between the J1/2 junction and ligand, between the J2/3 junction and ligand for the riboswitch + 3 ,3 -cGAMP (magentas dotted line) and riboswitch + c-di-GMP (blue dotted line) models in the two models. The yellow circles indicate the centroid of J1/2, J2/3 and ligand for the two models.
Structural Characteristics of the 3 ,3 -cGAMP Riboswitch in the Ligand-Free and 3 ,3 -cGAMP-Bound State
To address the structural change of the 3 ,3 -cGAMP riboswitch in the ligand-free and 3 ,3 -cGAMP-bound state, the angle changes among the centroid of P2, P3 and P1 of 3 ,3 -cGAMP riboswitch of the apo riboswitch and riboswitch + 3 ,3 -cGAMP systems have been analyzed and are shown in Figure 7A, and the average structures are shown in Figure 7B. It can be seen that the average angle among the centriod of P2, P3 and P1 decrease from 92.0 • in the riboswitch + 3 ,3 -cGAMP system to 80.8 • in the apo riboswitch system, which indicates that the 3 ,3 -cGAMP riboswitch leads to the rotation of P1 helix with 11.2 • induced by the 3 ,3 -cGAMP molecule unbinding ( Figure 7A,B). Such rotation results in the tighter packing of riboswitch in its free state that is validated by the radius of gyration (Rg) analysis of the 3 ,3 -cGAMP riboswitch in the apo riboswitch and riboswitch + 3 ,3 -cGAMP systems (Figure 8). The Rg of the apo riboswitch system was lower than that of the riboswitch + 3 ,3 -cGAMP system (Figure 8). The P1 helix rotation in the apo riboswitch system may be because of the destruction of hydrogen bonds and base stacking interactions compared with the riboswitch + 3 ,3 -cGAMP system. The destruction of these interactions also disturbs the 3 ,3 -cGAMP binding pocket conformation. To investigate the binding pocket conformational change of the 3′,3′-cGAMP riboswitch in the apo riboswitch and riboswitch + 3′,3′-cGAMP systems, the distances between J1/2 and J2/3, and between J1/2 and J1/3 in the riboswitch over the simulation times for the apo riboswitch and riboswitch + 3′,3′-cGAMP models has been also analyzed ( Figure 9A,B). The average distance between the J1/2 junction and the J2/3 junction decreases from 9.95 Å in the riboswitch + 3′,3′-cGAMP system to 6.86 Å in the apo riboswitch system ( Figure 9A). At the same time, the average distance between the J1/2 junction and the J1/3 junction is nearly same in two models ( Figure 9B). This distance with a decrease indicates that the J1/2 junction and the J2/3 junction of the 3′,3′-cGAMP riboswitch move close to each other, which may favor the smaller pocket in the apo riboswitch system and rotation of P1 helix. To investigate the binding pocket conformational change of the 3 ,3 -cGAMP riboswitch in the apo riboswitch and riboswitch + 3 ,3 -cGAMP systems, the distances between J1/2 and J2/3, and between J1/2 and J1/3 in the riboswitch over the simulation times for the apo riboswitch and riboswitch + 3 ,3 -cGAMP models has been also analyzed ( Figure 9A,B). The average distance between the J1/2 junction and the J2/3 junction decreases from 9.95 Å in the riboswitch + 3 ,3 -cGAMP system to 6.86 Å in the apo riboswitch system ( Figure 9A). At the same time, the average distance between the J1/2 junction and the J1/3 junction is nearly same in two models ( Figure 9B). This distance with a decrease indicates that the J1/2 junction and the J2/3 junction of the 3 ,3 -cGAMP riboswitch move close to each other, which may favor the smaller pocket in the apo riboswitch system and rotation of P1 helix. To investigate the binding pocket conformational change of the 3′,3′-cGAMP riboswitch in the apo riboswitch and riboswitch + 3′,3′-cGAMP systems, the distances between J1/2 and J2/3, and between J1/2 and J1/3 in the riboswitch over the simulation times for the apo riboswitch and riboswitch + 3′,3′-cGAMP models has been also analyzed ( Figure 9A,B). The average distance between the J1/2 junction and the J2/3 junction decreases from 9.95 Å in the riboswitch + 3′,3′-cGAMP system to 6.86 Å in the apo riboswitch system ( Figure 9A). At the same time, the average distance between the J1/2 junction and the J1/3 junction is nearly same in two models ( Figure 9B). This distance with a decrease indicates that the J1/2 junction and the J2/3 junction of the 3′,3′-cGAMP riboswitch move close to each other, which may favor the smaller pocket in the apo riboswitch system and rotation of P1 helix. (A)
Principal Component Analysis of the 3′,3′-cGAMP Riboswitch in the Ligand-Free and 3′,3′-cGAMP-Bound State
Principal component analysis was used to analyze the trajectories from the corresponding simulations to examine the dominant 3′,3′-cGAMP riboswitch dynamic motions in the apo riboswitch and riboswitch + 3′,3′-cGAMP models ( Table 3). The first 3 principal components, PC1-PC3, account for about 90% of all the motion modes. In the riboswitch + 3′,3′-cGAMP system, the first three principal modes clearly observed a bending motion between P3 and P1 helices, which may cause the closing and opening of P1 helix along the vertical plane and have no obvious rotation of P1 helix on the horizontal plane (Movie S1, S2 and S3). However, in the apo riboswitch system, the first three principal modes obviously showed a twist motion between P3 and P1 helix (Movie S4, S5 and S6), which may induce the rotation of P1 helix on the horizontal plane. Average structure analyses of the 3′,3′-cGAMP riboswitch support the PCA dominant motions (Figure 7). Table 3. Percentages of occupancy times of the first three principal components during the simulations of the apo riboswitch and riboswitch + 3′,3′-cGAMP models.
Principal Component Analysis of the 3 ,3 -cGAMP Riboswitch in the Ligand-Free and 3 ,3 -cGAMP-Bound State
Principal component analysis was used to analyze the trajectories from the corresponding simulations to examine the dominant 3 ,3 -cGAMP riboswitch dynamic motions in the apo riboswitch and riboswitch + 3 ,3 -cGAMP models ( Table 3). The first 3 principal components, PC1-PC3, account for about 90% of all the motion modes. In the riboswitch + 3 ,3 -cGAMP system, the first three principal modes clearly observed a bending motion between P3 and P1 helices, which may cause the closing and opening of P1 helix along the vertical plane and have no obvious rotation of P1 helix on the horizontal plane (Movie S1, S2 and S3). However, in the apo riboswitch system, the first three principal modes obviously showed a twist motion between P3 and P1 helix (Movie S4, S5 and S6), which may induce the rotation of P1 helix on the horizontal plane. Average structure analyses of the 3 ,3 -cGAMP riboswitch support the PCA dominant motions (Figure 7). Table 3. Percentages of occupancy times of the first three principal components during the simulations of the apo riboswitch and riboswitch + 3 ,3 -cGAMP models.
Allosteric Communication of the 3 ,3 -cGAMP Riboswitch from Binding Sites Pocket to the P1 Helix
To explore the allosteric communications, dynamic cross-correlation map (DCCM) of the allosteric process of 3 ,3 -cGAMP riboswitch induced by the 3 ,3 -cGAMP molecule have been analyzed ( Figure 10). The correlation values vary from −1 (high anticorrelation, blue) to 1 (high correlations, red). Because the 3 ,3 -cGAMP molecule binding sites mainly occurred at the nucleotides G8, A9, A11, A12, A14, C15 around the J1/2 junction, G40, A41, A42 around the J2/3 junction, C75 of the J1/3 junction, the DCCM also appeared in those regions of the 3 ,3 -cGAMP riboswitch. It can be found from Figure 10 Simultaneously, the DCCMs of the 3′,3′-cGAMP riboswitch for equilibrated simulation of the riboswitch + 3′,3′-cGAMP, riboswitch + c-di-GMP and apo riboswitch systems are shown in Figure S5A-C. It can be seen from Figures 10 and S5A,B that the DCCMs of the 3′,3′-cGAMP riboswitch extracted from the equilibrated simulation of the riboswitch + 3′,3′-cGAMP, riboswitch + c-di-GMP systems show very similar features with that from the allosteric process induced by the 3′,3′-cGAMP molecule, which indicate that the similar allosteric communication networks discussed above also exist in the equilibrated riboswitch + 3′,3′-cGAMP and riboswitch + c-di-GMP systems. Comparison of the DCCM of the equilibrated simulations of the apo riboswitch ( Figure S5C) system with that of the riboswitch + 3′,3′-cGAMP ( Figure S5A) system show distinct features. Namely, in the case of riboswitch + 3′,3′-cGAMP simulation ( Figure S5A), a large correlated motion is observed, in contrast, in the apo riboswitch system, the correlated motions have diminished, which indicate that the allosteric communication network from the 3′,3′-cGAMP binding sites to P1 helix discussed above was reduced or abolished in the apo riboswitch system ( Figure S5C). Overall, the allosteric communication between the 3′,3′-cGAMP binding pocket and the P1 helix can obviously occur induced by the ligand binding, such as the 3′,3′-cGAMP molecule or the c-di-GMP molecule.
Allosteric Communication Network from 3′,3′-cGAMP Binding Sites to P1 Helix
As discussed, the indirect and direct allosteric communication occurred at the 3′,3′-cGAMP riboswitch induced by the 3′,3′-cGAMP molecule binding, which relates to the structural changes/ interactions around the J1/2, J2/3, J1/3 junctions and P1 helix. Namely, the 3′,3′-cGAMP molecule binding to the 3′,3′-cGAMP riboswitch causes the movement of the J1/2, J2/3 and J1/3 junctions and the larger binding pocket, which favors the combination of the 3′,3′-cGAMP riboswitch with the Simultaneously, the DCCMs of the 3 ,3 -cGAMP riboswitch for equilibrated simulation of the riboswitch + 3 ,3 -cGAMP, riboswitch + c-di-GMP and apo riboswitch systems are shown in Figure S5A-C. It can be seen from Figure 10 and Figure S5A,B that the DCCMs of the 3 ,3 -cGAMP riboswitch extracted from the equilibrated simulation of the riboswitch + 3 ,3 -cGAMP, riboswitch + c-di-GMP systems show very similar features with that from the allosteric process induced by the 3 ,3 -cGAMP molecule, which indicate that the similar allosteric communication networks discussed above also exist in the equilibrated riboswitch + 3 ,3 -cGAMP and riboswitch + c-di-GMP systems. Comparison of the DCCM of the equilibrated simulations of the apo riboswitch ( Figure S5C) system with that of the riboswitch + 3 ,3 -cGAMP ( Figure S5A) system show distinct features. Namely, in the case of riboswitch + 3 ,3 -cGAMP simulation ( Figure S5A), a large correlated motion is observed, in contrast, in the apo riboswitch system, the correlated motions have diminished, which indicate that the allosteric communication network from the 3 ,3 -cGAMP binding sites to P1 helix discussed above was reduced or abolished in the apo riboswitch system ( Figure S5C). Overall, the allosteric communication between the 3 ,3 -cGAMP binding pocket and the P1 helix can obviously occur induced by the ligand binding, such as the 3 ,3 -cGAMP molecule or the c-di-GMP molecule.
When the binding of the 3 ,3 -cGAMP riboswitch to the noncognate ligand, the c-di-GMP molecule, the same indirect allosteric and direct allosteric communication networks have been detected versus to the cognate ligand, the 3 ,3 -cGAMP molecule. However, the structural change and interaction differences of binding pocket have been found between the riboswitch + 3 ,3 -cGAMP and riboswitch + c-di-GMP systems. When the c-di-GMP molecule binding to the 3 ,3 -cGAMP riboswitch causes the movement of the J1/2 junction away from the c-di-GMP molecule and the decrease of hydrogen bonds at nucleotides G8, A9, A11, A12, A14 and C15 of J1/2 versus to the 3 ,3 -cGAMP molecule, which leads to the binding energy decrease of the J1/2 junction. Though the binding energy of the J2/3 junction in the 3 ,3 -cGAMP riboswitch has a slight increase, due to the structural change of the J2/3 junction when the binding of the 3 ,3 -cGAMP riboswitch to the c-di-GMP molecule versus to the 3 ,3 -cGAMP molecule, it does not affect the binding ranking. Finally, the results revealed that the combination of 3 ,3 -cGAMP riboswitch with the 3 ,3 -cGAMP molecule is energetically favorable for the combination of the 3 ,3 -cGAMP riboswitch with the c-di-GMP molecule. The 3 ,3 -cGAMP riboswitch binding to the c-di-GMP molecule causes the difference of structure and interactions of the binding pocket (i.e., J1/2, J2/3, J1/3) versus to the 3 ,3 -cGAMP molecule. Furthermore, the correlations of the J1/2, J2/3 and J1/3 junctions to the P1 helix induced by the c-di-GMP molecule binding causes the rotation of P1 helix with 5.8 • versus that by the 3 ,3 -cGAMP binding.
Initial Structures
From previous studies, the initial structure of the 3 ,3 -cGAMP riboswitch in the 3 ,3 -cGAMP-bound state (assigned as riboswitch + 3 ,3 -cGAMP model) was taken from the crystal structure published by Ren et al. (PDB code: 4YAZ) [39] (Figure 1). To obtain the starting structure of the apo 3 ,3 -cGAMP riboswitch, the riboswitch + 3 ,3 -cGAMP model was edited to strip off the 3 ,3 -cGAMP molecule and was assigned as the apo riboswitch model. In order to investigate the specificity of ligand recognition, the structure of the 3 ,3 -cGAMP riboswitch in the c-di-GMP-bound state (assigned as riboswitch + c-di-GMP) was constructed from the crystal structure (PDB code: 4yb0) [39]. The initial structure of the 3 ,3 -cGAMP riboswitch in the c-di-AMP-bound state (assigned as riboswitch + c-di-AMP) was constructed based on the structure of the riboswitch + c-di-GMP model. The coordinates of the c-di-AMP molecule extracted from the crystal structure (PDB code: 3MUV) [39] were superimposed onto the riboswitch + c-di-GMP model as the substitute for the c-di-GMP molecule. To elucidate the 3 ,3 -cGAMP molecule binding to the class I GEMM riboswitch, the initial structure of the class I GEMM riboswitch with G20A mutation in the 3 ,3 -cGAMP-bound state (assigned as G20A-GEMM-I + 3 ,3 -cGAMP model) was taken from the crystal structure (PDB code: 4YB1) [39]. In the five models, we retained all ions and water coordinates. Na + were added to the negative position of RNA in each model to achieve electroneutrality. The systems were solvated in a rectangular box of TIP3P waters, with 10 Å padding of the solvent shell in all directions. Three MD simulations were repeated for each model to check robustness of the results although the starting structure was constructed from the X-ray crystal result, which yield a non-distinguishable result, e.g., the similar binding energy and average structure results (Table S2 and Figure S6A-C), when simulations reach equilibrium.
Ligand Force Field Parameter
The coordinates of the 3 ,3 -cGAMP and c-di-GMP ligand were extracted from riboswitch + 3 ,3 -cGAMP and riboswitch + c-di-GMP models, respectively, and the ligand structure was optimized and RESP [72] charges were calculated at the HF/6-31G* level of theory using the Gaussian09 program [73]. Generalized Amber force field parameters [74] of the 3 ,3 -cGAMP ligand were generated using the Antechamber program in the Amber.
Molecular Dynamics Simulation Protocols
All energy minimization and MD simulations were performed using the sander module in AMBER16 package [75] with the parm99 [76,77] and parmbsc0 force field [78] for RNA nucleotide. MD simulation in the NPT ensemble at the constant pressure and temperature of 1 bar and 300 K was carried out for each system. For each simulation, a 2 fs integration step was used. The MD protocols details are available in the Supplementary Materials and our previous studies [65,[79][80][81].
Principal Component Analysis
Principal component analysis (PCA) can be used to probe the most prominent characteristic of the dynamics of the studied system. The PCA method details are available in the Supplementary Materials and our previous studies [65].
where ∆G binding is the binding free energy, G complex , G ligand and G receptor are the free energy of complex, ligand and receptor, respectively. Computational details can be found in the Supplementary Materials and our previous studies [65,79,80].
Trajectory Analyses
The root-mean-square deviation (RMSD), the hydrogen interaction, the base-stacking interaction and the correlation coefficients [75,86] were calculated by Cpptraj and Ptraj in the AMBER16 program [75] in this work. Details of the correlation coefficients analyses are available in our previous studies [65,[79][80][81].
Conclusions
Molecular dynamics simulations have been performed for the aptamer domain of the 3 ,3 -cGAMP riboswitch in the ligand-free, the cognate ligand-bound (i.e., 3 ,3 -cGAMP-bound) and the noncognate ligand-bound (i.e., c-di-GMP-bound and c-di-AMP-bound) states. The results demonstrated that in the absence of ligand, the distance between J1/2 and J2/3 with a decrease of 3.09 Å causes a smaller binding pocket containing the J1/2, J2/3 and J1/3 junctions. When the binding of the 3 ,3 -cGAMP riboswitch to the 3 ,3 -cGAMP molecule, the hydrogen bonds and stacking interactions between the nucleotides G8, A9, A11, A12, A14, C15 around the J1/2 junction, G40-A42 around the J2/3 junction, C75 of the J1/3 junction and the 3 ,3 -cGAMP molecule increase the size of the 3 ,3 -cGAMP binding site pocket. Furthermore, the structural change of the binding pocket, due to the 3 ,3 -cGAMP binding causes the P1 helix rotation with 11.2 • versus to the apo 3 ,3 -cGAMP structure. The correlation and interaction analysis presents the direct and indirect allosteric communication networks in the | v3-fos-license |
2016-03-22T00:56:01.885Z | 2012-03-01T00:00:00.000 | 2669039 | {
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"Chemistry",
"Medicine"
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} | pes2o/s2orc | Pyrazole Carbohydrazide Derivatives of Pharmaceutical Interest
The main purpose of this paper is to provide an insight into the biological activities of pyrazole derivatives which contain the carbohydrazide moiety.
Introduction
Clinically biological activity is the result of a chemical compound's interaction with a human organism. The biological activity is dependent upon the compound's structure and its physical-chemical characteristics, as well as the biological entity and its mode of therapeutic treatment. Nonetheless, many times chemical compounds reveal a spectrum of different types of biological activity. Some of them are useful in the treatment of definite diseases, where others may cause various side and toxic effects. This complex of activities caused by the chemical compound in biological entities is called the "biological activity spectrum of the substance".
Hydrazides, carbohydrazides and similar compounds are well known as useful building blocks for the synthesis of a variety of heterocyclic rings. A large number of heterocyclic carbohydrazides and their derivatives are reported to exhibit significant biological activities [1,2] and the carbohydrazide function represents an important pharmacophoric group in several classes of therapeutically useful substances [1,[3][4][5][6][7][8]. In this paper, the authors will discuss the biological activity spectrum of various pyrazole derivatives containing carbohydrazide moieties.
Pyrazole Compounds with Carbohydrazide Moiety Internalized
The core pyrazole structure in general has attracted widespread attention because of the diversity of biological activity shown by derivatives of this nucleus. In fact, one of the first synthetic organic compounds to find use as an important drug was a pyrazolone. Antipyrine (1), first prepared in 1887, was used as antipyretic, analgesic and anti-inflammatory. Modifications in the pyrazolone nucleus led to the discovery of dipyrone (2), an analgesic and antipyretic drug, still used in several countries in Central and South America as well as in Europe, Asia, and Africa.
Biological Activities of Carbohydrazides Derived from Pyrazole
An investigation of the relationship between the pyrazole nucleus and the position of carbohydrazide moiety substituent with biological activity was one of the goals of this study. Substitution with carbohydrazide moiety at position C-3 of the pyrazole ring ( Figure 3) seems to provide derivatives that exhibit biological activities such as antitumor (compounds 11-13) and cannabinoid antagonist (compound 14) [13][14][15][16]. Figure 4) is a potent CB1 cannabinoid receptor antagonist. This activity can be investigated to provide a chemical tool for further characterization of the cannabinoid pharmacophore and its relationship to the binding domain of cannabinoid antagonists [15,16]. Its main effect is reduction in appetite [17,18]. The anticancer activity of the 4-hydroxypyrazole compounds 12 and 13 was revealed by the broad spectrum of antitumor potential against tumor cell lines at the GI50 and TGI levels, together with a mild cytotoxic activity [14]. The 1-phenylindeno[1,2-c]pyrazole 11 also showed potent cytotoxic activity [13].
The research for novel targeted therapies that can induce death in cancer cells or sensitize them to cytotoxic agents involves some 1H-pyrazole-5-carbohydrazide derivatives that can inhibit the growth of A549 cells in dosage-dependent and time-dependent manners [19,20]. Salicylaldehyde-pyrazolecarbohydrazide derivatives (compound 16, Figure 3) have been investigated in inhibition of the proliferation of A549 lung cancer cells [8,21,22,25,26]. In light of the increased anticancer activities of some copper complexes, salicylaldehyde-pyrazole-carbohydrazide derivatives shown to be potent growth inhibitors to A549 cells via inducing apoptosis [22].
On the other hand, substitution with a carbohydrazide moiety at position C-4 of the pyrazole ring seems to provide derivatives that exhibit different biological activities such as antinociceptive, antibacterial and antiparasital activities.
The insertion of a spacer unit (ethylenic moiety) between the aromatic subunit (R) and carbohydrazide function ( Figure 5) increases the distance from the aromatic units (R and R') that were previously separated only by the carbohydrazide moiety. A phenyl aromatic ring with different substituents inserted into the ethylenic portion increased the lipophilicity and changed the stereoelectronic factors of these derivatives. These compounds presented trypanocidal activity, especially the compound 21 that was shown to be the most potent and to have the lowest toxicity from this series [33]. The reversal of position of the aromatic units (R and R') as in derivative 22 exhibited anticonvulsant effects when evaluated by the standard pentylenetetrazole test and maximum electroshock seizure models [34].
Conclusions
The biological activity of the various pyrazole derivatives containing carbohydrazide moieties discussed in this paper includes antidepressant, anticonvulsant, analgesic, anti-inflammatory, anticancer, antimicrobial, antinociceptive, and antiparasitic activity, such as, antimalarial, trypanocidal, and leishmanicidal. Even though they have a high significance in the pharmaceutical and biotechnological field with a wide spectrum of biological activities for their various derivatives, one must highlight that the carbohydrazide pyrazole chemical class could possess other biological profiles as they are found in many pharmaceutical lead molecules. | v3-fos-license |
2019-04-07T13:07:18.913Z | 2015-01-10T00:00:00.000 | 101201321 | {
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"year": 2015
} | pes2o/s2orc | Effects of Citrus Peel Amendment on the Mobility of Heavy Metals in Contaminated Soil
To estimate the effect of aging on the mobility of heavy metals in soil, relationships between mobility of metals in soil and time after contamination were investigated. Copper, nickel and zinc were added as chloride solution to soil sample. Citrus peel as soil amendments was used at the rate of 5% to immobilize copper, nickel and zinc in contaminated soil. The soils were incubated at field capacity for 3 h, 1, 3, 7, 14, 21 and 28 days at 25oC. Mobility of copper, nickel and zinc from control soils and soils amended with citrus peel were studied with the aid of reduction of metals concentration in the exchangeable fraction. Results indicated that the citrus peel reduced the exchangeable fraction of metals in contaminated soil after 28 days. Application of citrus peel amendment with acidic pH value showed the low immobilizing efficiency of copper, nickel and zinc in the beginning of incubation time in this study due to decrease in soil pH. Batch adsorption of copper, nickel and zinc was conducted to evaluate the effect of contact time, from aqueous solution to understand the mechanism of sorption involved. Fit of the sorption experimental data was tested on the pseudo-first and pseudo-second-order kinetics mathematical equations, which was noted to follow the pseudo-second-order kinetics better, with coefficient of correlation ≥0.99.
INTRODUCTION
The environment can become polluted with organic and inorganic chemicals as a result of intended, accidental or naturally occurring events such as manufacturing processes, mineral extraction, poor environmental management and waste disposal activities, application of fertilizer, illegal dumping of wastes, leaking underground storage tanks, abandonment of mines and other industrial activities [1].
Given the widespread distribution of heavy metals in soil, it is desirable to develop costeffective remediation strategies for these metals.
There are relatively few fully developed, in situ methods available for remediation of metals in soils, and often, remediation at a given site consists of traditional alternatives such as excavation (for treatment/disposal) or containment. However, excavation of metalcontaminated soils may be impracticable due to the excessive cost involved, the magnitude (area, depth, volume) of the soil contamination, and the degree of disruption incurred at the site. Containment alternatives, such as soil caps, are often inconsistent with the desired end use for the site and may be viewed negatively by the regulatory community and the public. As with any remediation, the overall objective of an in situ remediation approach is to create a final solution that is protective of human health and the environment. Metal contamination of soils can represent a threat to human and ecological health through direct contact, can be a source for metal transport to critical resources (e.g., groundwater and surface water bodies), and can hinder the ability of the soil to support vegetation and a functioning ecosystem.
As a result, in situ remediation of metalcontaminated soils typically concerns reduce metal leaching and metal bioavailability to human and ecological receptors. At many sites, there is a potential for metals to be leached from soils by infiltrating water, spreading the contamination and potentially affecting groundwater and other down gradient resources.
The soil environment is able to store, transform and remove pollutants through a combination of biological, chemical and physical processes [1]. But, a suitable method of mitigation is required to render pollutants less mobile and harmful to receptors; this can potentially be achieved by the addition of amendments, to contaminated soils. Bio-solids have been increasingly used in soil management for improving soil quality. The initial measurements of contaminants during site investigation are usually total concentrations of contaminants. However, it is usually the "bioavailable" fraction that is relevant to whether the contaminants present in a soil pose a hazard or not. Bio-solids applications have been shown to be capable of reducing the bioavailability and phytotoxicity of lead, cadmium, and zinc in contaminated soils [2,3].
The land application of bio-solids significantly increases the organic matter content of the soil, enhancing its adsorptive capacity for heavy metals [4,5]. The organic matter adsorbs to inorganic soil constituents, providing additional complexation sites for heavy metals. However, much of the organic matters added to the soil as a result of the land application of bio-solids eventually decompose [6]. Addition of organic matter amendments is a common practice for immobilization of heavy metals and soil amelioration of contaminated soils [7]. The effect of organic matter amendments on heavy metal bioavailability depends on the nature of the organic matter, their microbial degradability, as well as on the particular soil type and metals concerned [8,9]. Cochrane et al. [10] investigated the use of three biosorbents (crab carapace, macroalgae Fucus vesiculosus, peat) for the removal of copper from aqueous media. The maximum adsorption capacity values were 79.4, 114.9 and 71.4 mg g -1 for crab carapace, F. vesiculosus and ion-exchange resin, respectively. In other study, Abdel Salam et al. [11] showed the peanut husk can adsorbe copper and zinc from wastewater. Peanut husk charcoal can adsorbe metal in average of 0.36 mg g -1 . A strange adsorbent material (straw) was used by Kumar et al. [12] in order to remove heavy metals from aqueous systems. The results of many biosorption studies vary widely because of the different criteria used by the authors in searching for suitable materials.
Certain waste products, natural materials and biosorbents have been tested and proposed for metal removal. It is evident from the discussion so far that each low-cost adsorbent has its specific physical and chemical characteristics such as porosity, surface area and physical strength, as well as inherent advantages and disadvantages in wastewater treatment. In addition, adsorption capacities of sorbents also vary, depending on the experimental conditions.
There is evidence that in bio-solid amendments soils, heavy metals remain in the top layer of the soil. Other researchers report either no or little movement of metals into the sub layers of the soil profile [13][14][15]. While soils tend to bind the various species of heavy metals to some degree, the specific mechanisms, and thus both the degree and strength of retention, may vary greatly and may change temporally. The binding mechanism will therefore influence the extent to which contaminant transport is attenuated and the availability of contaminants for uptake by biological organisms. Sequential extraction procedures have been used by many researchers in order to study the forms of heavy metals in soils. Chang et al. [16] found that land application of bio-solids tended to increase the fraction of total metal found in every fraction, especially the organic fractions of the soil. These results were substantiated by Sims and Une and Taylor et al. [17,18]. Swami and Buddhi reviewed the removal of toxic metals including Chrome, Copper, Mercury and nickle from industrial waste water using the agricultural by-products [19]. Compared to the conventional processes for the metal removal in waste water, the device was more cost effective. Also Inbaraj and Sulochana demonstrated fruit shell of Terminalia catappa as a useful sorbent for heavy metal removal from waste water [20].
The present study follow the objective to broaden and increase the knowledge of the effect of citrus peel additives on the quantity of exchangeable forms of copper, nickel and zinc in soil.
Soil Samples
The native soil sample (Typic xerochrepts) was taken from the 0 to 30 cm layer of agricultural soil (X:287441.21 Y:3819829.39 WGS 1984 UTM Zone 39N). The soil was air-dried and passed through a 2 mm sieve before being stored in polyethylene bags. The chemical and physical properties [21] of the studied soil are shown in (Table 1).
Batch Sorption Studies
The batch sorption experiments were run by adding 0.5 g of biosorbent (citrus peel) to 25 ml of solutions containing heavy metal ions of desired concentrations (100 mg l -1 ) for each metal at constant temperature (25±0.1ºC) in 50 ml polyethylene tubes. The tubes were shaken in an orbital shaker at 150 rpm form 10 min. to 24 h and solutions containing heavy metals were filtered through Whatman filter paper (No. 42). Blank samples (without bioasorbent) were prepared for all experiments to investigate copper, nickel and zinc sorption to tube walls and filters.
The percent metal ions removal (R, %) was calculated for each run by following expression: where C i and C e were the initial and final concentration of metal ions in the solution, respectively.
Kinetic Model
The pseudo-first and second order kinetic models were used to describe the sorption kinetic data of cadmium, copper and zinc measured on sorbent [22]: The linear form of the models is shown below where q e and q t are the amounts of metal ions adsorbed onto the citrus peel (mg g −1 ) at equilibrium and at time t, respectively. k 1 is the rate constant of first-order (min −1 ), and k 2 is the rate constant of second-order sorption (g mg −1 min −1 ).
Soil Amendment
The soil used in the experiment was artificially polluted by adding salts of copper, nickel and zinc using CuCl 2 .2H 2 O, NiCl 2 .6H 2 O and ZnCl 2 , to pollute the soil, respectively. All chemicals were of the analytical grade purchased from Merck Company (Darmstadt, Germany).
For this, 0.8 g CuCl 2 .2H 2 O, 1.2 g NiCl 2 .6H 2 O and 0.6 g ZnCl 2 were dissolved in 1 L distilled water separately and there after mixed uniformly with 1 kg soil. After uniform mixing, the suspensions were transferred to plastic boxes and left uncovered at ambient temperature for two weeks. Over the course of these two weeks, the soil samples lost their water content. The reason for leaving them to dry was to allow the test metal to stabilise and speciate before the experiment started.
The citrus peel was washed with distilled water; oven dried at 70ºC and powdered with electric grinder, then mixed whit soil samples.
The citrus peel stabilized soil sample was prepared by adding 5% citrus peel (5 g citrus peel added to 100 g soil). The samples were incubated for 3 h, 1, 3, 7, 14, 21 and 28 days at 25ºC. The appropriate amount of deionized water was added to bring the soil to the estimated field capacity. The samples were kept moist by adding distilled water as needed. All incubation experiments were carried out in duplicate. In order to investigate the heavy metals sorption mechanism onto soil, soil samples without citrus peel and artificially polluted with heavy metals were prepared as control soil and kept at the same condition of soil amended with citrus peel [23]. Also native soil sample without amended with heavy metals were prepared as investigate the heavy metal distribution in parent material.
After incubation the samples were air dried and ground through a 2 mm sieve.
Extraction Procedure
A standardized procedure with aqua regia was used to determine the total contents of heavy metals in soils (both amended and control) [24]. Exchangeable and Mobile fraction extractions extract was performed with 20 ml of 1 M NH 4 OAc at pH 7 for 2 h at room temperature [25]. The copper, nickel and zinc Concentrations in the extracts were determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES) (ES-710).
3.1Batch Sorption Experiment
The rate of sorption of copper, nickel and zinc by citrus peel was determined for different intervals of time. The equilibrium was reached within 60, 90 and 120 min for copper, nickel and zinc respectively (Fig. 1). The uptake of metal ions, as a function of time, was noted to occur in two phases. The first phase involved a rapid uptake of metals during the first 30-60 min of the sorbate-sorbent contact, which was followed by a slow phase of metal removal spread over a significantly longer period of time until the equilibrium was reached. The two-stage sorption, the first rapid and quantitatively predominant and the second slower and quantitatively insignificant, has been extensively reported in literature [26].
The rapid stage, furthermore, may last for several minutes to a few hours, while the slow one continues for several hours to a day [27]. The rapid phase is probably due to the abundant availability of active sites on the sorbent, whereas with the gradual occupancy of these sites the sorption process becomes less efficient during the slower phase [28].
To examine the controlling mechanism of the adsorption process, such as mass transfer and chemical reaction, the pseudo-first-order and the pseudo-second-order kinetics models were used to test the experimental data of copper, nickel and zinc by citrus peel.
A good linear plot of t/q t against t for the pseudosecond-order kinetics model shows good fit on the model (Fig. 2). The low correlation coefficient value (r 2 = 0.47, 0.52 and 0.21 for copper, nickel and zinc respectively), as obtained for the pseudo-first-order model, indicates that sorption of studied metals did not follow the pseudo-firstorder reaction.
The insufficiency of the pseudo-first-order model to fit the kinetics data could possibly be due to the limitations of the boundary layer controlling the sorption process. The experimental data were observed to fit well the pseudo-second order equation. The high correlation coefficient value (r 2 = 0.99), as obtained for the pseudosecond-order equation, was observed to be close to 1. This suggests that the process of sorption kinetics of copper, nickel and zinc by citrus peel follows the pseudo-second-order equation and the process controlling the rate may be controlled by chemical sorption involving valence forces through sharing or exchange of electrons between sorbent and sorbate [30].
Incubation Expriments
The soils amended with citrus peel were analyzed after the for 3 h, 1, 3, 7, 14, 21 and 28 day incubation period and their properties were compared with those of the control soil. The pH values of the soils are listed in (Table 2). As can be seen, an addition of citrus peel resulted in a relative decrease of the pH values. The pH values play a crucial role in the mobility of metals in soils, as they govern directly the acid-base equilibria, and affect (indirectly) both the solution and surface complexation reactions of metal cations, ion-exchange and other metal-binding processes, as well as solubility and metal-binding ability of humic substances as main organic soil constituents.
Typically, the decrease pH values promote the solution of metal cations (including a surface precipitation, in some cases) and increase the metal mobility (contents of easily extractable fractions) [31]. Introduced stabilization materials had a different influence on soil pH in respect to the nature of amendment and soil reaction [32]. Stabilizers exert an influence on physical and chemical properties of soil and metal by changing the conditions of soil environment to those more favorable for metal retention or precipitation.
The total (aqua regia extractable) contents of copper, nickel and zinc in the control soils and soils amended with citrus peel are listed in (Table 3) together with the results of the mobile fraction tests. As can be seen, the total content of copper, nickel and zinc were not affected significantly by an addition of citrus peel amendment, which is consistent with low contents of copper, nickel and zinc in the citrus peel.
Total metal concentration in the soil reflects the degree of contamination and natural differences in soil genesis. A comparison of the total metal levels with World Health Organization (WHO), United States Environmental Protection Agency (USEPA), and the Toxicity Characteristic Leaching Procedure (TCLP) presented in (Table 4). The data evidenced much enhanced levels for three metals than various standard values. Total nickle levels of native soil samples surpassed the limits set by the WHO, USEPA, and TCLP. The high concentration of nickel may cause toxicity not only to plants but also to humans [33].
The mobility's of the metals in soil samples were evaluated on the basis of the absolute and relative content of fractions bound weakly to soil components. The relative index of metal mobility (mobility factor, MF) was calculated on the basis of the ratio of proportion of mobile to the total content of metals [34].
The MF for copper, nickel and zinc in citrus peel treatment was 0.15, 0.25 and 0.15 and in control soil was 0.19, 0.34 and 0.21 respectively. A comparative evaluation of mobilities of different metals evidenced maximum mobility index for nickel. The high MF values are interpreted as symptoms of relatively high lability and biological availability of respective metal in soils [35,36]. Moreover, the mobility index of the metals was not found to exceed 15% evidencing the high stability of these metals in the soil matrix. [3][4][5]. Among metals, rates of exchangeable fraction were in the order of copper < zinc < nickel (There isn't any difference between the native and amended soil), reflecting their intrinsic chemical reactivity in soil. There was slight increase during the first days of incubation in citrus peel amended soil in contrast with control soil and then decrease overcome after the period for all three metals. It was concluded that decreasing the pH due to presence of citrus peel result in solution and availability of metals especially nickel. The mobile fraction properties depended on soil reaction, type of metal and properties of amendments [32]. It was concluded, therefore, that risk assessment of soil contamination can be accomplished by the determination of incubation time and pH of soils. According to Kumar et al., the optimum pH for nickel adsorption for all the husks is 5 to 5.5 [12]. The results of effect of pH on the removal of metals reveal that irrespective of the husk (adsorbent) metal ions were adsorbed. Also the results of Rieuwerts et al. [37] studies showed that metal solubility tends to increase at lower pH and adsorption reactions become more important than precipitation and complexation reactions. As their results, adsorption reactions of Pb are significant at soil pH 3-5 and of Zn at pH 5-6.5. Precipitation and complexation reactions of both Pb and Zn predominate at soil pH 6-7. According to McLean and Bledsoe nickel does not form insoluble precipitates in soils and retention for nickel is, therefore, exclusively through adsorption mechanisms [38]. Nickel will adsorb to clays, iron and manganese oxides, and organic matter and is thus removed from the soil solution.
When the incubation time was prolonged metal concentration of the exchangeable fraction decreased, especially for copper in citrus peel amended soil. In control soil the decrease of metals in exchangeable fraction was less than citrus peel amended soil. The newly added copper existed mainly as exchangeable fraction, but After 7 day incubation, copper in exchange fraction decreased and after 28 day was 46.9%.
Sayed and Zayed investigated adsorption capacity and the effectiveness of s garlic and onion peels, in oil spill clean-ups [39]. It was found that the adsorption capacities were high for garlic and onion peels.
Wilson and Yang converted peanut shells with pyrolysis to activated carbons [40]. It was reported that shells could be used as an adsorbent for metal ions such as: cadmium, copper, lead, nickel and zinc.
Fitzmorris et al. [41] evaluated the removal of calcium, copper, chrome, lead, nickel, zinc, arsenic and selenium from solution using the fruit stone and showed the good adsorption of studied amendment.
CONCLUSION
The application of citrus peel altered the mobile fraction of copper, nickel and zinc to less mobile fraction in contaminated soil in studied period (4 weeks). This resulted in a diminished uptake of metals to plants and lower lechabaility to ground water.
The citrus peel can be considered as an effective immobilization agent for remediation of copper, nickel and zinc in contaminated soils in longer period, because in short time decrease in pH value of soil led to increase the mobile fraction of metals.
COMPETING INTERESTS
Authors have declared that no competing interests exist. | v3-fos-license |
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} | pes2o/s2orc | New Advances in Titanium-Mediated Free Radical Reactions
Titanium complexes have been widely used as catalysts for C-C bond-forming processes via free-radical routes. Herein we provide an overview of some of the most significant contributions in the field, that covers the last decade, emphasizing the key role played by titanium salts in the promotion of selective reactions aimed at the synthesis of multifunctional organic compounds, including nucleophilic radical additions to imines, pinacol and coupling reactions, ring opening of epoxides and living polymerization.
Introduction
Since the pioneering work of Barton et al. [1] of 1960, the potential of radical reactions has been extensively explored, thanks also to the growth of advanced techniques for the detection of radicals and the definition of their structure and reactivity. This ongoing research has allowed the development of innovative selective free-radical reactions, occurring under mild conditions and tolerated by common functional groups, making such processes suitable for the synthesis and functionalization of organic compounds [2,3]. In the last decade, important improvements in this filed have been achieved OPEN ACCESS by combining radical chemistry with transition-metal catalysis, due to the key role of transition metal complexes both in promoting and highly controlling sophisticated free-radical synthetic routes [4][5][6][7]. This approach has been adopted for different applications, ranging from oxidative [8][9][10] and reductive [11] radical processes to the selective C-C and C-N bond formation [12][13][14][15][16][17][18][19], passing through living polymerization [20][21][22].
Among the transition metals widely employed for these purposes, in the present review we focus on titanium and address the recent advances in selective free-radical processes mediated by Ti(III) and Ti(IV) complexes. Titanium, the seventh most abundant metal on Earth, is one of the cheapest transition metals and a lot of titanium compounds are nontoxic and environmentally friendly. The high added value of using titanium salts to promote radical synthetic routes not only relies on their selective intervention in the initiation and the termination steps of the radical chain, but also on their remarkable coordinating role shown, due to their Lewis acid character.
Limiting our overview to the results reported in the literature in the past twelve years, Section 2 describes the potential of Ti(III)/PhN 2 + and Ti(III)/hydroperoxide systems in promoting one-pot, multicomponent, nucleophilic radical additions to aldimines and ketimines generated in situ. Section 3 deals with the most significant examples achieved in pinacol and coupling reactions while, in Section 4, ample discussion is devoted to the ring-opening of epoxides mediated and catalyzed by titanocenes. Finally, in Section 5, we briefly disclose the recent results reported in the field of living polymerization. It is noteworthy that titanium dioxide is also able to promote free-radical processes by photoactivation at suitable wavelengths, but this chemistry is not the object of the present review. The combined action of light and TiO 2 , under oxidative conditions, finds interesting applications in the field of pollutant photodegradation, selective organic photosynthesis and antibacterial activity. For a comprehensive overview on the TiO 2 photocatalytic activity, we refer the reader to some very recent reviews [23-26].
Nucleophilic Radical Addition to Imines
Nucleophilic radical addition to imines, iminium salts or hydrazones, mediated by transition metal salts such as manganese [27], copper [28][29][30], and zirconium [31-33], represents a valuable alternative to the classical ionic routes for the selective C-C bond formation and the synthesis of high added value polifunctional molecules [12][13][14][15][16][17]. This approach becomes even more intriguing if the transition metal is able to promote, in one-pot, the in situ formation of the imine followed by nucleophilic addition, in accordance with the multicomponent reaction (MCR) strategy. In the last decade TiCl 3 , because of this particular specificity, has attracted the increasing interest of our research group, enabling us to develop quite a number of free-radical MCRs.
TiCl 3 /ArN 2 + System
The selective arylative amination of aromatic aldehydes using the TiCl 3 /ArN 2 + system (Scheme 1) was first reported by Clerici and Porta in 1990 [34]. According to this procedure, Ti(III) promotes the aryldiazonium salt decomposition to an aryl radical. Simultaneously, the Ti(IV) formed, due to its oxophilicity, favors the in situ formation of aldimines from anilines and aldehydes. Furthermore, as a strong Lewis acid, Ti(IV) enhances, via N-complexation, the electrophilicity of the C=N bond carbon atom towards nucleophilic attack by the aryl radical.
This reaction occurs in an aqueous environment, where it is well known that imines, easily hydrolysable, are quite unstable and less prone to a nucleophilic attack. Later, in 2005, this approach was successfully extended to a wider range of nucleophilic radical sources by introducing a fourth component in the cascade reaction. In fact, in the presence of alkyl iodides [35], the phenyl radical, deriving from the corresponding diazonium salt decomposition, promotes the fast iodide-atom abstraction, generating an alkyl radical which, in turn, undergoes a nucleophilic attack onto aldimines generated in situ (Scheme 2).
According to a similar procedure, the nucleophilic addition of ethers onto aldimines was performed via α-H atom abstraction via the same phenyl radical source (Scheme 2) [36], providing the first radical version of the Mannich reaction. The resulting iminium radical is then reduced by a second equivalent of TiCl 3 , rendering the overall mechanism irreversible towards the formation of the desired product.
TiCl 3 /ROOH and TiCl 4 -Zn/ROOH Systems
In 2006, we reported a series of new one-pot multicomponent reactions mediated by TiCl 3 or TiCl 4 -Zn hydroperoxide systems, which overcome some limits of the previously disclosed approach. The choice of the right hydroperoxide allows the formation of more practical, efficient and selective precursors of the radical cascade. As a consequence, the new procedure can be extended to a wider range of nucleophilic sources and can be applied even onto ketimines generated in situ, allowing us to create different building blocks for bioactive molecules (Scheme 3). Moreover, this reaction occurs under mild conditions in 30-60 min. Finally, the proposed approach requires neither the pre-formation of the imine nor the protection of the amino group and may easily be conducted under aqueous conditions. . The TiCl 3 -tert-butylhydroperoxide (t-BuOOH) system was revealed to be a more practical, efficient and selective precursor of -alkoxyalkyl radicals from ethers. Accordingly, TiCl 3 /t-BuOOH readily assemble an aniline, an aldehyde, and an ether, leading to 1,2-aminoethers (Scheme 4).
Scheme 4. Aminoalkylation of ethers promoted by the TiCl 3 /t-BuOOH system.
In agreement with the general proposed mechanism (Scheme 5), the dropwise addition of TiCl 3 into the reaction medium promotes the decomposition of the hydroperoxide, leading to the formation of tert-butoxyl radical (t-BuO·). The latter undergoes an α-H abstraction from the ether, with higher selectivity with respect to the phenyl radical generated by the TiCl 3 /PhN 2 + system (no β-hydrogen abstraction was observed). Once formed, the nucleophilic ketyl radical adds to the aldimine generated in situ, in analogy with the mechanism indicated in Scheme 2.
Scheme 5. Nucleophilic radical generation promoted by the TiCl 3 /t-BuOOH system. The reaction goes like a titration and can be easily monitored by considering the different color of the species involved: Ti(III) (violet) and Ti(IV) (orange). Thus, the addition of the TiCl 3 solution stops when a pale blue color is barely maintained to ensure the complete decomposition of the peroxide. This methodology was then extended to the selective carbamoylation of aldimines in formamide [38], providing a radical version of the Strecker synthesis of α-amino amides (Scheme 6a), and to the aminoalkylation of methanol [39], leading to the formation of α,β-amino alcohols (Scheme 6b). When the same protocol is used in the presence of an alcoholic solvent different from methanol, a domino reaction competes with the one-pot process (Scheme 7) [40]. Scheme 7. Domino reaction in alcoholic solvent promoted by the TiCl 3 /t-BuOOH system.
In fact, in this case the ketyl radical formed behaves as a strong reducing agent [41] and competes with Ti(III) in the reduction of t-BuOOH, leading to the formation of the corresponding aldehyde. The latter, when in the presence of an aniline, promotes the formation of an aldimine, which is prone to nucleophilic attack by a second ketyl radical. As a consequence, when the reaction is carried out in the absence of aldehydes, it proceeds to the formation of β-amino alcohols by in situ generation of an aldehyde.
TiCl 4 -Zn/ROOH
Since ketimines are less stable in aqueous medium and less reactive than aldimines, studies involving the reductive radical addition to ketimines are scant in comparison with those conducted on their aldimine counterparts. Moreover, in almost all cases the preformation of ketimines is required. It is because of this low stability under non-anhydrous conditions that the TiCl 3 /ROOH system resulted ineffective when an aniline, a ketone and formamide were assembled in one-pot with the intention of promoting the formation of quaternary α-amino amides. Nevertheless, we recently reported an optimization of the previously discussed protocol, consisting in the replacement of the aqueous TiCl 3 with an anhydrous solution of TiCl 4 in CH 2 Cl 2 in combination with Zn powder (Scheme 8a) [42]. The same protocol was successfully applied in methanol solvent for the selective hydroxymetylation of ketimines, affording the corresponding ,-disubstituted--amino alcohols (Scheme 8b) [43]. The mechanism is analogous to that previously discussed and Zn metal has the unique role of sacrificial reductant for the in situ generation of Ti(III) from Ti(IV) (Scheme 9). Scheme 9. Nucleophilic addition to ketimines promoted by the TiCl 4 -Zn/ROOH system. Surprisingly, the TiCl 4 -Zn/t-BuOOH combination was also revealed to be even more effective and selective with respect to the TiCl 3 /t-BuOOH approach in promoting domino reactions. 1,2-Amino alcohols were obtained in high yields by simply combining two molecules of ethanol or propanol in the presence of aromatic and aliphatic amines, without requiring the addition of aldehydes or ketones (Scheme 10) [44]. Even more interesting is that an analogous reaction occurred by operating in cyclic ether solvents [44]. Scheme 10. Domino reaction in alcohols promoted by the TiCl 4 -Zn/ROOH system.
According to the proposed mechanism (Scheme 11), it was possible to synthetize amino alcohols by electrophilic amination of ketyl radicals, promoting the formation of the imine via ring opening of the ether cyclic and the subsequent nucleophilic radical addition by a second ketyl radical. Scheme 11. Domino reaction in cyclic ethers promoted by the TiCl 4 -Zn/ROOH system.
Cp 2 TiCl 2 /Zn Systems
In 2011, Saha and Roy reported a selective radical-induced allylation of pre-formed aldimines for the synthesis of homoallyl amines [45]. The combination of titanocene(IV) dichloride and Zn dust in THF under argon leads to the formation of Cp 2 TiCl in situ. The latter acts as radical initiator, promoting the formation of the allyl radical from the corresponding allyl bromide (Scheme 12). This approach was successfully applied to the synthesis of deoxy aza-disaccharides and for the preparation of the bicyclic skeleton of alkaloids. Scheme 12. Cp 2 TiCl-mediated allylation of aromatic aldimines.
Coupling Reactions
Radical coupling mediated by transition metal salts represents one of the pillars of free-radical synthesis. Once again, simple titanium salts and titanium complexes bearing specific organic ligands result particularly effective in promoting intramolecular as well as intermolecular coupling with high stereo-, diastero-and even enantioselectivity [46,47]. Among the several protocols developed, pinacolization, allylation and cross-coupling reactions have attracted particular attention in the last decade.
Pinacol Reactions
The pinacolization of aldehydes is one of the most studied reactions in the area of radical coupling for C-C bond formation. This interest arises from the usefulness of hydrobenzoins as chiral auxiliaries and ligands for stereoselective organic synthesis. Moreover, the pinacol reaction represents an ideal tool for studying the enatioselective catalyzed formation of radicals [48].
This reaction can be accomplished with a variety of one-or two-electron metal reducing agents. Our group has widely investigated the role that TiCl 3 and TiCl 4 play in promoting pinacolization of aromatic aldehydes. In particular, a dl-stereodirecting pinacolization was accomplished by using TiCl 3 in a strictly anhydrous non-coordinating solvent (CH 2 Cl 2 ), favored by Ti(III)-complexation of the oxygen atom of the carbonyl group, which lowers the reduction potential of the aromatic aldehyde (Scheme 13) [49].
Scheme 13. dl-Stereodirecting pinacolization of aromatic aldehydes promoted by TiCl 3 .
Later on, in the same CH 2 Cl 2 solvent, an analogous reactivity was observed when TiCl 4 is coupled with diisopropylethylamine (DIPEA). Porta and co-workers [50] revealed how, in this case, the TiCl 4 /DIPEA complexation is the driving force of the overall process, causing the instantaneous formation of Ti(III) by an inner-sphere electron transfer from Ti(IV) to DIPEA. Once generated, Ti(III) coordinates the carbonyl oxygen of an added aromatic aldehyde, promoting its reductive dimerization.
More recently we have unexpectedly found that, despite the non-anhydrous conditions, the aqueous TiCl 3 /t-BuOOH redox system promotes the hydrodimerization of aromatic aldehydes, when used in combination with an alcoholic co-solvent like ethanol [41].
In accordance with the proposed mechanism (Scheme 14), the explanation of the paradox relies in the reducing power of the -hydroxyalkyl radicals generated in situ via H-atom abstraction from the alcoholic solvent by tert-butoxy radical. These ketyl radicals, instead of Ti(III), are the actual reductants of the aromatic aldehydes.
Pinacolization of aromatic ketones like acetophenone can be performed in good yields and high stereoselectivity (dl/meso ratio = 9/1) in the presence of Cp 2 TiCl, generated in situ by combining Cp 2 TiCl 2 with an excess of Mn dust [51].
Moreover, new families of Ti(IV) complexes bearing chiral ligands (1, 2 and 3, Figure 1) have been developed in the last decade. These derivatives, when employed in combination with co-reducing agents, revealed to be ideal precursors of chiral Ti(III) catalysts for the enantioselective pinacol coupling of aldehydes. In 2001 Riant and co-workers [52] reported the combined use of stoichiometric amounts of the titanium-Schiff base complex 1 and Mn to promote the asymmetric dl-diastereoselective pinacolization with high yields and enantioselectivity up to 91% (Scheme 15). In 2005, Knoop and Studer reported a new method for the in situ generation of Ti(III) complexes without requiring a co-reducing reagent [54]. Cyclohexadienyl-Ti(IV) compounds, prepared from the corresponding lithiated cyclohexadienes, led to the corresponding Ti(III) complexes upon thermal C-Ti bond homolysis (Scheme 17). Following this procedure, complex 3 was readily prepared and employed to perform pinacol coupling of aromatic aldehydes with diastereomeric and enantiomeric excess.
Allylation Reactions
Among radical coupling reactions, the Barbier-type allylation of carbonyl derivatives, mediated by different transition metals, has attracted particular attention due to its considerable synthetic relevance (Scheme 18). The generic strategy for this process consists in the transformation of allyl halides into the corresponding allyl radicals which, in turn, would add onto the carbonyl compounds present in the reaction medium. Scheme 18. Barbier-type allylation of carbonyl derivatives.
Very recently, Justicia et al. have shown that an excess of titanocene(III) chloride, generated in situ by the Cp 2 TiCl 2 /Mn-dust system, allows to extend this protocol to the crotylation of carbonyl compounds [55].
However, most of the reported protocols usually require stoichiometric amounts of these metals, which is detrimental for the sustainability of the process. Stoichiometric quantities are generally necessary even when operating in the presence of Ti complexes, limiting the applicability of this approach to enatioselective additions [46]. In this context, in 2009 a comprehensive contribution by Oltra and co-workers has shown how catalytic proportions of titanocene(III) complexes, when combined with the regenerating agent 4 (obtained by mixing TMSCl and 2,4,6-collidine), are able to promote the Barbier-type allylation, in accordance with the mechanism reported in (Scheme 19) [56].
Scheme 19.
Barbier-type allylation mechanism promoted by catalytic titanocene. This approach has been successfully applied for the allyl radical addition onto aldehydes and ketones (including functionalized carbonyl substrates), for cyclization reactions and for prenylation of aldehdyes (Scheme 20).
The titanocene(III)-mediated Barbier-type prenylation to α,β-unsaturated aldehydes, which allows the introduction of hydroxyl groups at desiderable positions, and the subsequent radical cyclization promoted by the same titanium complex, open the straightforward access to polyhydroxylated terpenoids [57].
The main limitation of the Barbier-type approach is that it requires the use of allylic halides as reactive substrates, while allylic carbonates and carboxylates, which are easily prepared and handled, are inert against titanocene(III) complexes. To overcome this limit, Oltra and co-workers proposed the combination of Cp 2 TiCl with palladium and nickel complexes [58]. Indeed, it is well known that these metal derivatives are able to activate allylic carbonates and carboxylates by forming 3 -allylmetal complexes. The authors have demonstrated how the choice of the metal is crucial in modulating the Cp 2 TiCl activity in the allylation reaction. In fact, while palladium salts promote the intermolecular coupling with electrophilic reagents (Scheme 21a), the intramolecular allylation of alkenes with allylic carbonates occurs in the presence of nichel complexes, leading to carbocylic derivatives (Scheme 21b).
Scheme 21.
Divergent Ti-mediated allylations with modulation by Palladium or Nickel.
The titanium/palladium-mediated system has been successfully applied by the same research group for the selective allylation, crotylation and prenylation of aldehydes and ketones [59] (Scheme 22), for the synthesis of homopropargyl alcohols using propargyl carbonates as pronucleophiles [60] (Scheme 23), and to promote the intramolecular Michael-type addition of allylic carboxylates to activated alkenes [61] (Scheme 24). More recently, the efficient allylation of carbonyl compounds starting with allyl carbonates has been performed by replacing Pd with Ni [62].
Other Coupling Reactions
Several other coupling reactions promoted by titanium complexes have been recently reported. Roy and co-workers have proposed the use of the Cp 2 TiCl 2 /Zn-dust system to promote the synthesis of furan derivatives such as (±)-evodone from α-bromo-β-keto enolethers [63]. The same approach has been successfully applied to the asymmetric synthesis of α-methylene bis-γ-butyrolactone [64].
The Cp 2 TiCl-catalyzed homolytic opening of ozonides, reported by Rosales et al. [65], provides carbon centered radicals suitable to form C-C bonds via both homocoupling and cross-coupling processes (Scheme 25).
Scheme 25.
Homocoupling versus cross-coupling in the Ti-catalyzed opening of ozonides.
Scheme 26. Reductive cyclization of ketonitriles.
An example of the potential of radical chemistry is the use of the Ingold-Fisher "Persistent Radical Effect" (PRE) which has found ample employment in cross-coupling reactions for the selective synthesis of organic compounds. According to PRE, the cross-coupling between two radicals occurs when the two species are generated at similar rates and one is more persistent than the other, that is with a lifetime significantly greater than that for a methyl radical under the same conditions [67].
In 2008 we reported how α,β-dihydroxy ketones can be used as a source of stabilized radicals via selective C-C bond cleavage promoted by TiCl 4 [68] the coordinating effect of the metal ion induces an increased stabilization onto the new radicals, driven by the captodative effect (Scheme 27).
Scheme 27. C-C bond cleavage of α,β-dihydroxy ketones promoted by TiCl 4 .
This observation prompted us to develop a new one-pot multicomponent route leading to the synthesis of polyfunctional derivatives of high added value. The generation, in the same reaction medium, of transient radicals via thermal decomposition of 2,2′-azo-bis-isobutyronitrile (AIBN) yielded an innovative methodology for the synthesis of β-hydroxynitriles by cross-combination between Ti(IV)-stabilized radicals and the α-cyanoisopropyl radical (Scheme 28).
Epoxide Reactions Mediated by Titanocene
Bis (cyclopentadienyl) titanocene III chloride (Cp 2 TiCl) and substituted titanocenes are well known titanium complexes able to generate regioselective radical epoxide opening through single electron transfer (ET), both in stoichiometric and catalytic amounts [69,70]. The method of radical generation combines both the Lewis acid catalysis and the radical chemistry with interesting advantages: the initial ET by the metal, tuning the acidity of the metal center, avoids SN-type reactions, while the ensuing radical reaction can be controlled by the metal complex and his ligand. Cyclic voltammetry and kinetic measurements have disclosed the structure and mechanism of epoxide opening by Cp 2 TiCl reagents in solution: the activation of Cp 2 TiCl 2 species by Mn or Zn reducing metals in THF solution leads to a mixture composed by the monomer Cp 2 TiCl and the chlorine bridged dimer (Cp 2 TiCl) 2 Typical radical reactions mediated by titanocene reagents regard selective reduction and deoxygenation of epoxides, C-C bond formation processes such as cyclizations, and intermolecular additions to activated olefins [73,74].
It is worth noting that, as the reaction mechanism generally proceeds via the most substituted (i.e., energetically most stable) radical, the regiochemistry is the opposite (and complementary) to that normally expected for conventional SN 2 -type epoxide openings with carbon nucleophiles or hydride reagents [73,74].
Epoxide Deoxygenation
Though epoxides are usually prepared from olefins, their deoxygenation for the generation of C=C double bonds finds ample application in highly oxidized substrates like carbohydrates and other natural derivatives [75,76]. The process proceeds in good to excellent yields, with exceptional chemo-selectivity.
It has been shown that the deoxygenation occurs when operating in the absence of radical acceptors and that it depends on the substitution pattern [77]. On the basis of these observations, Fernández-Mateos and co-workers have recently reported the selective synthesis of terpenoids starting from pinene-oxide derivatives [78].
Doris and co-workers reported in 2002 the Cp 2 TiCl-mediated deoxigenation of the alkaloid leurosine (from Catharantus roseus), leading to anhydrovinblastine (Scheme 30), the key intermediate in the symthesis of the anticancer drug navelbine [79]. Furthermore, short and straightforward access to analogues of the natural onoceranes mediated by Cp 2 TiCl 2 has been proposed by Barrero and his group [80]. The dimerization of vinyl-epoxides to 1,5-dienes occurs via generation of very stable β-titanoxy allylradicals. The latter undergo dimerization to homo-onoceranes, followed by oxidation (Scheme 31).
Scheme 31. Dimerization of vinylepoxides for the preparation of homo-onecerans.
In the same year, Justicia et coworkers [82] showed that β-titanoxy radicals may rearrange to new trisubstituted radicals which undergo a mixed disproportionation process (via hydrogen atom transfer, Scheme 32) leading to allylic alcohols.
Scheme 32. Disproportionation of epoxide through hydrogen atom transfer.
Reductive Epoxide Opening
The catalytic reductive epoxide opening via electron transfer from titanocenes has emerged as an attractive alternative to the classical nucleophilic substitution strategies. The titanium-mediated mechanism combines the well-established advantages of radical chemistry with a regioselectivity of ring opening opposite to that of nucleophilic substitutions, leading to a number of interesting and unusual applications in the synthesis of complex molecules [83][84][85][86][87].
Synthesis of Alcohols
According to mechanistic studies, the depicted β-tytanoxyl radical epoxide complexes constitute the first intermediate of ring opening. These radicals are then reduced by hydrogen atom donor species, like for example 1,4-cyclohexadiene (Scheme 33).
If the use of catalytic amounts of titanocenes is attractive for reagent controlled radical reactions [88], on the other hand this process presents the disadvantage deriving from the generation of stoichiometric amounts of highly toxic reducing waste, such as benzene (Scheme 26). However, it has been demonstrated that titanocene reagents are able to activate water and methanol toward hydrogen atom transfer, by substantially lowering the bond dissociation energy (BDE) of the OH bond [89][90][91]. This combination is chemically more efficient and environmentally benign. Cp 2 TiCl-mediated reductive epoxide ring opening using water as a hydrogen source, allows access to alcohols with anti-Markovnikov regiochemistry from different epoxides. The use of D 2 O as a deuterium source, leads to an efficient synthesis of β-deuterated alcohols, which find successful application as internal standards in food analysis [92]. Another suitable hydrogen donor is H 2 [93,94].
Titanocene-catalyzed procedure has been immediately extended by Gansäuer et al. to the enantioselective opening of meso-epoxides. Titanocene complexes with chiral ligands, such as those reported in Figure 2, were employed in combination with manganese dust and collidine hydrochloride, the former as a stoichiometric reductant and the latter having the role of regenerating the alcohol and titanocenes [95][96][97][98][99][100][101][102] (Scheme 34). By operating in the presence of collidinium hydrochloride, the catalytic reaction could be also performed in water. This approach was exploited for the selective reduction of aromatic ketones [103,104] and the selective synthesis of 2-methyl-1,3-diol frameworks, using THF as hydrogen donor [105]. Also α,β-epoxy ketones could be opened with Cp 2 TiCl, affording β-hydroxy ketones in a better fashion than with SmI 2 (Scheme 35) [106].
Scheme 35. Example of elective reduction of α,β-epoxyketones.
The ring-opening reaction of tri-substituted epoxides promoted by Cp 2 TiCl on carvone derivatives has led to exo-methylene allylic alcohols as major compounds [107]. The generation of exocyclic olefins has been reported in the course of radical cyclizations of epoxy alkenes leading to monocyclic terpens as achilleol A [108], oxobicylic drimanes [109], eudesmanolides [110], and different ring synthons of paclitaxel [111]. In these cases, termination of the radical cycle is not reductive, but rather it involves β-hydrogen elimination providing an alkene function.
Very recently Gansäuer et al. have reported a new protocol to perform the radical reduction of epoxides by hydrogen atom transfer catalyzed by the active specie [Cp 2 TiH] [112]. The active catalyst is generated in situ from [Cp 2 TiOC 2 H 5 ) 2 ] in the presence of (CH 3 )PhSiH 2 (Scheme 36).
Intermolecular C-C Bond Formation
Epoxide-derived radicals generated in the presence of titanocenes are also intermediates for intermolecular C-C bond forming reactions [113]. The resultant products, like δ-hydroxyketones, esters, amides, nitriles and δ-lactones, constitute important classes of intermediates in organic synthesis (Scheme 37). It has been reported that the C-C bond formation employing α,β-unsaturated tungsten and chromium carbenes, as radical acceptors of epoxide-derived radicals, occurs with high chemoselectivity. This approach has been applied to both sugar-derived and α,β-unsaturated carbenes [114] (Scheme 38).
Scheme 39. 3-exo and 4-exo cyclization with aldehydes as radical traps.
While gem-dialkyl or gem-dialkoxyl substitution is usually required to maintain an efficient propagation of the radical chain, very recently Gansäuer et al. have reported the first example of 4-exo cyclization on unsaturated epoxides without gem disubstitution [124]. This result was achieved by using cationic functionalized titanocenes as template catalysts (Scheme 40). According to the proposed mechanism [126], the two-point binding which occurs between the substituted radical and the template forces the radical center and the radical acceptor into close spatial proximity (Figure 3). 5-exo cyclizations are usually conducted by using alkenes and alkynes as radical traps and this approach has been widely applied for several synthetic routes [127][128][129][130][131][132][133][134][135][136][137]. Following an analogous procedure, a 6-endo cyclization employing Cp 2 TiCl was reported in 2001 by Takahaschi and co-workers for the synthesis of (±)-smenospondiol [138]. Titanocene(III) chloride mediated 5-exo and 6-exo cyclization has been recently applied for the regio-and diastereoselective synthesis of highly functionalized terpenic cyclopentanes [139], and for the synthesis of (−)-methylenolactocin and (−)-protolichesterinic acid [140]. Titanocene-promoted cyclization of unsaturated epoxylactones was also reported as a key step for diastereoselective synthesis of limonoid CDE fragments [141].
Scheme 40. 4-exo-cyclization of unsaturated epoxides.
Epoxypolyene cyclizations were performed in the presence of Cp 2 TiCl 2 using the collidinechlorotrimethylsilane system, leading to exo-cyclic olefins by hydrogen atom abstraction (Scheme 41) [142][143][144]. These cyclization reactions proceed with high stereoselectivity giving only one of the many possible stereoisomers. Moreover, some of the cyclization products can be readily elaborated into natural products or key intermediates such as in the preparation of zonarol, a natural antifungal (Scheme 42).
Following the same procedure, 7-endo cyclizations can be performed in surprisingly high yields (Scheme 43) [145]. Finally, 8-endo cyclizations have been reported by Roy and his group for the preparation of aromatic ethers [146]. Scheme 43. 7-endo cyclization.
Living Polymerizations
Several metal complexes were designed and tested to effectively control the radical polymerization of a number of polar olefins, leading to the synthesis of complex macromolecular architectures [147,148].
The use of titanium complexes for this purpose is relatively rare. Nevertheless, in recent years it has been emphasized the Ti(III)/Ti(IV) capacity of modulating the equilibrium in atom transfer reactions. As a consequence, this redox pair was reported as a very active catalytic system for atom transfer radical polymerization [149].
Radical controlled polymerization reactions by epoxide radical ring opening (RRO) have been performed in the presence of Cp 2 TiCl 2 in combination with Zn powder [150][151][152]. According to this approach, methyl methacrylate (MMA) polymerization has been performed with high-molecular weight in aqueous conditions (Mw = 51,000-73,000). An analogous route has been recently reported for the radical polymerization of styrene [151]. The Ti(III)Cp 2 Cl-catalyzed RRO of epoxides produces Ti-alkoxides, which initiate the ring opening polymerization of cyclic esters like ε-caprolactone (Scheme 44) [153], the radical polymerization of isoprene [154] and styrene/isoprene copolymers [155].
The ligand effect in Ti-mediated living radical styrene polymerizations initiated by epoxide RRO has been widely investigated by Asandei and Moran, focusing in particular onto alkoxide and bisketonate titanium complexes [156], scorpionate and half-sandwich LTiCl 3 derivatives [157] and substituted sandwich metallocenes [158]. Moreover, the same research group disclosed in detail the role of solvents and additives for this polymerization reactions [159], the effect of the reducing agent and temperature, as well as of the ratio titanium/epoxide and titanium/zinc [160]. Excellent results in the living radical polymerization were also achieved by initiating the process via single electron transfer reduction of aldehydes promoted by Cp 2 TiCl [161]. This approach has been successfully applied also for the polymerization of ε-caprolactone [162].
Very recently, Cp 2 TiCl 2 has been applied in the field of photopolymerization for applications requiring safe conditions (Scheme 45) [163]. Cp 2 TiCl 2 /silane (tris(trimethylsilyl)silane TTMSS)/ iodonium salt (Ph 2 I + ) resulted to be a good photoinitiating system in the free radical promoted cationic photopolymerization process of an epoxide monomer (EPOX) upon long wavelength excitation (λ > 400 nm). Under visible LED bulbs, xenon lamp and laser diodes, the reactions exhibit an excellent performance and a remarkable final polymerization close to 100%. The basic idea is to produce a radical (from a photoinitiating system) which, in turn, should be oxidized by the iodonium salt: Cp 2 TiCl 2 generates under argon cyclopentadienyl radicals (Cp • ) and/or metal-centered radicals CpTi • Cl 2 . In the presence of TTMSS, Cp • can rapidly react by hydrogen abstraction with the silane Si-H generating a silyl radical. The latter can be easily oxidized by Ph 2 I + with a rate constant of 2.6 × 10 6 M −1 s −1 , leading to the formation of a silylium cation, which, in turn, can initiate the cationic polymerization by ring-opening of EPOX. Scheme 45. Polymerization of EPOX mediated by titanocene.
Conclusions
This study covers the literature over the past twelve years emphasizing the key role that titanium salts and complexes play in promoting synthetic free-radical processes. While several excellent reviews, focusing on specific Ti-mediated reactions and defined product targets or on the general role of transition metals in promoting radical chains, have been recently published, to the best of our knowledge this work represents the first comprehensive contribution which outlines the potential of titanium catalysis in radical reactions, analytically overviewing the recent progresses in selective freeradical organic synthesis.
Though multicomponent procedures are of great interest for the development of new synthetic routes, examples in the field of radical reactions are in general scant. Among the few protocols recently reported, the nucleophilic radical addition to imines mediated by Ti(III)/Ti(IV) systems represents an innovative one-pot approach leading to a wide range of intriguing functionalized derivatives under very mild experimental conditions. Pinacolization of aldehydes is particularly attractive, not only for the importance of the final diols, but also because it represents a significant model for investigating enantioselectivity in radical chemistry. Other radical coupling reactions, when mediated by titanium complexes, lead to high added value products with chemo-and stereo-selectivity.
Finally, deoxygenation or reductive ring opening of epoxides by means of titanocenes find ample use in the synthesis of natural and biologically active molecules. Ti-mediated radical ring opening of epoxides is also successfully applied to promote living radical polymerization of olefins (such as styrene and isoprene) and lactones (for example ε-caprolactone).
The development and optimization of titanium ligands, including the introduction of chiral complexes, allows in many cases to perform stero-and/or enantio-selective syntheses of the desired products. The ongoing research for the design, synthesis, and application of these new ligands guarantees the development of new Ti-based catalytic systems for selective transformations under very mild conditions. | v3-fos-license |
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