Chitosan Hydrogel Supplemented with Metformin Promotes Neuron-like Cell Differentiation of Gingival Mesenchymal Stem Cells.

Human gingival mesenchymal stem cells (GMSCs) are derived from migratory neural crest stem cells and have the potential to differentiate into neurons. Metformin can inhibit stem-cell aging and promotes the regeneration and development of neurons. In this study, we investigated the potential of metformin as an enhancer on neuronal differentiation of GMSCs in the growth environment of chitosan hydrogel. The crosslinked chitosan/ß-glycerophosphate hydrogel can form a perforated microporous structure that is suitable for cell growth and channels to transport water and macromolecules. GMSCs have powerful osteogenic, adipogenic and chondrogenic abilities in the induction medium supplemented with metformin. After induction in an induction medium supplemented with metformin, Western blot and immunofluorescence results showed that GMSCs differentiated into neuron-like cells with a significantly enhanced expression of neuro-related markers, including Nestin (NES) and ß-Tubulin (TUJ1). Proteomics was used to construct protein profiles in neural differentiation, and the results showed that chitosan hydrogels containing metformin promoted the upregulation of neural regeneration-related proteins, including ATP5F1, ATP5J, NADH dehydrogenase (ubiquinone) Fe-S protein 3 (NDUFS3), and Glutamate Dehydrogenase 1 (GLUD1). Our results help to promote the clinical application of stem-cell neural regeneration.

Oxidative Kinetic Resolution of Cyclic Benzylic Ethers.

A manganese-catalyzed oxidative kinetic resolution of cyclic benzylic ethers through asymmetric C(sp3 )-H oxidation is reported. The practical approach is applicable to a wide range of 1,3-dihydroisobenzofurans bearing diverse functional groups and substituent patterns at the α position with extremely efficient enantiodiscrimination. The generality of the strategy was further demonstrated by efficient oxidative kinetic resolution of another type of five-membered cyclic benzylic ether, 2,3-dihydrobenzofurans, and six-membered 6H-benzo[c]chromenes. Direct late-stage oxidative kinetic resolution of bioactive molecules that are otherwise difficult to access was further explored.

Site- and Enantiodifferentiating C(sp3)-H Oxidation Enables Asymmetric Access to Structurally and Stereochemically Diverse Saturated Cyclic Ethers.

A manganese-catalyzed site- and enantiodifferentiating oxidation of C(sp3)-H bonds in saturated cyclic ethers has been described. The mild and practical method is applicable to a range of tetrahydrofurans, tetrahydropyrans, and medium-sized cyclic ethers with multiple stereocenters and diverse substituent patterns in high efficiency with extremely efficient site- and enantiodiscrimination. Late-stage application in complex biological active molecules was further demonstrated. Mechanistic studies by combined experiments and computations elucidated the reaction mechanism and origins of stereoselectivity. The ability to employ ether substrates as the limiting reagent, together with a broad substrate scope, and a high level of chiral recognition, represent a valuable demonstration of the utility of asymmetric C(sp3)-H oxidation in complex molecule synthesis.

Protein O-mannosylation affects protein secretion, cell wall integrity and morphogenesis in Trichoderma reesei.

Protein O-mannosyltransferases (PMTs) initiate O-mannosylation of proteins in the ER. Trichoderma reesei strains displayed a single representative of each PMT subfamily, Trpmt1, Trpmt2 and Trpmt4. In this work, two knockout strains ΔTrpmt1and ΔTrpmt4were obtained. Both mutants showed retarded growth, defective cell walls, reduced conidiation and decreased protein secretion. Additionally, the ΔTrpmt1strain displayed a thermosensitive growth phenotype, while the ΔTrpmt4 strain showed abnormal polarity. Meanwhile, OETrpmt2 strain, in which the Trpmt2 was over-expressed, exhibited increased conidiation, enhanced protein secretion and abnormal polarity. Using a lectin enrichment method and MS/MS analysis, 173 O-glycoproteins, 295 O-glycopeptides and 649 O-mannosylation sites were identified as the targets of PMTs in T. reesei. These identified O-mannoproteins are involved in various physiological processes such as protein folding, sorting, transport, quality control and secretion, as well as cell wall integrity and polarity. By comparing proteins identified in the mutants and its parent strain, the potential specific protein substrates of PMTs were identified. Based on our results, TrPMT1 is specifically involved inO-mannosylation of intracellular soluble proteins and secreted proteins, specially glycosidases. TrPMT2 is involved inO-mannosylation of secreted proteins and GPI-anchor proteins, and TrPMT4 mainly modifies multiple transmembrane proteins. The TrPMT1-TrPMT4 complex is responsible for O-mannosylation of proteins involved in cell wall integrity. Overexpression of TrPMT2 enhances protein secretion, which might be a new strategy to improve expression efficiency in T. reesei.

Genetical and O-glycoproteomic analyses reveal the roles of three protein O-mannosyltransferases in phytopathogen Fusarium oxysporum f.sp. cucumerinum.

Protein O-mannosyltransferases (PMTs) have been identified in fungi but not in plants and nematodes, which makes PMTs become attractive targets for developing a new strategy against phytopathogens. Three PMTs have been identified in Fusarium oxysporum, a fungal pathogen that causes vascular wilt in a broad range of economical crops. By deletion or suppression of the pmt genes, we showed that all mutants displayed retarded growth, reduced conidiation, cell wall defects, ER stress and attenuated virulence in F. oxysporum f.sp. cucumerinum. In addition, the Δpmt1 exhibited reduced thermotolerance, while the Δpmt4 and the pmt2 conditional mutant exhibited abnormal polarized growth. Comparative glycoproteome analysis of these pmt mutants revealed that PMTs preferentially modified random coils with flanking regions rich in Ser, Thr, Ala, Glu, Asp and Lys at the stem region of membrane proteins, the N-terminal region close to signal peptide of secreted proteins, or surface of soluble proteins. PMT1 specifically acted on nuclear proteins and proteins that are responsible for protein folding, which might contribute to thermotolerance. PMT4 specifically acted on the membrane and soluble proteins in secretory pathways, especially the GPI anchoring pathway, which might contribute to synthesis and transportation of GPI anchored proteins and thus polarized growth. PMT2 was responsible for modification of proteins that are required for protein folding and cell wall synthesis, which might make PMT2 essential. Our results gave an insight to understanding of the roles of each O-mannosyltransferase in F. oxysporum f.sp. cucumerinum and provide a new perspective to prevent Fusarium wilt.

δ-Cyano substituted para-quinone methides enable access to unsymmetric tri- and tetraarylmethanes containing all-carbon quaternary stereocenters.

para-Quinone methides bearing an electron-withdrawing cyano group at the exocyclic methylene δ-position were identified as valuable 1,6-conjugate addition building blocks for acyclic all-carbon quaternary stereocenter construction. A wide variety of electron-rich arenes as nucleophiles were tolerated, effectively furnishing diverse unsymmetrical triarylmethanes bearing all-carbon quaternary stereocenters. The robust transformable abilities of the cyano group provide a platform to access other valuable functional group-containing unsymmetrical tri- and tetraarylmethanes that are otherwise difficult to be prepared. Computational studies supported the hypothesis that the cyano group at the δ-position tunes the molecular electron-density distribution, and the stability of para-quinone methides is enhanced by lowering their polymerizability.

CerR, a Single-Domain Regulatory Protein of the LuxR Family, Promotes Cerecidin Production and Immunity in Bacillus cereus.

Cerecidins are small lantibiotics from Bacillus cereus that were obtained using a semi-in vitro biosynthesis strategy and showed prominent antimicrobial activities against certain Gram-positive bacteria. However, the parental strain B. cereus As 1.1846 is incapable of producing cerecidins, most probably due to the transcriptional repression of the cerecidin gene cluster. Located in the cerecidin gene cluster, cerR encodes a putative response regulator protein that belongs to the LuxR family transcriptional regulators. CerR (84 amino acids) contains only a conserved DNA binding domain and lacks a conventional phosphorylation domain, which is rarely found in lantibiotic gene clusters. To investigate its function in cerecidin biosynthesis, cerR was constitutively expressed in B. cereus As 1.1846. Surprisingly, Constitutive expression of cerR enabled the production of cerecidins and enhanced self-immunity of B. cereus toward cerecidins. Reverse transcription-PCR analysis and electrophoresis mobility shift assays indicated, respectively, that the cer cluster was transcribed in two transcripts (cerAM and cerRTPFE) and that CerR regulated the cerecidin gene cluster directly by binding to the two predicted promoter regions of cerA and cerR DNase I footprinting experiments further confirmed that CerR specifically bound to the two promoter regions at a conserved inverted repeat sequence that was designated a CerR binding motif (cerR box). The present study demonstrated that CerR, as the first single-domain LuxR family transcriptional regulator, serves as a transcriptional activator in cerecidin biosynthesis and activates the cerecidin gene cluster, which was otherwise cryptic in B. cereusIMPORTANCE Lantibiotics with intriguing and prominent bioactivities are potential peptide antibiotics that could be applied in many areas, including food and pharmaceutical industries. The biosynthesis of lantibiotics is generally controlled by two-component regulatory systems consisting of histidine kinases and response regulators, while some unique and interesting regulatory systems are also revealed with the ever-increasing discovery of lantibiotic gene clusters among diverse microorganisms. Dissection of diverse lantibiotic regulation machineries would permit deep understanding of the biological functions of lantibiotics in different niches and even enable genetic activation of lantibiotic gene clusters that are otherwise cryptic. The significance of our study is to illuminate the regulatory mechanism of a special single-domain protein, CerR, in regulating cerecidin biosynthesis in Bacillus cereus, providing a possible novel approach to activate cryptic lantibiotic clusters.

Iron(II)-Catalyzed Site-Selective Functionalization of Unactivated C(sp3 )-H Bonds Guided by Alkoxyl Radicals.

An alkoxyl radical guided strategy for site-selective functionalization of unactivated methylene and methine C-H bonds enabled by an FeII -catalyzed redox process is described. The mild, expeditious, and modular protocol allows efficient remote aliphatic fluorination, chlorination, amination, and alkynylation of structurally and electronically varied primary, secondary, and tertiary hydroperoxides with excellent functional-group tolerance. The application for one-pot 1,4-hydroxyl functionalization of non-oxygenated alkane substrates initiated by aerobic C-H oxygenation is also demonstrated.

The Thioredoxin GbNRX1 Plays a Crucial Role in Homeostasis of Apoplastic Reactive Oxygen Species in Response to Verticillium dahliae Infection in Cotton.

Examining the proteins that plants secrete into the apoplast in response to pathogen attack provides crucial information for understanding the molecular mechanisms underlying plant innate immunity. In this study, we analyzed the changes in the root apoplast secretome of the Verticillium wilt-resistant island cotton cv Hai 7124 (Gossypium barbadense) upon infection with Verticillium dahliae Two-dimensional differential gel electrophoresis and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry analysis identified 68 significantly altered spots, corresponding to 49 different proteins. Gene ontology annotation indicated that most of these proteins function in reactive oxygen species (ROS) metabolism and defense response. Of the ROS-related proteins identified, we further characterized a thioredoxin, GbNRX1, which increased in abundance in response to V. dahliae challenge, finding that GbNRX1 functions in apoplastic ROS scavenging after the ROS burst that occurs upon recognition of V. dahliae Silencing of GbNRX1 resulted in defective dissipation of apoplastic ROS, which led to higher ROS accumulation in protoplasts. As a result, the GbNRX1-silenced plants showed reduced wilt resistance, indicating that the initial defense response in the root apoplast requires the antioxidant activity of GbNRX1. Together, our results demonstrate that apoplastic ROS generation and scavenging occur in tandem in response to pathogen attack; also, the rapid balancing of redox to maintain homeostasis after the ROS burst, which involves GbNRX1, is critical for the apoplastic immune response.

Organocatalytic Redox Deracemization of Cyclic Benzylic Ethers Enabled by An Acetal Pool Strategy.

The first redox deracemization of a series of cyclic benzylic ethers, including 6H-benzo[c]chromenes, isochromans, and 1H-isochromenes, is described. An "acetal pool" strategy was adopted to harmonize the complete oxidation of secondary ethers with imidodiphosphoric acid catalyzed asymmetric transfer hydrogenation. The synthetic utility of the process was demonstrated by the effective deracemization of biologically active molecules of interest that are difficult to prepare by other methods.

Chromium(II)-catalyzed enantioselective arylation of ketones.

The chromium-catalyzed enantioselective addition of carbo halides to carbonyl compounds is an important transformation in organic synthesis. However, the corresponding catalytic enantioselective arylation of ketones has not been reported to date. Herein, we report the first Cr-catalyzed enantioselective addition of aryl halides to both arylaliphatic and aliphatic ketones with high enantioselectivity in an intramolecular version, providing facile access to enantiopure tetrahydronaphthalen-1-ols and 2,3-dihydro-1H-inden-1-ols containing a tertiary alcohol.

Thiosulfate transfer mediated by DsrE/TusA homologs from acidothermophilic sulfur-oxidizing archaeon Metallosphaera cuprina.

Conserved clusters of genes encoding DsrE and TusA homologs occur in many archaeal and bacterial sulfur oxidizers. TusA has a well documented function as a sulfurtransferase in tRNA modification and molybdenum cofactor biosynthesis in Escherichia coli, and DsrE is an active site subunit of the DsrEFH complex that is essential for sulfur trafficking in the phototrophic sulfur-oxidizing Allochromatium vinosum. In the acidothermophilic sulfur (S(0))- and tetrathionate (S4O6(2-))-oxidizing Metallosphaera cuprina Ar-4, a dsrE3A-dsrE2B-tusA arrangement is situated immediately between genes encoding dihydrolipoamide dehydrogenase and a heterodisulfide reductase-like complex. In this study, the biochemical features and sulfur transferring abilities of the DsrE2B, DsrE3A, and TusA proteins were investigated. DsrE3A and TusA proved to react with tetrathionate but not with NaSH, glutathione persulfide, polysulfide, thiosulfate, or sulfite. The products were identified as protein-Cys-S-thiosulfonates. DsrE3A was also able to cleave the thiosulfate group from TusA-Cys(18)-S-thiosulfonate. DsrE2B did not react with any of the sulfur compounds tested. DsrE3A and TusA interacted physically with each other and formed a heterocomplex. The cysteine residue (Cys(18)) of TusA is crucial for this interaction. The single cysteine mutants DsrE3A-C(93)S and DsrE3A-C(101)S retained the ability to transfer the thiosulfonate group to TusA. TusA-C(18)S neither reacted with tetrathionate nor was it loaded with thiosulfate with DsrE3A-Cys-S-thiosulfonate as the donor. The transfer of thiosulfate, mediated by a DsrE-like protein and TusA, is unprecedented not only in M. cuprina but also in other sulfur-oxidizing prokaryotes. The results of this study provide new knowledge on oxidative microbial sulfur metabolism.

Identification of heat shock protein 27 as a novel autoantigen of Behçet's disease.

OBJECTIVE: The aim of this study was to identify candidate pathogenic autoantigens of Behçet's disease (BD) in pathogen-stimulated target cells. METHODS: First, three cell lines were used as target cells to screen autoantibody. Second, selected target cells were simulated with pathogens. Third, western blotting was used for detecting the auto-antigens in cell extracts. Next, immunoprecipitation was performed and the amino-acid sequences of target antigens were analyzed by LC-MALDI-TOF/TOF. Then, the potential target antigen was expressed, purified, and immunologically confirmed. And finally, an ELISA kit was developed and clinically validated through the assessments of 456 clinical samples with BD. RESULTS: One antigen with a molecular weight of approximately 27-kDa was identified as heat shock protein 27 (HSP27). The reactivity of serum IgG against recombinant human HSP27 was detected in 52 of 91 BD patients (57%), 66 of 92 rheumatoid arthritis (RA) patients (72%), 32 of 90 Sjogren syndrome (SS) patients (36%), 22 of 92 systemic lupus erythematosus (SLE) patients (24%) and 0 of 91 healthy controls (HC). The reactivity of BD serum IgG antibodies against HSP27 was significantly higher than SLE (P<0.0001) SS (P<0.0001) and HC (P<0.0001). CONCLUSIONS: This study identified HSP27 as a candidate endothelial cell autoantigen of BD, which is interesting and probably worth further exploration.

[Semi-in vitro biosynthesis of cryptic lanthipeptide CLA 124 from Streptomyces clavuligerus].

OBJECTIVE: To obtain the cryptic lanthipeptide from Streptomyces clavuligerus by semi-in vitro biosynthesis that is a novel method for mining lanthipeptides resource from Streptomyces. METHODS: The core peptide of cryptic lanthipeptide was modified in E. coli by nisin modification system, and purified by affinity chromatography and High Performance Liquid Chromatography (HPLC). After the leader peptide was removed, the core peptide was obtained and its dehydration and cyclic structure were analyzed by MALDI-TOF MS and tandem MS. RESULTS: A novel lanthipeptide named CLA 124 with 4-fold dehydration 2 thioether bridges and one disulfide bridge was produced. CONCLUSION: Cryptic lanthipeptides from Streptomyces could be produced by semi-in vitro biosynthesis.

The periplasmic PDZ domain-containing protein Prc modulates full virulence, envelops stress responses, and directly interacts with dipeptidyl peptidase of Xanthomonas oryzae pv. oryzae.

PDZ domain-containing proteases, also known as HtrA family proteases, play important roles in bacterial cells by modulating disease pathogenesis and cell-envelope stress responses. These proteases have diverse functions through proteolysis- and nonproteolysis-dependent modes. Here, we report that the genome of the causative agent of rice bacterial blight, Xanthomonas oryzae pv. oryzae, encodes seven PDZ domain-containing proteins. Systematic inactivation of their encoding genes revealed that PXO_01122 and PXO_04290 (prc) are involved in virulence. prc encodes a putative HtrA family protease that localizes in the bacterial periplasm. Mutation of prc also resulted in susceptibility to multiple environmental stresses, including H2O2, sodium dodecylsulfate, and osmolarity stresses. Comparative subproteomic analyses showed that the amounts of 34 periplasmic proteins were lower in the prc mutant than in wild-type. These proteins were associated with proteolysis, biosynthesis of macromolecules, carbohydrate or energy metabolism, signal transduction, and protein translocation or folding. We provide in vivo and in vitro evidence demonstrating that Prc stabilizes and directly binds to one of these proteins, DppP, a dipeptidyl peptidase contributing to full virulence. Taken together, our results suggest that Prc contributes to bacterial virulence by acting as a periplasmic modulator of cell-envelope stress responses.

Cerecidins, novel lantibiotics from Bacillus cereus with potent antimicrobial activity.

Lantibiotics are ribosomally synthesized and posttranslationally modified antimicrobial peptides that are widely produced by Gram-positive bacteria, including many species of the Bacillus group. In the present study, one novel gene cluster coding lantibiotic cerecidins was unveiled in Bacillus cereus strain As 1.1846 through genomic mining and PCR screening. The designated cer locus is different from that of conventional class II lantibiotics in that it included seven tandem precursor cerA genes, one modification gene (cerM), two processing genes (cerT and cerP), one orphan regulator gene (cerR), and two immunity genes (cerF and cerE). In addition, one unprecedented quorum sensing component, comQXPA, was inserted between cerM and cerR. The expression of cerecidins was not detected in this strain of B. cereus, which might be due to repressed transcription of cerM. We constitutively coexpressed cerA genes and cerM in Escherichia coli, and purified precerecidins were proteolytically processed with the endoproteinase GluC and a truncated version of putative serine protease CerP. Thus, two natural variants of cerecidins A1 and A7 were obtained which contained two terminal nonoverlapping thioether rings rarely found in lantibiotics. Both cerecidins A1 and A7 were active against a broad spectrum of Gram-positive bacteria. Cerecidin A7, especially its mutant Dhb13A, showed remarkable efficacy against multidrug-resistant Staphylococcus aureus (MDRSA), vancomycin-resistant Enterococcus faecalis (VRE), and even Streptomyces.

Restoration of bioactive lantibiotic suicin from a remnant lan locus of pathogenic Streptococcus suis serotype 2.

Lantibiotics are ribosomally synthesized, posttranslationally modified antimicrobial peptides. Their biosynthesis genes are usually organized in gene clusters, which are mainly found in Gram-positive bacteria, including pathogenic streptococci. Three highly virulent Streptococcus suis serotype 2 strains (98HAH33, 05ZYH33, and SC84) have been shown to contain an 89K pathogenicity island. Here, on these islands, we unveiled and reannotated a putative lantibiotic locus designated sui which contains a virulence-associated two-component regulator, suiK-suiR. In silico analysis revealed that the putative lantibiotic modification gene suiM was interrupted by a 7.9-kb integron and that other biosynthesis-related genes contained various frameshift mutations. By reconstituting the intact suiM in Escherichia coli together with a semi-in vitro biosynthesis system, a putative lantibiotic named suicin was produced with bactericidal activities against a variety of Gram-positive strains, including pathogenic streptococci and vancomycin-resistant enterococci. Ring topology dissection indicated that the 34-amino-acid lantibiotic contained two methyllanthionine residues and one disulfide bridge, which render suicin in an N-terminal linear and C-terminal globular shape. To confirm the function of suiK-suiR, SuiR was overexpressed and purified. In vitro analysis showed that SuiR could specifically bind to the suiA gene promoter. Its coexpression with suiK could activate suiA gene promoter in Lactococcus lactis NZ9000. Conclusively, we obtained a novel lantibiotic suicin by restoring its production from the remnant sui locus and demonstrated that virulence-associated SuiK-SuiR regulates its production.

An economical and environmentally friendly oxidative biaryl coupling promoted by activated MnO2.

An activated manganese dioxide (MnO2)-BF3·OEt2 oxidation system was developed to efficiently mediate the intramolecular as well as intermolecular biaryl coupling. The oxidative coupling proceeds smoothly at ambient temperature to deliver the corresponding five- to eight-membered tricyclic products in good to excellent yields. The employment of the combination of MnO2 and BF3·OEt2 is attractive on the basis of economical and environmental issues.

Catalytic enantioselective oxidative cross-coupling of benzylic ethers with aldehydes.

The first one-pot enantioselective oxidative coupling of cyclic benzylic ethers with aldehydes has been developed. A variety of benzylic ethers were transformed into the corresponding oxygen heterocycles with high enantioselectivity. Mechanistic experiments were conducted to determine the nature of the reaction intermediates. The application of this strategy to coupling reactions with other nucleophiles besides aldehydes was also explored.

Catalytic Asymmetric Access to Structurally Diverse N-Alkoxy Amines via a Kinetic Resolution Strategy.

Chiral N-alkoxy amines are increasingly vital substrates in bioscience. However, asymmetric synthetic strategies for these compounds remain scarce. Catalytic kinetic resolution represents an attractive approach to prepare structurally diverse enantiopure N-alkoxy amines, which has remained elusive due to the notably reduced nucleophilicity of the nitrogen atom together with the low bond dissociation energies of labile NO-C and N-O bonds. We here report a general kinetic resolution of N-alkoxy amines through chemo- and enantioselective oxygenation. The mild and green titanium-catalyzed approach features broad substrate scope (55 examples), noteworthy functional group compatibility, high catalyst turnover number (up to 5200), excellent selectivity factor (s > 150), and scalability.