Melatonin alleviates hypoxia-induced injury of PC12 cells by inhibiting SOX2 expression / 国际生物医学工程杂志

Objective:To investigate the effect of melatonin (MT) on hypoxia-induced injury of PC12 cells and its mechanism.Methods:PC12 cells cultured in vitro were divided into the control, the hypoxic, and three groups of hypoxic cells combined with different doses (low, medium, and high) of MT at 12.5, 25, and 50 μmol/L, respectively. Cell viability was measured by MTT. The expression of cysteine-containing aspartate 3 (cleaved caspase-3) and SOX2 proteins was detected by Western blot. The expression of SOX2 mRNA was detected by real-time fluorescence quantitative polymerase chain reaction (PCR). The activity of superoxide dismutase (SOD) and content of malondialdehyde (MDA) were detected by the kit. The combined treatment group with the most significant effect was selected as the hypoxia+MT group, and then the effects of hypoxia+MT, hypoxia+siRNA-NC, hypoxia+siRNA-SOX2, hypoxia+MT+pcDNA, and hypoxia+MT+pcDNA-SOX2 on cell viability, apoptosis, protein expression level of cleaved-caspase-3, protein and mRNA levels of SOX2, SOD activity, and MDA levels, respectively, were investigated.Results:Compared with the control group, cell survival rate and SOD activity in the hypoxia group were significantly decreased, while cell apoptosis rate, cleaved caspase-3 protein expression level, MDA content, SOX2 mRNA, and protein expression level were significantly increased (all P<0.05). The effects of low, medium, and high levels of MT and the low expression of SOX2 could ameliorate the above changes induced by hypoxia (all P<0.05), while the overexpression of SOX2 could alleviate the effects of MT on the viability, apoptosis, and oxidative stress of hypoxia-induced PC12 cells (all P<0.05). Conclusions:MT can reduce hypoxia-induced injury to PC12 cells by inhibiting the expression of SOX2.

Discovery of a new nosiheptide-producing strain and its fermentation optimization for nosiheptide production / 中国药科大学学报

@#Nosiheptide is a typical thiopeptide antibiotic displaying potent activity toward various drug-resistant strains of Gram-positive pathogens.Although nosiheptide lacks in vivo activity, and good water-solubility with a series of uncontrollable analogues, which may limit its clinical application, glycosylated analogues may overcome problem of low activity and may improve its druggability.In search of novel glycosylated nosiheptide producers, we applied a genome mining strategy that identified Actinoalloteichus sp.AHMU CJ021 that contains all genes required.However, despite the presence of a predicted glycosyltransferase, glycosylated derivatives of nosiheptide were not detected, after following one strain many compounds (OSMAC) strategy and heterologous expression of a regulatory protein NocP.Nevertheless, nosiheptide produced by this strain was remarkably pure, and further experiments were conducted to improve its production by optimization of the culture medium.Under optimal conditions, 58.73 mg/L nosiheptide was produced, representing an almost 6-fold improvement compared to the original fermentation medium.Therefore, we consider Actinoalloteichus sp.AHMU CJ021 a suitable potential candidate for industrial production of nosiheptide, which provides the basis for solving the problem of nosiheptide structural analogues.

Genome mining combined metabolic shunting and OSMAC strategy of an endophytic fungus leads to the production of diverse natural products

Endophytic fungi are promising producers of bioactive small molecules. Bioinformatic analysis of the genome of an endophytic fungus

Genome sequencing of Streptomyces aureofaciens DM-1 and analysis of 6-demethylchlortetracycline biosynthesis gene cluster / 生物工程学报

Streptomyces aureofaciens DM-1 is a high-yielding 6-demethylchlortetracycline producer. The genome sequencing of DM-1 reveals a linear chromosome containing 6 824 334 bps nucleotides with GC content of 72.6%. In this genome, a total of 6 431 open reading frames were predicted by using glimmer 3.02, Genemark and Z-Curve softwares. Twenty-eight secondary metabolite biosynthetic gene clusters were uncovered by using AntiSMASH gene prediction software, including the complete 6-demethylchlortetracycline biosynthetic gene cluster. A frame-shift mutation in methyltransferase coding region was detected, which may result in the demethylation of chlortetracycline. The complete genome sequence of S. aureofaciens DM-1 provides basic information for functional genomics studies and selection of high-yielding strains for 6-demethylchlortetracycline.

Characterization of inthomycin biosynthetic gene cluster revealing new insights into carboxamide formation / 中国天然药物

Inthomycins are polyketide antibiotics which contain a terminal carboxamide group and a triene chain. Inthomycin B (1) and its two new analogues 2 and 3 were isolated from the crude extract of Streptomyces pactum L8. Identification of the gene cluster for inthomycin biosynthesis as well as the N-labeled glycine incorporation into inthomycins are described. Combined with the gene deletion of the rare P450 domain in the NRPS module, a formation mechanism of carboxamide moiety in inthomycins was proposed via an oxidative release of the assembly chain assisted by the P450 domain.

Activation of anthrachamycin biosynthesis in Streptomyces chattanoogensis L10 by site-directed mutagenesis of rpoB / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Genome sequencing projects revealed massive cryptic gene clusters encoding the undiscovered secondary metabolites in Streptomyces. To investigate the metabolic products of silent gene clusters in Streptomyces chattanoogensis L10 (CGMCC 2644), we used site-directed mutagenesis to generate ten mutants with point mutations in the highly conserved region of rpsL (encoding the ribosomal protein S12) or rpoB (encoding the RNA polymerase β-subunit). Among them, L10/RpoB (H437Y) accumulated a dark pigment on a yeast extract-malt extract-glucose (YMG) plate. This was absent in the wild type. After further investigation, a novel angucycline antibiotic named anthrachamycin was isolated and determined using nuclear magnetic resonance (NMR) spectroscopic techniques. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis and electrophoretic mobility shift assay (EMSA) were performed to investigate the mechanism underlying the activation effect on the anthrachamycin biosynthetic gene cluster. This work indicated that the rpoB-specific missense H437Y mutation had activated anthrachamycin biosynthesis in S. chattanoogensis L10. This may be helpful in the investigation of the pleiotropic regulation system in Streptomyces.

Activation of anthrachamycin biosynthesis in Streptomyces chattanoogensis L10 by site-directed mutagenesis of rpoB / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Genome sequencing projects revealed massive cryptic gene clusters encoding the undiscovered secondary metabolites in Streptomyces. To investigate the metabolic products of silent gene clusters in Streptomyces chattanoogensis L10 (CGMCC 2644), we used site-directed mutagenesis to generate ten mutants with point mutations in the highly conserved region of rpsL (encoding the ribosomal protein S12) or rpoB (encoding the RNA polymerase β-subunit). Among them, L10/RpoB (H437Y) accumulated a dark pigment on a yeast extract-malt extract-glucose (YMG) plate. This was absent in the wild type. After further investigation, a novel angucycline antibiotic named anthrachamycin was isolated and determined using nuclear magnetic resonance (NMR) spectroscopic techniques. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis and electrophoretic mobility shift assay (EMSA) were performed to investigate the mechanism underlying the activation effect on the anthrachamycin biosynthetic gene cluster. This work indicated that the rpoB-specific missense H437Y mutation had activated anthrachamycin biosynthesis in S. chattanoogensis L10. This may be helpful in the investigation of the pleiotropic regulation system in Streptomyces.

Exploring tRNA gene cluster in archaea

BACKGROUND Shared traits between prokaryotes and eukaryotes are helpful in the understanding of the tree of life evolution. In bacteria and eukaryotes, it has been shown a particular organisation of tRNA genes as clusters, but this trait has not been explored in the archaea domain. OBJECTIVE Explore the occurrence of tRNA gene clusters in archaea. METHODS In-silico analyses of complete and draft archaeal genomes based on tRNA gene isotype and synteny, tRNA gene cluster content and mobilome elements. FINDINGS We demonstrated the prevalence of tRNA gene clusters in archaea. tRNA gene clusters, composed of archaeal-type tRNAs, were identified in two Archaea class, Halobacteria and Methanobacteria from Euryarchaeota supergroup. Genomic analyses also revealed evidence of the association between tRNA gene clusters to mobile genetic elements and intra-domain horizontal gene transfer. MAIN CONCLUSIONS tRNA gene cluster occurs in the three domains of life, suggesting a role of this type of tRNA gene organisation in the biology of the living organisms.

Progress in fungal polyketide biosynthesis / 生物工程学报

Fungal polyketides display complex structures and variously biological activities. Their biosynthetic pathways generally contain novel enzyme-catalyzed reactions. This review provides a summary of recent research advances in molecular mechanism of the biosynthesis of fungal polyketides including highly-reducing polyketide synthases (HR-PKSs), non-reducing polyketide synthases (NR-PKSs), as well as polyketide-nonribosomal peptide synthase (PKS-NRPSs) and reducingnon- reducing polyketide synthase (HR-NR PKSs) hybrids. The elucidation of biosynthetic mechanism of many fungal polyketides provides guidance on the discovery of new biosynthetic gene cluster of fungal polyketide natural products and compounds with novel structures as well as their analogue.

Study of the Polymorphisms rs1491033A/C and rs1813389A/G in the miR-17-92 Gene Cluster Promoter Region in a Guangxi Population / 中国医科大学学报

Objective To investigate the distribution of the polymorphisms rs1491033A/C and rs1813389A/G in the promoter region of the miR-17-92 gene cluster in a normal population of Guangxi and compare this distribution with that in different ethnic groups. Methods We detected the genotypes of rs1491033A/C and rs1813389A/G of 275 people from Guangxi using the SNaPshot technique and DNA sequencing. We then analyzed the genotype and allele frequency distribution differences among different genders and groups. Results Three genotypes,AA,AC,and CC,were found for rs1491033A/C with frequency distributions of 21. 1％,53. 1％,and 25. 8％, respectively. There were significant differences in genotype and allele frequencies of rs1491033A/C between the Guangxi population and those of Europe and Africa,as published in the International HapMap project (P < 0. 05). Three genotypes,AA,AG,and GG were observed for rs1813389A/G with frequency distributions of 40. 0％,50. 2％,and 9. 8％,respectively. There were significant differences in the genotype and allele frequencies of rs1813389A/G between the Guangxi population and those of Europe,Beijing,Japan,and Africa (P <0. 05). However,there were no significant differences in genotype or allele frequencies of the rs1491033A/C or rs1813389A/G polymorphisms between genders in the Guangxi population (P > 0. 05). Conclusion There are different distributions of the rs1491033A/C and rs1813389A/G polymorphisms in the miR-17-92 gene cluster in different races and regions.

Distribution of polymorphisms at promoter region of miR-17-92 in Guangxi population and association of its polymorphisms and lymphocytes / 中国免疫学杂志

Objective:To investigate the distribution characteristics of polymorphisms of rs9515692C/T and rs1352743A/G in the promoter region of miR-17-92 gene cluster in Guangxi people and compare them with those of other ethnic groups and explore the association of its polymorphisms and lymphocytes.Methods:The rs9515692C/T and rs1352743A/G of miR-17-92 gene cluster were genotyped by using SNaPshot technique and DNA sequencing.Detection of the number of lymphocytes using flow cytometry.The differences of polymorphisms between groups were analyzed statistically.Results:No significant differences of genotype and allele frequency in the two SNPs was observed between different gender in the Guangxi population(P>0.05).However,there were significant differences in the distribution frequencies of genotype and allele of Europeans,Japanese and Africans in rs9515692C/T and rs1352743A/G (P<0.05).Conclusion:Polymorphisms of rs9515692C/T and rs1352743A/G are different in different people.In addition,rs9515692C/T polymorphism may be associated with the number of B cells.

van genotype and molecular typing of clinical isolates of vancomycin-resistant Enterococcus / 中国感染与化疗杂志

Objective To determine the genotype and molecular typing of vancomycin-resistant Enterococcus (VRE).Methods Seventeen clinical isolates of VRE were collected in 2016.The strains were identified to species and confirmed by 16S rRNA sequencing.The minimum inhibitory concentration of antimicrobial agents was determined by microdilution method and agar dilution method.Multilocus sequence typing (MLST) was used for molecular typing.Results The VRE strains were confirmed as Enterococcusfaecium by 16S rRNA sequencing.All strains were resistant to vancomycin,but only 12 strains were resistant to teicoplanin.The vanA gene was identified in 13 of the 17 strains.The vanM gene was detected in 9 strains.Both vanA and vanM genes were identified in five of the 17 strains.Six MLST types were identified in the 17 strains,including ST78 (n=8),ST80 (n=4),ST555 (n=2),and one each for ST117,ST262 and ST341.Conclusions The van genotype was primarily vanA (76.5％) and vanM (52.9％) in clinical isolates of VRE.The VRE strains carrying both vanA and vanM were found for the first time.

Biosynthesis of antibiotic chuangxinmycin from

Chuangxinmycin is an antibiotic isolated from CPCC 200056 in the 1970s with a novel indole-dihydrothiopyran heterocyclic skeleton. Chuangxinmycin showed antibacterial activity and efficacy in mouse infection models as well as preliminary clinical trials. But the biosynthetic pathway of chuangxinmycin has been obscure since its discovery. Herein, we report the identification of a stretch of DNA from the genome of CPCC 200056 that encodes genes for biosynthesis of chuangxinmycin by bioinformatics analysis. The designated cluster was then confirmed to be responsible for chuangxinmycin biosynthesis by direct cloning and heterologous expressing in M1146. The cytochrome P450 CxnD was verified to be involved in the dihydrothiopyran ring closure reaction by the identification of seco-chuangxinmycin in M1146 harboring the gene cluster with an inactivated . Based on these results, a plausible biosynthetic pathway for chuangxinmycin biosynthesis was proposed, by hijacking the primary sulfur transfer system for sulfur incorporation. The identification of the biosynthetic gene cluster of chuangxinmycin paves the way for elucidating the detail biochemical machinery for chuangxinmycin biosynthesis, and provides the basis for the generation of novel chuangxinmycin derivatives by means of combinatorial biosynthesis and synthetic biology.

Activation of an unconventional meroterpenoid gene cluster in leads to the production of new berkeleyacetals

Fungal genomes carry many gene clusters seemingly capable of natural products biosynthesis, yet most clusters remain cryptic or down-regulated. Genome mining revealed an unconventional paraherquonin-like meroterpenoid biosynthetic gene cluster in the chromosome of . The cryptic or down-regulated pathway was activated by constitutive expression of pathway-specific regulator gene encoded within biosynthetic gene cluster. Chemical analysis of mutant -OE: extracts enabled the isolation of four berkeleyacetal congeners, in which two of them are new. On the basis of careful bioinformatic analysis of the coding enzymes in the gene cluster, the biosynthetic pathway of berkeleyacetals was proposed. These results indicate that this approach would be valuable for discovery of novel natural products and will accelerate the exploitation of prodigious natural products in filamentous fungi.

Application of TAR cloning in microbial secondary metabolites / 中国药科大学学报

@#Microbial secondary metabolites have been a major source for drug discovery and development due to their structural novelty and diversity. Microbial secondary metabolites, typically encoded by specific biosynthetic gene cluster(BGC), are non-essential for the growth and propagation of the microbes. Despite the abundant existence of microbes, the majority of them are unculturable under laboratory conditions. Moreover, given that most of the BGCs from culturable microbes are silent, the discovery of novel microbial secondary metabolites has been hampered. Recently, the heterologous expression of BGCs has become an attractive approach to discover various microbial secondary metabolites, among which TAR-based heterologous expression is one of the important tools. This review summarized the principle of TAR, the applications and the advanced strategies of TAR-based methods for heterologous expression of secondary metabolites, which may help the advancements of drug discovery and development from microbial sources.

Influence of Temperature and Water Activity on Deleterious Fungi and Mycotoxin Production during Grain Storage

Cereal grains are the most important food source for humans. As the global population continues to grow exponentially, the need for the enhanced yield and minimal loss of agricultural crops, mainly cereal grains, is increasing. In general, harvested grains are stored for specific time periods to guarantee their continuous supply throughout the year. During storage, economic losses due to reduction in quality and quantity of grains can become very significant. Grain loss is usually the result of its deterioration due to fungal contamination that can occur from preharvest to postharvest stages. The deleterious fungi can be classified based on predominance at different stages of crop growth and harvest that are affected by environmental factors such as water activity (a(w)) and eco-physiological requirements. These fungi include species such as those belonging to the genera Aspergillus and Penicillium that can produce mycotoxins harmful to animals and humans. The grain type and condition, environment, and biological factors can also influence the occurrence and predominance of mycotoxigenic fungi in stored grains. The main environmental factors influencing grain fungi and mycotoxins are temperature and a(w). This review discusses the effects of temperature and a(w) on fungal growth and mycotoxin production in stored grains. The focus is on the occurrence and optimum and minimum growth requirements for grain fungi and mycotoxin production. The environmental influence on aflatoxin production and hypothesized mechanisms of its molecular suppression in response to environmental changes are also discussed. In addition, the use of controlled or modified atmosphere as an environmentally safe alternative to harmful agricultural chemicals is discussed and recommended future research issues are highlighted.

In silico dissection of Type VII Secretion System components across bacteria: New directions towards functional characterization.

Type VII Secretion System (T7SS) is one of the factors involved in virulence of Mycobacteriun tuberculosis H37Rv. Numerous research efforts have been made in the last decade towards characterizing the components of this secretion system. An extensive genome-wide analysis through compilation of isolated information is required to obtain a global view of diverse characteristics and pathogenicity-related aspects of this machinery. The present study suggests that differences in structural components (of T7SS) between Actinobacteria and Firmicutes, observed earlier in a few organisms, is indeed a global trend. A few hitherto uncharacterized T7SS-like clusters have been identified in the pathogenic bacteria Enterococcus faecalis, Saccharomonospora viridis, Streptococcus equi, Streptococcuss gordonii and Streptococcus sanguinis. Experimental verification of these clusters can shed lights on their role in bacterial pathogenesis. Similarly, verification of the identified variants of T7SS clusters consisting additional membrane components may help in unraveling new mechanism of protein translocation through T7SS. A database of various components of T7SS has been developed to facilitate easy access and interpretation of T7SS related data.

New methods for cloning large gene clusters based on CRISPR/cas9 / 生物工程学报

Cloning of large genomic sequences is an enabling technology in synthetic biology. To obtain large gene fragments, traditional cloning methods are faced with various defects, for instance, random library cloning relies always on high-throughput screening. It is difficult to get gene fragments more than 10 kb by PCR amplification. Assembly of small fragments is labor intensive with high mutation rates. It is difficult to find suitable cleavage sites on the fragment ends by restriction endonuclease. Recently genome-wide editing creates a new high-performance large fragments cloning methods. For example, CRISPR/cas9 system can identify and cut 20 bp nucleic acid sequences recognition sites used to obtain any desired gene fragments; if combined with Gibson or transformation associated recombination (TAR) assembly technology, these methods can efficiently clone large fragments. This article introduces large fragments cloning technology by classification, then proposes the choice criteria of methods for cloning gene fragments of different sizes.

Comparative analysis of rapamycin biosynthesis clusters between Actinoplanes sp. N902-109 and Streptomyces hygroscopicus ATCC29253 / 中国天然药物

The present study was designed to identify the difference between two rapamycin biosynthetic gene clusters from Streptomyces hygroscopicus ATCC29253 and Actinoplanes sp. N902-109 by comparing the sequence and organization of the gene clusters. The biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus ATCC29253 was reported in 1995. The second rapamycin producer, Actinoplanes sp. N902-109, which was isolated in 1995, could produce more rapamycin than Streptomyces hygroscopicus ATCC29253. The genomic map of Actinoplanes sp. N902-109 has been elucidated in our laboratory. Two gene clusters were compared using the online software anti-SMASH, Glimmer 3.02 and Subsystem Technology (RAST). Comparative analysis revealed that the organization of the multifunctional polyketide synthases (PKS) genes: RapA, RapB, RapC, and NRPS-like RapP were identical in the two clusters. The genes responsible for precursor synthesis and macrolactone modification flanked the PKS core region in N902-109, while the homologs of those genes located downstream of the PKS core region in ATCC29253. Besides, no homolog of the gene encoding a putative type II thioesterase that may serve as a PKS "editing" enzyme accounted for over-production of rapamycin in N902-109, was found in ATCC29253. Furthermore, no homologs of genes rapQ (encoding a methyltransferase) and rapG in N902-109 were found in ATCC29253, however, an extra rapM gene encoding methyltransferase was discovered in ATCC29253. Two rapamycin biosynthetic gene clusters displayed overall high homology as well as some differences in gene organization and functions.

Analysis of tellurite resistance level and resistance gene cluster in non-O157 Shiga toxin-producing Escherichia coli isolates / 中华微生物学和免疫学杂志

Objective To investigate the tellurite resistance level,the presence of tellurite resistance (ter) gene cluster and their relationships in non-O157 Shiga toxin-producing Escherichia coli(STEC) isolates.Methods Tellurite resistance level was evaluated by plate dilution method and the ter gene cluster was tested by PCR.Results Only 5 of 39 non-O157 STEC isolates tested in this study were identified to have ter gene cluster,which showed relatively high levels of tellurite resistance ranging from 128 μg/ml to 512 μg/ml.In contrast,the other 34 isolates without ter gene cluster were sensitive to potassium tellurite and showed very low levels of tellurite resistance,the minimal inhibitory concentration (MIC) was ＜1 μg/ml for 29 isolates,8 μg/ml for 2 isolates and 2 μg/ml for 3 isolates.Conclusion Most non-O157 STEC isolates were sensitive to potassium tellurite.It could be concluded that much attention should be paid when screening the non-O157 STEC isolates using the selective medium supplemented with potassium tellurite.