Alginate oligosaccharide assimilation by gut microorganisms and the potential role in gut inflammation alleviation.

Dietary fiber metabolism by gut microorganisms plays important roles in host physiology and health. Alginate, the major dietary fiber of daily diet seaweeds, is drawing more attention because of multiple biological activities. To advance the understanding of alginate assimilation mechanism in the gut, we show the presence of unsaturated alginate oligosaccharides (uAOS)-specific alginate utilization loci (AUL) in human gut microbiome. As a representative example, a working model of the AUL from the gut microorganism Bacteroides clarus was reconstructed from biochemistry and transcriptome data. The fermentation of resulting monosaccharides through Entner-Doudoroff pathway tunes the metabolism of short-chain fatty acids and amino acids. Furthermore, we show that uAOS feeding protects the mice against dextran sulfate sodium-induced acute colitis probably by remodeling gut microbiota and metabolome. IMPORTANCE: Alginate has been included in traditional Chinese medicine and daily diet for centuries. Recently discovered biological activities suggested that alginate-derived alginate oligosaccharides (AOS) might be an active ingredient in traditional Chinese medicine, but how these AOS are metabolized in the gut and how it affects health need more information. The study on the working mechanism of alginate utilization loci (AUL) by the gut microorganism uncovers the role of unsaturated alginate oligosaccharides (uAOS) assimilation in tuning short-chain fatty acids and amino acids metabolism and demonstrates that uAOS metabolism by gut microorganisms results in a variation of cell metabolites, which potentially contributes to the physiology and health of gut.

A medicine and food homology formula prevents cognitive deficits by inhibiting neuroinflammation and oxidative stress via activating AEA-Trpv1-Nrf2 pathway.

Alzheimer's disease (AD) is a neurodegenerative disorder frequently accompanied by neuroinflammation and oxidative stress. The medicine and food homology (MFH) has shown potential for treating neuroinflammation and oxidative stress. This study aimed to provide a safe and efficient therapy for AD based on MFH. In this study, we develop a MFH formula consisting of egg yolk oil, perilla seed oil, raphani seed oil, cinnamon oil, and noni puree (EPRCN). To evaluate the ameliorative effects of EPRCN on AD-related symptoms, a mouse model of AD was constructed using intraperitoneal injection of scopolamine in ICR mice. Experimental results demonstrated that EPRCN supplement restored behavioral deficits and suppressed neuroinflammation and oxidative stress in the hippocampus of scopolamine-induced mice. An in vitro study was then performed using induction of Aß(25-35) in glial (BV-2 and SW-1783) and neuron (SH-SY5Y) cell lines to examine the improvement mechanism of EPRCN on cognitive deficits. Multi-omics and in vitro studies demonstrated that these changes were driven by the anandamide (AEA)-Trpv1-Nrf2 pathway, which was inhibited by AM404 (an AEA inhibitor), AMG9810 (a Trpv1 inhibitor), and BT (an Nrf2 inhibitor). Consequently, EPRCN is an effective therapy on preventing cognitive deficits in mouse models of AD. In contrast to donepezil, EPRCN exhibits a novel modes action for ameliorating neuroinflammation. The mechanism of EPRCN on preventing cognitive deficits is mediated by improving neuroinflammation and oxidative stress via activating the AEA-Trpv1-Nrf2 pathway.

Complete genome sequences and comparative genome analysis of Lactobacillus plantarum strain 5-2 isolated from fermented soybean.

Lactobacillus plantarum is an important probiotic and is mostly isolated from fermented foods. We sequenced the genome of L. plantarum strain 5-2, which was derived from fermented soybean isolated from Yunnan province, China. The strain was determined to contain 3114 genes. Fourteen complete insertion sequence (IS) elements were found in 5-2 chromosome. There were 24 DNA replication proteins and 76 DNA repair proteins in the 5-2 genome. Consistent with the classification of L. plantarum as a facultative heterofermentative lactobacillus, the 5-2 genome encodes key enzymes required for the EMP (Embden-Meyerhof-Parnas) and phosphoketolase (PK) pathways. Several components of the secretion machinery are found in the 5-2 genome, which was compared with L. plantarum ST-III, JDM1 and WCFS1. Most of the specific proteins in the four genomes appeared to be related to their prophage elements.

Characterization of the IncA/C plasmid pSCEC2 from Escherichia coli of swine origin that harbours the multiresistance gene cfr.

OBJECTIVES: To determine the complete nucleotide sequence of the multidrug resistance plasmid pSCEC2, isolated from a porcine Escherichia coli strain, and to analyse it with particular reference to the cfr gene region. METHODS: Plasmid pSCEC2 was purified from its E. coli J53 transconjugant and then sequenced using the 454 GS-FLX System. After draft assembly, predicted gaps were closed by PCR with subsequent sequencing of the amplicons. RESULTS: Plasmid pSCEC2 is 135 615 bp in size and contains 200 open reading frames for proteins of ≥100 amino acids. Analysis of the sequence of pSCEC2 revealed two resistance gene segments. The 4.4 kb cfr-containing segment is flanked by two IS256 elements in the same orientation, which are believed to be involved in the dissemination of the rRNA methylase gene cfr. The other segment harbours the resistance genes floR, tet(A)-tetR, strA/strB and sul2, which have previously been found on other IncA/C plasmids. Except for these two resistance gene regions, the pSCEC2 backbone displayed >99% nucleotide sequence identity to that of other IncA/C family plasmids isolated in France, Chile and the USA. CONCLUSIONS: The cfr gene was identified on an IncA/C plasmid, which is well known for its broad host range and transfer and maintenance properties. The location on such a plasmid will further accelerate the dissemination of cfr and co-located resistance genes among different Gram-negative bacteria. The genetic context of cfr on plasmid pSCEC2 underlines the complexity of cfr transfer events and confirms the role that insertion sequences play in the spread of cfr.

ASPG and DAD1 are potential placental-derived biomarkers for ASD-like symptom severity levels in male/female offspring.

INTRODUCTION: An early evaluating system for autism spectrum disorder (ASD) severity is crucial. Questionnaire survey is challenging for accurately assessing the severity levels for ASD in children. METHODS: Offspring with ASD-like phenotypes were induced by treating pregnant mice with Poly (I:C) at GD12.5 and the placentae corresponding to the offspring were obtained by caesarean. The autism severity composite score (ASCS) for offspring was calculated through behavioral tests. HE staining and immunohistochemistry were used to observe the morphology of placenta. Candidate biomarkers were identified by weighted protein co-expression network analysis (WPCNA) combined with machine learning and further validated by ELISA. Sperman's was used to analyze the correlation between biomarkers and metabolome. RESULTS: The placental weight and mean vascular area of male offspring with ASD-like phenotypes were significantly decreased compared with typical mice. According to the WPCNA, four modules were identified and significantly correlated with ASCS of offspring. Two biomarkers (ASPG and DAD1) with high correlation with ASCS in offspring were identified. DISCUSSION: VEGF pathway may contribute to sexual dimorphism in placental morphology within mice with ASD-like phenotypes in term. The placental ASPG and DAD1 levels could reflect ASD-like symptom severity levels in male/female mice offspring.

[Molecular cloning and characterization of a novel Clonorchis sinensis antigenic protein containing tandem repeat sequences].

OBJECTIVE: To find and clone new antigen genes from the lambda-ZAP cDNA expression library of adult Clonorchis sinensis, and determine the immunological characteristics of the recombinant proteins. METHODS: The cDNA expression library of adult C. sinensis was screened by pooled sera of clonorchiasis patients. The sequences of the positive phage clones were compared with the sequences in EST database, and the full-length sequence of the gene (Cs22 gene) was obtained by RT-PCR. cDNA fragments containing 2 and 3 times tandem repeat sequences were generated by jumping PCR. The sequence encoding the mature peptide or the tandem repeat sequence was respectively cloned into the prokaryotic expression vector pET28a (+), and then transformed into E. coli Rosetta DE3 cells for expression. The recombinant proteins (rCs22-2r, rCs22-3r, rCs22M-2r, and rCs22M-3r) were purified by His-bind-resin (Ni-NTA) affinity chromatography. The immunogenicity of rCs22-2r and rCs22-3r was identified by ELISA. To evaluate the immunological diagnostic value of rCs22-2r and rCs22-3r, serum samples from 35 clonorchiasis patients, 31 healthy individuals, 15 schistosomiasis patients, 15 paragonimiasis westermani patients and 13 cysticercosis patients were examined by ELISA. To locate antigenic determinants, the pooled sera of clonorchiasis patients and healthy persons were analyzed for specific antibodies by ELISA with recombinant protein rCs22M-2r and rCs22M-3r containing the tandem repeat sequences. RESULTS: The full-length sequence of Cs22 antigen gene of C. sinensis was obtained. It contained 13 times tandem repeat sequences of EQQDGDEEGMGGDGGRGKEKGKVEGEDGAGEQKEQA. Bioinformatics analysis indicated that the protein (Cs22) belonged to GPI-anchored proteins family. The recombinant proteins rCs22-2r and rCs22-3r showed a certain level of immunogenicity. The positive rate by ELISA coated with the purified PrCs22-2r and PrCs22-3r for sera of clonorchiasis patients both were 45.7% (16/35), and 3.2% (1/31) for those of healthy persons. There was no cross reaction with sera of schistosomiasis and cysticercosis patients. The cross reaction with sera of paragonimiasis westermani patients was 1/15. The recombinant proteins rCs22M-2r and rCs22M-3r which only contained tandem repeats were specifically recognized by pooled sera of clonorchiasis patients. CONCLUSION: The Cs22 antigen gene of Clonorchis sinensis is obtained, and the recombinant proteins have certain diagnostic value. The antigenic determinant is located in tandem repeat sequences.

Cellular differentiation into hyphae and spores in halophilic archaea.

Several groups of bacteria have complex life cycles involving cellular differentiation and multicellular structures. For example, actinobacteria of the genus Streptomyces form multicellular vegetative hyphae, aerial hyphae, and spores. However, similar life cycles have not yet been described for archaea. Here, we show that several haloarchaea of the family Halobacteriaceae display a life cycle resembling that of Streptomyces bacteria. Strain YIM 93972 (isolated from a salt marsh) undergoes cellular differentiation into mycelia and spores. Other closely related strains are also able to form mycelia, and comparative genomic analyses point to gene signatures (apparent gain or loss of certain genes) that are shared by members of this clade within the Halobacteriaceae. Genomic, transcriptomic and proteomic analyses of non-differentiating mutants suggest that a Cdc48-family ATPase might be involved in cellular differentiation in strain YIM 93972. Additionally, a gene encoding a putative oligopeptide transporter from YIM 93972 can restore the ability to form hyphae in a Streptomyces coelicolor mutant that carries a deletion in a homologous gene cluster (bldKA-bldKE), suggesting functional equivalence. We propose strain YIM 93972 as representative of a new species in a new genus within the family Halobacteriaceae, for which the name Actinoarchaeum halophilum gen. nov., sp. nov. is herewith proposed. Our demonstration of a complex life cycle in a group of haloarchaea adds a new dimension to our understanding of the biological diversity and environmental adaptation of archaea.

Evaluation of four candidate VNTR Loci for genotyping 225 Chinese clinical Mycobacterium tuberculosis complex strains.

OBJECTIVE: To evaluate four candidate variable number tandem repeat (VNTR) loci for genotyping Mycobacterium tuberculosis complex strains. METHODS: Genomic sequences for two M. tuberculosis strains (CCDC5079 and CCDC5180) were generated, and using published sequence data, four candidate VNTR loci were identified. The VNTRs were used to genotype 225 Chinese clinical M. tuberculosis complex strains. The discriminatory power of the VNTRs was evaluated using BioNumerics 5.0 software. RESULTS: The Hunter-Gaston Index (HGI) for BJ1, BJ2, BJ3, and BJ4 loci was 0.634, 0.917, 0.697, and 0.910, respectively. Combining all four loci gave an HGI value of 0.995, thus confirming that the genotyping had good discriminatory power. The HGI values for BJ1, BJ2, BJ3, and BJ4, obtained from Beijing family strain genotyping, were 0.447, 0.878, 0.315, and 0.850, respectively. Combining all four loci produced an HGI value of 0.988 for genotyping the Beijing family strains. We observed unique patterns for M. bovis and M. africanum strains from the four loci. CONCLUSION: We have shown that the four VNTR loci can be successfully used for genotyping M. tuberculosis complex strains. Notably, these new loci may provide additional information about Chinese M. tuberculosis isolates than that currently afforded by established VNTR loci typing.

Signatures of Positive Selection in the Genome of Apis mellifera carnica: A Subspecies of European Honeybees.

The technology of long reads substantially improved the contingency of the genome assembly, particularly resolving contiguity of the repetitive regions. By integrating the interactive fragment using Hi-C, and the HiFi technique, a solid genome of the honeybee Apis mellifera carnica was assembled at the chromosomal level. A distinctive pattern of genes involved in social evolution was found by comparing it with social and solitary bees. A positive selection was identified in genes involved with cold tolerance, which likely underlies the adaptation of this European honeybee subspecies in the north hemisphere. The availability of this new high-quality genome will foster further studies and advances on genome variation during subspeciation, honeybee breeding and comparative genomics.

Complete genome sequence of Lactococcus lactis subsp. lactis CV56, a probiotic strain isolated from the vaginas of healthy women.

Lactic acid bacteria that exist in the urinogenital system play an important role in maintaining the health of the host. Here, we report the finished and annotated genome of a Lactococcus strain that was isolated from the vaginas of healthy women and shows probiotic properties, including nisin A production and adhesion to vaginal epithelial cells.

Complete genome sequence of the plant pathogen Ralstonia solanacearum strain Po82.

Ralstonia solanacearum strain Po82, a phylotype IIB/sequevar 4 strain, was found to be pathogenic to both solanaceous plants and banana. Here, we report the complete genome sequence of Po82 and its comparison with seven published R. solanacearum genomes.

TB-QUICK: CRISPR-Cas12b-assisted rapid and sensitive detection of Mycobacterium tuberculosis.

OBJECTIVES: Tuberculosis (TB) remains one of the public health problems worldwide. Rapid, sensitive and cost-effective diagnosis of Mycobacterium tuberculosis (M.tb) is critical for TB control. METHODS: We developed a novel M.tb DNA detection platform (nominated as TB-QUICK) which combined loop-mediated isothermal amplification (LAMP) and CRISPR-Cas12b detection. TB-QUICK was performed on pulmonary or plasma samples collected from 138 pulmonary TB (PTB) patients, 21 non-TB patients and 61 close contacts to TB patients. Acid-fast bacillus (AFB) smear, M.tb culture and GeneXpert MTB/RIF (Xpert) assays were routinely conducted in parallel. RESULTS: By targeting M.tb IS6110, TB-QUICK platform could detect as low as 1.3 copy/µL M.tb DNA within 2 h. In pulmonary TB samples, TB-QUICK exhibited improved overall sensitivity of 86.8% over M.tb culture (66.7%) and Xpert (70.4%), with the specificity of 95.2%. More significantly, TB-QUICK exhibited a superior sensitivity in AFB-negative samples (80.5%) compared to Xpert (57.1%) and M.tb culture (46.2%). In the detection of plasma M.tb DNA by TB-QUICK, 41.2% sensitivity for AFB-positive and 31.7% for AFB-negative patients were achieved. CONCLUSION: In conclusion, TB-QUICK exhibits rapidity and sensitivity for M.tb DNA detection with the superiority in smear-negative paucibacillary TB patients. The clinical application of TB-QUICK in TB diagnosis needs to be further validated in larger cohort.

A Chromosome-Scale Assembly of the Asian Honeybee Apis cerana Genome.

Apis cerana is one of the main honeybee species in artificial farming, which is widely distributed in Asian countries. The genome of A. cerana has been sequenced by several different research groups using second generation sequencing technologies. However, it is still necessary to obtain more complete and accurate genome sequences. Here we present a chromosome-scale assembly of the A. cerana genome using single-molecule real-time (SMRT) Pacific Biosciences sequencing and high-throughput chromatin conformation capture (Hi-C) genome scaffolding. The updated assembly is 215.67 Mb in size with a contig N50 of 4.49 Mb, representing an 212-fold improvement over the previous Illumina-based version. Hi-C scaffolding resulted in 16 pseudochromosomes occupying 97.85% of the assembled genome sequences. A total of 10,741 protein-coding genes were predicted and 9,627 genes were annotated. Besides, 314 new genes were identified compared to the previous version. The improved high-quality A. cerana reference genome will provide precise sequence information for biological research of A. cerana.

Complete genome sequence of Lactobacillus plantarum JDM1.

Lactobacillus plantarum is a lactic acid bacterium (LAB) species commonly used as a probiotic. We have sequenced the genome of Lactobacillus plantarum JDM1, which is a Chinese commercial LAB with several probiotic functions, using a GS 20 system. We recommend that each commercial probiotic strain should undergo complete genome sequencing to ensure safety and stability.

The Oral Microbiota May Have Influence on Oral Cancer.

The oral microbiota plays an important role in the human microbiome and human health, and imbalances between microbes and their hosts can lead to oral and systemic diseases and chronic inflammation, which is usually caused by bacteria and contributes to cancer. There may be a relationship between oral bacteria and oral squamous cell carcinoma (OSCC); however, this relationship has not been thoroughly characterized. Therefore, in this study, we compared the microbiota compositions between tumor sites and opposite normal tissues in buccal mucosal of 50 patients with OSCC using the 16S rDNA sequencing. Richness and diversity of bacteria were significantly higher in tumor sites than in the control tissues. Cancer tissues were enriched in six families (Prevotellaceae, Fusobacteriaceae, Flavobacteriaceae, Lachnospiraceae, Peptostreptococcaceae, and Campylobacteraceae) and 13 genera, including Fusobacterium, Alloprevotella and Porphyromonas. At the species level, the abundances of Fusobacterium nucleatum, Prevotella intermedia, Aggregatibacter segnis, Capnocytophaga leadbetteri, Peptostreptococcus stomatis, and another five species were significantly increased, suggesting a potential association between these bacteria and OSCC. Furthermore, the functional prediction revealed that genes involved in bacterial chemotaxis, flagellar assembly and lipopolysaccharide (LPS) biosynthesis which are associated with various pathological processes, were significantly increased in the OSCC group. Overall, oral bacterial profiles showed significant difference between cancer sites and normal tissue of OSCC patients, which might be onsidered diagnostic markers and treatment targets. Our study has been registered in the Chinese clinical trial registry (ChiCTR1900025253, http://www.chictr.org.cn/index.aspx).

miR-3074-5p Promotes the Apoptosis but Inhibits the Invasiveness of Human Extravillous Trophoblast-Derived HTR8/SVneo Cells In Vitro.

OBJECTIVE: The objective of this study was to observe the effects of the overexpression of miR-3074-5p in human trophoblast cells in vitro. DESIGN: Experimental in vitro study in HTR8/SVneo cells. METHODS: HTR8/SVneo cells were transfected with miR-3074-5p mimic. The cell apoptosis and invasion were measured via flow cytometry and transwell assay, respectively. The expression levels of P53, Cyclin Dependent Kinase Inhibitor 1B (P27), BCL-2, BCL2 associated X (BAX), and BCL2 like 14 (BCL-G) in HTR8/SVneo cells were determined by Western blot. The alterations in gene expression profile of HTR8/SVneo cells were evaluated by complementary DNA microarray assay, and the differential expressions of dihydrolipoamide S-succinyltransferase (DLST), growth-associated protein 43 (GAP43), runt-related transcription factor 2 (RUNX2), and C-C type chemokine receptor 3 (CCR3) were validated by Western blot. Biofunctions of these differentially expressed genes were enriched by Gene Ontology analysis. RESULTS: The overexpression of miR-3074-5p in HTR8/SVneo cells promoted cell apoptosis but inhibited cell invasion, being accompanied by the significantly elevated expressions of P27, BCL-2, and BCL-G. Meanwhile, an increased expression of P27 and P57 was also detected in a small sample size of placental villi of recurrent miscarriage (RM) patients. Totally, 411 genes and 397 genes were screened out, respectively, to be downregulated or upregulated at least by 2-folds in miR-3074-5p overexpressed HTR8/SVneo cells. These differentially expressed genes were involved in several important functions related to pregnancy. Subsequently, the reduced expressions of DLST and GAP43 proteins, as well as the increased expressions of CCR3 and RUNX2 proteins, were validated in miR-3074-5p overexpressed HTR8/SVneo cells. CONCLUSION: These data suggested a potential contribution of miR-3074-5p in the pathogenesis of RM by disturbing the normal activities of trophoblast cells.

Comparative transcriptome analysis between csrA-disruption Clostridium acetobutylicum and its parent strain.

The genome of Clostridium acetobutylicum contains the gene encoding CsrA, a carbon storage regulator. We investigated the function of CsrA in C. acetobutylicum by insertionally inactivating the encoding gene, CA_C2209 using the ClosTron. Disruption of csrA obviously decreases the growth of the organism and reduces the yield of acetone, butanol and ethanol (ABEs). Like the csrA in Escherichia coli, RNA-seq and ß-galactosidase analysis revealed that csrA in C. acetobutylicum was closely involved in regulating multiple pathways including flagella assembly, oligopeptide transporting, iron uptake, and central carbon metabolism. It has also been newly demonstrated that csrA in C. acetobutylicum is related to the regulation of pathways involved in the phosphotransferase transporting systems, synthesis of riboflavin, and stage III sporulation. This research represented the first investigation of global regulation by CsrA in the strain belonging to Gram-positive bacteria through transcriptome analysis and provided the important theoretical evidence for improving solvent production by transcriptor engineering in C. acetobutylicum.

The novel helicase helG (DHX30) is expressed during gastrulation in mice and has a structure similar to a human DExH box helicase.

The gene trap method for embryonic stem cells is an efficient method for identifying new genes that are involved in development. Using this method, we identified a novel gene called helicase family gene related to gastrulation (helG). Helicase family proteins regulate many systems in the body that are related to cell survival. HelG encodes a protein of 137 kDa, which contains a DExH helicase motif that is now named DHX30. HelG is strongly expressed in neural cells (ie, in the headfold, neural plate, neural tube, and brain) and somites during embryogenesis. Growing homozygous mutant embryos have neither differentiated somites nor brains. In these mutants, development was retarded by embryonic day 7.5 (E7.5), and the mutants died at E9.5. After the purification of HelG, an untwisting experiment was performed to confirm the helicase activity of HelG for DNA in vitro. We report for the first time that a helicase family gene is required for differentiation during embryogenesis; this gene might interact with polynucleotides to regulate some genes that are important for early development and has a structure similar to that of a human DExH box helicase.

Complete genome sequence of the molybdenum-resistant bacterium Bacillus subtilis strain LM 4-2.

Bacillus subtilis LM 4-2, a Gram-positive bacterium was isolated from a molybdenum mine in Luoyang city. Due to its strong resistance to molybdate and potential utilization in bioremediation of molybdate-polluted area, we describe the features of this organism, as well as its complete genome sequence and annotation. The genome was composed of a circular 4,069,266 bp chromosome with average GC content of 43.83 %, which included 4149 predicted ORFs and 116 RNA genes. Additionally, 687 transporter-coding and 116 redox protein-coding genes were identified in the strain LM 4-2 genome.