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Journal of Biochemistry Aug 2000A novel type of heparinase (heparin lyase, no EC number) has been purified from Bacteroides stercoris HJ-15, isolated from human intestine, which produces three kinds of...
A novel type of heparinase (heparin lyase, no EC number) has been purified from Bacteroides stercoris HJ-15, isolated from human intestine, which produces three kinds of heparinases. The enzyme was purified to apparent homogeneity by a combination of QAE-cellulose, DEAE-cellulose, CM-Sephadex C-50, hydroxyapatite, and HiTrap SP chromatographies with a final specific activity of 19.5 mmol/min/mg. It showed optimal activity at pH 7.2 and 45 degrees C and the presence of 300 mM KCl greatly enhanced its activity. The purified enzyme activity was inhibited by Cu(2+), Pb(2+), and some agents that modify histidine and cysteine residues, and activated by reducing agents such as dithiothreitol and 2-mercaptoethanol. This purified Bacteroides heparinase is an eliminase that shows its greatest activity on bovine intestinal heparan sulfate, and to a lesser extent on porcine intestinal heparan sulfate and heparin. This enzyme does not act on acharan sulfate but de-O-sulfated acharan sulfate and N-sulfoacharan sulfate were found to be poor substrates. The substrate specificity of this enzyme is similar to that of Flavobacterial heparinase II. However, an internal amino acid sequence of the purified Bacteroides heparinase shows significant (73%) homology to Flavobacterial heparinase III and only 43% homology to Flavobacterial heparinase II. These findings suggest that the Bacteroidal heparinase is a novel enzyme degrading GAGs.
Topics: Amino Acids; Bacteroides; Electrophoresis, Polyacrylamide Gel; Heparin Lyase; Heparitin Sulfate; Humans; Intestines; Kinetics; Substrate Specificity
PubMed: 10920269
DOI: 10.1093/oxfordjournals.jbchem.a022756 -
Brain, Behavior, and Immunity May 2023Recent evidence suggests that there is a link between neurodevelopmental disorders, such as attention-deficit hyperactivity disorder (ADHD), and the gut microbiome....
Recent evidence suggests that there is a link between neurodevelopmental disorders, such as attention-deficit hyperactivity disorder (ADHD), and the gut microbiome. However, most studies to date have had low sample sizes, have not investigated the impact of psychostimulant medication, and have not adjusted for potential confounders, including body mass index, stool consistency and diet. To this end, we conducted the largest, to our knowledge, fecal shotgun metagenomic sequencing study in ADHD, with 147 well-characterized adult and child patients. For a subset of individuals, plasma levels of inflammatory markers and short-chain fatty acids were also measured. In adult ADHD patients (n = 84), compared to controls (n = 52), we found a significant difference in beta diversity both regarding bacterial strains (taxonomic) and bacterial genes (functional). In children with ADHD (n = 63), we found that those on psychostimulant medication (n = 33 on medication vs. n = 30 not on medication) had (i) significantly different taxonomic beta diversity, (ii) lower functional and taxonomic evenness, (iii) lower abundance of the strain Bacteroides stercoris CL09T03C01 and bacterial genes encoding an enzyme in vitamin B synthesis, and (iv) higher plasma levels of vascular inflammatory markers sICAM-1 and sVCAM-1. Our study continues to support a role for the gut microbiome in neurodevelopmental disorders and provides additional insights into the effects of psychostimulant medication. However, additional studies are needed to replicate these findings and examine causal relationships with the disorder.
Topics: Humans; Child; Adult; Attention Deficit Disorder with Hyperactivity; Gastrointestinal Microbiome; Central Nervous System Stimulants; Diet; Feces
PubMed: 36940753
DOI: 10.1016/j.bbi.2023.03.012 -
European Journal of Biochemistry Jun 2002Two novel chondroitinases, chondroitin ABC lyase (EC 4.2.2.4) and chondroitin AC lyase (EC 4.2.2.5), have been purified from Bacteroides stercoris HJ-15, which was...
Two novel chondroitinases, chondroitin ABC lyase (EC 4.2.2.4) and chondroitin AC lyase (EC 4.2.2.5), have been purified from Bacteroides stercoris HJ-15, which was isolated from human intestinal bacteria with glycosaminoglycan degrading enzymes. Chondroitin ABC lyase was purified to apparent homogeneity by a combination of QAE-cellulose, CM-Sephadex C-50, hydroxyapatite and Sephacryl S-300 column chromatography with a final specific activity of 45.7 micromol.min-1.mg-1. Chondroitin AC lyase was purified to apparent homogeneity by a combination of QAE-cellulose, CM-Sephadex C-50, hydroxyapatite and phosphocellulose column chromatography with a final specific activity of 57.03 micromol.min-1.mg-1. Chondroitin ABC lyase is a single subunit of 116 kDa by SDS/PAGE and gel filtration. Chondroitin AC lyase is composed of two identical subunits of 84 kDa by SDS/PAGE and gel filtration. Chondroitin ABC and AC lyases showed optimal activity at pH 7.0 and 40 degrees C, and 5.7-6.0 and 45-50 degrees C, respectively. Both chondroitin lyases were potently inhibited by Cu2+, Zn2+, and p-chloromercuriphenyl sulfonic acid. The purified Bacteroidal chondroitin ABC lyase acted to the greatest extent on chondroitin sulfate A (chondroitin 4-sulfate), to a lesser extent on chondroitin sulfate B (dermatan sulfate) and C (chondroitin 6-sulfate). The purified chondroitin AC lyase acted to the greatest extent on chondroitin sulfate A, and to a lesser extent on chondroitin C and hyaluronic acid. They did not act on heparin and heparan sulfate. These findings suggest that the biochemical properties of these purified chondroitin lyases are different from those of the previously purified chondroitin lyases.
Topics: Bacteria, Anaerobic; Bacteroides; Chondroitin ABC Lyase; Chondroitin Lyases; Chondroitin Sulfates; Dermatan Sulfate; Humans; Intestines; Kinetics; Substrate Specificity
PubMed: 12071957
DOI: 10.1046/j.1432-1033.2002.02967.x -
AIDS (London, England) Jan 2022The rectal microbiome was examined to assess the relationship between the microbiome and liver disease in HIV-infection.
OBJECTIVE
The rectal microbiome was examined to assess the relationship between the microbiome and liver disease in HIV-infection.
DESIGN
Eighty-two HIV-1 mono-infected individuals from the PROSPEC-HIV-study (NCT02542020) were grouped into three liver health categories based on results of controlled attenuation parameter (CAP) and liver stiffness measurement (LSM) of transient elastography: normal (n = 30), steatosis (n = 30), or fibrosis (n = 22).
METHODS
Liver steatosis and fibrosis were defined by CAP at least 248 dB/m and LSM at least 8.0 kPa, respectively. 16S rRNA gene and whole genome shotgun metagenomic sequencing were performed on rectal swabs. Bacterial differences were assessed using zero-inflated negative binomial regression and random forests modeling; taxonomic drivers of functional shifts were identified using FishTaco.
RESULTS
Liver health status explained four percentage of the overall variation (r2 = 0.04, P = 0.003) in bacterial composition. Participants with steatosis had depletions of Akkermansia muciniphila and Bacteroides dorei and enrichment of Prevotella copri, Finegoldia magna, and Ruminococcus bromii. Participants with fibrosis had depletions of Bacteroides stercoris and Parabacteroides distasonis and enrichment of Sneathia sanguinegens. In steatosis, functional analysis revealed increases in primary and secondary bile acid synthesis encoded by increased Eubacterium rectale, F. magna, and Faecalibacterium prausnitzii and decreased A. muciniphila, Bacteroides fragilis and B. dorei. Decreased folate biosynthesis was driven by similar changes in microbial composition.
CONCLUSION
HIV mono-infection with steatosis or fibrosis had distinct microbial profiles. Some taxa are similar to those associated with non-alcoholic fatty liver disease in HIV-negative populations. Further studies are needed to define the role of the gut microbiota in the pathogenesis of liver disease in HIV-infected persons.
Topics: Brazil; Elasticity Imaging Techniques; Fatty Liver; HIV Infections; Humans; Liver; Liver Cirrhosis; Non-alcoholic Fatty Liver Disease; Pilot Projects; RNA, Ribosomal, 16S
PubMed: 34873092
DOI: 10.1097/QAD.0000000000003084 -
Microbiology Spectrum May 2024Esophageal squamous cell carcinoma (ESCC) is one of the most predominant subtypes of esophageal cancer. The characteristics of the gut microbiome and its metabolites...
Esophageal squamous cell carcinoma (ESCC) is one of the most predominant subtypes of esophageal cancer. The characteristics of the gut microbiome and its metabolites from patients with ESCC have not been adequately studied and discussed. In this study, 40 fecal samples (20 from ESCC patients and 20 from healthy controls) were analyzed by 16S rRNA gene sequencing and untargeted metabolomics. The data sets were analyzed individually and synthesized using various bioinformatics methods. Alpha and beta diversity indicated significant differences in microbial diversity and abundance between ESCC and healthy control feces. At the genus level, the abundance of , , and was significantly increased in ESCC. At the genus level, linear discriminant analysis effect size identified two biomarkers: and . Untargeted metabolomics analysis revealed 307 differential metabolites between ESCC and healthy control feces, with indoles and derivatives, tropane alkaloids, lipids, and lipid-like molecules in higher relative abundance in ESCC feces than in healthy control feces. Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that unsaturated fatty acids (FAs), ascorbate and aldarate metabolism, and hypoxia-inducible factor 1 signaling pathway were significantly associated with differential metabolite. Phenylethanolamine and despropionyl p-fluoro fentanyl could be used as reliable biomarkers to differentiate ESCC from healthy control. The correlation analysis showed that may be involved in the synthesis of fatty acyl, carboxylic acids and derivatives, benzenes and substituted derivatives, organic oxygenates, and indoles and derivatives as metabolites. and may be involved in the degradation of indoles and derivatives. , , and may be involved in the synthesis of indoles and derivatives with strong contributions. There is an intricate relationship between the gut microbiome and the levels of several metabolites (e.g., fatty acyls, carboxylic acids and derivatives, indoles, and derivatives). Microbial-associated metabolites can be used as diagnostic biomarkers in therapeutic exploration. Further analysis revealed that , , , and might promote ESCC by regulating the synthesis of indoles and their derivatives. The results of this study provide favorable evidence for the early diagnosis of ESCC and subsequent individualized treatment and targeted interventions.IMPORTANCEWe describe for the first time the differences in fecal microbiome composition and metabolites between patients with esophageal squamous cell carcinoma (ESCC) and healthy controls by 16S rRNA gene sequencing and untargeted metabolomics. The results of this study provide a favorable basis for the early diagnosis of ESCC and subsequent targeted interventional therapy.
Topics: Humans; Feces; Esophageal Squamous Cell Carcinoma; Metabolomics; Gastrointestinal Microbiome; Esophageal Neoplasms; Male; Female; Middle Aged; Bacteria; RNA, Ribosomal, 16S; Biomarkers, Tumor; Aged; Adult
PubMed: 38497715
DOI: 10.1128/spectrum.04012-23 -
Frontiers in Cellular and Infection... 2023There is no direct evidence of gut microbiota disturbance in children with gastroesophageal reflux disease (GERD). This study aimed to provide direct evidence and a...
BACKGROUND
There is no direct evidence of gut microbiota disturbance in children with gastroesophageal reflux disease (GERD). This study aimed to provide direct evidence and a comprehensive understanding of gut microbiota disturbance in children with GERD through combined metagenomic and metabolomic analysis.
METHODS
30 children with GERD and 30 healthy controls (HCs) were continuously enrolled, and the demographic and clinical characteristics of the subjects were collected. First, 16S rRNA sequencing was used to evaluate differences in the gut microbiota between children with GERD and HC group, and 10 children with GERD and 10 children in the HC group were selected for metagenomic analysis. Nontargeted metabolomic analysis was performed using liquid chromatography/mass spectrometry (LC/MS), and metagenomic and metabolomic data were analyzed together.
RESULTS
There were significant differences in the gut microbiota diversity and composition between children with GERD and HCs. The dominant bacteria in children with GERD were Proteobacteria and Bacteroidota. At the species level, the top three core bacterial groups were , and . The main differential pathways were identified to be related to energy, amino acid, vitamin, carbohydrate and lipid metabolism. LC/MS detected 288 different metabolites in the positive and negative ion modes between children with GERD and HCs, which were mainly involved in arachidonic acid (AA), tyrosine, glutathione and caffeine metabolism.
CONCLUSION
This study provides new evidence of the pathogenesis of GERD. There are significant differences in the gut microbiota, metabolites and metabolic pathways between HCs and children with GERD, and the differences in metabolites are related to specific changes in bacterial abundance. In the future, GERD may be treated by targeting specific bacteria related to AA metabolism.
Topics: Humans; Child; Gastrointestinal Microbiome; RNA, Ribosomal, 16S; Metabolomics; Bacteria; Metagenomics; Gastroesophageal Reflux
PubMed: 37900308
DOI: 10.3389/fcimb.2023.1267192 -
Journal of Applied Microbiology Jan 2010This study focused on the cloning, expression and characterization of recombinant heparinase II (rHepII) from Bacteroides stercoris HJ-15.
AIMS
This study focused on the cloning, expression and characterization of recombinant heparinase II (rHepII) from Bacteroides stercoris HJ-15.
METHODS AND RESULTS
The heparinase II gene from Bact. stercoris HJ-15 was identified by Southern blotting and the sequence was deposited in GenBank. The gene was cloned and overexpressed in Escherichia coli, and rHepII was purified using two simple ion-exchange column chromatography steps. Enzymatic properties and substrate specificities of rHepII were assessed and its kinetic constants were calculated. Heparin-like glycosaminoglycans (HLGAGs) were digested with rHepII under optimal reaction conditions, and the products were analysed by SAX-HPLC.
CONCLUSIONS
The heparinase II gene is 2322-bp long and consists of 773 amino acids. rHepII is most active in 50 mmol l(-1) sodium phosphate buffer with 75 mmol l(-1) NaCl (pH 7.4) at 32 degrees C, and the activity is stable at 4 degrees C for 15 days on storage. Acharan sulfate is the best substrate for rHepII, followed by heparan sulfate and heparin. The major degradation products were verified as highly sulfated disaccharides through SAX-HPLC analysis. It means that rHepII prefers iduronic acid over glucuronic acid on the HLGAG structure.
SIGNIFICANCE AND IMPACT OF THE STUDY
This study provides easy and certain means for obtaining large amounts of pure rHepII and also provides important information regarding the tendencies of this enzyme and its digested products. rHepII digests HLGAGs in a different manner than heparinases from Flavobacterium heparinum; therefore, we anticipate that rHepII will be a powerful tool for studies of GAGs and GAGs lyases.
Topics: Bacterial Proteins; Bacteroides; Base Sequence; Chromatography, Ion Exchange; Cloning, Molecular; Escherichia coli; Gene Expression; Glycosaminoglycans; Heparin Lyase; Molecular Sequence Data; Recombinant Proteins; Substrate Specificity
PubMed: 19566715
DOI: 10.1111/j.1365-2672.2009.04418.x -
British Journal of Cancer Nov 2022Emerging evidence indicates the potential clinical significance of specific microbial signatures as diagnostic and prognostic biomarkers, in multiple cancers. However,...
BACKGROUND
Emerging evidence indicates the potential clinical significance of specific microbial signatures as diagnostic and prognostic biomarkers, in multiple cancers. However, to date, no studies have systematically interrogated circulating metagenome profiling in oesophageal adenocarcinoma (EAC) patients, particularly as novel non-invasive, early detection, surveillance and prognostic classifiers.
METHODS
Metagenome sequencing was performed on 81 serum specimens collected across EAC spectrum, with sequencing reads classified using Bracken and MetaPhlAn3. Followed by the Linear Discriminant Analysis effect size (LEfSe) method to identify microbial profiles between groups. Logistic regression and Kaplan-Meier analyses were used to build classifiers.
RESULTS
A significant loss of alpha and beta diversity was identified in serum specimens from EAC patients. We observed a shift in microbial taxa between each group-at the phylum, genus, and species level-with Lactobacillus sakei as the most prominent species in gastroesophageal reflux (GERD) vs other patient groups. Interestingly, LEfSe analysis identified a complete loss of Lactobacillus (L. Sakei and L. Curvatus), Collinsella stercoris and Bacteroides stercoris but conversely a significant increase in Escherichia coli in patients with EAC. Finally, we developed a metagenome panel that discriminated EAC from GERD patients with an AUC value of 0.89 (95% CI: 0.78-0.95; P < 0.001) and this panel in conjunction with the TNM stage was a robust predictor of overall survival (≥24 months; AUC = 0.84 (95% CI: 0.66-0.92; P = 0.006)).
CONCLUSION
This study firstly describes unique blood-based microbial profiles in patients across EAC carcinogenesis, that are further utilised to establish a novel circulating diagnostic and prognostic metagenomic signature for EAC.
TRANSLATIONAL RELEVANCE
Accumulating data indicates the clinical relevance of specific microbial signatures as diagnostic and prognostic biomarkers, in multiple cancers. However, to date, no studies have systematically interrogated circulating metagenome profiling in patients with oesophageal adenocarcinoma (EAC). Herein, we performed metagenome sequencing in serum specimens from EAC patients 81 collected across EAC spectrum and observed a significant loss of alpha and beta diversity, with a shift in microbial taxa between each group-at the phylum, genus, and species level-with Lactobacillus sakei as the most prominent species in gastroesophageal reflux (GERD) vs other patient groups. Interestingly, LEfSe analysis identified a complete loss of Lactobacillus (L. Sakei and L. Curvatus), Collinsella stercoris and Bacteroides stercoris but conversely a significant increase in Escherichia coli in patients with EAC. Finally, we developed a metagenome panel that discriminated EAC from GERD patients with an AUC value of 0.89 and this panel, in conjunction with the TNM stage, was a robust predictor of overall survival. This study for the first time describes unique blood-based microbial profiles in patients across EAC carcinogenesis, that are further utilised to establish a novel circulating diagnostic and prognostic metagenomic signature for EAC.
Topics: Humans; Metagenome; Early Detection of Cancer; Esophageal Neoplasms; Adenocarcinoma; Prognosis; Gastroesophageal Reflux; Carcinogenesis; Escherichia coli; Biomarkers
PubMed: 36097175
DOI: 10.1038/s41416-022-01974-5 -
Scientific Reports Oct 2022Observational studies have shown that the composition of the human gut microbiome in children diagnosed with Autism Spectrum Disorder (ASD) differs significantly from... (Meta-Analysis)
Meta-Analysis
Observational studies have shown that the composition of the human gut microbiome in children diagnosed with Autism Spectrum Disorder (ASD) differs significantly from that of their neurotypical (NT) counterparts. Thus far, reported ASD-specific microbiome signatures have been inconsistent. To uncover reproducible signatures, we compiled 10 publicly available raw amplicon and metagenomic sequencing datasets alongside new data generated from an internal cohort (the largest ASD cohort to date), unified them with standardized pre-processing methods, and conducted a comprehensive meta-analysis of all taxa and variables detected across multiple studies. By screening metadata to test associations between the microbiome and 52 variables in multiple patient subsets and across multiple datasets, we determined that differentially abundant taxa in ASD versus NT children were dependent upon age, sex, and bowel function, thus marking these variables as potential confounders in case-control ASD studies. Several taxa, including the strains Bacteroides stercoris t__190463 and Clostridium M bolteae t__180407, and the species Granulicatella elegans and Massilioclostridium coli, exhibited differential abundance in ASD compared to NT children only after subjects with bowel dysfunction were removed. Adjusting for age, sex and bowel function resulted in adding or removing significantly differentially abundant taxa in ASD-diagnosed individuals, emphasizing the importance of collecting and controlling for these metadata. We have performed the largest (n = 690) and most comprehensive systematic analysis of ASD gut microbiome data to date. Our study demonstrated the importance of accounting for confounding variables when designing statistical comparative analyses of ASD- and NT-associated gut bacterial profiles. Mitigating these confounders identified robust microbial signatures across cohorts, signifying the importance of accounting for these factors in comparative analyses of ASD and NT-associated gut profiles. Such studies will advance the understanding of different patient groups to deliver appropriate therapeutics by identifying microbiome traits germane to the specific ASD phenotype.
Topics: Autism Spectrum Disorder; Bacteria; Child; Gastrointestinal Microbiome; Humans; Metagenome; Microbiota
PubMed: 36220843
DOI: 10.1038/s41598-022-21327-9 -
Frontiers in Microbiology 2023The association between gut microbes and short-chain fatty acids (SCFAs) and therapeutic responses of patients with lung cancer (LC) receiving therapy remains unknown.
BACKGROUND
The association between gut microbes and short-chain fatty acids (SCFAs) and therapeutic responses of patients with lung cancer (LC) receiving therapy remains unknown.
METHODS
Fecal and serum samples were prospectively collected from patients with LC, classified as responders, if they presented durable clinical benefits, and non-responders, if not. The composition of gut microbes was analyzed using 16S ribosomal DNA sequencing. Serum SCFA concentrations were detected using gas chromatography. Cell proliferation, migration, invasion, cell cycle, and apoptosis assays were performed on isobutyric acid-treated A549 cells. Reverse transcription-quantitative PCR, Western blotting, immunocytochemistry, and immunofluorescence staining experiments have been performed to investigate the expression of associated genes or proteins.
RESULTS
Non-responders harbored higher microbiome α-diversity but lower β-diversity compared with responders. Compared to the patients with low α-diversity, those with high α-diversity showed significantly shorter progression-free survival. Additionally, β-diversity has also been observed between these two groups. Specifically, , and were more abundant among responders, whereas and were more abundant in non-responders. The serum SCFA (especially acetate and isobutyrate) levels tended to be higher in responders. Isobutyric acid inhibited the proliferation, migration, and invasion of A549 cells by inducing apoptosis and G1/S arrest while upregulating the expression of GPR41, GPR43, and GPR5C and downregulating that of PAR1, and increasing the activity of histone acetyltransferases.
CONCLUSION
We revealed the influence of gut microbiota and SCFAs on the therapeutic responses in patients with LC and the anti-tumor effect of isobutyric acid, indicating their potential use as therapeutic targets.
PubMed: 37564290
DOI: 10.3389/fmicb.2023.1165360