Propionate is produced in the human large intestine by microbial fermentation and may help maintain human health. We have examined the distribution of three different pathways used by bacteria for ...propionate formation using genomic and metagenomic analysis of the human gut microbiota and by designing degenerate primer sets for the detection of diagnostic genes for these pathways. Degenerate primers for the acrylate pathway (detecting the lcdA gene, encoding lactoyl-CoA dehydratase) together with metagenomic mining revealed that this pathway is restricted to only a few human colonic species within the Lachnospiraceae and Negativicutes. The operation of this pathway for lactate utilisation in Coprococcus catus (Lachnospiraceae) was confirmed using stable isotope labelling. The propanediol pathway that processes deoxy sugars such as fucose and rhamnose was more abundant within the Lachnospiraceae (based on the pduP gene, which encodes propionaldehyde dehydrogenase), occurring in relatives of Ruminococcus obeum and in Roseburia inulinivorans. The dominant source of propionate from hexose sugars, however, was concluded to be the succinate pathway, as indicated by the widespread distribution of the mmdA gene that encodes methylmalonyl-CoA decarboxylase in the Bacteroidetes and in many Negativicutes. In general, the capacity to produce propionate or butyrate from hexose sugars resided in different species, although two species of Lachnospiraceae (C. catus and R. inulinivorans) are now known to be able to switch from butyrate to propionate production on different substrates. A better understanding of the microbial ecology of short-chain fatty acid formation may allow modulation of propionate formation by the human gut microbiota.
Full text
Available for:
IZUM, KILJ, NUK, PILJ, PNG, SAZU, SBMB, SBNM, UL, UM, UPUK
Feces from dogs in an unexplained outbreak of diarrhea were analyzed by viral metagenomics revealing the genome of a novel parvovirus. The parvovirus was named cachavirus and was classified within ...the proposed
genus. Using PCR, cachavirus DNA was detected in two of nine tested dogs from that outbreak. In order to begin to elucidate the clinical impact of this virus, 2,053 canine fecal samples were screened using real-time PCR. Stool samples from 203 healthy dogs were positive for cachavirus DNA at a rate of 1.47%, while 802 diarrhea samples collected in 2017 and 964 samples collected in 2018 were positive at rates of 4.0% and 4.66% frequencies, respectively (healthy versus 2017-2018 combined diarrhea
-value of 0.05). None of 83 bloody diarrhea samples tested positive. Viral loads were generally low with average real-time PCR Ct values of 36 in all three positive groups. The species tropism and pathogenicity of cachavirus, the first chapparvovirus reported in feces of a placental carnivore, remains to be fully determined.
Full text
Available for:
IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Roseburia inulinivorans is a recently identified motile representative of the Firmicutes that contributes to butyrate formation from a variety of dietary polysaccharide substrates in the human large ...intestine. Microarray analysis was used here to investigate substrate-driven gene-expression changes in R. inulinivorans A2-194. A cluster of fructo-oligosaccharide/inulin utilization genes induced during growth on inulin included one encoding a β-fructofuranosidase protein that was prominent in the proteome of inulin-grown cells. This cluster also included a 6-phosphofructokinase and an ABC transport system, whereas a distinct inulin-induced 1-phosphofructokinase was linked to a fructose-specific phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS II transport enzyme). Real-time PCR analysis showed that the β-fructofuranosidase and adjacent ABC transport protein showed greatest induction during growth on inulin, whereas the 1-phosphofructokinase enzyme and linked sugar phosphotransferase transport system were most strongly up-regulated during growth on fructose, indicating that these two clusters play distinct roles in the use of inulin. The R. inulinivorans β-fructofuranosidase was overexpressed in Escherichia coli and shown to hydrolyze fructans ranging from inulin down to sucrose, with greatest activity on fructo-oligosaccharides. Genes induced on starch included the major extracellular α-amylase and two distinct α-glucanotransferases together with a gene encoding a flagellin protein. The latter response may be concerned with improving bacterial access to insoluble starch particles.
Full text
Available for:
BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Colon cancers are characterized by aberrant gene expression signatures associated with disease initiation and progression. Identification of aberrant gene expression associated with colon ...carcinogenesis has increased significantly with application of gene array technologies. Downstream processing of these data has been hindered by the lack of robust multiplexed gene quantitative technologies facilitating study of the identified multiple gene targets. The GenomeLab Genetic Analysis System presents a novel technology platform for quantitative multiplexed gene expression analysis. This report describes the custom design of a GeXP multiplexed assay used to assess expression profiles of 14 inflammatory gene targets in normal, polyp, and tumor tissue. Characteristic normal, polyp, and tumor tissue gene expression profiles were obtained. Statistical analysis confirmed comparable relative quantitation of gene expression using the GeXP, macroarray, and single-plex real-time polymerase chain reaction assays. GeXP assays may be usefully applied in clinical and regulatory studies of multiple gene targets. This system permits custom-design options for relative quantification of multiple gene target expression, simultaneously in a single reaction, using nanogram quantities of total RNA template. The system provides an approach to advance the study of multiple targets identified from gene array analysis with potential for characterizing gene expression signatures in clinical diagnostics.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
BackgroundInadequate checking of safety-critical issues can compromise care quality in general practice (GP) work settings. Adopting a systemic, methodical approach may lead to improved ...standardisation of processes and reliability of task performance, strengthening the safety systems concerned. This study aimed to revise, modify and test the content and relevance of a previously validated safety checklist to the current GP context.MethodsA multimethod study was undertaken in Scottish GP involving: consensus building workshops with users and ‘experts’ to revise checklist content; regional testing of the modified checklist and follow-up usability evaluation survey of users. Quantitative data underwent descriptive statistical analyses and selected survey free-text comments are presented.ResultsA redesigned checklist tool consisting of eight themes (eg, medication safety) and 61 items (eg, out-of-date stock is appropriately disposed) was agreed by 53 users/experts with items reclassified as: mandatory (n=25), essential (n=24) and advisory (n=12). Totally 42/55 GPs tested the tool and submitted checklist data (76.4%). The mean aggregated results demonstrated 92.0% compliance with all 61 checklist items (range: 83.0%–98.0%) and 25/42 GP managers responded to the survey (59.5%) and reported high mean levels of agreement on the usefulness of the checklist (77.0%), ease of use (89.0%), learnability (94.0%) and satisfaction (78.4%).ConclusionsThe checklist was comprehensively redesigned as a practical safety monitoring and improvement tool for potential implementation in Scottish GP. Testing and evaluation demonstrated high levels of checklist content compliance and strong usability feedback, but some variation was evident indicating room for improvement in current safety-critical checking processes. The checklist should be of interest in similar GP settings internationally and to other areas of primary care practice.
Bacterial butyryl-CoA CoA-transferase activity plays a key role in butyrate formation in the human colon, but the enzyme and corresponding gene responsible for this activity have not previously been ...identified. A novel CoA-transferase gene is described from the colonic bacterium Roseburia sp. A2-183, with similarity to acetyl-CoA hydrolase as well as 4-hydroxybutyrate CoA-transferase sequences. The gene product, overexpressed in an Escherichia coli lysate, showed activity with butyryl-CoA and to a lesser degree propionyl-CoA in the presence of acetate. Butyrate, propionate, isobutyrate and valerate competed with acetate as the co-substrate. Despite the sequence similarity to 4-hydroxybutyrate CoA-transferases, 4-hydroxybutyrate did not compete with acetate as the co-substrate. Thus the CoA-transferase preferentially uses butyryl-CoA as substrate. Similar genes were identified in other butyrate-producing human gut bacteria from clostridial clusters IV and XIVa, while other candidate CoA-transferases for butyrate formation could not be detected in Roseburia sp. A2-183. This suggests strongly that the newly identified group of CoA-transferases described here plays a key role in butyrate formation in the human colon.
Summary
Bacterial β‐glucuronidase in the human colon plays an important role in cleaving liver conjugates of dietary compounds and xenobiotics, while other glycosidase activities are involved in the ...conversion of dietary plant glycosides. Here we detected an increase in β‐glucuronidase activity in faecal samples from obese volunteers following a high‐protein moderate carbohydrate weight‐loss diet, compared with a weight maintenance diet, but little or no changes were observed when the type of fermentable carbohydrate was varied. Other faecal glycosidase activities showed little or no change over a fivefold range of dietary NSP intake, although α‐glucosidase increased on a resistant starch‐enriched diet. Two distinct groups of gene, gus and BG, have been reported to encode β‐glucuronidase activity among human colonic bacteria. Degenerate primers were designed against these genes. Overall, Firmicutes were found to account for 96% of amplified gus sequences, with three operational taxonomic units particularly abundant, whereas 59% of amplified BG sequences belonged to Bacteroidetes and 41% to Firmicutes. A similar distribution of operational taxonomic units was found in a published metagenome dataset involving a larger number of volunteers. Seven cultured isolates of human colonic bacteria that carried only the BG gene gave relatively low β‐glucuronidase activity that was not induced by 4‐nitrophenyl‐β‐D‐glucuronide. By comparison, in three of five isolates that possessed only the gus gene, β‐glucuronidase activity was induced.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
This volume of essays examines the empirical evidence on school choice in different countries across Europe, North America, sub-Saharan Africa and South Asia.
A viral metagenomic analysis of feces from an unexplained outbreak of feline diarrhea revealed the presence of Lyon-IARC polyomavirus (LIPyV) DNA. LIPyV, whose genome was originally sequenced from ...swabs of human skin, was fecally shed by three out of five diarrheic cats.
PDF
