Colonic microbiota is associated with inflammation and host epigenomic alterations in inflammatory bowel disease.

Studies of inflammatory bowel disease (IBD) have been inconclusive in relating microbiota with distribution of inflammation. We report microbiota, host transcriptomics, epigenomics and genetics from matched inflamed and non-inflamed colonic mucosa [50 Crohn's disease (CD); 80 ulcerative colitis (UC); 31 controls]. Changes in community-wide and within-patient microbiota are linked with inflammation, but we find no evidence for a distinct microbial diagnostic signature, probably due to heterogeneous host-microbe interactions, and show only marginal microbiota associations with habitual diet. Epithelial DNA methylation improves disease classification and is associated with both inflammation and microbiota composition. Microbiota sub-groups are driven by dominant Enterbacteriaceae and Bacteroides species, representative strains of which are pro-inflammatory in vitro, are also associated with immune-related epigenetic markers. In conclusion, inflamed and non-inflamed colonic segments in both CD and UC differ in microbiota composition and epigenetic profiles.

Long-term colonisation with donor bacteriophages following successful faecal microbial transplantation.

BACKGROUND: Faecal microbiota transplantation (FMT) is used in the treatment of recurrent Clostridium difficile infection. Its success is typically attributed to the restoration of a diverse microbiota. Viruses (including bacteriophages) are the most numerically dominant and potentially the most diverse members of the microbiota, but their fate following FMT has not been well studied. RESULTS: We studied viral transfer following FMT from 3 donors to 14 patients. Recipient viromes resembled those of their donors for up to 12 months. Tracking individual bacteriophage colonisation revealed that engraftment of individual bacteriophages was dependent on specific donor-recipient pairings. Specifically, multiple recipients from a single donor displayed highly individualised virus colonisation patterns. CONCLUSIONS: The impact of viruses on long-term microbial dynamics is a factor that should be reviewed when considering FMT as a therapeutic option.

Regulation of prefrontal cortex myelination by the microbiota.

The prefrontal cortex (PFC) is a key region implicated in a range of neuropsychiatric disorders such as depression, schizophrenia and autism. In parallel, the role of the gut microbiota in contributing to these disorders is emerging. Germ-free (GF) animals, microbiota-deficient throughout life, have been instrumental in elucidating the role of the microbiota in many aspects of physiology, especially the role of the microbiota in anxiety-related behaviours, impaired social cognition and stress responsivity. Here we aim to further elucidate the mechanisms of the microbial influence by investigating changes in the homeostatic regulation of neuronal transcription of GF mice within the PFC using a genome-wide transcriptome profiling approach. Our results reveal a marked, concerted upregulation of genes linked to myelination and myelin plasticity. This coincided with upregulation of neural activity-induced pathways, potentially driving myelin plasticity. Subsequent investigation at the ultrastructural level demonstrated the presence of hypermyelinated axons within the PFC of GF mice. Notably, these changes in myelin and activity-related gene expression could be reversed by colonization with a conventional microbiota following weaning. In summary, we believe we demonstrate for the first time that the microbiome is necessary for appropriate and dynamic regulation of myelin-related genes with clear implications for cortical myelination at an ultrastructural level. The microbiota is therefore a potential therapeutic target for psychiatric disorders involving dynamic myelination in the PFC.

Genetic diversity and structure of the narrow endemic Wyethia reticulata and its congener W. bolanderi (Asteraceae) using RAPD and allozyme techniques.

Wyethia reticulata is an edaphic endemic in the Sierra Nevada foothills. Its sympatric congener, W. bolanderi, is also restricted to the foothills, but has a north-south range of 275 km, compared to 14 km for W. reticulata. The goals of this study were to determine clonal diversity, population size, genetic variation, and spatial and generic structure for each species from paired populations in El Dorado County, California, using allozyme and RAPD (random amplified polymorphic DNA) methodologies. Wyethia reticulata, spreading by rhizomes, had populations dominated by a few large individuals, while W. bolanderi, with a basal caudex, had populations of a few hundred evenly sized individuals. Genetic analyses indicated that W. reticulata, compared to its congener, had somewhat less genetic diversity (H(T): 0.28 vs. 0.38), had more of its genetic variation partitioned among populations (F(ST): 0.25 vs. 0.07), and showed a complete absence of inbreeding (F(IS): -0.03 vs. 0.22). Population membership in accord with populations defined by geographical location resulted only when all markers were included in the analysis. Ecological limits on recruitment of genets appears to result in small population size in W. reticulata. Limited gene flow, drift within small populations, and sexual reproductive dominance of large clones result in the genetic divergence of populations in this species, while genetic diversity is maintained by the longevity of clones and outbreeding.

Drug testing effectiveness in identifying and preventing drug use.

The effectiveness of drug testing in identifying and preventing drug use was assessed by a study of intercollegiate athletes required to participate in a urine testing program. Five hundred athletes who underwent testing were contrasted with a comparison group of 124 athletes not tested. Results show that some drug-using athletes avoided detection. Although many reduced their drug usage, some continued in the same pattern as before; a few actually increased drug usage.

Interference in immunoenzymometric assays caused by IgM anti-mouse IgG antibodies.

Serum samples from a female patient gave falsely elevated results in nine of ten immunoenzymometric assays (IEMAs) that used mouse monoclonal antibodies specific for human chorionic gonadotropin (n = 8), thyrotropin, or creatine kinase-MB isozyme. In contrast, normal results were obtained in five radioimmunoassays that used either mouse monoclonal antibodies or antisera specific for human chorionic gonadotropin (n = 3) or thyrotropin (n = 2). Incubation of her serum with IgG from different species or with F(ab)'2 from mouse IgG prior to IEMA showed that the interference was markedly inhibited by mouse IgG, indicating an antibody specific for the Fc portion of mouse IgG. The interfering activity was bound to and eluted from a column containing Protein A-Sepharose CL-4B. Fractionation of the eluted protein over another column containing Sephacryl S300 showed the activity was enriched in the first protein peak, which contained predominantly IgM. A model is proposed to explain how IgM anti-mouse IgG antibody selectively interferes in IEMAs that use mouse monoclonal antibodies.

Simple and precise method for liquid chromatographic determination of chloramphenicol in serum using a phase separation extraction.

A high-performance liquid chromatographic method for the determination of chloramphenicol using an alkaline salt-induced phase separation extraction is described. The extraction procedure is as simple as miscible organic solvent deproteinization methods, yet produces extracts that have negligible protein and minimal interference from acidic compounds. The use of 2,4-dinitroacetanilide as an internal standard and of microprocessor-stored standard relative response factors results in a more precise assay without need for daily routine standardization. Serum is mixed with an equal volume of acetonitrile containing the internal standard, and then solid sodium chloride containing 20% of sodium phosphate by weight is added and mixed. The resulting upper phase is chromatographed on a C-18 reversed-phase column and monitored at 278 nm. Between-day precision is excellent. Comparison with an alternate method and with drug-monitoring proficiency samples showed the method to have excellent accuracy. The possible use of this phase separation extraction in other assays in which miscible solvent extraction is used is discussed.

A total and direct bilirubin determination using an SDS procedure on the Abbott Bichromatic Analyser.

A procedure to measure total and direct bilirubin using the Abbott Bichromatic Analyser is described. The method is an adaptation of a sodium dodecyl sulfate method. Reaction products are measured at an acid pH in a buffered matrix using a 600-650 filter wheel. The method has very little spectral interference from either hemolysis or lipemia and correlates well with the manual Jendrassik-Grof method.

A consortium to coordinate public and voluntary sectors under contract in child welfare.

A group of voluntary agencies and other contractors for service with the Washington, DC. Department of Human Services has developed a plan for coordinating work on a comprehensive program for child care.

Ribulose diphosphate carboxylase/oxygenase. III. Isolation and properties.

Similarities in properties of ribulose diphosphate carboxylase and oxygenase activities further substantiate the hypothesis that the same protein catalyzes both reactions. The Km (ribulose diphosphate) is 0.33 mM for the ribulose diphosphate oxygenase, when assayed in air with an oxygen electrode. Maximum activity is obtained with 10 to 35 mM MgCl2. Higher MgCl2 concentrations are inhibitory, but they shift the pH optimum from 9.3 or 9.4 to 8.7 or 9.0. MnCl2 is an effective cofactor of the oxygenase and some activity is obtained with CoCl2. Both the ribulose diphosphate carboxylase and oxygenase activity of the purified protein from spinach leaves are slowly inactivated by storage at 0 degrees and reactivated in 10 min at 50 degrees, provided both 25 mM MgCl2 and 1 mM dithiothreitol are present. The sulfhydryl groups of the enzyme which react rapidly with 5,5'-dithiobis(2-nitrobenzoic acid) are approximately 4 at pH 7.8 and 11 at pH 9.4. At both pH values ribulose diphosphate prevents two of these sulfhydryl groups from reacting with this reagent. About 50% inhibition of the oxygenase activity at pH 9.0 occurs with 50 mM bicarbonate in the presence of 3 mM ribulose diphosphate, and from variations in these parameters the inhibition is attributed to the CO2 species. The purified enzyme of acrylamide gels prevented the reduction of nitroblue tetrazolium in the presence of the superoxide radical, but the enzyme in solution did not react as a superoxide dismutase.

Ribulose diphosphate carboxylase/oxygenase. IV. Regulation by phosphate esters.

The stimulation or inhibition of ribulose diphosphate oxygenase by a variety of compounds is compared with the reported effects on these compounds on the ribulose diphosphate carboxylase activity. A possible transition state analog of ribulose diphosphate, 2-carboxyribitol 1, 5-diphosphate, at a molar ratio of inhibitor to enzyme of 10 to 1, irreversibly inactivates the oxygenase and carboxylase activities. This is consistent with the hypothesis that there may be a single active site for both the carboxylase and oxygenase activities. Several compounds of the reductive pentose photosynthetic carbon cycle act as effectors of the ribulose diphosphate oxygenase in a manner complementary to their reported effect upon the carboxylase. Ribose 5-phosphate inhibits the oxygenase with an apparent Ki of 1.8 mM, but it is reported to activate the carboxylase; fructose 6-phosphate and glucose 6-phosphate act similarly but are less effective than ribose 5-phosphate. Fructose 1. 6-diphosphate stimulates the oxygenase at low magnesium ion concentrations. The stimulatory effect of 6-phosphogluconate on the oxygenase is associated with a 3-fold reduction of the Km (Mg2+). ATP inhibits the oxygenase but has been reported to stimulate the carboxylase; pyrophosphate acts in an opposite manner. From these results it appears that the ratio of carboxylase to oxygenase activity may be a variable factor with predictable subsequent alteration in the ratio between photosynthetic CO2 fixation and photorespiration.