A Bayesian method for identifying associations between response variables and bacterial community composition.

Determining associations between intestinal bacteria and continuously measured physiological outcomes is important for understanding the bacteria-host relationship but is not straightforward since abundance data (compositional data) are not normally distributed. To address this issue, we developed a fully Bayesian linear regression model (BRACoD; Bayesian Regression Analysis of Compositional Data) with physiological measurements (continuous data) as a function of a matrix of relative bacterial abundances. Bacteria can be classified as operational taxonomic units or by taxonomy (genus, family, etc.). Bacteria associated with the physiological measurement were identified using a Bayesian variable selection method: Stochastic Search Variable Selection. The output is a list of inclusion probabilities ([Formula: see text]) and coefficients that indicate the strength of the association ([Formula: see text]) for each bacterial taxa. Tests with simulated communities showed that adopting a cut point value of [Formula: see text] ≥ 0.3 for identifying included bacteria optimized the true positive rate (TPR) while maintaining a false positive rate (FPR) of ≤ 5%. At this point, the chances of identifying non-contributing bacteria were low and all well-established contributors were included. Comparison with other methods showed that BRACoD (at [Formula: see text] ≥ 0.3) had higher precision and a higher TPR than a commonly used center log transformed LASSO procedure (clr-LASSO) as well as higher TPR than an off-the-shelf Spike and Slab method after center log transformation (clr-SS). BRACoD was also less likely to include non-contributing bacteria that merely correlate with contributing bacteria. Analysis of a rat microbiome experiment identified 47 operational taxonomic units that contributed to fecal butyrate levels. Of these, 31 were positively and 16 negatively associated with butyrate. Consistent with their known role in butyrate metabolism, most of these fell within the Lachnospiraceae and Ruminococcaceae. We conclude that BRACoD provides a more precise and accurate method for determining bacteria associated with a continuous physiological outcome compared to clr-LASSO. It is more sensitive than a generalized clr-SS algorithm, although it has a higher FPR. Its ability to distinguish genuine contributors from correlated bacteria makes it better suited to discriminating bacteria that directly contribute to an outcome. The algorithm corrects for the distortions arising from compositional data making it appropriate for analysis of microbiome data.

Development of an Improved Standard Reference Material for Vitamin D Metabolites in Human Serum.

The National Institute of Standards and Technology (NIST) has developed Standard Reference Material (SRM) 972a Vitamin D Metabolites in Frozen Human Serum as a replacement for SRM 972, which is no longer available. SRM 972a was developed in collaboration with the National Institutes of Health's Office of Dietary Supplements. In contrast to the previous reference material, three of the four levels of SRM 972a are composed of unmodified human serum. This SRM has certified and reference values for the following 25-hydroxyvitamin D [25(OH)D] species: 25(OH)D2, 25(OH)D3, and 3-epi-25(OH)D3. The value assignment and certification process included three isotope-dilution mass spectrometry approaches, with measurements performed at NIST and at the Centers for Disease Control and Prevention (CDC). The value assignment methods employed have been modified from those utilized for the previous SRM, and all three approaches now incorporate chromatographic resolution of the stereoisomers, 25(OH)D3 and 3-epi-25(OH)D3.

Impact of ß2-1 fructan on faecal community change: results from a placebo-controlled, randomised, double-blinded, cross-over study in healthy adults.

Healthy adults (n 30) participated in a placebo-controlled, randomised, double-blinded, cross-over study consisting of two 28 d treatments (ß2-1 fructan or maltodextrin; 3×5 g/d) separated by a 14-d washout. Subjects provided 1 d faecal collections at days 0 and 28 of each treatment. The ability of faecal bacteria to metabolise ß2-1 fructan was common; eighty-seven species (thirty genera, and four phyla) were isolated using anaerobic medium containing ß2-1 fructan as the sole carbohydrate source. ß2-1 fructan altered the faecal community as determined through analysis of terminal restriction fragment length polymorphisms and 16S rRNA genes. Supplementation with ß2-1 fructan reduced faecal community richness, and two patterns of community change were observed. In most subjects, ß2-1 fructan reduced the content of phylotypes aligning within the Bacteroides, whereas increasing those aligning within bifidobacteria, Faecalibacterium and the family Lachnospiraceae. In the remaining subjects, supplementation increased the abundance of Bacteroidetes and to a lesser extent bifidobacteria, accompanied by decreases within the Faecalibacterium and family Lachnospiraceae. ß2-1 Fructan had no impact on the metagenome or glycoside hydrolase profiles in faeces from four subjects. Few relationships were found between the faecal bacterial community and various host parameters; Bacteroidetes content correlated with faecal propionate, subjects whose faecal community contained higher Bacteroidetes produced more caproic acid independent of treatment, and subjects having lower faecal Bacteroidetes exhibited increased concentrations of serum lipopolysaccharide and lipopolysaccharide binding protein independent of treatment. We found no evidence to support a defined health benefit for the use of ß2-1 fructans in healthy subjects.

ß2-1 Fructan supplementation alters host immune responses in a manner consistent with increased exposure to microbial components: results from a double-blinded, randomised, cross-over study in healthy adults.

ß2-1 Fructans are purported to improve health by stimulating growth of colonic bifidobacteria, increasing host resistance to pathogens and stimulating the immune system. However, in healthy adults, the benefits of supplementation remain undefined. Adults (thirteen men, seventeen women) participated in a double-blinded, placebo-controlled, randomised, cross-over study consisting of two 28-d treatments separated by a 14-d washout period. Subjects' regular diets were supplemented with ß2-1 fructan or placebo (maltodextrin) at 3×5 g/d. Fasting blood and 1-d faecal collections were obtained at the beginning and at the end of each phase. Blood was analysed for clinical, biochemical and immunological variables. Determinations of well-being and general health, gastrointestinal (GI) symptoms, regularity, faecal SCFA content, residual faecal ß2-1 fructans and faecal bifidobacteria content were undertaken. ß2-1 Fructan supplementation had no effect on blood lipid or cholesterol concentrations or on circulating lymphocyte and macrophage numbers, but significantly increased serum lipopolysaccharide, faecal SCFA, faecal bifidobacteria and indigestion. With respect to immune function, ß2-1 fructan supplementation increased serum IL-4, circulating percentages of CD282+/TLR2+ myeloid dendritic cells and ex vivo responsiveness to a toll-like receptor 2 agonist. ß2-1 Fructans also decreased serum IL-10, but did not affect C-reactive protein or serum/faecal Ig concentrations. No differences in host well-being were associated with either treatment, although the self-reported incidence of GI symptoms and headaches increased during the ß2-1 fructan phase. Although ß2-1 fructan supplementation increased faecal bifidobacteria, this change was not directly related to any of the determined host parameters.

Risk of bias in cross-sectional studies: Protocol for a scoping review of concepts and tools.

Cross-sectional studies are commonly used to study human health and disease, but are especially susceptible to bias. This scoping review aims to identify and describe available tools to assess the risk of bias (RoB) in cross-sectional studies and to compile the key bias concepts relevant to cross-sectional studies into an item bank. Using the JBI scoping review methodology, the strategy to locate relevant RoB concepts and tools is a combination of database searches, prospective review of PROSPERO registry records; and consultation with knowledge users and content experts. English language records will be included if they describe tools, checklists, or instruments which describe or permit assessment of RoB for cross-sectional studies. Systematic reviews will be included if they consider eligible RoB tools or use RoB tools for RoB of cross-sectional studies. All records will be independently screened, selected, and extracted by one researcher and checked by a second. An analytic framework will be used to structure the extraction of data. Results for the scoping review are pending. Results from this scoping review will be used to inform future selection of RoB tools and to consider whether development of a new RoB tool for cross-sectional studies is needed.

A scoping review shows that no single existing risk of bias assessment tool considers all sources of bias for cross-sectional studies.

OBJECTIVES: Different tools to assess the potential risk of bias (RoB) for cross-sectional studies have been developed, but it is unclear whether all pertinent bias concepts are addressed. We aimed to identify RoB concepts applicable to cross-sectional research validity and to explore coverage for each in existing appraisal tools. STUDY DESIGN AND SETTING: This scoping review followed the Joanna Briggs Institute methodology. We included records of any study design describing or reporting methods, concepts or tools used to consider RoB in health research reported to be descriptive/prevalence survey or analytic/association (cross-sectional) study designs. Synthesis included quantitative and qualitative analysis. RESULTS: Of the 4556 records screened, 90 were selected for inclusion; 67 (74%) described the development of, or validation process for, appraisal tools, 15 (17%) described methodological content or theory relevant to RoB for cross-sectional studies and 8 (9%) records of methodological systematic reviews. Review of methodological reports identified important RoB concepts for both descriptive/prevalence and analytic/association studies. Tools identified (n = 64 unique tools) were either intended to appraise quality or assess RoB in multiple study designs including cross-sectional studies (n = 21; 33%) or cross-sectional designs alone (n = 43; 67%). Several existing tools were modified (n = 17; 27%) for application to cross-sectional studies. The RoB items most frequently addressed in the RoB tools were validity and reliability of the exposure (53%) or outcome (65%) measurement and representativeness of the study population (59%). Most tools did not consider nonresponse or missingness appropriately or at all. CONCLUSION: Assessing cross-sectional studies involve unique RoB considerations. We identified RoB tools designed for broad applicability across various study designs as well as those specifically tailored for cross-sectional studies. However, none of the identified tools comprehensively address all potential biases pertinent to cross-sectional studies. Our findings indicate a need for continued improvement of RoB tools and suggest that the development of context-specific or more precise tools for this study design may be necessary.

Identification of Methanoculleus spp. as active methanogens during anoxic incubations of swine manure storage tank samples.

Methane emissions represent a major environmental concern associated with manure management in the livestock industry. A more thorough understanding of how microbial communities function in manure storage tanks is a prerequisite for mitigating methane emissions. Identifying the microorganisms that are metabolically active is an important first step. Methanogenic archaea are major contributors to methanogenesis in stored swine manure, and we investigated active methanogenic populations by DNA stable isotope probing (DNA-SIP). Following a preincubation of manure samples under anoxic conditions to induce substrate starvation, [U-(13)C]acetate was added as a labeled substrate. Fingerprint analysis of density-fractionated DNA, using length-heterogeneity analysis of PCR-amplified mcrA genes (encoding the alpha subunit of methyl coenzyme M reductase), showed that the incorporation of (13)C into DNA was detectable at in situ acetate concentrations (~7 g/liter). Fingerprints of DNA retrieved from heavy fractions of the (13)C treatment were primarily enriched in a 483-bp amplicon and, to a lesser extent, in a 481-bp amplicon. Analyses based on clone libraries of the mcrA and 16S rRNA genes revealed that both of these heavy DNA amplicons corresponded to Methanoculleus spp. Our results demonstrate that uncultivated methanogenic archaea related to Methanoculleus spp. were major contributors to acetate-C assimilation during the anoxic incubation of swine manure storage tank samples. Carbon assimilation and dissimilation rate estimations suggested that Methanoculleus spp. were also major contributors to methane emissions and that the hydrogenotrophic pathway predominated during methanogenesis.

Dietary folate does not significantly affect the intestinal microbiome, inflammation or tumorigenesis in azoxymethane-dextran sodium sulphate-treated mice.

Inflammatory bowel disease (IBD) is a risk factor for the development of colon cancer. Environmental factors including diet and the microflora influence disease outcome. Folate and homocysteine have been associated with IBD-mediated colon cancer but their roles remain unclear. We used a model of chemically induced ulcerative colitis (dextran sodium sulphate (DSS)) with or without the colon carcinogen azoxymethane (AOM) to determine the impact of dietary folic acid (FA) on colonic microflora and the development of colon tumours. Male mice (n 15 per group) were fed a FA-deficient (0 mg/kg), control (2 mg/kg) or FA-supplemented (8 mg/kg) diet for 12 weeks. Folate status was dependent on the diet (P< 0·001) and colitis-induced treatment (P= 0·04) such that mice with colitis had lower circulating folate. FA had a minimal effect on tumour initiation, growth and progression, although FA-containing diets tended to be associated with a higher tumour prevalence in DSS-treated mice (7-20 v. 0%, P= 0·08) and the development of more tumours in the distal colon of AOM-treated mice (13-83% increase, P= 0·09). Folate deficiency was associated with hyperhomocysteinaemia (P< 0·001) but homocysteine negatively correlated with tumour number (r - 0·58, P= 0·02) and load (r - 0·57, P= 0·02). FA had no effect on the intestinal microflora. The present data indicate that FA intake has no or little effect on IBD or IBD-mediated colon cancer in this model and that hyperhomocysteinaemia is a biomarker of dietary status and malabsorption rather than a cause of IBD-mediated colon cancer.

Summary of an NIH workshop to identify research needs to improve the monitoring of iodine status in the United States and to inform the DRI.

The Office of Dietary Supplements (ODS) at the NIH sponsored a workshop on May 12-13, 2011, to bring together representatives from various NIH institutes and centers as a first step in developing an NIH iodine research initiative. The workshop also provided an opportunity to identify research needs that would inform the dietary reference intakes for iodine, which were last revised in 2001. Iodine is required throughout the life cycle, but pregnant women and infants are the populations most at risk of deficiency, because iodine is required for normal brain development and growth. The CDC monitors iodine status of the population on a regular basis, but the status of the most vulnerable populations remains uncertain. The NIH funds very little investigator-initiated research relevant to iodine and human nutrition, but the ODS has worked for several years with a number of other U.S. government agencies to develop many of the resources needed to conduct iodine research of high quality (e.g., validated analytical methods and reference materials for multiple types of samples). Iodine experts, scientists from several U.S. government agencies, and NIH representatives met for 2 d to identify iodine research needs appropriate to the NIH mission.

Prebiotics and probiotics: some thoughts on demonstration of efficacy within the regulatory sphere.

Probiotics and prebiotics present regulators with challenges because they require a demonstrated positive health outcome and proof that the prebiotic or probiotic is the agent of action once safety aspects have been satisfied. Thus, probiotic and prebiotic definitions are important because they will set the criteria by which these materials will be judged within the regulatory sphere. Use of the terms probiotic and prebiotic are, themselves, considered health claims in some jurisdictions, so that both product health claims and product content labeling may be regulated. Currently accepted definitions of prebiotic and probiotic make it easier to draw a straight line between ingestion and health outcome for probiotics but much more difficult for prebiotics, where a health outcome must be linked to changes in specific bacterial species within the gut microbial community. These challenges highlight the difficulties facing regulatory bodies and the scientific community when emerging science is turned into consumable product.

Molecular methods to measure intestinal bacteria: a review.

The intestine is an exceptionally rich ecosystem encompassing a complex interaction among microorganisms, influenced by host factors, ingested food, and liquid. Characterizing the intestinal microbiota is currently an active area of research. Various molecular-based methods are available to characterize the intestinal microbiota, but all methods possess relative strengths, as well as salient weaknesses. It is important that researchers are cognizant of the limitations of these methods, and that they take the appropriate steps to mitigate weaknesses. Here, we discuss methodologies used to monitor intestinal bacteria including: (i) traditional clone libraries; (ii) direct sequencing using next-generation parallel sequencing technology; (iii) denaturing gradient gel electrophoresis and temperature gradient gel electrophoresis; (iv) terminal restriction fragment length polymorphism analysis; (v) fluorescent in situ hybridization; and (vi) quantitative PCR. In addition, we also discuss experimental design, sample collection and storage, DNA extraction, gene targets, PCR bias, and methods to reduce PCR bias.

NUQUEST-NUtrition QUality Evaluation Strengthening Tools: development of tools for the evaluation of risk of bias in nutrition studies.

BACKGROUND: Dietary exposure assessments are a critical issue in evaluating human nutrition studies; however, nutrition-specific criteria are not consistently included in existing bias assessment tools. OBJECTIVES: Our objective was to develop a set of risk of bias (RoB) tools that integrated nutrition-specific criteria into validated generic assessment tools to address RoB issues, including those specific to dietary exposure assessment. METHODS: The Nutrition QUality Evaluation Strengthening Tools (NUQUEST) development and validation process included 8 steps. The first steps identified 1) a development strategy; 2) generic assessment tools with demonstrated validity; and 3) nutrition-specific appraisal issues. This was followed by 4) generation of nutrition-specific items and 5) development of guidance to aid users of NUQUEST. The final steps used established ratings of selected studies and feedback from independent raters to 6) assess reliability and validity; 7) assess formatting and usability; and 8) finalize NUQUEST. RESULTS: NUQUEST is based on the Scottish Intercollegiate Guidelines Network checklists for randomized controlled trials, cohort studies, and case-control studies. Using a purposive sample of 45 studies representing the 3 study designs, interrater reliability was high (Cohen's κ: 0.73; 95% CI: 0.52, 0.93) across all tools and at least moderate for individual tools (range: 0.57-1.00). The use of a worksheet improved usability and consistency of overall interrater agreement across all study designs (40% without worksheet, 80%-100% with worksheet). When compared to published ratings, NUQUEST ratings for evaluated studies demonstrated high concurrent validity (93% perfect or near-perfect agreement). Where there was disagreement, the nutrition-specific component was a contributing factor in discerning exposure methodological issues. CONCLUSIONS: NUQUEST integrates nutrition-specific criteria with generic criteria from assessment tools with demonstrated reliability and validity. NUQUEST represents a consistent and transparent approach for evaluating RoB issues related to dietary exposure assessment commonly encountered in human nutrition studies.

Perspective: Human Milk Composition and Related Data for National Health and Nutrition Monitoring and Related Research.

National health and nutrition monitoring is an important federal effort in the United States and Canada, and the basis for many of their nutrition and health policies. Understanding of child exposures through human milk (HM) remains out of reach due to lack of current and representative data on HM's composition and intake volume. This article provides an overview of the current national health and nutrition monitoring activities for HM-fed children, HM composition (HMC) and volume data used for exposure assessment, categories of potential measures in HM, and associated variability factors. In this Perspective, we advocate for a framework for collection and reporting of HMC data for national health and nutrition monitoring and programmatic needs, including a shared vision for a publicly available Human Milk Composition Data Repository (HMCD-R) to include essential metadata associated with HMC. HMCD-R can provide a central, integrated platform for researchers and public health officials for compiling, evaluating, and sharing HMC data. The compiled compositional and metadata in HMCD-R would provide pertinent measures of central tendency and variability and allow use of modeling techniques to approximate compositional profiles for subgroups, providing more accurate exposure assessments for purposes of monitoring and surveillance. HMC and related metadata could facilitate understanding the complexity and variability of HM composition, provide crucial data for assessment of infant and maternal nutritional needs, and inform public health policies, food and nutrition programs, and clinical practice guidelines.

Dietary fructooligosaccharides and wheat bran elicit specific and dose-dependent gene expression profiles in the proximal colon epithelia of healthy Fischer 344 rats.

Proximal colon epithelial gene responses to diets containing increasing levels of dietary fermentable material (FM) from 2 different sources were measured to determine whether gene expression patterns were independent of the source of FM. Male Fischer 344 rats (10/group) were fed for 6 wk a control diet containing 10% (g/g) cellulose (0% FM); or a 2, 5, or 10% wheat bran (WB) diet (1, 2, 5% FM); or a 2, 5, or 8% fructooligosaccharides (FOS) diet (2, 5, 8% FM). WB and FOS were substituted for cellulose to give a final 10% nondigestible material content including FM. Gene responses were relative to expression in rats fed the control diet. The gene response patterns associated with feeding ∼2% FM (5% WB and 2% FOS) were similar (∼10 gene changes ≥ 1.6-fold; P ≤ 0.01) and involved genes associated with transport (Scnn1g, Mt1a), transcription (Zbtb16, Egr1), immunity (Fkbp5), a gut hormone (Retn1ß), and lipid metabolism (Scd2, Insig1). These changes were also similar to those associated with 5% FM but only in rats fed the 10% WB diet. In contrast, the 5% FOS diet (~5% FM) was associated with 68 gene expression changes ≥ 1.6-fold (P ≤ 0.01). The diet with the highest level of fermentation (8% FOS, ~8% FM) was associated with 132 changes ≥ 1.6-fold (P ≤ 0.01), including genes associated with transport, cellular proliferation, oncogene and tumor metastasis, the cell cycle, apoptosis, signal transduction, transcript regulation, immunity, gut hormones, and lipid metabolic processes. These results show that both the amount and source of FM determine proximal colon epithelial gene response patterns in rats.

Inadequate vitamin D status is associated with lower food plus supplemental intake of vitamin D in children of South Asian ethnicity living in the National Capital Region of Canada.

Vitamin D status, measured in a Vitamin D Standardization Program certified laboratory, was assessed among children of South Asian and European ethnicity living in the national capital region of Canada to explore factors that may account for inadequate status. Demographic information, dietary and supplemental vitamin D over 30 d prior to measurement of serum 25-hydroxyvitamin D (25OHD), and anthropometry were measured (age 6.0-18.9 y; n = 58/group; February-March 2015). No group related differences in age, height and body mass index (BMI) Z-scores or in food vitamin D intakes were observed. Standardized serum 25OHD was lower in South Asian children (mean ± SD: 39.0 ± 16.8 nmol/L vs. European: 58.4 ± 15.8 nmol/L). A greater proportion of South Asian children had serum 25OHD <40 nmol/L (56.9 vs. 8.6%, P < 0.0001) and fewer took supplements (31 vs. 50%, P = 0.0389). In a multi-factorial model (r2 = 0.54), lower vitamin D status was associated with overweight/obese BMI and older age (14-18 y); no interaction with ethnicity was observed. Lower vitamin D status was associated with lower total vitamin D intake only in South Asian children. This study reinforces the importance of public health actions towards meeting vitamin D intake recommendations among those of high-risk deficiency. Novelty: A higher proportion of South Asian vs. European children had inadequate vitamin D status. Lower vitamin D status was associated with a BMI in the overweight/obese range. Lower vitamin D status was associated with lower total vitamin D intake in South Asian but not European children.

Early prenatal use of a multivitamin diminishes the risk for inadequate vitamin D status in pregnant women: results from the Maternal-Infant Research on Environmental Chemicals (MIREC) cohort study.

BACKGROUND: Reports on the adequacy of vitamin D status of pregnant women are not available in Canada. OBJECTIVES: The objectives of this study were to examine vitamin D status across pregnancy and identify the correlates of vitamin D status of pregnant women in Canada. METHODS: Pregnant women (≥18 years) from 6 provinces (2008-2011) participating in a longitudinal cohort were studied. Sociodemographic data, obstetrical histories, and dietary and supplemental vitamin D intakes were surveyed. Plasma 25-hydroxyvitamin D (25OHD) was measured using an immunoassay standardized to LC-MS/MS from samples collected during the first (n = 1905) and third trimesters (n = 1649) and at delivery (n = 1543). The proportion of women with ≥40 nmol/L of plasma 25OHD (adequate status) was estimated at each time point, and factors related to achieving this cut point were identified using repeated-measures logistic regression. Differences in 25OHD concentrations across trimesters and at delivery were tested a using repeated-measures ANOVA with a post hoc Tukey's test. RESULTS: In the first trimester, 93.4% (95% CI: 92.3%-94.5%) of participants had 25OHD ≥40 nmol/L. The mean plasma 25OHD concentration increased from the first to the third trimester and then declined by delivery (69.8 ± 0.5 nmol/L, 78.6 ± 0.7 nmol/L, and 75.7 ± 0.7 nmol/L, respectively; P < 0.0001). A lack of multivitamin use early in pregnancy reduced the odds of achieving 25OHD ≥40 nmol/L (ORadj = 0.33; 95% CI: 0.25-0.42) across all time points. Factors associated with not using a prenatal multivitamin included multiparity (ORadj = 2.08; 95% CI: 1.42-3.02) and a below-median income (ORadj = 1.39; 95% CI: 1.02-1.89). CONCLUSIONS: The results from this cohort demonstrate the importance of early multivitamin supplement use to achieve an adequate vitamin D status in pregnant women.

Spatial distribution of some microbial trophic groups in a plug-flow-type anaerobic bioreactor treating swine manure.

Anaerobic digestion of swine manure is carried out by a consortium of microbial species, including volatile fatty acid (VFA) producers, VFA-degraders and methanogens. The distribution of five phylogenetic groups within a plug-flow-type anaerobic bioreactor consisting of eight serially-connected tanks was examined through the sequential digestion of swine manure. Quantification was carried out using reverse transcription real-time PCR (RT-Q-PCR) assays targeting the 16S rRNA of Clostridium (cluster XIVa), Peptostreptococcus, Syntrophomonas, Methanosaeta, and Methanosarcina spp. The VFA producers Peptostreptococcus spp. and Clostridium spp. were found predominantly in compartments where hydrolysis/acidogenesis took place. The spatial distribution of the aceticlastic methanogens, Methanosaeta and Methanosarcina, within the bioreactor was not correlated with methanogenic activity. In contrast the VFA-degrading genus Syntrophomonas spp. was more abundant in compartments with elevated methanogenic activity. Multivariate statistical analyses of the RT-Q-PCR data have provided new insights into our understanding of how the various trophic groups were distributed within this bioreactor system. While the distribution of clostridia, peptostreptococci and Syntrophomonas corresponded to their known metabolic functions, aceticlastic methanogens were not apparently linked to the methanogenesis stage occurring in latter compartments, suggesting that hydrogenotrophic methanogens were the primary methane generators in this bioreactor. However, aceticlastic methanogens could be involved in compartments related to the hydrolysis/acidogenesis stage.

Calcium exacerbates the inhibitory effects of phytic acid on zinc bioavailability in rats.

BACKGROUND: Complementary feeding of breastfed infants with foods high in bioavailable zinc (Zn) can help meet physiological requirements for Zn. Some infant cereals contain high concentrations of phytic acid (PA) and calcium (Ca) that may reduce absorbable Zn. OBJECTIVES: This study measured PA, Zn and Ca concentrations in selected infant cereals sold in Canada and investigated the effects of dietary PA and Ca at concentrations present in infant cereals on Zn bioavailability in rats. METHODS AND RESULTS: Male Sprague-Dawley rats (36-day old) were fed a control diet containing normal Zn (29.1 mg/kg) and Ca (4.95 g/kg) or six test diets (n = 12/diet group). Test diets were low in Zn (8.91-9.74 mg/kg) and contained low (2.16-2.17 g/kg), normal (5.00-5.11 g/kg) or high (14.6-14.9 g/kg) Ca without or with added PA (8 g/kg). After 2 weeks, rats were killed and Zn status of the rats was assessed. PA, Zn and Ca concentrations in infant cereals (n = 20) differed widely. PA concentrations ranged from undetectable to 16.0 g/kg. Zn and Ca concentrations ranged from 7.0-29.1 mg/kg and 0.8-13.4 g/kg, respectively. The [PA]/[Zn] and [PA × Ca]/[Zn] molar ratios in infants cereals with detectable PA (16 of 20 cereals) ranged from 22-75 and 0.9-14.9 mol/kg, respectively, predicting low Zn bioavailability. Body weight, body composition (lean and fat mass), right femur weight and length measurements and Zn concentrations in serum and femur indicated that diets higher in Ca had a more pronounced negative effect on Zn status of rats fed a PA-supplemented diet. Addition of PA to the diet had a greater negative effect on Zn status when Ca concentration in the diet was higher. CONCLUSION: These results show that, in rats, higher concentrations of dietary Ca and PA interact to potentiate a decrease in bioavailable Zn and may suggest lower Zn bioavailability in infant cereals with higher PA and Ca concentrations.

Diets enriched in oat bran or wheat bran temporally and differentially alter the composition of the fecal community of rats.

A clear understanding of how diet alters gastrointestinal communities is important given the suggested link between gut community composition and a wide variety of disease pathologies. To characterize this link for commonly consumed dietary fiber sources, we investigated the change in the fecal community of rats fed diets containing 5% nonnutritive fiber (control), 3% (wt:wt) oat bran plus 2% nonnutritive fiber (OB), or 5% (w/w) wheat bran (WB) over a 28-d feeding trial using both molecular- and cultivation-based methodologies. Pooled fecal samples from 8 rats fed the same diet were analyzed at 4 time points. On d 28, bran-fed rats had approximately twice the total cultivable bacteria than rats fed the control diet. Over the course of feeding, the cultivable community was initially dominated by bacteroides, then by bifidobacteria, lactobacilli, enterococci, and various enterics. In contrast, molecular analysis revealed the appearance of new operational taxonomic units (phylotypes) that were both temporally and inequitably distributed throughout the fecal community. The majority of change occurred in 2 major lineages within the Firmicutes: the Clostridium coccoides group and the Clostridium leptum subgroup. The time course of change depended on the source of bran, with the majority of new phylotypes appearing by d 14 (OB) or d 28 (WB), although adaptation of the fecal community was slow and continued over the entire feeding trial. Bacterial community richness was higher in bran-fed rats than in those fed the control diet. Change within the C. coccoides and C. leptum lineages likely reflect their high abundance within the gut bacterial community and the role of clostridia in fiber digestion. The results illustrate the limitations of relying solely on cultivation to assess bacterial changes and illustrate that community changes are complex in an ecosystem containing high numbers of interdependent and competing species of bacteria.

Ctr2 is partially localized to the plasma membrane and stimulates copper uptake in COS-7 cells.

Ctr1 (copper transporter 1) mediates high-affinity copper uptake. Ctr2 (copper transporter 2) shares sequence similarity with Ctr1, yet its function in mammalian cells is poorly understood. In African green monkey kidney COS-7 cells and rat tissues, Ctr2 migrated as a predominant band of approximately 70 kDa and was most abundantly expressed in placenta and heart. A transiently expressed hCtr2-GFP (human Ctr2-green fluorescent protein) fusion protein and the endogenous Ctr2 in COS-7 cells were mainly localized to the outer membrane of cytoplasmic vesicles, but were also detected at the plasma membrane. Biotinylation of Ctr2 with the membrane-impermeant reagent sulfo-NHS-SS-biotin [sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate] confirmed localization at the cell surface. Cells expressing hCtr2-GFP hyperaccumulated copper when incubated in medium supplemented with 10 microM CuSO(4), whereas cells depleted of endogenous Ctr2 by siRNAs (small interfering RNAs) accumulated lower levels of copper. hCtr2-GFP expression did not affect copper efflux, suggesting that hCtr2-GFP increased cellular copper concentrations by promoting uptake at the cell surface. Kinetic analyses showed that hCtr2-GFP stimulated saturable copper uptake with a K(m) of 11.0+/-2.5 microM and a K(0.5) of 6.9+/-0.7 microM when data were fitted to a rectangular hyperbola or Hill equation respectively. Competition experiments revealed that silver completely inhibited hCtr2-GFP-dependent copper uptake, whereas zinc, iron and manganese had no effect on uptake. Furthermore, increased copper concentrations in hCtr2-GFP-expressing cells were inversely correlated with copper chaperone for Cu/Zn superoxide dismutase protein expression. Collectively, these results suggest that Ctr2 promotes copper uptake at the plasma membrane and plays a role in regulating copper levels in COS-7 cells.