Evaluating Established Methods for Rumen 16S rRNA Amplicon Sequencing With Mock Microbial Populations

peer-reviewedThe rumen microbiome scientific community has utilized amplicon sequencing as an aid in identifying potential community compositional trends that could be used as an estimation of various production and performance traits including methane emission, animal protein production efficiency, and ruminant health status. In order to translate rumen microbiome studies into executable application, there is a need for experimental and analytical concordance within the community. The objective of this study was to assess these factors in relation to selected currently established methods for 16S phylogenetic community analysis on a microbial community standard (MC) and a DNA standard (DS; ZymoBIOMICSTM). DNA was extracted from MC using the RBBC method commonly used for microbial DNA extraction from rumen digesta samples. 16S rRNA amplicon libraries were generated for the MC and DS using primers routinely used for rumen bacterial and archaeal community analysis. The primers targeted the V4 and V3–V4 region of the 16S rRNA gene and samples were subjected to both 20 and 28 polymerase chain reaction (PCR) cycles under identical cycle conditions. Sequencing was conducted using the Illumina MiSeq platform. As the bacteria contained in the microbial mock community were well-classified species, and for ease of explanation, we used the results of the Basic Local Alignment Search Tool classification to assess the DNA, PCR cycle number, and primer type. Sequence classification methodology was assessed independently. Spearman’s correlation analysis indicated that utilizing the repeated bead beating and column method for DNA extraction in combination with primers targeting the 16S rRNA gene using 20 first-round PCR cycles was sufficient for amplicon sequencing to generate a relatively accurate depiction of the bacterial communities present in rumen samples. These results also emphasize the requirement to develop and utilize positive mock community controls for all rumen microbiomic studies in order to discern errors which may arise at any step during a next-generation sequencing protocol

Characterization of Anodic Fuel Cell Catalysts Utilizing Various Surface Sensitive Techniques

117 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2004.The spontaneously deposited Pt/Pd catalyst was optimized. The optimum palladium coverage for unsupported platinum black was theta = 0.79, whereas for carbon-supported platinum black it was theta = 1.39. The catalyst was also inspected via EC-IRAS, which indicated the presence of palladium on the surface and smaller particle sizes. It also revealed a potential tool for determining the poison from formic acid oxidation on palladium nanoparticles. Further, the efficacy of methyl formate oxidation on the catalyst was examined. This fuel was determined to hold no advantages over formic acid for oxidation with the Pt/Pd catalyst. This study also revealed the inadequacy of methanol oxidation on the Pt/Pd catalyst. Radiolabeling with electrochemistry was used to examine the poison/potential and poison/environment relationships. Poison from methanol and formic acid was determined to be strongly adsorbed CO, which was replaceable by CO passed through the electrolyte. The potentiometric differences between methanol and formic acid oxidation were also studied on a platinum black substrate. Formic acid oxidation was determined to go through the direct CO2 pathway, with poison buildup from CO formation only observed in a dynamic experiment in the HUPD region, indicating a reverse water-gas shift. Finally, we attempted to identify the poison species from formic acid oxidation on palladium. The experiments revealed that palladium is not significantly poisoned by carbon-containing species during formic acid oxidation.Ope

Plane of nutrition affects the phylogenetic diversity and relative abundance of transcriptionally active methanogens in the bovine rumen

peer-reviewedMethane generated during enteric fermentation in ruminant livestock species is a major contributor to global anthropogenic greenhouse gas emissions. A period of moderate feed restriction followed by ad libitum access to feed is widely applied in cattle management to exploit the animal’s compensatory growth potential and reduce feed costs. In the present study, we utilised microbial RNA from rumen digesta samples to assess the phylogenetic diversity of transcriptionally active methanogens from feed-restricted and non-restricted animals. To determine the contribution of different rumen methanogens to methanogenesis during dietary restriction of cattle, we conducted high-throughput mcrA cDNA amplicon sequencing on an Illumina MiSeq and analysed both the abundance and phylogenetic origin of different mcrA cDNA sequences. When compared to their unrestricted contemporaries, in feed-restricted animals, the methanogenic activity, based on mcrA transcript abundance, of Methanobrevibacter gottschalkii clade increased while the methanogenic activity of the Methanobrevibacter ruminantium clade and members of the Methanomassiliicoccaceae family decreased. This study shows that the quantity of feed consumed can evoke large effects on the composition of methanogenically active species in the rumen of cattle. These data potentially have major implications for targeted CH4 mitigation approaches such as anti-methanogen vaccines and/or tailored dietary management

Counting atoms in a deep optical microtrap

We demonstrate a method to count small numbers of atoms held in a deep, microscopic optical dipole trap by collecting fluorescence from atoms exposed to a standing wave of light that is blue detuned from resonance. While scattering photons, the atoms are also cooled by a Sisyphus mechanism that results from the spatial variation in light intensity. The use of a small blue detuning limits the losses due to light assisted collisions, thereby making the method suitable for counting several atoms in a microscopic volume

Prevalence and correlates of substance use in Black, White, and biracial Black–White adolescents: Evidence for a biracial intermediate phenomena.

Most substance-use prevention interventions are based on the implicit assumption that risk and protective factors for substance use are the same for biracial and monoracial youth. However, preliminary research suggests this assumption may be untrue. This study compared the prevalence and correlates of substance use among Black, White, and biracial Black-White youth. Data were derived from the National Longitudinal Study of Adolescent and Adult Health (Add Health), which is a longitudinal investigation using stratified random sampling to study health behaviors. After controlling for sociodemographic factors and using weighted Poisson and logistic regression, we found the substance-use prevalence rates of Black-White youth to be intermediate to the higher rates of Whites and lower rates of Blacks. In addition, Black-White youth’s scores on most covariates were intermediate to those of the monoracial groups. Family factors were more important in explaining higher substance use than other contextual factors. School factors seem to be important in explaining lower substance use for Black-White youth. Correlates of substance use for Black-White youth were not identical to those of either Black or White youth. More research on the observed intermediate phenomena among biracial youth vis-à-vis prevalence, correlates, and causes of substance use is needed

The Structural and Functional Capacity of Ruminal and Cecal Microbiota in Growing Cattle Was Unaffected by Dietary Supplementation of Linseed Oil and Nitrate

peer-reviewedMicroorganisms in the digestive tract of ruminants differ in their functionality and ability to use feed constituents. While cecal microbiota play an important role in post-rumen fermentation of residual substrates undigested in the rumen, limited knowledge exists regarding its structure and function. In this trial we investigated the effect of dietary supplementation with linseed oil and nitrate on methane emissions and on the structure of ruminal and cecal microbiota of growing bulls. Animals were allocated to either a CTL (control) or LINNIT (CTL supplemented with 1.9% linseed and 1.0% nitrates) diet. Methane emissions were measured using the GreenFeed system. Microbial diversity was assessed using amplicon sequencing of microbial genomic DNA. Additionally, total RNA was extracted from ruminal contents and functional mcrA and mtt genes were targeted in amplicon sequencing approach to explore the diversity of functional gene expression in methanogens. LINNIT had no effect on methane yield (g/kg DMI) even though it decreased methane production by 9% (g/day; P < 0.05). Methanobrevibacter- and Methanomassiliicoccaceae-related OTUs were more abundant in cecum (72 and 24%) compared to rumen (60 and 11%) irrespective of the diet (P < 0.05). Feeding LINNIT reduced the relative abundance of Methanomassiliicoccaceae mcrA cDNA reads in the rumen. Principal component analysis revealed significant differences in taxonomic composition and abundance of bacterial communities between rumen and cecum. Treatment decreased the relative abundance of a few Ruminococcaceae genera, without affecting global bacterial community structure. Our research confirms a high level of heterogeneity in species composition of microbial consortia in the main gastrointestinal compartments where feed is fermented in ruminants. There was a parallel between the lack of effect of LINNIT on ruminal and cecal microbial community structure and functions on one side and methane emission changes on the other. These results suggest that the sequencing strategy used here to study microbial diversity and function accurately reflected the absence of effect on methane phenotypes in bulls treated with linseed plus nitrate.This experiment is a part of a large collaborative project led by INRA granted by 11 companies: Adisseo France SAS, Agrial, Apis Gene, Deltavit, DSM Nutritional Products AG, Institut de l'Elevage, Lallemand, Moy Park Beef Orléans, Neovia, Techna France Nutrition, Valorex. This project aims to reduce enteric methane emission by nutrition. MP was the recipient of a PHC Ulysses travel scholarship to Ireland, provided by the French ministry of Foreign Affairs and International Development (Ministères des Affaires Etrangères et du Développement International, MAEDI) and the ministry of National Education, Higher Education, and Research (Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche, MENESR). EM was the recipient of a FACCE-JPI scholarship

Promoting Alcohol Reduction in Non-Treatment Seeking parents (PAReNTS):a protocol for a pilot feasibility cluster randomised controlled trial of alcohol screening and brief interventions to reduce parental alcohol use disorders in vulnerable families

Brief alcohol advice intervention. (DOCX 1500 kb

Molecular classification of Crohn's disease reveals two clinically relevant subtypes

The clinical presentation and course of Crohn’s disease (CD) is highly variable. We sought to better understand the cellular and molecular mechanisms that guide this heterogeneity, and characterize the cellular processes associated with disease phenotypes

Alterations to chromatin in intestinal macrophages link IL-10 deficiency to inappropriate inflammatory responses

Intestinal macrophages are uniquely programmed to tolerate exposure to bacteria without mounting potent inflammatory responses. The cytokine IL-10 maintains the macrophage anti-inflammatory response such that loss of IL-10 results in chronic intestinal inflammation. To investigate how IL-10-deficiency alters intestinal macrophage programming and bacterial tolerance, we studied changes in chromatin accessibility in response to bacteria in macrophages from two distinct niches, the intestine and bone-marrow, from both wild-type and IL-10-deficient mice. In both bone-marrow-derived and intestinal macrophages, we identified chromatin accessibility changes associated with bacterial exposure and IL-10-deficiency. Surprisingly, IL-10-deficient intestinal macrophages adopted chromatin and gene expression patterns characteristic of an inflammatory response, even in the absence of bacteria. Further, if IL-10 protein was added to cells that had previously been IL-10-deficient, it could not revert the chromatin landscape to a normal state. Our results demonstrate that IL-10 deficiency results in stable chromatin alterations in macrophages, even in the absence of bacteria. This supports a model where IL-10-deficiency leads to chromatin alterations that contribute to a loss of intestinal macrophage tolerance to bacteria, which is a primary initiating event in chronic intestinal inflammation

Hematopoietic origin of Langerhans cell histiocytosis and Erdheim-Chester disease in adults.

Langerhans cell histiocytosis (LCH) and Erdheim-Chester disease (ECD) are rare histiocytic disorders induced by somatic mutation of MAPK pathway genes. BRAFV600E mutation is the most common mutation in both conditions and also occurs in the hematopoietic neoplasm hairy cell leukemia (HCL). It is not known if adult LCH or ECD arises from hematopoietic stem cells (HSCs), nor which potential blood borne precursors lead to the formation of histiocytic lesions. In this study, BRAFV600E allele-specific polymerase chain reaction was used to map the neoplastic clone in 20 adults with LCH, ECD, and HCL. BRAFV600E was tracked to classical monocytes, nonclassical monocytes, and CD1c+ myeloid dendritic cells (DCs) in the blood, and mutations were observed in HSCs and myeloid progenitors in the bone marrow of 4 patients. The pattern of involvement of peripheral blood myeloid cells was indistinguishable between LCH and ECD, although the histiocytic disorders were distinct to HCL. As reported in children, detection of BRAFV600E in peripheral blood of adults was a marker of active multisystem LCH. The healthy counterparts of myeloid cells affected by BRAF mutation had a range of differentiation potentials depending on exogenous signals. CD1c+ DCs acquired high langerin and CD1a with granulocyte-macrophage colony-stimulating factor and transforming growth factor β alone, whereas CD14+ classical monocytes required additional notch ligation. Both classical and nonclassical monocytes, but not CD1c+ DCs, made foamy macrophages easily in vitro with macrophage colony-stimulating factor and human serum. These studies are consistent with a hematopoietic origin and >1 immediate cellular precursor in both LCH and ECD