Isotopic and genetic methods reveal the role of the gut microbiome in mammalian host essential amino acid metabolism.

Intestinal microbiota perform many functions for their host, but among the most important is their role in metabolism, especially the conversion of recalcitrant biomass that the host is unable to digest into bioavailable compounds. Most studies have focused on the assistance gut microbiota provide in the metabolism of carbohydrates, however, their role in host amino acid metabolism is poorly understood. We conducted an experiment on Mus musculus using 16S rRNA gene sequencing and carbon isotope analysis of essential amino acids (AAESS) to quantify the community composition of gut microbiota and the contribution of carbohydrate carbon used by the gut microbiome to synthesize AAESS that are assimilated by mice to build skeletal muscle tissue. The relative abundances of Firmicutes and Bacteroidetes inversely varied as a function of dietary macromolecular content, with Firmicutes dominating when mice were fed low-protein diets that contained the highest proportions of simple carbohydrates (sucrose). Mixing models estimated that the microbial contribution of AAESS to mouse muscle varied from less than 5% (threonine, lysine, and phenylalanine) to approximately 60% (valine) across diet treatments, with the Firmicute-dominated microbiome associated with the greatest contribution. Our results show that intestinal microbes can provide a significant source of the AAESS their host uses to synthesize structural tissues. The role that gut microbiota play in the amino acid metabolism of animals that consume protein-deficient diets is likely a significant but under-recognized aspect of foraging ecology and physiology

Teaching prescribing: just what the doctor ordered? A thematic analysis of the views of newly qualified doctors

Undergraduate medical education has been criticised for failing to adequately prepare doctors for the task of prescribing. Pharmacists have been shown to improve medication use in hospitals. This study aims to elicit the views of intern doctors on the challenges of prescribing, and to suggest changes in education to enhance prescribing practice and potential role of the pharmacist. Semi-structured, qualitative interviews were conducted with intern doctors in their first year post qualification in an Irish hospital. Data collection was conducted until no new themes emerged and thematic analysis was performed. Thirteen interviews took place. Interns described training in practical prescribing as limited and felt the curriculum failed to convey the reality of actual prescribing. Pharmacists were perceived to be a useful, but underutilised, information source in the prescribing process. They requested an earlier introduction, and repeated exposure, to prescribing, and suggested the involvement of peers and pharmacists in this teaching. Intern doctors reported difficulties in applying knowledge gained in medical school to clinical practice. New strategies are needed to enhance the clinical relevance of the medical curriculum by rethinking the learning outcomes regarding prescribing practice and the involvement of pharmacists in prescribing education

Radionuclide method for evaluating the performance of hemodialysis in vivo

Radionuclide method for evaluating the performance of hemodialysis in vivo.BackgroundSpecifications of dialyzer performance are generally based on in vitro measurements. There is, however, a shortage of data on dialyzer performance in vivo. The aim of this study was to use continuous measurement of technetium-99m-diethyltriaminepentaacetic acid (Tc-99m-DTPA) blood concentration as a means of continuously monitoring dialyzer function in vivo in patients undergoing routine hemodialysis.MethodsThe study population comprised 15 patients (45 to 80 years old; 13 males). Tc-99m-DTPA was administered intravenously 90 minutes before obtaining a blood sample and starting dialysis. Blood Tc-99m-DTPA activity was continuously monitored by passing the line carrying blood from the patient to the dialyzer close to a scintillation probe mounted in a shielded housing. At the end of hemodialysis, lasting 180 to 300 minutes, chromium-51-ethylenediaminetetraacetic acid (Cr-51-EDTA) was given intravenously and a blood sample taken 90 minutes later. Baseline dialyzer blood flow (Qb) and dialysate flow (Qd) were 250 to 350mL/min and 500mL/min, respectively. The rate constant, α, of the decrease in blood Tc-99m-DTPA activity was used as the measure of moment-to-moment dialyzer function. Pre- and postdialysis extracellular fluid volumes were calculated from the blood Tc-99m-DTPA and Cr-51-EDTA concentrations (VDTPA and VEDTA) before and after dialysis. Tc-99m-DTPA clearance was measured as the product of α and VDTPA. Dialyzer urea clearance was calculated from pre- and postdialysis urea nitrogen concentrations and the time of dialysis. The effects of brief changes in Qb and Qd on dialyzer function were assessed from the associated changes in α.ResultsThe Tc-99m-DTPA clearance profile was biexponential, becoming monoexponential about 1 hour after starting hemodialysis, with α remaining constant for as long as dialysis continued in five patients in whom Qb and Qd were left unaltered. Mean (SEM) plasma Tc-99m-DTPA clearance averaged over the entire period of dialysis in all 15 patients was 110 (3.1)mL/min. It correlated with urea clearance (r = 0.71) (P < 0.01) which was 225 (9.5)mL/min based on a total body water of 2.5 that of VDTPA and 212 (13)mL/min scaled to 40 L/1.73m2. Extracellular fluid volume decreased by 1.73 (0.74) l over dialysis, which was comparable to the change in weight [1.48 (0.57) kg]. The extraction fraction of Tc-99m-DTPA across the artificial kidney, directly measured from afferent and efferent blood samples under baseline Qb and Qd, was 0.5 (0.013). Average extraction fraction indirectly estimated from Tc-99m-DTPA blood clearance and Qb was 0.54 (0.019). These two measurements of extraction fraction correlated with each other under conditions of varying Qb and Qd (r = 0.74) (N = 27) (P < 0.001). Changes in α resulting from changes in Qb and Qd were similar to changes predicted from computerized modeling. The ratio of mass transfer coefficients of urea and Tc-99m-DTPA with respect to the dialyzer, calculated as if they were permeability-surface area products, was 3.3, similar to the ratio, obtained from the literature, in continuous capillary endothelium.ConclusionTc-99m-DTPA is a useful agent for continuously monitoring dialyzer function in vivo and provides a platform for the use of other radio-pharmaceuticals of different molecular sizes that could be used in an analogous fashion

Laparoscopic Management of Chemical Peritonitis Caused by Dermoid Cyst Spillage

Early recognition and prompt treatment with removal of dermoid cyst content and peritoneal lavage can be successful in the management of‘ chemical peritonitis secondary to spillage of cyst content during surgery

Novel components of the Toxoplasma inner membrane complex revealed by BioID.

UNLABELLED:The inner membrane complex (IMC) of Toxoplasma gondii is a peripheral membrane system that is composed of flattened alveolar sacs that underlie the plasma membrane, coupled to a supporting cytoskeletal network. The IMC plays important roles in parasite replication, motility, and host cell invasion. Despite these central roles in the biology of the parasite, the proteins that constitute the IMC are largely unknown. In this study, we have adapted a technique named proximity-dependent biotin identification (BioID) for use in T.&nbsp;gondii to identify novel components of the IMC. Using IMC proteins in both the alveoli and the cytoskeletal network as bait, we have uncovered a total of 19 new IMC proteins in both of these suborganellar compartments, two of which we functionally evaluate by gene knockout. Importantly, labeling of IMC proteins using this approach has revealed a group of proteins that localize to the sutures of the alveolar sacs that have been seen in their entirety in Toxoplasma species only by freeze fracture electron microscopy. Collectively, our study greatly expands the repertoire of known proteins in the IMC and experimentally validates BioID as a strategy for discovering novel constituents of specific cellular compartments of T.&nbsp;gondii. IMPORTANCE:The identification of binding partners is critical for determining protein function within cellular compartments. However, discovery of protein-protein interactions within membrane or cytoskeletal compartments is challenging, particularly for transient or unstable interactions that are often disrupted by experimental manipulation of these compartments. To circumvent these problems, we adapted an in vivo biotinylation technique called BioID for Toxoplasma species to identify binding partners and proximal proteins within native cellular environments. We used BioID to identify 19 novel proteins in the parasite IMC, an organelle consisting of fused membrane sacs and an underlying cytoskeleton, whose protein composition is largely unknown. We also demonstrate the power of BioID for targeted discovery of proteins within specific compartments, such as the IMC cytoskeleton. In addition, we uncovered a new group of proteins localizing to the alveolar sutures of the IMC. BioID promises to reveal new insights on protein constituents and interactions within cellular compartments of Toxoplasma

Essential role of Notch signaling in effector memory CD8+ T cell–mediated airway hyperresponsiveness and inflammation

Adoptive transfer of in vivo–primed CD8+ T cells or in vitro–generated effector memory CD8+ T (TEFF) cells restores airway hyperresponsiveness (AHR) and airway inflammation in CD8-deficient (CD8−/−) mice. Examining transcription levels, there was a strong induction of Notch1 in TEFF cells compared with central memory CD8+ T cells. Treatment of TEFF cells with a γ-secretase inhibitor (GSI) strongly inhibited Notch signaling in these cells, and after adoptive transfer, GSI-treated TEFF cells failed to restore AHR and airway inflammation in sensitized and challenged recipient CD8−/− mice, or to enhance these responses in recipient wild-type (WT) mice. These effects of GSI were also associated with increased expression of the Notch ligand Delta1 in TEFF cells. Treatment of sensitized and challenged WT mice with Delta1-Fc resulted in decreased AHR and airway inflammation accompanied by higher levels of interferon γ in bronchoalveolar lavage fluid. These results demonstrate a role for Notch in skewing the T cell response from a T helper (Th)2 to a Th1 phenotype as a consequence of the inhibition of Notch receptor activation and the up-regulation of the Notch ligand Delta1. These data are the first to show a functional role for Notch in the challenge phase of CD8+ T cell–mediated development of AHR and airway inflammation, and identify Delta1 as an important regulator of allergic airway inflammation

Contemporary evidence: baseline data from the D2B Alliance

© 2008 Bradley et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

Transcriptional profiling of MnSOD-mediated lifespan extension in Drosophila reveals a species-general network of aging and metabolic genes.

BACKGROUND: Several interventions increase lifespan in model organisms, including reduced insulin/insulin-like growth factor-like signaling (IIS), FOXO transcription factor activation, dietary restriction, and superoxide dismutase (SOD) over-expression. One question is whether these manipulations function through different mechanisms, or whether they intersect on common processes affecting aging. RESULTS: A doxycycline-regulated system was used to over-express manganese-SOD (MnSOD) in adult Drosophila, yielding increases in mean and maximal lifespan of 20%. Increased lifespan resulted from lowered initial mortality rate and required MnSOD over-expression in the adult. Transcriptional profiling indicated that the expression of specific genes was altered by MnSOD in a manner opposite to their pattern during normal aging, revealing a set of candidate biomarkers of aging enriched for carbohydrate metabolism and electron transport genes and suggesting a true delay in physiological aging, rather than a novel phenotype. Strikingly, cross-dataset comparisons indicated that the pattern of gene expression caused by MnSOD was similar to that observed in long-lived Caenorhabditis elegans insulin-like signaling mutants and to the xenobiotic stress response, thus exposing potential conserved longevity promoting genes and implicating detoxification in Drosophila longevity. CONCLUSION: The data suggest that MnSOD up-regulation and a retrograde signal of reactive oxygen species from the mitochondria normally function as an intermediate step in the extension of lifespan caused by reduced insulin-like signaling in various species. The results implicate a species-conserved net of coordinated genes that affect the rate of senescence by modulating energetic efficiency, purine biosynthesis, apoptotic pathways, endocrine signals, and the detoxification and excretion of metabolites.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Enhancing the genome editing toolbox: genome wide CRISPR arrayed libraries.

CRISPR-Cas9 technology has accelerated biological research becoming routine for many laboratories. It is rapidly replacing conventional gene editing techniques and has high utility for both genome-wide and gene-focussed applications. Here we present the first individually cloned CRISPR-Cas9 genome wide arrayed sgRNA libraries covering 17,166 human and 20,430 mouse genes at a complexity of 34,332 sgRNAs for human and 40,860 sgRNAs for the mouse genome. For flexibility in generating stable cell lines the sgRNAs have been cloned in a lentivirus backbone containing PiggyBac transposase recognition elements together with fluorescent and drug selection markers. Over 95% of tested sgRNA induced specific DNA cleavage as measured by CEL-1 assays. Furthermore, sgRNA targeting GPI anchor protein pathway genes induced loss of function mutations in human and mouse cell lines measured by FLAER labelling. These arrayed libraries offer the prospect for performing screens on individual genes, combinations as well as larger gene sets. They also facilitate rapid deconvolution of signals from genome-wide screens. This set of vectors provide an organized comprehensive gene editing toolbox of considerable scientific value

The Sloan Digital Sky Survey Reverberation Mapping Project: Technical Overview

The Sloan Digital Sky Survey Reverberation Mapping project (SDSS-RM) is a dedicated multi-object RM experiment that has spectroscopically monitored a sample of 849 broad-line quasars in a single 7 deg$^2$ field with the SDSS-III BOSS spectrograph. The RM quasar sample is flux-limited to i_psf=21.7 mag, and covers a redshift range of 0.1<z<4.5. Optical spectroscopy was performed during 2014 Jan-Jul dark/grey time, with an average cadence of ~4 days, totaling more than 30 epochs. Supporting photometric monitoring in the g and i bands was conducted at multiple facilities including the CFHT and the Steward Observatory Bok telescopes in 2014, with a cadence of ~2 days and covering all lunar phases. The RM field (RA, DEC=14:14:49.00, +53:05:00.0) lies within the CFHT-LS W3 field, and coincides with the Pan-STARRS 1 (PS1) Medium Deep Field MD07, with three prior years of multi-band PS1 light curves. The SDSS-RM 6-month baseline program aims to detect time lags between the quasar continuum and broad line region (BLR) variability on timescales of up to several months (in the observed frame) for ~10% of the sample, and to anchor the time baseline for continued monitoring in the future to detect lags on longer timescales and at higher redshift. SDSS-RM is the first major program to systematically explore the potential of RM for broad-line quasars at z>0.3, and will investigate the prospects of RM with all major broad lines covered in optical spectroscopy. SDSS-RM will provide guidance on future multi-object RM campaigns on larger scales, and is aiming to deliver more than tens of BLR lag detections for a homogeneous sample of quasars. We describe the motivation, design and implementation of this program, and outline the science impact expected from the resulting data for RM and general quasar science.Comment: 25 pages, submitted to ApJS; project website at http://www.sdssrm.or