144 research outputs found

    Omadacycline gut microbiome exposure does not induce Clostridium difficile proliferation or toxin production in a model that simulates the proximal, medial and distal human colon

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    A clinically reflective model of the human colon was used to investigate the effects of the broad-spectrum antibiotic omadacycline on the gut microbiome, and subsequent potential to induce simulated Clostridium difficile infection (CDI). Triple stage chemostat gut models were inoculated with pooled human faecal slurry from healthy volunteers (age ≥60 years). Models were challenged twice with 107 cfu C. difficile spores (PCR ribotype 027). Omadacycline effects were assessed in a single gut model. Observations were confirmed in a parallel study with omadacycline and moxifloxacin. Antibiotic instillation was performed once daily for 7 days. The models were observed for 3 weeks post-antibiotic challenge. Gut microbiota populations and C. difficile total viable and spore counts were enumerated daily by culture. Cytotoxin titres and antibiotic concentrations were also measured. Gut microbiota populations were stable before antibiotic challenge. Moxifloxacin instillation caused a ∼4 log10 cfu/mL decline in enterococci and Bacteroides fragilis group populations, and a ∼3 log10 cfu/mL decline in bifidobacteria and lactobacilli, followed by simulated CDI (vegetative cell proliferation and detectable toxin). In both models, omadacycline instillation decreased populations of bifidobacteria (∼8 log10 cfu/mL), B. fragilis group populations (7-8 log10 cfu/mL), lactobacilli (2-6 log10 cfu/mL), and enterococci (4-6 log10 cfu/mL). Despite these microbial shifts, there was no evidence of C. difficile germination or toxin production. In contrast to moxifloxacin, omadacycline exposure did not facilitate simulated CDI, suggesting this antibiotic may have a low propensity to induce CDI in the clinical setting

    The clock genes Period 2 and Cryptochrome 2 differentially balance bone formation

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    Background: Clock genes and their protein products regulate circadian rhythms in mammals but have also been implicated in various physiological processes, including bone formation. Osteoblasts build new mineralized bone whereas osteoclasts degrade it thereby balancing bone formation. To evaluate the contribution of clock components in this process, we investigated mice mutant in clock genes for a bone volume phenotype. Methodology/Principal Findings: We found that Per2Brdm1 mutant mice as well as mice lacking Cry2-/- displayed significantly increased bone volume at 12 weeks of age, when bone turnover is high. Per2Brdm1 mutant mice showed alterations in parameters specific for osteoblasts whereas mice lacking Cry2-/- displayed changes in osteoclast specific parameters. Interestingly, inactivation of both Per2 and Cry2 genes leads to normal bone volume as observed in wild type animals. Importantly, osteoclast parameters affected due to the lack of Cry2, remained at the level seen in the Cry2-/- mutants despite the simultaneous inactivation of Per2. Conclusions/Significance: This indicates that Cry2 and Per2 affect distinct pathways in the regulation of bone volume with Cry2 influencing mostly the osteoclastic cellular component of bone and Per2 acting on osteoblast parameters

    Caspase-8 binding to cardiolipin in giant unilamellar vesicles provides a functional docking platform for bid

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    Caspase-8 is involved in death receptor-mediated apoptosis in type II cells, the proapoptotic programme of which is triggered by truncated Bid. Indeed, caspase-8 and Bid are the known intermediates of this signalling pathway. Cardiolipin has been shown to provide an anchor and an essential activating platform for caspase-8 at the mitochondrial membrane surface. Destabilisation of this platform alters receptor-mediated apoptosis in diseases such as Barth Syndrome, which is characterised by the presence of immature cardiolipin which does not allow caspase-8 binding. We used a simplified in vitro system that mimics contact sites and/or cardiolipin-enriched microdomains at the outer mitochondrial surface in which the platform consisting of caspase-8, Bid and cardiolipin was reconstituted in giant unilamellar vesicles. We analysed these vesicles by flow cytometry and confirm previous results that demonstrate the requirement for intact mature cardiolipin for caspase-8 activation and Bid binding and cleavage. We also used confocal microscopy to visualise the rupture of the vesicles and their revesiculation at smaller sizes due to alteration of the curvature following caspase-8 and Bid binding. Biophysical approaches, including Laurdan fluorescence and rupture/tension measurements, were used to determine the ability of these three components (cardiolipin, caspase-8 and Bid) to fulfil the minimal requirements for the formation and function of the platform at the mitochondrial membrane. Our results shed light on the active functional role of cardiolipin, bridging the gap between death receptors and mitochondria

    Accounting for a Quantitative Trait Locus for Plasma Triglyceride Levels: Utilization of Variants in Multiple Genes

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    For decades, research efforts have tried to uncover the underlying genetic basis of human susceptibility to a variety of diseases. Linkage studies have resulted in highly replicated findings and helped identify quantitative trait loci (QTL) for many complex traits; however identification of specific alleles accounting for linkage remains elusive. The purpose of this study was to determine whether with a sufficient number of variants a linkage signal can be fully explained.We used comprehensive fine-mapping using a dense set of single nucleotide polymorphisms (SNPs) across the entire quantitative trait locus (QTL) on human chromosome 7q36 linked to plasma triglyceride levels. Analyses included measured genotype and combined linkage association analyses.Screening this linkage region, we found an over representation of nominally significant associations in five genes (MLL3, DPP6, PAXIP1, HTR5A, INSIG1). However, no single genetic variant was sufficient to account for the linkage. On the other hand, multiple variants capturing the variation in these five genes did account for the linkage at this locus. Permutation analyses suggested that this reduction in LOD score was unlikely to have occurred by chance (p = 0.008).With recent findings, it has become clear that most complex traits are influenced by a large number of genetic variants each contributing only a small percentage to the overall phenotype. We found that with a sufficient number of variants, the linkage can be fully explained. The results from this analysis suggest that perhaps the failure to identify causal variants for linkage peaks may be due to multiple variants under the linkage peak with small individual effect, rather than a single variant of large effect

    Minimum Criteria for DNA Damage-Induced Phase Advances in Circadian Rhythms

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    Robust oscillatory behaviors are common features of circadian and cell cycle rhythms. These cyclic processes, however, behave distinctively in terms of their periods and phases in response to external influences such as light, temperature, nutrients, etc. Nevertheless, several links have been found between these two oscillators. Cell division cycles gated by the circadian clock have been observed since the late 1950s. On the other hand, ionizing radiation (IR) treatments cause cells to undergo a DNA damage response, which leads to phase shifts (mostly advances) in circadian rhythms. Circadian gating of the cell cycle can be attributed to the cell cycle inhibitor kinase Wee1 (which is regulated by the heterodimeric circadian clock transcription factor, BMAL1/CLK), and possibly in conjunction with other cell cycle components that are known to be regulated by the circadian clock (i.e., c-Myc and cyclin D1). It has also been shown that DNA damage-induced activation of the cell cycle regulator, Chk2, leads to phosphorylation and destruction of a circadian clock component (i.e., PER1 in Mus or FRQ in Neurospora crassa). However, the molecular mechanism underlying how DNA damage causes predominantly phase advances in the circadian clock remains unknown. In order to address this question, we employ mathematical modeling to simulate different phase response curves (PRCs) from either dexamethasone (Dex) or IR treatment experiments. Dex is known to synchronize circadian rhythms in cell culture and may generate both phase advances and delays. We observe unique phase responses with minimum delays of the circadian clock upon DNA damage when two criteria are met: (1) existence of an autocatalytic positive feedback mechanism in addition to the time-delayed negative feedback loop in the clock system and (2) Chk2-dependent phosphorylation and degradation of PERs that are not bound to BMAL1/CLK

    Antagonism of the discriminative effects of ethylketazocine, cyclazocine, and nalorphine in macaques

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    dl -Ethylketazocine (EKC, 0.01 mg/kg) and saline were established as discriminative stimuli for food-maintained responding in macaque monkeys. Thirty consecutive presses on a right or left lever were reinforced with food, contingent on whether EKC or saline were administered before the session. For tests of antagonism, naltrexone, or UM 979 [( l )-5,9-alpha-dimethyl-2-(3-furylmethyl)-2′-hydroxy-6,7-benzomorphan] was administered concomitantly with EKC, dl -cyclazocine, or nalorphine. Both antagonists blocked completely the EKC discriminative stimulus. The antagonism of the stimulus and rate-altering effects of EKC was surmountable, with considerable intersubject variability in the magnitude of the EKC dose increase required to overcome the blockade. Cyclazocine and nalophine, mixed agonist-antagonist opioids that share stimulus properties with EKC, were also susceptible to antagonism. Naltrexone antagonized completely the EKC stimulus effects of nalorphine; naltrexone and UM 979 antagonized completely the EKC stimulus effects of cyclazocine. Naltrexone antagonism of the cyclazocine stimulus was not surmountable, due to a lack of antagonism of the rate-decreasing effects of high cyclazocine doses.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46433/1/213_2004_Article_BF00555213.pd

    GWAS for discovery and replication of genetic loci associated with sudden cardiac arrest in patients with coronary artery disease

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    <p>Abstract</p> <p>Background</p> <p>Epidemiologic evidence suggests a heritable component to risk for sudden cardiac arrest independent of risk for myocardial infarction. Recent candidate gene association studies for community sudden cardiac arrests have focused on a limited number of biological pathways and yielded conflicting results. We sought to identify novel gene associations for sudden cardiac arrest in patients with coronary artery disease by performing a genome-wide association study.</p> <p>Methods</p> <p>Tagging SNPs (n = 338,328) spanning the genome were typed in a case-control study comparing 89 patients with coronary artery disease and sudden cardiac arrest due to ventricular tachycardia or ventricular fibrillation to 520 healthy controls.</p> <p>Results</p> <p>Fourteen SNPs including 7 SNPs among 7 genes (ACYP2, AP1G2, ESR1, DGES2, GRIA1, KCTD1, ZNF385B) were associated with sudden cardiac arrest (all p < 1.30 × 10<sup>-7</sup>), following Bonferroni correction and adjustment for population substructure, age, and sex; genetic variation in ESR1 (p = 2.62 × 10<sup>-8</sup>; Odds Ratio [OR] = 1.43, 95% confidence interval [CI]:1.277, 1.596) has previously been established as a risk factor for cardiovascular disease. In tandem, the role of 9 genes for monogenic long QT syndrome (LQT1-9) was assessed, yielding evidence of association with CACNA1C (LQT8; p = 3.09 × 10<sup>-4</sup>; OR = 1.18, 95% CI:1.079, 1.290). We also assessed 4 recently published gene associations for sudden cardiac arrest, validating NOS1AP (p = 4.50 × 10<sup>-2</sup>, OR = 1.15, 95% CI:1.003, 1.326), CSMD2 (p = 6.6 × 10<sup>-3</sup>, OR = 2.27, 95% CI:1.681, 2.859), and AGTR1 (p = 3.00 × 10<sup>-3</sup>, OR = 1.13, 95% CI:1.042, 1.215).</p> <p>Conclusion</p> <p>We demonstrate 11 gene associations for sudden cardiac arrest due to ventricular tachycardia/ventricular fibrillation in patients with coronary artery disease. Validation studies in independent cohorts and functional studies are required to confirm these associations.</p

    The Framingham Heart Study 100K SNP genome-wide association study resource: overview of 17 phenotype working group reports

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    Background: The Framingham Heart Study (FHS), founded in 1948 to examine the epidemiology of cardiovascular disease, is among the most comprehensively characterized multi-generational studies in the world. Many collected phenotypes have substantial genetic contributors; yet most genetic determinants remain to be identified. Using single nucleotide polymorphisms (SNPs) from a 100K genome-wide scan, we examine the associations of common polymorphisms with phenotypic variation in this community-based cohort and provide a full-disclosure, web-based resource of results for future replication studies. Methods: Adult participants (n = 1345) of the largest 310 pedigrees in the FHS, many biologically related, were genotyped with the 100K Affymetrix GeneChip. These genotypes were used to assess their contribution to 987 phenotypes collected in FHS over 56 years of follow up, including: cardiovascular risk factors and biomarkers; subclinical and clinical cardiovascular disease; cancer and longevity traits; and traits in pulmonary, sleep, neurology, renal, and bone domains. We conducted genome-wide variance components linkage and population-based and family-based association tests. Results: The participants were white of European descent and from the FHS Original and Offspring Cohorts (examination 1 Offspring mean age 32 ± 9 years, 54% women). This overview summarizes the methods, selected findings and limitations of the results presented in the accompanying series of 17 manuscripts. The presented association results are based on 70,897 autosomal SNPs meeting the following criteria: minor allele frequency ≥ 10%, genotype call rate ≥ 80%, Hardy-Weinberg equilibrium p-value ≥ 0.001, and satisfying Mendelian consistency. Linkage analyses are based on 11,200 SNPs and short-tandem repeats. Results of phenotype-genotype linkages and associations for all autosomal SNPs are posted on the NCBI dbGaP website at http:// www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?id=phs000007. Conclusion: We have created a full-disclosure resource of results, posted on the dbGaP website, from a genome-wide association study in the FHS. Because we used three analytical approaches to examine the association and linkage of 987 phenotypes with thousands of SNPs, our results must be considered hypothesis-generating and need to be replicated. Results from the FHS 100K project with NCBI web posting provides a resource for investigators to identify high priority findings for replication.Molecular and Cellular Biolog
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