The effect of low-abundance OTU filtering methods on the reliability and variability of microbial composition assessed by 16S rRNA amplicon sequencing

PCR amplicon sequencing may lead to detection of spurious operational taxonomic units (OTUs), inflating estimates of gut microbial diversity. There is no consensus in the analytical approach as to what filtering methods should be applied to remove low-abundance OTUs; moreover, few studies have investigated the reliability of OTU detection within replicates. Here, we investigated the reliability of OTU detection (% agreement in detecting OTU in triplicates) and accuracy of their quantification (assessed by coefficient of variation (CV)) in human stool specimens. Stool samples were collected from 12 participants 22–55 years old. We applied several methods for filtering low-abundance OTUs and determined their impact on alpha-diversity and beta-diversity metrics. The reliability of OTU detection without any filtering was only 44.1% (SE=0.9) but increased after filtering low-abundance OTUs. After filtering OTUs with <0.1% abundance in the dataset, the reliability increased to 87.7% (SE=0.6) but at the expense of removing 6.97% reads from the dataset. When filtering was based on individual sample, the reliability increased to 73.1% after filtering OTUs with <10 copies while removing only 1.12% of reads. High abundance OTUs (>10 copies in sample) had lower CV, indicating better accuracy of quantification than low-abundance OTUs. Excluding very low-abundance OTUs had a significant impact on alpha-diversity metrics sensitive to the presence of rare species (observed OTUs, Chao1) but had little impact on relative abundance of major phyla and families and alpha-diversity metrics accounting for both richness and evenness (Shannon, Inverse Simpson). To increase the reliability of microbial composition, we advise removing OTUs with <10 copies in individual samples, particularly in studies where only one subsample per specimen is available for analysis

Reduced Retinal Function in the Absence of Nav1.6

Background: Mice with a function-blocking mutation in the Scn8a gene that encodes Nav1.6, a voltage-gated sodium channel (VGSC) isoform normally found in several types of retinal neurons, have previously been found to display a profoundly abnormal dark adapted flash electroretinogram. However the retinal function of these mice in light adapted conditions has not been studied. Methodology/Principal Findings: In the present report we reveal that during light adaptation these animals are shown to have electroretinograms with significant decreases in the amplitude of the a- and b-waves. The percent decrease in the a-and b-waves substantially exceeds the acute effect of VGSC block by tetrodotoxin in control littermates. Intravitreal injection of CoCl 2 or CNQX to isolate the a-wave contributions of the photoreceptors in littermates revealed that at high background luminance the cone-isolated component of the a-wave is of the same amplitude as the a-wave of mutants. Conclusions/Significance: Our results indicate that Scn8a mutant mice have reduced function in both rod and the cone retinal pathways. The extent of the reduction in the cone pathway, as quantified using the ERG b-wave, exceeds the reduction seen in control littermates after application of TTX, suggesting that a defect in cone photoreceptors contributes to the reduction. Unless the postreceptoral component of the a-wave is increased in Scn8a mutant mice, the reduction in the b-wave is larger than can be accounted for by reduced photoreceptor function alone. Our data suggests that th

Expression of survivin detected by immunohistochemistry in the cytoplasm and in the nucleus is associated with prognosis of leiomyosarcoma and synovial sarcoma patients

<p>Abstract</p> <p>Background</p> <p>Survivin, a member of the inhibitor of apoptosis-protein family suppresses apoptosis and regulates cell division. It is strongly overexpressed in the vast majority of cancers. We were interested if survivin detected by immunohistochemistry has prognostic relevance especially for patients of the two soft tissue sarcoma entities leiomyosarcoma and synovial sarcoma.</p> <p>Methods</p> <p>Tumors of leiomyosarcoma (n = 24) and synovial sarcoma patients (n = 26) were investigated for their expression of survivin by immunohistochemistry. Survivin expression was assessed in the cytoplasm and the nucleus of tumor cells using an immunoreactive scoring system (IRS).</p> <p>Results</p> <p>We detected a survivin expression (IRS > 2) in the cytoplasm of 20 leiomyosarcomas and 22 synovial sarcomas and in the nucleus of 12 leiomyosarcomas and 9 synovial sarcomas, respectively. There was no significant difference between leiomyosarcoma and synovial sarcoma samples in their cytoplasmic or nuclear expression of survivin. Next, all sarcoma patients were separated in four groups according to their survivin expression in the cytoplasm and in the nucleus: group 1: negative (IRS 0 to 2); group 2: weak (IRS 3 to 4); group 3: moderate (IRS 6 to 8); group 4: strong (IRS 9 to 12). In a multivariate Cox's regression hazard analysis survivin expression detected in the cytoplasm or in the nucleus was significantly associated with overall survival of patients in group 3 (RR = 5.7; P = 0.004 and RR = 5.7; P = 0.022, respectively) compared to group 2 (reference). Patients whose tumors showed both a moderate/strong expression of survivin in the cytoplasm and a moderate expression of survivin in the nucleus (in both compartments IRS ≥ 6) possessed a 24.8-fold increased risk of tumor-related death (P = 0.003) compared to patients with a weak expression of survivin both in the cytoplasm and in the nucleus.</p> <p>Conclusion</p> <p>Survivin protein expression in the cytoplasma and in the nucleus detected by immunohistochemistry is significantly associated with prognosis of leiomyosarcoma and synovial sarcoma patients.</p

Medical students’ preparedness for professional activities in early clerkships

Background Sufficient preparedness is important for transitions to workplace participation and learning in clinical settings. This study aims to analyse medical students’ preparedness for early clerkships using a three-dimensional, socio-cognitive, theory-based model of preparedness anchored in specific professional activities and their supervision level. Methods Medical students from a competency-based undergraduate curriculum were surveyed about preparedness for 21 professional activities and level of perceived supervision during their early clerkships via an online questionnaire. Preparedness was operationalized by the three dimensions of confidence to carry out clerkship activities, being prepared through university teaching and coping with failure by seeking support. Factors influencing preparedness and perceived stress as outcomes were analysed through step-wise regression. Results Professional activities carried out by the students (n = 147; 19.0%) and their supervision levels varied. While most students reported high confidence to perform the tasks, the activity-specific analysis revealed important gaps in preparation through university teaching. Students regularly searched for support in case of difficulty. One quarter of the variance of each preparedness dimension was explained by self-efficacy, supervision quality, amount of prior clerkship experience and nature of professional activities. Preparedness contributed to predicting perceived stress. Conclusions The applied three-dimensional concept of preparedness and the task-specific approach provided a detailed and meaningful view on medical students’ workplace participation and experiences in early clerkships

Postnatal exposure to ambient air pollutants is associated with the composition of the infant gut microbiota at 6-months of age

Epidemiological studies in adults have shown that exposure to ambient air pollution (AAP) is associated with the composition of the adult gut microbiome, but these relationships have not been examined in infancy. We aimed to determine if 6-month postnatal AAP exposure was associated with the infant gut microbiota at 6&nbsp;months of age in a cohort of Latino mother-infant dyads from the Southern California Mother&rsquo;s Milk Study (n&nbsp;=&nbsp;103). We estimated particulate matter (PM2.5 and PM10) and nitrogen dioxide (NO2) exposure from birth to 6-months based on residential address histories. We characterized the infant gut microbiota using 16S rRNA amplicon sequencing at 6-months of age. At 6-months, the gut microbiota was dominated by the phyla Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria. Our results show that, after adjusting for important confounders, postnatal AAP exposure was associated with the composition of the gut microbiota. As an example, PM10 exposure was positively associated with Dialister, Dorea, Acinetobacter, and Campylobacter while PM2.5 was positively associated with Actinomyces. Further, exposure to PM10 and PM2.5 was inversely associated with Alistipes and NO2 exposure was positively associated with Actinomyces, Enterococcus, Clostridium, and Eubacterium. Several of these taxa have previously been linked with systemic inflammation, including the genera Dialister and Dorea. This study provides the first evidence of significant associations between exposure to AAP and the composition of the infant gut microbiota, which may have important implications for future infant health and development. &nbsp;</p

Alloxan-Induced Diabetes Triggers the Development of Periodontal Disease in Rats

BACKGROUND: Periodontal disease in diabetic patients presents higher severity and prevalence; and increased severity of ligature-induced periodontal disease has been verified in diabetic rats. However, in absence of aggressive stimuli such as ligatures, the influence of diabetes on rat periodontal tissues is incompletely explored. The aim of this study was to evaluate the establishment and progression of periodontal diseases in rats only with diabetes induction. METHODOLOGY/PRINCIPAL FINDINGS: Diabetes was induced in Wistar rats (n = 25) by intravenous administration of alloxan (42 mg/kg) and were analyzed at 1, 3, 6, 9 and 12 months after diabetes induction. The hemimandibles were removed and submitted to radiographical and histopathological procedures. A significant reduction was observed in height of bone crest in diabetic animals at 3, 6, 9 and 12 months, which was associated with increased numbers of osteoclasts and inflammatory cells. The histopathological analyses of diabetic rats also showed a reduction in density of collagen fibers, fibroblasts and blood vessels. Severe caries were also detected in the diabetic group. CONCLUSIONS/SIGNIFICANCE: The results demonstrate that diabetes induction triggers, or even co-induces the onset of alterations which are typical of periodontal diseases even in the absence of aggressive factors such as ligatures. Therefore, diabetes induction renders a previously resistant host into a susceptible phenotype, and hence diabetes can be considered a very important risk factor to the development of periodontal disease

Static versus dynamic fracturing in shallow carbonate fault zones

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