Filtering for increased power for microarray data analysis

<p>Abstract</p> <p>Background</p> <p>Due to the large number of hypothesis tests performed during the process of routine analysis of microarray data, a multiple testing adjustment is certainly warranted. However, when the number of tests is very large and the proportion of differentially expressed genes is relatively low, the use of a multiple testing adjustment can result in very low power to detect those genes which are truly differentially expressed. Filtering allows for a reduction in the number of tests and a corresponding increase in power. Common filtering methods include filtering by variance, average signal or MAS detection call (for Affymetrix arrays). We study the effects of filtering in combination with the Benjamini-Hochberg method for false discovery rate control and q-value for false discovery rate estimation.</p> <p>Results</p> <p>Three case studies are used to compare three different filtering methods in combination with the two false discovery rate methods and three different preprocessing methods. For the case studies considered, filtering by detection call and variance (on the original scale) consistently led to an increase in the number of differentially expressed genes identified. On the other hand, filtering by variance on the log₂scale had a detrimental effect when paired with MAS5 or PLIER preprocessing methods, even when the testing was done on the log₂scale. A simulation study was done to further examine the effect of filtering by variance. We find that filtering by variance leads to higher power, often with a decrease in false discovery rate, when paired with either of the false discovery rate methods considered. This holds regardless of the proportion of genes which are differentially expressed or whether we assume dependence or independence among genes.</p> <p>Conclusion</p> <p>The case studies show that both detection call and variance filtering are viable methods of filtering which can increase the number of differentially expressed genes identified. The simulation study demonstrates that when paired with a false discovery rate method, filtering by variance can increase power while still controlling the false discovery rate. Filtering out 50% of probe sets seems reasonable as long as the majority of genes are not expected to be differentially expressed.</p

Predoctoral Interns\u27 Nondisclosure in Supervision

In interviews with 14 counseling center predoctoral interns regarding a significant nondisclosure in supervision, eight interns reported good supervisory relationships and six indicated that they experienced problematic supervisory relationships. Nondisclosures for the interns in good supervisory relationships related to personal reactions to clients, whereas nondisclosures for interns in problematic supervisory relationships related to global dissatisfaction with the supervisory relationship. In both groups, interns mentioned concerns about evaluation and negative feelings as typical reasons for nondisclosure. Additional reasons for nondisclosure for interns in problematic supervision were power dynamics, inhibiting demographic or cultural variables, and the supervisor\u27s theoretical orientation. Both groups described negative effects of nondisclosure on themselves and their relationships with clients. Interns in problematic supervision also reported that nondisclosures had negative effects on the supervisory relationship

Non-Coding RNA Sequencing of Equine Endometrium During Maternal Recognition of Pregnancy.

Maternal recognition of pregnancy (MRP) in the mare is not well defined. In a non-pregnant mare, prostaglandin F2α (PGF) is released on day 14 post-ovulation (PO) to cause luteal regression, resulting in loss of progesterone production. Equine MRP occurs prior to day 14 to halt PGF production. Studies have failed to identify a gene candidate for MRP, so attention has turned to small, non-coding RNAs. The objective of this study was to evaluate small RNA (&lt;200 nucleotides) content in endometrium during MRP. Mares were used in a cross-over design with each having a pregnant and non-mated cycle. Each mare was randomly assigned to collection day 11 or 13 PO (n = 3/day) and endometrial biopsies were obtained. Total RNA was isolated and sequencing libraries were prepared using a small RNA library preparation kit and sequenced on a HiSeq 2000. EquCab3 was used as the reference genome and DESeq2 was used for statistical analysis. On day 11, 419 ncRNAs, representing miRNA, snRNA, snoRNA, scaRNA, and vaultRNA, were different between pregnancy statuses, but none on day 13. Equine endometrial ncRNAs with unknown structure and function were also identified. This study is the first to describe ncRNA transcriptome in equine endometrium. Identifying targets of these ncRNAs could lead to determining MRP

Chemical Study of the Interstitial Water Dissolved Organic Matter and Gases in Lake Erie, Cleveland Harbor, and Hamilton Harbour Bottom Sediments - Composition and Fluxes to Overlying Waters

The research on which this report is based was financed in part by the U.S. Department of the Interior, as authorized by the Water Research and Development Act of 1978 (P.L. 95-467).(print) iv, 167, [45] p. : ill., maps ; 29 cm.FINAL REPORT FOR OWRT GRANT A-O59-OHIOItem lacks publication date. Issue date supplied from hand-written year on coverIntroduction -- The Study Area -- Methods and Materials -- Results -- Discussion -- Conclusions -- Selected Bibliographic References -- Tables 1-32 -- Figures 1-36 -- Appendi

Small RNA profiling of Dengue virus-mosquito interactions implicates the PIWI RNA pathway in anti-viral defense

<p>Abstract</p> <p>Background</p> <p>Small RNA (sRNA) regulatory pathways (SRRPs) are important to anti-viral defence in mosquitoes. To identify critical features of the virus infection process in Dengue serotype 2 (DENV2)-infected <it>Ae. aegypti</it>, we deep-sequenced small non-coding RNAs. Triplicate biological replicates were used so that rigorous statistical metrics could be applied.</p> <p>Results</p> <p>In addition to virus-derived siRNAs (20-23 nts) previously reported for other arbovirus-infected mosquitoes, we show that PIWI pathway sRNAs (piRNAs) (24-30 nts) and unusually small RNAs (usRNAs) (13-19 nts) are produced in DENV-infected mosquitoes. We demonstrate that a major catalytic enzyme of the siRNA pathway, Argonaute 2 (Ago2), co-migrates with a ~1 megadalton complex in adults prior to bloodfeeding. sRNAs were cloned and sequenced from Ago2 immunoprecipitations. Viral sRNA patterns change over the course of infection. Host sRNAs were mapped to the published aedine transcriptome and subjected to analysis using edgeR (Bioconductor). We found that sRNA profiles are altered early in DENV2 infection, and mRNA targets from mitochondrial, transcription/translation, and transport functional categories are affected. Moreover, small non-coding RNAs (ncRNAs), such as tRNAs, spliceosomal U RNAs, and snoRNAs are highly enriched in DENV-infected samples at 2 and 4 dpi.</p> <p>Conclusions</p> <p>These data implicate the PIWI pathway in anti-viral defense. Changes to host sRNA profiles indicate that specific cellular processes are affected during DENV infection, such as mitochondrial function and ncRNA levels. Together, these data provide important progress in understanding the DENV2 infection process in <it>Ae. aegypti</it>.</p

Silent Phase of Johne’s Disease in Experimentally Infected Goats – A Study on New and Established Diagnostic Approaches Using Specific and Non-Specific Parameters

The current gold standard diagnostic test for Johne’s disease (JD) is detecting Mycobacterium avium subsp. paratuberculosis (MAP) from fecal samples via culture and/or PCR. Other commercially available JD diagnostic tests focus on the detection of specific antibodies within the serum or milk of infected ruminants. These tests have a high specificity but low their sensitivity and usually fail to diagnose the disease until later stages of the disease. The ideal diagnostic test should detect infected animals already during the silent phase. Here, we evaluate the use of new and established approaches to define the silent phase of JD in experimentally infected goats. None of the established diagnostic tests or new approaches for the detection of humoral and cellular immune responses were positive during the first year of infection. Only the characterization of various subsets of peripheral blood leukocytes and the weight development gave some indication for the presence of a chronic, but silent, infection. Weight differences were present throughout the first year. In addition, some of the subsets of leukocytes (WC1+ T cells, MHC class II+ leukocytes, CD1+ leukocytes, CD14+ granulocytes, and CD14+/MHC class II+ granulocytes) demonstrated significant differences, but only at certain time points

Deciphering ocean carbon in a changing world

Author Posting. © The Author(s), 2016. This is the author's version of the work. It is posted here for personal use, not for redistribution. The definitive version was published in Proceedings of the National Academy of Sciences of the United States of America 113 (2016): 3143-3151, doi:10.1073/pnas.1514645113.Dissolved organic matter (DOM) in the oceans is one of the largest pools of reduced carbon on Earth, comparable in size to the atmospheric CO2 reservoir. A vast number of compounds are present in DOM and they play important roles in all major element cycles, contribute to the storage of atmospheric CO2 in the ocean, support marine ecosystems, and facilitate interactions between organisms. At the heart of the DOM cycle lie molecular-level relationships between the individual compounds in DOM and the members of the ocean microbiome that produce and consume them. In the past, these connections have eluded clear definition because of the sheer numerical complexity of both DOM molecules and microorganisms. Emerging tools in analytical chemistry, microbiology and informatics are breaking down the barriers to a fuller appreciation of these connections. Here we highlight questions being addressed using recent methodological and technological developments in those fields and consider how these advances are transforming our understanding of some of the most important reactions of the marine carbon cycle.Support was provided by National Science Foundation grants OCE1356010, OCE1154320, and OCE1356890, and Gordon and Betty Moore Foundation Grant #3304

Cytometry profiling of ex vivo recall responses to Coxiella burnetii in previously naturally exposed individuals reveals long-term changes in both adaptive and innate immune cellular compartments

IntroductionQ fever, caused by the intracellular bacterium Coxiella burnetii, is considered an occupational and biodefense hazard and can result in debilitating long-term complications. While natural infection and vaccination induce humoral and cellular immune responses, the exact nature of cellular immune responses to C. burnetii is incompletely understood. The current study seeks to investigate more deeply the nature of long-term cellular recall responses in naturally exposed individuals by both cytokine release assessment and cytometry profiling.MethodsIndividuals exposed during the 2007-2010 Dutch Q fever outbreak were grouped in 2015, based on a C. burnetii-specific IFNγ release assay (IGRA), serological status, and self-reported clinical symptoms during initial infection, into asymptomatic IGRA-negative/seronegative controls, and three IGRA-positive groups (seronegative/asymptomatic; seropositive/asymptomatic and seropositive/symptomatic). Recall responses following in vitro re-stimulation with heat-inactivated C. burnetii in whole blood, were assessed in 2016/2017 by cytokine release assays (n=55) and flow cytometry (n=36), and in blood mononuclear cells by mass cytometry (n=36).ResultsCytokine release analysis showed significantly elevated IL-2 responses in all seropositive individuals and elevated IL-1β responses in those recovered from symptomatic infection. Comparative flow cytometry analysis revealed significantly increased IFNγ, TNFα and IL-2 recall responses by CD4 T cells and higher IL-6 production by monocytes from symptomatic, IGRA-positive/seropositive individuals compared to controls. Mass cytometry profiling and unsupervised clustering analysis confirmed recall responses in seropositive individuals by two activated CD4 T cell subsets, one characterized by a strong Th1 cytokine profile (IFNγ+IL-2+TNFα+), and identified C. burnetii-specific activation of CD8 T cells in all IGRA-positive groups. Remarkably, increased C. burnetii-specific responses in IGRA-positive individuals were also observed in three innate cell subpopulations: one characterized by an IFNγ+IL-2+TNFα+ Th1 cytokine profile and lack of canonical marker expression, and two IL-1β-, IL-6- and IL-8-producing CD14+ monocyte subsets that could be the drivers of elevated secretion of innate cytokines in pre-exposed individuals.DiscussionThese data highlight that there are long-term increased responses to C. burnetii in both adaptive and innate cellular compartments, the latter being indicative of trained immunity. These findings warrant future studies into the protective role of these innate responses and may inform future Q fever vaccine design