The Reproducibility of Lists of Differentially Expressed Genes in Microarray Studies

Reproducibility is a fundamental requirement in scientific experiments and clinical contexts. Recent publications raise concerns about the reliability of microarray technology because of the apparent lack of agreement between lists of differentially expressed genes (DEGs). In this study we demonstrate that (1) such discordance may stem from ranking and selecting DEGs solely by statistical significance (P) derived from widely used simple t-tests; (2) when fold change (FC) is used as the ranking criterion, the lists become much more reproducible, especially when fewer genes are selected; and (3) the instability of short DEG lists based on P cutoffs is an expected mathematical consequence of the high variability of the t-values. We recommend the use of FC ranking plus a non-stringent P cutoff as a baseline practice in order to generate more reproducible DEG lists. The FC criterion enhances reproducibility while the P criterion balances sensitivity and specificity

Enhanced sphingosine-1-phosphate receptor 2 expression underlies female CNS autoimmunity susceptibility

Multiple sclerosis (MS) is an inflammatory disease of the CNS that is characterized by BBB dysfunction and has a much higher incidence in females. Compared with other strains of mice, EAE in the SJL mouse strain models multiple features of MS, including an enhanced sensitivity of female mice to disease; however, the molecular mechanisms that underlie the sex- and strain-dependent differences in disease susceptibility have not been described. We identified sphingosine-1-phosphate receptor 2 (S1PR2) as a sex- and strain-specific, disease-modifying molecule that regulates BBB permeability by destabilizing adherens junctions. S1PR2 expression was increased in disease-susceptible regions of the CNS of both female SJL EAE mice and female patients with MS compared with their male counterparts. Pharmacological blockade or lack of S1PR2 signaling decreased EAE disease severity as the result of enhanced endothelial barrier function. Enhanced S1PR2 signaling in an in vitro BBB model altered adherens junction formation via activation of Rho/ROCK, CDC42, and caveolin endocytosis-dependent pathways, resulting in loss of apicobasal polarity and relocation of abluminal CXCL12 to vessel lumina. Furthermore, S1PR2-dependent BBB disruption and CXCL12 relocation were observed in vivo. These results identify a link between S1PR2 signaling and BBB polarity and implicate S1PR2 in sex-specific patterns of disease during CNS autoimmunity

Useful pharmacodynamic endpoints in children: selection, measurement, and next steps.

Pharmacodynamic (PD) endpoints are essential for establishing the benefit-to-risk ratio for therapeutic interventions in children and neonates. This article discusses the selection of an appropriate measure of response, the PD endpoint, which is a critical methodological step in designing pediatric efficacy and safety studies. We provide an overview of existing guidance on the choice of PD endpoints in pediatric clinical research. We identified several considerations relevant to the selection and measurement of PD endpoints in pediatric clinical trials, including the use of biomarkers, modeling, compliance, scoring systems, and validated measurement tools. To be useful, PD endpoints in children need to be clinically relevant, responsive to both treatment and/or disease progression, reproducible, and reliable. In most pediatric disease areas, this requires significant validation efforts. We propose a minimal set of criteria for useful PD endpoint selection and measurement. We conclude that, given the current heterogeneity of pediatric PD endpoint definitions and measurements, both across and within defined disease areas, there is an acute need for internationally agreed, validated, and condition-specific pediatric PD endpoints that consider the needs of all stakeholders, including healthcare providers, policy makers, patients, and families.Pediatric Research advance online publication, 11 April 2018; doi:10.1038/pr.2018.38

Kinetics and Mechanism of Bulk Polymerization of Vinyl Chloride in a Polymerization Reactor

Polyvinyl chloride (PVC) is the third most commonly produced polymer and is important because of its mechanical characteristics. The most common method of PVC manufacturing is the process of suspension. Although, there are several benefits associated with suspension, this study will focus on the bulk polymerization of vinyl chloride; highlight the physical and chemical properties of PVC, which can be changed through an estimation of the optimum ratio that exists between the hydrophilic and hydrophobic parts of the polymer’s surface, and propose a new mathematical model which will be helpful for the conversion of PVC into a useful form. The result will be the proposal of a new dynamic mathematical model for the three-phase structure model. All particles have been taken into account in the proposed model, which helped contribute to the reaction in gel, solid, and liquid phases, emphasizing the use of mercury (Hg) as a catalyst. The proposed mathematical model considers the heat and mass transfer between the liquid, gel, and solid phases with chemical reactions that occur between the liquid and solid phases, and between the gel and solid phases. The effect of the catalyst and volumetric flow rates of vinyl chloride monomer (VCM) on the system have been evaluated through the proposed mathematical model. Furthermore, the study’s experimental data have been compared with the findings of the suggested model in the context of concentration and temperature reaction. Obtained results show good agreement between the proposed mathematical model and the actual plant data

A pilot study to evaluate the application of a generic protein standard panel for quality control of biomarker detection technologies

<p>Abstract</p> <p>Background</p> <p>Protein biomarker studies are currently hampered by a lack of measurement standards to demonstrate quality, reliability and comparability across multiple assay platforms. This is especially pertinent for immunoassays where multiple formats for detecting target analytes are commonly used.</p> <p>Findings</p> <p>In this pilot study a generic panel of six non-human protein standards (50 - 10^7 pg/mL) of varying abundance was prepared as a quality control (QC) material. Simulated "normal" and "diseased" panels of proteins were prepared in pooled human plasma and incorporated into immunoassays using the Meso Scale Discovery^®(MSD^®) platform to illustrate reliable detection of the component proteins. The protein panel was also evaluated as a spike-in material for a model immunoassay involving detection of ovarian cancer biomarkers within individual human plasma samples. Our selected platform could discriminate between two panels of the proteins exhibiting small differences in abundance. Across distinct experiments, all component proteins exhibited reproducible signal outputs in pooled human plasma. When individual donor samples were used, half the proteins produced signals independent of matrix effects. These proteins may serve as a generic indicator of platform reliability.</p> <p>Each of the remaining proteins exhibit differential signals across the distinct samples, indicative of sample matrix effects, with the three proteins following the same trend. This subset of proteins may be useful for characterising the degree of matrix effects associated with the sample which may impact on the reliability of quantifying target diagnostic biomarkers.</p> <p>Conclusions</p> <p>We have demonstrated the potential utility of this panel of standards to act as a generic QC tool for evaluating the reproducibility of the platform for protein biomarker detection independent of serum matrix effects.</p

Generation and characterization of antibodies specific for caspase-cleaved neo-epitopes: a novel approach

Apoptosis research has been significantly aided by the generation of antibodies against caspase-cleaved peptide neo-epitopes. However, most of these antibodies recognize the N-terminal fragment and are specific for the protein in question. The aim of this project was to create antibodies, which could identify caspase-cleaved proteins without a priori knowledge of the cleavage sites or even the proteins themselves. We hypothesized that many caspase-cleavage products might have a common antigenic shape, given that they must all fit into the same active site of caspases. Rabbits were immunized with the eight most prevalent exposed C-terminal tetrapeptide sequences following caspase cleavage. After purification of the antibodies we demonstrated (1) their specificity for exposed C-terminal (but not internal) peptides, (2) their ability to detect known caspase-cleaved proteins from apoptotic cell lysates or supernatants from apoptotic cell culture and (3) their ability to detect a caspase-cleaved protein whose tetrapeptide sequence differs from the eight tetrapeptides used to generate the antibodies. These antibodies have the potential to identify novel neo-epitopes produced by caspase cleavage and so can be used to identify pathway-specific caspase cleavage events in a specific cell type. Additionally this methodology may be applied to generate antibodies against products of other proteases, which have a well-defined and non-promiscuous cleavage activity

The balance of reproducibility, sensitivity, and specificity of lists of differentially expressed genes in microarray studies

<p>Abstract</p> <p>Background</p> <p>Reproducibility is a fundamental requirement in scientific experiments. Some recent publications have claimed that microarrays are unreliable because lists of differentially expressed genes (DEGs) are not reproducible in similar experiments. Meanwhile, new statistical methods for identifying DEGs continue to appear in the scientific literature. The resultant variety of existing and emerging methods exacerbates confusion and continuing debate in the microarray community on the appropriate choice of methods for identifying reliable DEG lists.</p> <p>Results</p> <p>Using the data sets generated by the MicroArray Quality Control (MAQC) project, we investigated the impact on the reproducibility of DEG lists of a few widely used gene selection procedures. We present comprehensive results from inter-site comparisons using the same microarray platform, cross-platform comparisons using multiple microarray platforms, and comparisons between microarray results and those from TaqMan – the widely regarded "standard" gene expression platform. Our results demonstrate that (1) previously reported discordance between DEG lists could simply result from ranking and selecting DEGs solely by statistical significance (<it>P</it>) derived from widely used simple <it>t</it>-tests; (2) when fold change (FC) is used as the ranking criterion with a non-stringent <it>P</it>-value cutoff filtering, the DEG lists become much more reproducible, especially when fewer genes are selected as differentially expressed, as is the case in most microarray studies; and (3) the instability of short DEG lists solely based on <it>P</it>-value ranking is an expected mathematical consequence of the high variability of the <it>t</it>-values; the more stringent the <it>P</it>-value threshold, the less reproducible the DEG list is. These observations are also consistent with results from extensive simulation calculations.</p> <p>Conclusion</p> <p>We recommend the use of FC-ranking plus a non-stringent <it>P </it>cutoff as a straightforward and baseline practice in order to generate more reproducible DEG lists. Specifically, the <it>P</it>-value cutoff should not be stringent (too small) and FC should be as large as possible. Our results provide practical guidance to choose the appropriate FC and <it>P</it>-value cutoffs when selecting a given number of DEGs. The FC criterion enhances reproducibility, whereas the <it>P </it>criterion balances sensitivity and specificity.</p

First report of pediatric hematopoietic stem cell transplantation activities in the eastern mediterranean region from 1984 to 2011: On behalf of the pediatric cancer working committee of the eastern mediterranean blood and marrow transplantation group

To describe the hematopoietic stem cell transplantation (HSCT) activities for children in the Eastern Mediterranean (EM) region, data on transplants performed for children less than 18 years of age between 1984 and 2011 in eight EM countries (Egypt, Iran, Jordan, Lebanon, Oman, Pakistan, Saudi Arabia and Tunisia) were collected. A total of 5187 transplants were performed, of which 4513 (87%) were allogeneic and 674 (13%) were autologous. Overall, the indications for transplantation were malignant diseases in 1736 (38.5%) and non-malignant in 2777 (61.5%) patients. A myeloablative conditioning regimen was used in 88% of the allografts. Bone marrow (BM) was the most frequent source of stem cells (56.2%), although an increasing use of PBSC was observed in the last decade. The stem cell source of autologous HSCT has shifted over time from BM to PBSC, and 80.9% of autologous HSCTs were from PBSCs. The donors for allogeneic transplants were matched-related in 94.5% of the cases, and unrelated transplants, mainly cord blood (99%) in 239 (5.5%) cases. This is the first report to describe the pediatric HSCT activities in EM countries. Non-malignant disorders are the main indication for allogeneic transplantation. Frequency of alternate donor transplantation is low. © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved

Binding of an RNA trafficking response element to heterogeneous nuclear ribonucleoproteins A1 and A2

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 binds a 21-nucleotide myelin basic protein mRNA response element, the A2RE, and A2RE-like sequences in other localized mRNAs, and is a trans-acting factor in oligodendrocyte cytoplasmic RNA trafficking. Recombinant human hnRNPs A1 and A2 were used in a biosensor to explore interactions with A2RE and the cognate oligodeoxyribonucleotide. Both proteins have a single site that bound oligonucleotides with markedly different sequences but did not bind in the presence of heparin. Both also possess a second, specific site that bound only A2RE and was unaffected by heparin, hnRNP A2 bound A2RE in the latter site with a K-d near 50 nM, whereas the K-d for hnRNP A1 was above 10 muM. UV cross-linking assays led to a similar conclusion. Mutant A2RE sequences, that in earlier qualitative studies appeared not to bind hnRNP A2 or support RNA trafficking in oligodendrocytes, had dissociation constants above 5 muM for this protein. The two concatenated RNA recognition motifs (RRMs), but not the individual RRMs, mimicked the binding behavior of hnRNP A2. These data highlight the specificity of the interaction of A2RE with these hnRNPs and suggest that the sequence-specific A2RE-binding site on hnRNP A2 is formed by both RRMs acting in cis