A Direct Phenotypic Comparison of siRNA Pools and Multiple Individual Duplexes in a Functional Assay

Background: RNAi is a prominent tool for the identification of novel regulatory elements within complex cellular pathways. In invertebrates, RNAi is a relatively straightforward process, where large double-stranded RNA molecules initiate sequencespecific transcript destruction in target cells. In contrast, RNAi in mammalian cell culture assays requires the delivery of short interfering RNA duplexes to target cells. Due to concerns over off-target phenotypes and extreme variability in duplex efficiency, investigators typically deliver and analyze multiple duplexes per target. Currently, duplexes are delivered and analyzed either individually or as a pool of several independent duplexes. A choice between experiments based on siRNA pools or multiple individual duplexes has considerable implications for throughput, reagent requirements and data analysis in genome-wide surveys, yet there are relatively few data that directly compare the efficiency of the two approaches. Methodology/Principal Findings: To address this critical issue, we conducted a direct comparison of siRNA pools and multiple single siRNAs that target all human phosphatases in a robust functional assay. We determined the frequency with which both approaches uncover loss-of-function phenotypes and compared the phenotypic severity for siRNA pools and the constituent individual duplexes. Conclusions/Significance: Our survey indicates that screens with siRNA pools have several significant advantages over identical screens with the corresponding individual siRNA duplexes. Of note, we frequently observed greater phenotypi

The diverse roles of C-type lectin-like receptors in immunity

Our understanding of the C-type lectin-like receptors (CTLRs) and their functions in immunity have continued to expand from their initial roles in pathogen recognition. There are now clear examples of CTLRs acting as scavenger receptors, sensors of cell death and cell transformation, and regulators of immune responses and homeostasis. This range of function reflects an extensive diversity in the expression and signaling activity between individual CTLR members of otherwise highly conserved families. Adding to this diversity is the constant discovery of new receptor binding capabilities and receptor-ligand interactions, distinct cellular expression profiles, and receptor structures and signaling mechanisms which have expanded the defining roles of CTLRs in immunity. The natural killer cell receptors exemplify this functional diversity with growing evidence of their activity in other immune populations and tissues. Here, we broadly review select families of CTLRs encoded in the natural killer cell gene complex (NKC) highlighting key receptors that demonstrate the complex multifunctional capabilities of these proteins. We focus on recent evidence from research on the NKRP1 family of CTLRs and their interaction with the related C-type lectin (CLEC) ligands which together exhibit essential immune functions beyond their defined activity in natural killer (NK) cells. The ever-expanding evidence for the requirement of CTLR in numerous biological processes emphasizes the need to better understand the functional potential of these receptor families in immune defense and pathological conditions

An immune dysfunction score for stratification of patients with acute infection based on whole-blood gene expression

Dysregulated host responses to infection can lead to organ dysfunction and sepsis, causing millions of global deaths each year. To alleviate this burden, improved prognostication and biomarkers of response are urgently needed. We investigated the use of whole-blood transcriptomics for stratification of patients with severe infection by integrating data from 3149 samples from patients with sepsis due to community-acquired pneumonia or fecal peritonitis admitted to intensive care and healthy individuals into a gene expression reference map. We used this map to derive a quantitative sepsis response signature (SRSq) score reflective of immune dysfunction and predictive of clinical outcomes, which can be estimated using a 7- or 12-gene signature. Last, we built a machine learning framework, SepstratifieR, to deploy SRSq in adult and pediatric bacterial and viral sepsis, H1N1 influenza, and COVID-19, demonstrating clinically relevant stratification across diseases and revealing some of the physiological alterations linking immune dysregulation to mortality. Our method enables early identification of individuals with dysfunctional immune profiles, bringing us closer to precision medicine in infection.peer-reviewe

Development and Application of Improvements to the Tile-based Fisher Ratio Method and Fundamental Instrument Considerations for Non-targeted Analysis using Two-dimensional Gas Chromatography

Thesis (Ph.D.)--University of Washington, 2016-06Comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry GC×GC–TOFMS has arguably made possible the largest increase in gas chromatograph performance since the innovation of the wall-coated open tubular (WCOT) capillary analytical column in 1979. As with the adoption of capillary columns, taking advantage of the performance possible with GC×GC–TOFMS has required the carefully study of fundamental chromatographic parameters and the development of new data analysis strategies and software to leverage the data rich output of the platform for meaningful discovery in complex chemical matrices. This dissertation presents a thorough account the development and validation of the tile-based Fisher ratio software, which aims to provide a robust and efficient method for non-targeted analysis in experiments comprised of classes of GC×GC–TOFMS chromatograms. Additionally, the software is further developed and demonstrated in a unique application to a challenging problem of forensic interest: the chemical characterization of the illicit acid alteration of diesel fuel. Finally, the chromatographic methods used in the development, validation, and demonstration of the software are carefully examined in the context of instrument performance, and compared to alternative instrumental configurations that have the potential to further increase performance

Properties of PTFE tape as a semipermeable membrane in fluorous reactions

In a PTFE tape phase-vanishing reaction (PV-PTFE), a delivery tube sealed with PTFE tape is inserted into a vessel which contains the substrate. The reagent diffuses across the PTFE tape barrier into the reaction vessel. PTFE co-polymer films have been found to exhibit selective permeability towards organic compounds, which was affected by the presence of solvents. In this study, we attempted to establish general trends of permeability of PTFE tape to different compounds and to better describe the process of solvent transport in PV-PTFE bromination reactions. Though PTFE tape has been reported as impermeable to some compounds, such as dimethyl phthalate, solvent adsorption to the tape altered its permeability and allowed diffusion through channels of solvent within the PTFE tape. In this case, the solvent-filled pores of the PTFE tape are chemically more akin to the adsorbed solvent rather than to the PTFE fluorous structure. The solvent uptake effect, which was frequently observed in the course of PV-PTFE reactions, can be related to the surface tension of the solvent and the polarity of the solvent relative to the reagent. The lack of pores in bulk PTFE prevents solvents from altering its permeability and, therefore, bulk PTFE is impermeable to most solvents and reagents. However, bromine, which is soluble in liquid fluorous media, diffused through the bulk PTFE. A better understanding of the PTFE phase barrier will make it possible to further optimize the PV-PTFE reaction design

Detection, Characterization and Typing of Shiga toxin-producing Escherichia coli.

Shiga toxin-producing Escherichia coli (STEC) are responsible for gastrointestinal diseases reported in numerous outbreaks around the world. Given the public health importance of STEC, effective detection, characterization and typing is critical to any medical laboratory system. While non-O157 serotypes account for the majority of STEC infections, frontline microbiology laboratories may only screen for STEC using O157-specific agar-based methods. As a result, non-O157 STEC infections are significantly under-reported. This review discusses recent advances on the detection, characterization and typing of STEC with emphasis on work performed at the Alberta Provincial Laboratory for Public Health (ProvLab). Candidates for the detection of all STEC serotypes include chromogenic agars, enzyme immunoassays (EIA) and real-time polymerase chain reaction (qPCR). Culture methods allow further characterization of isolates, whereas qPCR provides the greatest sensitivity and specificity, followed by EIA. The virulence gene profiles using PCR arrays and stx gene subtypes can subsequently be determined. Different non-O157 serotypes exhibit markedly different virulence gene profiles and a greater prevalence of stx1 than stx2 subtypes compared to O157:H7 isolates. Finally, recent innovations in whole genome sequencing (WGS) have allowed it to emerge as a candidate for the characterization and typing of STEC in diagnostic surveillance isolates. Methods of whole genome analysis such as single nucleotide polymorphisms and k-mer analysis are concordant with epidemiological data and standard typing methods, such as pulsed-field gel electrophoresis and multiple-locus variable number tandem repeat analysis while offering additional strain differentiation. Together these findings highlight improved strategies for STEC detection using currently available systems and the development of novel approaches for future surveillance

Association of Ct Values from Real-Time PCR with Culture in Microbiological Clearance Samples for Shiga Toxin-Producing Escherichia coli (STEC)

Shiga toxin-producing Escherichia coli (STEC) are associated with acute gastroenteritis worldwide, which induces a high economic burden on both healthcare and individuals. Culture-independent diagnostic tests (CIDT) in frontline microbiology laboratories have been implemented in Alberta since 2019. The objectives of this study were to determine the association between gene detection and culture positivity over time using STEC microbiological clearance samples and also to establish the frequency of specimen submission. Both stx genes’ amplification by real-time PCR was performed with DNA extracted from stool samples using the easyMAG system. Stools were inoculated onto chromogenic agar for culture. An association between gene detection and culture positivity was found to be independent of which stx gene was present. CIDT can provide rapid reporting with less hands-on time and technical expertise. However, culture is still important for surveillance and early cluster detection. In addition, stool submissions could be reduced from daily to every 3–5 days until a sample is negative by culture

Need for an updated overview to assess the benefits of epidurals: Response

Philip J. Peyton, John A. Rigg, Konrad Jamrozik, Paul S. Myles, Brendan S. Silbert, and Richard Parson