Methods Of Gene Expression Monitoring

The invention provides methods of monitoring expression of a plurality of genes in a cell or small population of cells. Preferred methods entail contacting an array of probes with a population of nucleic acids derived from a population of fewer than 1000 cells then determining the relative hybridization of the probes to the population of nucleic acid as a measure of the relative representation of genes from the cells. The invention further provides methods of classifying cells. These preferred methods entail determining an expression profile of each of a plurality of cells then classifying the cells in clusters determined by similarity of expression profile. The invention further provides methods of monitoring differentiation of a cell lineage. These preferred methods entail determining an expression profile of each of a plurality of cells at different differentiation stages within the lineage. These cells can then be classified into clusters determined by similarity of expression profile. The clusters can then be ordered by similarity of expression profile. A time course of expression levels for each of the plurality of genes at different stages of differentiation in the cell lineage can then be determined

Cross-species gene expression analysis of species specific differences in the preclinical assessment of pharmaceutical compounds

Animals are frequently used as model systems for determination of safety and efficacy in pharmaceutical research and development. However, significant quantitative and qualitative differences exist between humans and the animal models used in research. This is as a result of genetic variation between human and the laboratory animal. Therefore the development of a system that would allow the assessment of all molecular differences between species after drug exposure would have a significant impact on drug evaluation for toxicity and efficacy. Here we describe a cross-species microarray methodology that identifies and selects orthologous probes after cross-species sequence comparison to develop an orthologous cross-species gene expression analysis tool. The assumptions made by the use of this orthologous gene expression strategy for cross-species extrapolation is that; conserved changes in gene expression equate to conserved pharmacodynamic endpoints. This assumption is supported by the fact that evolution and selection have maintained the structure and function of many biochemical pathways over time, resulting in the conservation of many important processes. We demonstrate this cross-species methodology by investigating species specific differences of the peroxisome proliferatoractivator receptor (PPAR) a response in rat and human

Internal and external cooling methods and their effect on body temperature, thermal perception and dexterity

© 2018 The Authors. Published by PLOS. This is an open access article available under a Creative Commons licence. The published version can be accessed at the following link on the publisher’s website: https://doi.org/10.1371/journal.pone.0191416© 2018 Maley et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Objective The present study aimed to compare a range of cooling methods possibly utilised by occupational workers, focusing on their effect on body temperature, perception and manual dexterity. Methods Ten male participants completed eight trials involving 30 min of seated rest followed by 30 min of cooling or control of no cooling (CON) (34C, 58% relative humidity). The cooling methods utilised were: ice cooling vest (CV0), phase change cooling vest melting at 14C (CV14), evaporative cooling vest (CVEV), arm immersion in 10C water (AI), portable water-perfused suit (WPS), heliox inhalation (HE) and ice slushy ingestion (SL). Immediately before and after cooling, participants were assessed for fine (Purdue pegboard task) and gross (grip and pinch strength) manual dexterity. Rectal and skin temperature, as well as thermal sensation and comfort, were monitored throughout. Results Compared with CON, SL was the only method to reduce rectal temperature (P = 0.012). All externally applied cooling methods reduced skin temperature (P0.05). Conclusion The present study observed that ice ingestion or ice applied to the skin produced the greatest effect on rectal and skin temperature, respectively. AI should not be utilised if workers require subsequent fine manual dexterity. These results will help inform future studies investigating appropriate pre-cooling methods for the occupational worker.This project is financially supported by the US Government through the Technical Support Working Group within the Combating Terrorism Technical Support Office.Published versio

The impact of Cochrane Systematic Reviews : a mixed method evaluation of outputs from Cochrane Review Groups supported by the UK National Institute for Health Research

© 2014 Bunn et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Background: There has been a growing emphasis on evidence-informed decision making in health care. Systematic reviews, such as those produced by the Cochrane Collaboration, have been a key component of this movement. The UK National Institute for Health Research (NIHR) Systematic Review Programme currently supports 20 Cochrane Review Groups (CRGs). The aim of this study was to identify the impacts of Cochrane reviews published by NIHR funded CRGs during the years 2007-11. Methods: We sent questionnaires to CRGs and review authors, interviewed guideline developers and used bibliometrics and documentary review to get an overview of CRG impact and to evaluate the impact of a sample of 60 Cochrane reviews. We used a framework with four categories (knowledge production, research targeting, informing policy development, and impact on practice/services). Results: A total of 1502 new and updated reviews were produced by the 20 NIHR funded CRGs between 2007-11. The clearest impacts were on policy with a total of 483 systematic reviews cited in 247 sets of guidance; 62 were international, 175 national (87 from the UK) and 10 local. Review authors and CRGs provided some examples of impact on practice or services, for example safer use of medication, the identification of new effective drugs or treatments and potential economic benefits through the reduction in the use of unproven or unnecessary procedures. However, such impacts are difficult to objectively document and the majority of reviewers were unsure if their review had produced specific impacts. Qualitative data suggested that Cochrane reviews often play an instrumental role in informing guidance although a poor fit with guideline scope or methods, reviews being out of date and a lack of communication between CRGs and guideline developers were barriers to their use. Conclusions: Health and economic impacts of research are generally difficult to measure. We found that to be the case with this evaluation. Impacts on knowledge production and clinical guidance were easier to identify and substantiate than those on clinical practice. Questions remain about how we define and measure impact and more work is needed to develop suitable methods for impact analysis.Peer reviewe

Phenotypic Variation and Bistable Switching in Bacteria

Microbial research generally focuses on clonal populations. However, bacterial cells with identical genotypes frequently display different phenotypes under identical conditions. This microbial cell individuality is receiving increasing attention in the literature because of its impact on cellular differentiation, survival under selective conditions, and the interaction of pathogens with their hosts. It is becoming clear that stochasticity in gene expression in conjunction with the architecture of the gene network that underlies the cellular processes can generate phenotypic variation. An important regulatory mechanism is the so-called positive feedback, in which a system reinforces its own response, for instance by stimulating the production of an activator. Bistability is an interesting and relevant phenomenon, in which two distinct subpopulations of cells showing discrete levels of gene expression coexist in a single culture. In this chapter, we address techniques and approaches used to establish phenotypic variation, and relate three well-characterized examples of bistability to the molecular mechanisms that govern these processes, with a focus on positive feedback.

B Cells Regulate Neutrophilia during Mycobacterium tuberculosis Infection and BCG Vaccination by Modulating the Interleukin-17 Response

We have previously demonstrated that B cells can shape the immune response to Mycobacterium tuberculosis, including the level of neutrophil infiltration and granulomatous inflammation at the site of infection. The present study examined the mechanisms by which B cells regulate the host neutrophilic response upon exposure to mycobacteria and how neutrophilia may influence vaccine efficacy. To address these questions, a murine aerosol infection tuberculosis (TB) model and an intradermal (ID) ear BCG immunization mouse model, involving both the μMT strain and B cell-depleted C57BL/6 mice, were used. IL (interleukin)-17 neutralization and neutrophil depletion experiments using these systems provide evidence that B cells can regulate neutrophilia by modulating the IL-17 response during M. tuberculosis infection and BCG immunization. Exuberant neutrophilia at the site of immunization in B cell-deficient mice adversely affects dendritic cell (DC) migration to the draining lymph nodes and attenuates the development of the vaccine-induced Th1 response. The results suggest that B cells are required for the development of optimal protective anti-TB immunity upon BCG vaccination by regulating the IL-17/neutrophilic response. Administration of sera derived from M. tuberculosis-infected C57BL/6 wild-type mice reverses the lung neutrophilia phenotype in tuberculous μMT mice. Together, these observations provide insight into the mechanisms by which B cells and humoral immunity modulate vaccine-induced Th1 response and regulate neutrophila during M. tuberculosis infection and BCG immunization. © 2013 Kozakiewicz et al

Gait stability and variability measures show effects of impaired cognition and dual tasking in frail people

<p>Abstract</p> <p>Background</p> <p>Falls in frail elderly are a common problem with a rising incidence. Gait and postural instability are major risk factors for falling, particularly in geriatric patients. As walking requires attention, cognitive impairments are likely to contribute to an increased fall risk. An objective quantification of gait and balance ability is required to identify persons with a high tendency to fall. Recent studies have shown that stride variability is increased in elderly and under dual task condition and might be more sensitive to detect fall risk than walking speed. In the present study we complemented stride related measures with measures that quantify trunk movement patterns as indicators of dynamic balance ability during walking. The aim of the study was to quantify the effect of impaired cognition and dual tasking on gait variability and stability in geriatric patients.</p> <p>Methods</p> <p>Thirteen elderly with dementia (mean age: 82.6 ± 4.3 years) and thirteen without dementia (79.4 ± 5.55) recruited from a geriatric day clinic, walked at self-selected speed with and without performing a verbal dual task. The Mini Mental State Examination and the Seven Minute Screen were administered. Trunk accelerations were measured with an accelerometer. In addition to walking speed, mean, and variability of stride times, gait stability was quantified using stochastic dynamical measures, namely regularity (sample entropy, long range correlations) and local stability exponents of trunk accelerations.</p> <p>Results</p> <p>Dual tasking significantly (p < 0.05) decreased walking speed, while stride time variability increased, and stability and regularity of lateral trunk accelerations decreased. Cognitively impaired elderly showed significantly (p < 0.05) more changes in gait variability than cognitive intact elderly. Differences in dynamic parameters between groups were more discerned under dual task conditions.</p> <p>Conclusions</p> <p>The observed trunk adaptations were a consistent instability factor. These results support the concept that changes in cognitive functions contribute to changes in the variability and stability of the gait pattern. Walking under dual task conditions and quantifying gait using dynamical parameters can improve detecting walking disorders and might help to identify those elderly who are able to adapt walking ability and those who are not and thus are at greater risk for falling.</p

Rapid tests and urine sampling techniques for the diagnosis of urinary tract infection (UTI) in children under five years: a systematic review

Background: Urinary tract infection (UTI) is one of the most common sources of infection in children under five. Prompt diagnosis and treatment is important to reduce the risk of renal scarring. Rapid, cost-effective, methods of UTI diagnosis are required as an alternative to culture. Methods: We conducted a systematic review to determine the diagnostic accuracy of rapid tests for detecting UTI in children under five years of age. Results: The evidence supports the use of dipstick positive for both leukocyte esterase and nitrite (pooled LR+ = 28.2, 95% CI: 17.3, 46.0) or microscopy positive for both pyuria and bacteriuria (pooled LR+ = 37.0, 95% CI: 11.0, 125.9) to rule in UTI. Similarly dipstick negative for both LE and nitrite (Pooled LR- = 0.20, 95% CI: 0.16, 0.26) or microscopy negative for both pyuria and bacteriuria (Pooled LR- = 0.11, 95% CI: 0.05, 0.23) can be used to rule out UTI. A test for glucose showed promise in potty-trained children. However, all studies were over 30 years old. Further evaluation of this test may be useful. Conclusion: Dipstick negative for both LE and nitrite or microscopic analysis negative for both pyuria and bacteriuria of a clean voided urine, bag, or nappy/pad specimen may reasonably be used to rule out UTI. These patients can then reasonably be excluded from further investigation, without the need for confirmatory culture. Similarly, combinations of positive tests could be used to rule in UTI, and trigger further investigation

Large scale analysis of positional effects of single-base mismatches on microarray gene expression data

<p>Abstract</p> <p>Background</p> <p>Affymetrix GeneChips utilize 25-mer oligonucleotides probes linked to a silica surface to detect targets in solution. Mismatches due to single nucleotide polymorphisms (SNPs) can affect the hybridization between probes and targets. Previous research has indicated that binding between probes and targets strongly depends on the positions of these mismatches. However, there has been substantial variability in the effect of mismatch type across studies.</p> <p>Methods</p> <p>By taking advantage of naturally occurring mismatches between rhesus macaque transcripts and human probes from the Affymetrix U133 Plus 2 GeneChip, we collected the largest 25-mer probes dataset with single-base mismatches at each of the 25 positions on the probe ever used in this type of analysis.</p> <p>Results</p> <p>A mismatch at the center of a probe led to a greater loss in signal intensity than a mismatch at the ends of the probe, regardless of the mismatch type. There was a slight asymmetry between the ends of a probe: effects of mismatches at the 3' end of a probe were greater than those at the 5' end. A cross study comparison of the effect of mismatch types revealed that results were not in good agreement among different reports. However, if the mismatch types were consolidated to purine or pyrimidine mismatches, cross study conclusions could be generated.</p> <p>Conclusion</p> <p>The comprehensive assessment of the effects of single-base mismatches on microarrays provided in this report can be useful for improving future versions of microarray platform design and the corresponding data analysis algorithms.</p

Reference values for methacholine reactivity (SAPALDIA study)

BACKGROUND: The distribution of airway responsiveness in a general population of non-smokers without respiratory symptoms has not been established, limiting its use in clinical and epidemiological practice. We derived reference equations depending on individual characteristics (i.e., sex, age, baseline lung function) for relevant percentiles of the methacholine two-point dose-response slope. METHODS: In a reference sample of 1567 adults of the SAPALDIA cross-sectional survey (1991), defined by excluding subjects with respiratory conditions, responsiveness during methacholine challenge was quantified by calculating the two-point dose-response slope (O'Connor). Weighted L1-regression was used to estimate reference equations for the 95(th ), 90(th ), 75(th )and 50(th )percentiles of the two-point slope. RESULTS: Reference equations for the 95(th ), 90(th ), 75(th )and 50(th )percentiles of the two-point slope were estimated using a model of the form a + b* Age + c* FEV(1 )+ d* (FEV(1))(2 ), where FEV(1 )corresponds to the pre-test (or baseline) level of FEV(1). For the central half of the FEV(1 )distribution, we used a quadratic model to describe the dependence of methacholine slope on baseline FEV(1). For the first and last quartiles of FEV(1), a linear relation with FEV(1 )was assumed (i.e., d was set to 0). Sex was not a predictor term in this model. A negative linear association with slope was found for age. We provide an Excel file allowing calculation of the percentile of methacholine slope of a subject after introducing age – pre-test FEV(1 )– and results of methacholine challenge of the subject. CONCLUSION: The present study provides equations for four relevant percentiles of methacholine two-point slope depending on age and baseline FEV(1 )as basic predictors in an adult reference population of non-obstructive and non-atopic persons. These equations may help clinicians and epidemiologists to better characterize individual or population airway responsiveness