A statistical analysis of murine incisional and excisional acute wound models

YesMice represent the most commonly used species for preclinical in vivo research. While incisional and excisional acute murine wound models are both frequently employed, there is little agreement on which model is optimum. Moreover, current lack of standardization of wounding procedure, analysis time point(s), method of assessment, and the use of individual wounds vs. individual animals as replicates makes it difficult to compare across studies. Here we have profiled secondary intention healing of incisional and excisional wounds within the same animal, assessing multiple parameters to determine the optimal methodology for future studies. We report that histology provides the least variable assessment of healing. Furthermore, histology alone (not planimetry) is able to detect accelerated healing in a castrated mouse model. Perhaps most importantly, we find virtually no correlation between wounds within the same animal, suggesting that use of wound (not animal) biological replicates is perfectly acceptable. Overall, these findings should guide and refine future studies, increasing the likelihood of detecting novel phenotypes while reducing the numbers of animals required for experimentation

Integrating Murine Gene Expression Studies to Understand Obstructive Lung Disease due to Chronic Inhaled Endotoxin

Rationale: Endotoxin is a near ubiquitous environmental exposure that that has been associated with both asthma and chronic obstructive pulmonary disease (COPD). These obstructive lung diseases have a complex pathophysiology, making them difficult to study comprehensively in the context of endotoxin. Genome-wide gene expression studies have been used to identify a molecular snapshot of the response to environmental exposures. Identification of differentially expressed genes shared across all published murine models of chronic inhaled endotoxin will provide insight into the biology underlying endotoxin-associated lung disease. Methods: We identified three published murine models with gene expression profiling after repeated low-dose inhaled endotoxin. All array data from these experiments were re-analyzed, annotated consistently, and tested for shared genes found to be differentially expressed. Additional functional comparison was conducted by testing for significant enrichment of differentially expressed genes in known pathways. The importance of this gene signature in smoking-related lung disease was assessed using hierarchical clustering in an independent experiment where mice were exposed to endotoxin, smoke, and endotoxin plus smoke. Results: A 101-gene signature was detected in three murine models, more than expected by chance. The three model systems exhibit additional similarity beyond shared genes when compared at the pathway level, with increasing enrichment of inflammatory pathways associated with longer duration of endotoxin exposure. Genes and pathways important in both asthma and COPD were shared across all endotoxin models. Mice exposed to endotoxin, smoke, and smoke plus endotoxin were accurately classified with the endotoxin gene signature. Conclusions: Despite the differences in laboratory, duration of exposure, and strain of mouse used in three experimental models of chronic inhaled endotoxin, surprising similarities in gene expression were observed. The endotoxin component of tobacco smoke may play an important role in disease development

Glucocorticoids rapidly inhibit cell migration through a novel, non-transcriptional HDAC6 pathway

Glucocorticoids (GCs) act through the glucocorticoid receptor (GR, also known as NR3C1) to regulate immunity, energy metabolism and tissue repair. Upon ligand binding, activated GR mediates cellular effects by regulating gene expression, but some GR effects can occur rapidly without new transcription. Here, we show that GCs rapidly inhibit cell migration, in response to both GR agonist and antagonist ligand binding. The inhibitory effect on migration is prevented by GR knockdown with siRNA, confirming GR specificity, but not by actinomycin D treatment, suggesting a non-transcriptional mechanism. We identified a rapid onset increase in microtubule polymerisation following GC treatment, identifying cytoskeletal stabilisation as the likely mechanism of action. HDAC6 overexpression, but not knockdown of αTAT1, rescued the GC effect, implicating HDAC6 as the GR effector. Consistent with this hypothesis, ligand-dependent cytoplasmic interaction between GR and HDAC6 was demonstrated by quantitative imaging. Taken together, we propose that activated GR inhibits HDAC6 function, and thereby increases the stability of the microtubule network to reduce cell motility. We therefore report a novel, non-transcriptional mechanism whereby GCs impair cell motility through inhibition of HDAC6 and rapid reorganization of the cell architecture. This article has an associated First Person interview with the first author of the paper

Phenotypic plasticity as a cause and consequence of population dynamics

Predicting complex species-environment interactions is crucial for guiding con- servation and mitigation strategies in a dynamically changing world. Phenotypic plasticity is a mechanism of trait variation that determines how individuals and populations adapt to changing and novel environments. For individuals, the ef- fects of phenotypic plasticity can be quantified by measuring environment–trait relationships, but it is often difficult to predict how phenotypic plasticity affects populations. The assumption that environment–trait relationships validated for in- dividuals indicate how populations respond to environmental change is commonly made without sufficient justification. Here we derive a novel general mathematical framework linking trait variation due to phenotypic plasticity to population dy- namics. Applying the framework to the classical example of Nicholson's blowflies, we show how seemingly sensible predictions made from environment–trait rela- tionships do not generalise to population responses. As a consequence, trait-based analyses that do not incorporate population feedbacks risk mischaracterising the effect of environmental change on populations

Pioglitazone for secondary prevention after ischemic stroke and transient ischemic attack: Rationale and design of the Insulin Resistance Intervention after Stroke Trial

Background: Recurrent vascular events remain a major source of morbidity and mortality after stroke or transient ischemic attack (TIA). The IRIS Trial is evaluating an approach to secondary prevention based on the established association between insulin resistance and increased risk for ischemic vascular events. Specifically, IRIS will test the effectiveness of pioglitazone, an insulin-sensitizing drug of the thiazolidinedione class, for reducing the risk for stroke and myocardial infarction (MI) among insulin resistant, nondiabetic patients with a recent ischemic stroke or TIA. Design: Eligible patients for IRIS must have had insulin resistance defined by a Homeostasis Model Assessment-Insulin Resistance \u3e3.0 without meeting criteria for diabetes. Within 6 months of the index stroke or TIA, patients were randomly assigned to pioglitazone (titrated from 15 to 45 mg/d) or matching placebo and followed for up to 5 years. The primary outcome is time to stroke or MI. Secondary outcomes include time to stroke alone, acute coronary syndrome, diabetes, cognitive decline, and all-cause mortality. Enrollment of 3,876 participants from 179 sites in 7 countries was completed in January 2013. Participant follow-up will continue until July 2015. Summary: The IRIS Trial will determine whether treatment with pioglitazone improves cardiovascular outcomes of nondiabetic, insulin-resistant patients with stroke or TIA. Results are expected in early 2016

Innate immune activation by inhaled lipopolysaccharide, independent of oxidative stress, exacerbates silica-induced pulmonary fibrosis in mice

Acute exacerbations of pulmonary fibrosis are characterized by rapid decrements in lung function. Environmental factors that may contribute to acute exacerbations remain poorly understood. We have previously demonstrated that exposure to inhaled lipopolysaccharide (LPS) induces expression of genes associated with fibrosis. To address whether exposure to LPS could exacerbate fibrosis, we exposed male C57BL/6 mice to crystalline silica, or vehicle, followed 28 days later by LPS or saline inhalation. We observed that mice receiving both silica and LPS had significantly more total inflammatory cells, more whole lung lavage MCP-1, MIP-2, KC and IL-1β, more evidence of oxidative stress and more total lung hydroxyproline than mice receiving either LPS alone, or silica alone. Blocking oxidative stress with N-acetylcysteine attenuated whole lung inflammation but had no effect on total lung hydroxyproline. These observations suggest that exposure to innate immune stimuli, such as LPS in the environment, may exacerbate stable pulmonary fibrosis via mechanisms that are independent of inflammation and oxidative stress. © 2012 Brass et al

A physical and regulatory map of host-influenza interactions reveals pathways in H1N1 infection

available in PMC 2010 June 28.During the course of a viral infection, viral proteins interact with an array of host proteins and pathways. Here, we present a systematic strategy to elucidate the dynamic interactions between H1N1 influenza and its human host. A combination of yeast two-hybrid analysis and genome-wide expression profiling implicated hundreds of human factors in mediating viral-host interactions. These factors were then examined functionally through depletion analyses in primary lung cells. The resulting data point to potential roles for some unanticipated host and viral proteins in viral infection and the host response, including a network of RNA-binding proteins, components of WNT signaling, and viral polymerase subunits. This multilayered approach provides a comprehensive and unbiased physical and regulatory model of influenza-host interactions and demonstrates a general strategy for uncovering complex host-pathogen relationships.National Institutes of Health (U.S.) (grant U01 AI074575)National Institutes of Health (U.S.) (grant U54 AI057159)National Institutes of Health (U.S.) (NIH New Innovator Award)Ford Foundation (Predoctoral Fellowship)Ellison Medical FoundationNational Institutes of Health (U.S.) (NIH grant R01 HG001715)National Institutes of Health (U.S.) (grant P50 HG004233)National Institutes of Health (U.S.) (PIONEER award)Howard Hughes Medical InstituteBurroughs Wellcome Fund (Career Award at the Scientific Interface)Alfred P. Sloan Foundatio

Strand Transfer and Elongation of HIV-1 Reverse Transcription Is Facilitated by Cell Factors In Vitro

Recent work suggests a role for multiple host factors in facilitating HIV-1 reverse transcription. Previously, we identified a cellular activity which increases the efficiency of HIV-1 reverse transcription in vitro. Here, we describe aspects of the activity which shed light on its function. The cellular factor did not affect synthesis of strong-stop DNA but did improve downstream DNA synthesis. The stimulatory activity was isolated by gel filtration in a single fraction of the exclusion volume. Velocity-gradient purified HIV-1, which was free of detectable RNase activity, showed poor reverse transcription efficiency but was strongly stimulated by partially purified cell proteins. Hence, the cell factor(s) did not inactivate an RNase activity that might degrade the viral genomic RNA and block completion of reverse transcription. Instead, the cell factor(s) enhanced first strand transfer and synthesis of late reverse transcription suggesting it stabilized the reverse transcription complex. The factor did not affect lysis of HIV-1 by Triton X-100 in the endogenous reverse transcription (ERT) system, and ERT reactions with HIV-1 containing capsid mutations, which varied the biochemical stability of viral core structures and impeded reverse transcription in cells, showed no difference in the ability to be stimulated by the cell factor(s) suggesting a lack of involvement of the capsid in the in vitro assay. In addition, reverse transcription products were found to be resistant to exogenous DNase I activity when the active fraction was present in the ERT assay. These results indicate that the cell factor(s) may improve reverse transcription by facilitating DNA strand transfer and DNA synthesis. It also had a protective function for the reverse transcription products, but it is unclear if this is related to improved DNA synthesis