Standardized high-throughput evaluation of cell-based compound screens

<p>Abstract</p> <p>Background</p> <p>High-throughput screening of pharmaceutical compound activity in tissue culture experiments requires time-consuming repeated analysis of the large amounts of data generated. Automation of the evaluation procedure and assessment of measurement accuracy can save time and improve the comparability of results.</p> <p>Results</p> <p>We present a tool for simultaneous evaluation of an arbitrary number of compound screens including a standardized statistical validation. It is provided as a novel R package with a Tcl/Tk-based GUI for convenient use in the lab and runs on usual platforms like Linux, Windows and Mac OS. In a compound screen of lung cancer cells, the tool was successfully and efficiently applied for data analysis.</p> <p>Conclusion</p> <p>The package provides an efficient and intuitive platform for automatic evaluation of compound screens, improving the performance and standardization of data analysis.</p

Delayed mGluR5 activation limits neuroinflammation and neurodegeneration after traumatic brain injury

<p>Abstract</p> <p>Background</p> <p>Traumatic brain injury initiates biochemical processes that lead to secondary neurodegeneration. Imaging studies suggest that tissue loss may continue for months or years after traumatic brain injury in association with chronic microglial activation. Recently we found that metabotropic glutamate receptor 5 (mGluR5) activation by (<it>RS</it>)-2-chloro-5-hydroxyphenylglycine (CHPG) decreases microglial activation and release of associated pro-inflammatory factors <it>in vitro</it>, which is mediated in part through inhibition of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Here we examined whether delayed CHPG administration reduces chronic neuroinflammation and associated neurodegeneration after experimental traumatic brain injury in mice.</p> <p>Methods</p> <p>One month after controlled cortical impact traumatic brain injury, C57Bl/6 mice were randomly assigned to treatment with single dose intracerebroventricular CHPG, vehicle or CHPG plus a selective mGluR5 antagonist, 3-((2-Methyl-4-thiazolyl)ethynyl)pyridine. Lesion volume, white matter tract integrity and neurological recovery were assessed over the following three months.</p> <p>Results</p> <p>Traumatic brain injury resulted in mGluR5 expression in reactive microglia of the cortex and hippocampus at one month post-injury. Delayed CHPG treatment reduced expression of reactive microglia expressing NADPH oxidase subunits; decreased hippocampal neuronal loss; limited lesion progression, as measured by repeated T2-weighted magnetic resonance imaging (at one, two and three months) and white matter loss, as measured by high field <it>ex vivo </it>diffusion tensor imaging at four months; and significantly improved motor and cognitive recovery in comparison to the other treatment groups.</p> <p>Conclusion</p> <p>Markedly delayed, single dose treatment with CHPG significantly improves functional recovery and limits lesion progression after experimental traumatic brain injury, likely in part through actions at mGluR5 receptors that modulate neuroinflammation.</p

Genomewide Analyses Define Different Modes of Transcriptional Regulation by Peroxisome Proliferator-Activated Receptor-β/δ (PPARβ/δ)

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors with essential functions in lipid, glucose and energy homeostasis, cell differentiation, inflammation and metabolic disorders, and represent important drug targets. PPARs heterodimerize with retinoid X receptors (RXRs) and can form transcriptional activator or repressor complexes at specific DNA elements (PPREs). It is believed that the decision between repression and activation is generally governed by a ligand-mediated switch. We have performed genomewide analyses of agonist-treated and PPARβ/δ-depleted human myofibroblasts to test this hypothesis and to identify global principles of PPARβ/δ-mediated gene regulation. Chromatin immunoprecipitation sequencing (ChIP-Seq) of PPARβ/δ, H3K4me3 and RNA polymerase II enrichment sites combined with transcriptional profiling enabled the definition of 112 bona fide PPARβ/δ target genes showing either of three distinct types of transcriptional response: (I) ligand-independent repression by PPARβ/δ; (II) ligand-induced activation and/or derepression by PPARβ/δ; and (III) ligand-independent activation by PPARβ/δ. These data identify PPRE-mediated repression as a major mechanism of transcriptional regulation by PPARβ/δ, but, unexpectedly, also show that only a subset of repressed genes are activated by a ligand-mediated switch. Our results also suggest that the type of transcriptional response by a given target gene is connected to the structure of its associated PPRE(s) and the biological function of its encoded protein. These observations have important implications for understanding the regulatory PPAR network and PPARβ/δ ligand-based drugs

Molecular evidence for increased regulatory conservation during metamorphosis, and against deleterious cascading effects of hybrid breakdown in Drosophila

<p>Abstract</p> <p>Background</p> <p>Speculation regarding the importance of changes in gene regulation in determining major phylogenetic patterns continues to accrue, despite a lack of broad-scale comparative studies examining how patterns of gene expression vary during development. Comparative transcriptional profiling of adult interspecific hybrids and their parental species has uncovered widespread divergence of the mechanisms controlling gene regulation, revealing incompatibilities that are masked in comparisons between the pure species. However, this has prompted the suggestion that misexpression in adult hybrids results from the downstream cascading effects of a subset of genes improperly regulated in early development.</p> <p>Results</p> <p>We sought to determine how gene expression diverges over development, as well as test the cascade hypothesis, by profiling expression in males of <it>Drosophila melanogaster</it>, <it>D. sechellia</it>, and <it>D. simulans</it>, as well as the <it>D. simulans </it>(♀) × <it>D. sechellia </it>(♂) male F1 hybrids, at four different developmental time points (3rd instar larval, early pupal, late pupal, and newly-emerged adult). Contrary to the cascade model of misexpression, we find that there is considerable stage-specific autonomy of regulatory breakdown in hybrids, with the larval and adult stages showing significantly more hybrid misexpression as compared to the pupal stage. However, comparisons between pure species indicate that genes expressed during earlier stages of development tend to be more conserved in terms of their level of expression than those expressed during later stages, suggesting that while Von Baer's famous law applies at both the level of nucleotide sequence and expression, it may not apply necessarily to the underlying overall regulatory network, which appears to diverge over the course of ontogeny and which can only be ascertained by combining divergent genomes in species hybrids.</p> <p>Conclusion</p> <p>Our results suggest that complex integration of regulatory circuits during morphogenesis may lead to it being more refractory to divergence of underlying gene regulatory mechanisms - more than that suggested by the conservation of gene expression levels between species during earlier stages. This provides support for a 'developmental hourglass' model of divergence of gene expression in <it>Drosophila </it>resulting in a highly conserved pupal stage.</p

How Do Human Cells React to the Absence of Mitochondrial DNA?

Mitochondrial biogenesis is under the control of two different genetic systems: the nuclear genome (nDNA) and the mitochondrial genome (mtDNA). The mtDNA is a circular genome of 16.6 kb encoding 13 of the approximately 90 subunits that form the respiratory chain, the remaining ones being encoded by the nDNA. Eukaryotic cells are able to monitor and respond to changes in mitochondrial function through alterations in nuclear gene expression, a phenomenon first defined in yeast and known as retrograde regulation. To investigate how the cellular transcriptome is modified in response to the absence of mtDNA, we used Affymetrix HG-U133A GeneChip arrays to study the gene expression profile of two human cell lines, 143BTK(-) and A549, which had been entirely depleted of mtDNA (rho(o) cells), and compared it with that of corresponding undepleted parental cells (rho(+) cells).Our data indicate that absence of mtDNA is associated with: i) a down-regulation of cell cycle control genes and a reduction of cell replication rate, ii) a down-regulation of nuclear-encoded subunits of complex III of the respiratory chain and iii) a down-regulation of a gene described as the human homolog of ELAC2 of E. coli, which encodes a protein that we show to also target to the mitochondrial compartment.Our results indicate a strong correlation between mitochondrial biogenesis and cell cycle control and suggest that some proteins could have a double role: for instance in controlling both cell cycle progression and mitochondrial functions. In addition, the finding that ELAC2 and maybe other transcripts that are located into mitochondria, are down-regulated in rho(o) cells, make them good candidates for human disorders associated with defective replication and expression of mtDNA

Effects of traumatic brain injury and posttraumatic stress disorder on Alzheimer's disease in veterans, using the Alzheimer's Disease Neuroimaging Initiative

Both traumatic brain injury (TBI) and posttraumatic stress disorder (PTSD) are common problems resulting from military service, and both have been associated with increased risk of cognitive decline and dementia resulting from Alzheimer's disease (AD) or other causes. This study aims to use imaging techniques and biomarker analysis to determine whether traumatic brain injury (TBI) and/or PTSD resulting from combat or other traumas increase the risk for AD and decrease cognitive reserve in Veteran subjects, after accounting for age. Using military and Department of Veterans Affairs records, 65 Vietnam War veterans with a history of moderate or severe TBI with or without PTSD, 65 with ongoing PTSD without TBI, and 65 control subjects are being enrolled in this study at 19 sites. The study aims to select subject groups that are comparable in age, gender, ethnicity, and education. Subjects with mild cognitive impairment (MCI) or dementia are being excluded. However, a new study just beginning, and similar in size, will study subjects with TBI, subjects with PTSD, and control subjects with MCI. Baseline measurements of cognition, function, blood, and cerebrospinal fluid biomarkers; magnetic resonance images (structural, diffusion tensor, and resting state blood-level oxygen dependent (BOLD) functional magnetic resonance imaging); and amyloid positron emission tomographic (PET) images with florbetapir are being obtained. One-year follow-up measurements will be collected for most of the baseline procedures, with the exception of the lumbar puncture, the PET imaging, and apolipoprotein E genotyping. To date, 19 subjects with TBI only, 46 with PTSD only, and 15 with TBI and PTSD have been recruited and referred to 13 clinics to undergo the study protocol. It is expected that cohorts will be fully recruited by October 2014. This study is a first step toward the design and statistical powering of an AD prevention trial using at-risk veterans as subjects, and provides the basis for a larger, more comprehensive study of dementia risk factors in veterans

Comparative Analysis of Gene Regulation by the Transcription Factor PPARα between Mouse and Human

Studies in mice have shown that PPARalpha is an important regulator of hepatic lipid metabolism and the acute phase response. However, little information is available on the role of PPARalpha in human liver. Here we set out to compare the function of PPARalpha in mouse and human hepatocytes via analysis of target gene regulation

A Selection Index for Gene Expression Evolution and Its Application to the Divergence between Humans and Chimpanzees

The importance of gene regulation in animal evolution is a matter of long-standing interest, but measuring the impact of selection on gene expression has proven a challenge. Here, we propose a selection index of gene expression as a straightforward method for assessing the mode and strength of selection operating on gene expression levels. The index is based on the widely used McDonald-Kreitman test and requires the estimation of four quantities: the within-species and between-species expression variances as well as the sequence heterozygosity and divergence of neutrally evolving sequences. We apply the method to data from human and chimpanzee lymphoblastoid cell lines and show that gene expression is in general under strong stabilizing selection. We also demonstrate how the same framework can be used to estimate the proportion of adaptive gene expression evolution