Genome-Wide microRNA Binding Site Variation between Extinct Wild Aurochs and Modern Cattle Identifies Candidate microRNA-Regulated Domestication Genes

peer-reviewedThe domestication of cattle from the now-extinct wild aurochs (Bos primigenius) involved selection for physiological and behavioral traits, with underlying genetic factors that remain largely unknown. Non-coding microRNAs have emerged as key regulators of the spatio-temporal expression of target genes controlling mammalian growth and development, including in livestock species. During the domestication process, selection of mutational changes in miRNAs and/or miRNA binding sites could have provided a mechanism to generate some of the traits that differentiate domesticated cattle from wild aurochs. To investigate this, we analyzed the open reading frame DNA sequence of 19,994 orthologous protein-coding gene pairs from extant Bos taurus genomes and a single extinct B. primigenius genome. We identified miRNA binding site polymorphisms in the 3′ UTRs of 1,620 of these orthologous genes. These 1,620 genes with altered miRNA binding sites between the B. taurus and B. primigenius lineages represent candidate domestication genes. Using a novel Score Site ratio metric we have ranked these miRNA-regulated genes according to the extent of divergence between miRNA binding site presence, frequency and copy number between the orthologous genes from B. taurus and B. primigenius. This provides an unbiased approach to identify cattle genes that have undergone the most changes in miRNA binding (i.e., regulation) between the wild aurochs and modern-day cattle breeds. In addition, we demonstrate that these 1,620 candidate domestication genes are enriched for roles in pigmentation, fertility, neurobiology, metabolism, immunity and production traits (including milk quality and feed efficiency). Our findings suggest that directional selection of miRNA regulatory variants was important in the domestication and subsequent artificial selection that gave rise to modern taurine cattle

Key Hub and Bottleneck Genes Differentiate the Macrophage Response to Virulent and Attenuated Mycobacterium bovis

Mycobacterium bovis is an intracellular pathogen that causes tuberculosis in cattle. Following infection, the pathogen resides and persists inside host macrophages by subverting host immune responses via a diverse range of mechanisms. Here, a high-density bovine microarray platform was used to examine the bovine monocyte-derived macrophage transcriptome response to M. bovis infection relative to infection with the attenuated vaccine strain, M. bovis Bacille Calmette–Guérin. Differentially expressed genes were identified (adjusted P-value ≤0.01) and interaction networks generated across an infection time course of 2, 6, and 24 h. The largest number of biological interactions was observed in the 24-h network, which exhibited scale-free network properties. The 24-h network featured a small number of key hub and bottleneck gene nodes, including IKBKE, MYC, NFKB1, and EGR1 that differentiated the macrophage response to virulent and attenuated M. bovis strains, possibly via the modulation of host cell death mechanisms. These hub and bottleneck genes represent possible targets for immuno-modulation of host macrophages by virulent mycobacterial species that enable their survival within a hostile environment

Antigen stimulation of peripheral blood mononuclear cells from Mycobacterium bovis infected cattle yields evidence for a novel gene expression program

<p>Abstract</p> <p>Background</p> <p>Bovine tuberculosis (BTB) caused by <it>Mycobacterium bovis </it>continues to cause substantial losses to global agriculture and has significant repercussions for human health. The advent of high throughput genomics has facilitated large scale gene expression analyses that present a novel opportunity for revealing the molecular mechanisms underlying mycobacterial infection. Using this approach, we have previously shown that innate immune genes in peripheral blood mononuclear cells (PBMC) from BTB-infected animals are repressed <it>in vivo </it>in the absence of exogenous antigen stimulation. In the present study, we hypothesized that the PBMC from BTB-infected cattle would display a distinct gene expression program resulting from exposure to <it>M. bovis</it>. A functional genomics approach was used to examine the immune response of BTB-infected (<it>n </it>= 6) and healthy control (<it>n </it>= 6) cattle to stimulation with bovine tuberculin (purified protein derivative – PPD-b) <it>in vitro</it>. PBMC were harvested before, and at 3 h and 12 h post <it>in vitro </it>stimulation with bovine tuberculin. Gene expression changes were catalogued within each group using a reference hybridization design and a targeted immunospecific cDNA microarray platform (BOTL-5) with 4,800 spot features representing 1,391 genes.</p> <p>Results</p> <p>250 gene spot features were significantly differentially expressed in BTB-infected animals at 3 h post-stimulation contrasting with only 88 gene spot features in the non-infected control animals (<it>P </it>≤ 0.05). At 12 h post-stimulation, 56 and 80 gene spot features were differentially expressed in both groups respectively. The results provided evidence of a proinflammatory gene expression profile in PBMC from BTB-infected animals in response to antigen stimulation. Furthermore, a common panel of eighteen genes, including transcription factors were significantly expressed in opposite directions in both groups. Real-time quantitative reverse transcription PCR (qRT-PCR) demonstrated that many innate immune genes, including components of the TLR pathway and cytokines were differentially expressed in BTB-infected (<it>n </it>= 8) versus control animals (<it>n </it>= 8) after stimulation with bovine tuberculin.</p> <p>Conclusion</p> <p>The PBMC from BTB-infected animals exhibit different transcriptional profiles compared with PBMC from healthy control animals in response to <it>M. bovis </it>antigen stimulation, providing evidence of a novel gene expression program due to <it>M. bovis </it>exposure.</p

Screening Fluorescent Voltage Indicators with Spontaneously Spiking HEK Cells

Development of improved fluorescent voltage indicators is a key challenge in neuroscience, but progress has been hampered by the low throughput of patch-clamp characterization. We introduce a line of non-fluorescent HEK cells that stably express NaV 1.3 and KIR 2.1 and generate spontaneous electrical action potentials. These cells enable rapid, electrode-free screening of speed and sensitivity of voltage sensitive dyes or fluorescent proteins on a standard fluorescence microscope. We screened a small library of mutants of archaerhodopsin 3 (Arch) in spiking HEK cells and identified two mutants with greater voltage-sensitivity than found in previously published Arch voltage indicators

Global Gene Expression and Systems Biology Analysis of Bovine Monocyte-Derived Macrophages in Response to In Vitro Challenge with Mycobacterium bovis

peer-reviewedBackground Mycobacterium bovis, the causative agent of bovine tuberculosis, is a major cause of mortality in global cattle populations. Macrophages are among the first cell types to encounter M. bovis following exposure and the response elicited by these cells is pivotal in determining the outcome of infection. Here, a functional genomics approach was undertaken to investigate global gene expression profiles in bovine monocyte-derived macrophages (MDM) purified from seven age-matched non-related females, in response to in vitro challenge with M. bovis (multiplicity of infection 2:1). Total cellular RNA was extracted from non-challenged control and M. bovis-challenged MDM for all animals at intervals of 2 hours, 6 hours and 24 hours post-challenge and prepared for global gene expression analysis using the Affymetrix® GeneChip® Bovine Genome Array. Results Comparison of M. bovis-challenged MDM gene expression profiles with those from the non-challenged MDM controls at each time point identified 3,064 differentially expressed genes 2 hours post-challenge, with 4,451 and 5,267 differentially expressed genes detected at the 6 hour and 24 hour time points, respectively (adjusted P-value threshold ≤0.05). Notably, the number of downregulated genes exceeded the number of upregulated genes in the M. bovis-challenged MDM across all time points; however, the fold-change in expression for the upregulated genes was markedly higher than that for the downregulated genes. Systems analysis revealed enrichment for genes involved in: (1) the inflammatory response; (2) cell signalling pathways, including Toll-like receptors and intracellular pathogen recognition receptors; and (3) apoptosis. Conclusions The increased number of downregulated genes is consistent with previous studies showing that M. bovis infection is associated with the repression of host gene expression. The results also support roles for MyD88-independent signalling and intracellular PRRs in mediating the host response to M. bovis.Science Foundation Ireland (www.sfi.ie) Investigator grants (Nos: SFI/01/F.1/B028 and SFI/08/IN.1/B2038); Department of Agriculture, Fisheries and Food (www.agriculture.ie) Research Stimulus Grant (No: RSF 06 405); European Union Framework 7 (http://cordis.europa.eu/fp7) Project Grant (No: KBBE-211602-MACROSYS); Irish Research Council for Science, Engineering and Technology (IRCSET) funded Bioinformatics and Systems Biology PhD Programme (http://bioinfo-casl.ucd.ie/PhD)

G(alpha)11 signaling through ARF6 regulates F-actin mobilization and GLUT4 glucose transporter translocation to the plasma membrane

The action of insulin to recruit the intracellular GLUT4 glucose transporter to the plasma membrane of 3T3-L1 adipocytes is mimicked by endothelin 1, which signals through trimeric G(alpha)q or G(alpha)11 proteins. Here we report that murine G(alpha)11 is most abundant in fat and that expression of the constitutively active form of G(alpha)11 [G(alpha)11(Q209L)] in 3T3-L1 adipocytes causes recruitment of GLUT4 to the plasma membrane and stimulation of 2-deoxyglucose uptake. In contrast to the action of insulin on GLUT4, the effects of endothelin 1 and G(alpha)11 were not inhibited by the phosphatidylinositol 3-kinase inhibitor wortmannin at 100 nM. Signaling by insulin, endothelin 1, or G(alpha)11(Q209L) also mobilized cortical F-actin in cultured adipocytes. Importantly, GLUT4 translocation caused by all three agents was blocked upon disassembly of F-actin by latrunculin B, suggesting that the F-actin polymerization caused by these agents may be required for their effects on GLUT4. Remarkably, expression of a dominant inhibitory form of the actin-regulatory GTPase ARF6 [ARF6(T27N)] in cultured adipocytes selectively inhibited both F-actin formation and GLUT4 translocation in response to endothelin 1 but not insulin. These data indicate that ARF6 is a required downstream element in endothelin 1 signaling through G(alpha)11 to regulate cortical actin and GLUT4 translocation in cultured adipocytes, while insulin action involves different signaling pathways

The Organic Crystallizing Agent 2-Methyl-2,4-pentanediol Reduces DNA Curvature by Means of Structural Changes in A-tracts

Contemporary predictive models for sequence-dependent DNA structure provide a good estimation of overall DNA curvature in most cases. However, the two current models differ fundamentally in their view of the origin of DNA curvature. An earlier model that associates DNA bending primarily, although not exclusively, with stretches of adenines (A-tracts) is based on results of comparative gel retardation, cyclization kinetics, hydroxyl radical cutting, and other solution measurements. It represents an intersection of wedge and junction models. More recently, a non-A-tract bending model has been proposed, built on structural results from x-ray crystallography and molecular modeling. In this view, A-tracts are proposed to be straight and rigid, whereas mixed sequence DNA is bent. Because a key premise of the non-A-tract bending model is the crystallographic observation that A-tracts are straight, we have examined the effect in solution of 2-methyl-2,4-pentanediol (MPD), an organic solvent used in crystal preparation for crystallographic DNA structure determinations. Using cyclization analysis, DNase I cutting, chemical probing, and electron microscopy on DNA oligomers with and without A-tracts, we show that the presence of MPD in solution dramatically affects A-tracts and that the effect is specific to these sequence elements. Combined with the previous observation that MPD affects gel mobility of curved sequences with A-tracts, our findings support the bent A-tract model and call for caution in the interpretation of crystallographic results on DNA structure as these are presently obtained

Generalized messengers of supersymmetry breaking and the sparticle mass spectrum

We investigate the sparticle spectrum in models of gauge-mediated supersymmetry breaking. In these models, supersymmetry is spontaneously broken at an energy scale only a few orders of magnitude above the electroweak scale. The breakdown of supersymmetry is communicated to the standard model particles and their superpartners by "messenger" fields through their ordinary gauge interactions. We study the effects of a messenger sector in which the supersymmetry-violating F-term contributions to messenger scalar masses are comparable to the supersymmetry-preserving ones. We also argue that it is not particularly natural to restrict attention to models in which the messenger fields lie in complete SU(5) GUT multiplets, and we identify a much larger class of viable models. Remarkably, however, we find that the superpartner mass parameters in these models are still subject to many significant contraints.Comment: 24 pages, LaTeX, uses epsf.sty, 4 figures. Assumptions clarified, numerical bounds tweaked, typos correcte

Early and efficient detection of Mycobacterium tuberculosis in sputum by microscopic observation of broth cultures.

Early, efficient and inexpensive methods for the detection of pulmonary tuberculosis are urgently needed for effective patient management as well as to interrupt transmission. These methods to detect M. tuberculosis in a timely and affordable way are not yet widely available in resource-limited settings. In a developing-country setting, we prospectively evaluated two methods for culturing and detecting M. tuberculosis in sputum. Sputum samples were cultured in liquid assay (micro broth culture) in microplate wells and growth was detected by microscopic observation, or in Löwenstein-Jensen (LJ) solid media where growth was detected by visual inspection for colonies. Sputum samples were collected from 321 tuberculosis (TB) suspects attending Bugando Medical Centre, in Mwanza, Tanzania, and were cultured in parallel. Pulmonary tuberculosis cases were diagnosed using the American Thoracic Society diagnostic standards. There were a total of 200 (62.3%) pulmonary tuberculosis cases. Liquid assay with microscopic detection detected a significantly higher proportion of cases than LJ solid culture: 89.0% (95% confidence interval [CI], 84.7% to 93.3%) versus 77.0% (95% CI, 71.2% to 82.8%) (p = 0.0007). The median turn around time to diagnose tuberculosis was significantly shorter for micro broth culture than for the LJ solid culture, 9 days (interquartile range [IQR] 7-13), versus 21 days (IQR 14-28) (p<0.0001). The cost for micro broth culture (labor inclusive) in our study was US $4.56 per sample, versus US$11.35 per sample for the LJ solid culture. The liquid assay (micro broth culture) is an early, feasible, and inexpensive method for detection of pulmonary tuberculosis in resource limited settings