A Human Cytomegalovirus-Encoded microRNA Regulates Expression of Multiple Viral Genes Involved in Replication

Although multiple studies have documented the expression of over 70 novel virus-encoded microRNAs (miRNAs), the targets and functions of most of these regulatory RNA species are unknown. In this study a comparative bioinformatics approach was employed to identify potential human cytomegalovirus (HCMV) mRNA targets of the virus-encoded miRNA miR-UL112-1. Bioinformatics analysis of the known HCMV mRNA 3′ untranslated regions (UTRs) revealed 14 potential viral transcripts that were predicted to contain functional target sites for miR-UL112-1. The potential target sites were screened using luciferase reporters that contain the HCMV 3′UTRs in co-transfection assays with miR-UL112-1. Three of the 14 HCMV miRNA targets were validated, including the major immediate early gene encoding IE72 (UL123, IE1), UL112/113, and UL120/121. Further analysis of IE72 regulation by miR-UL112-1 with clones encoding the complete major immediate early region revealed that the IE72 3′UTR target site is necessary and sufficient to direct miR-UL112-1-specific inhibition of expression in transfected cells. In addition, miR-UL112-1 regulation is mediated through translational inhibition rather than RNA degradation. Premature expression of miR-UL112-1 during HCMV infection resulted in a significant decrease in genomic viral DNA levels, suggesting a functional role for miR-UL112-1 in regulating the expression of genes involved in viral replication. This study demonstrates the ability of a viral miRNA to regulate multiple viral genes

Genetic Differences in Behavioral Sensitivity to a Neuroactive Steroid

ABSTRACT Recent work found that lower endogenous levels of the ␥-aminobutyric acid-agonist, neuroactive steroid 3␣-hydroxy-5␣-pregnan-20-one (3␣,5␣-THP) may be correlated with increased ethanol withdrawal severity in the selectively bred Withdrawal Seizure-Prone and -Resistant mice. The present studies were conducted to determine whether decreased sensitivity to 3␣,5␣-THP was correlated with ethanol withdrawal hyperexcitability in another genetic mouse model, namely the C57BL/6 (B6) and DBA/2 (D2) inbred strains. These strains also differ in ethanol withdrawal severity (D2 Ͼ Ͼ B6). B6 and D2 male mice were injected with 3␣,5␣-THP (0 -10 mg/kg i.p.) 15 min before the timed tail vein infusion of pentylenetetrazol. B6 mice were more sensitive than D2 animals to the anticonvulsant effect of 3␣,5␣-THP. Subsequent studies measured sensitivity to several of the pharmacological effects of 3␣,5␣-THP. B6 and D2 male mice were injected with 3␣,5␣-THP (0 -32 mg/kg) before testing for locomotor activation (total number of entries) and anxiolysis (percent open arm entries) on the elevated plus maze, muscle relaxation (impairment of forelimb grip strength), ataxia (impairment of Rotarod performance) and seizure susceptibility to pentylenetetrazol. B6 mice were more sensitive than D2 animals to the anxiolytic, locomotor stimulant and anticonvulsant effects of 3␣,5␣-THP. In contrast, D2 mice were more sensitive than B6 mice to 3␣,5␣-THP-induced muscle relaxation and ataxia. Plasma 3␣,5␣-THP levels did not differ in the B6 and D2 mice injected with this steroid, suggesting that the strain differences were not pharmacokinetic. Collectively, the results in selectively bred Withdrawal Seizure-Prone and -Resistant mice and B6 and D2 inbred strains suggest that genetic differences in neuroactive steroid sensitivity and biosynthesis may contribute to ethanol withdrawal severity

Pharmacological insights into the role of P2X4 receptors in behavioural regulation: lessons from ivermectin

This is the publisher's version, also available electronically from http://journals.cambridge.org/action/displayAbstract?fromPage=online&aid=8920271&fileId=S1461145712000909Purinergic ionotropic P2X receptors are a family of cation-permeable channels that bind extracellular adenosine 5′-triphosphate. In particular, convergent lines of evidence have recently highlighted P2X4 receptors as a potentially critical target in the regulation of multiple nervous and behavioural functions, including pain, neuroendocrine regulation and hippocampal plasticity. Nevertheless, the role of the P2X4 receptor in behavioural organization remains poorly investigated. To study the effects of P2X4 activation, we tested the acute effects of its potent positive allosteric modulator ivermectin (IVM, 2.5–10 mg/kg i.p.) on a broad set of paradigms capturing complementary aspects of perceptual, emotional and cognitive regulation in mice. In a novel open field, IVM did not induce significant changes in locomotor activity, but increased the time spent in the peripheral zone. In contrast, IVM produced anxiolytic-like effects in the elevated plus maze and marble burying tasks, as well as depression-like behaviours in the tail-suspension and forced swim tests. The agent induced no significant behavioural changes in the conditioned place preference test and in the novel object recognition task. Finally, the drug induced a dose-dependent decrease in sensorimotor gating, as assessed by pre-pulse inhibition (PPI) of the acoustic startle reflex. In P2X4 knockout mice, the effects of IVM in the open field and elevated plus maze were similar to those observed in wild type mice; conversely, the drug significantly increased startle amplitude and failed to reduce PPI. Taken together, these results suggest that P2X4 receptors may play a role in the regulation of sensorimotor gatin

Sex and the Lab: An Alcohol-Focused Commentary on the NIH Initiative to Balance Sex in Cell and Animal Studies

In May 2014, Dr. Francis Collins, the director of US National Institutes of Health, and Dr. Janine Clayton, the director of the US National Institutes of Health Office of Research on Women’s Health (ORWH) published a commentary in the journal Nature announcing new policies to ensure that preclinical research funded by the NIH consider both males and females. While these policies are still developing, they have already generated great interest by the scientific community and triggered both criticism and applause. This review provides a description and interpretation of the NIH guidelines and it traces the history that led to their implementation. As expected, this NIH initiative generated some anxiety in the scientific community. The use of female animals in the investigation of basic mechanisms is perceived to increase variability in the results, and the use of both sexes has been claimed to slow the pace of scientific discoveries and to increase the cost at a time characterized by declining research support

Genetic risk and a primary role for cell-mediated immune mechanisms in multiple sclerosis.

Multiple sclerosis is a common disease of the central nervous system in which the interplay between inflammatory and neurodegenerative processes typically results in intermittent neurological disturbance followed by progressive accumulation of disability. Epidemiological studies have shown that genetic factors are primarily responsible for the substantially increased frequency of the disease seen in the relatives of affected individuals, and systematic attempts to identify linkage in multiplex families have confirmed that variation within the major histocompatibility complex (MHC) exerts the greatest individual effect on risk. Modestly powered genome-wide association studies (GWAS) have enabled more than 20 additional risk loci to be identified and have shown that multiple variants exerting modest individual effects have a key role in disease susceptibility. Most of the genetic architecture underlying susceptibility to the disease remains to be defined and is anticipated to require the analysis of sample sizes that are beyond the numbers currently available to individual research groups. In a collaborative GWAS involving 9,772 cases of European descent collected by 23 research groups working in 15 different countries, we have replicated almost all of the previously suggested associations and identified at least a further 29 novel susceptibility loci. Within the MHC we have refined the identity of the HLA-DRB1 risk alleles and confirmed that variation in the HLA-A gene underlies the independent protective effect attributable to the class I region. Immunologically relevant genes are significantly overrepresented among those mapping close to the identified loci and particularly implicate T-helper-cell differentiation in the pathogenesis of multiple sclerosis

EcoliWiki: a wiki-based community resource for Escherichia coli

EcoliWiki is the community annotation component of the PortEco (http://porteco.org; formerly EcoliHub) project, an online data resource that integrates information on laboratory strains of Escherichia coli, its phages, plasmids and mobile genetic elements. As one of the early adopters of the wiki approach to model organism databases, EcoliWiki was designed to not only facilitate community-driven sharing of biological knowledge about E. coli as a model organism, but also to be interoperable with other data resources. EcoliWiki content currently covers genes from five laboratory E. coli strains, 21 bacteriophage genomes, F plasmid and eight transposons. EcoliWiki integrates the Mediawiki wiki platform with other open-source software tools and in-house software development to extend how wikis can be used for model organism databases. EcoliWiki can be accessed online at http://ecoliwiki.net

Regulation of Hemocytes in Drosophila Requires dappled Cytochrome b5

A major category of mutant hematopoietic phenotypes in Drosophila is melanotic tumors or nodules, which consist of abnormal and overproliferated blood cells, similar to granulomas. Our analyses of the melanotic mutant dappled have revealed a novel type of gene involved in blood cell regulation. The dappled gene is an essential gene that encodes cytochrome b5, a conserved hemoprotein that participates in electron transfer in multiple biochemical reactions and pathways. Viable mutations of dappled cause melanotic nodules and hemocyte misregulation during both hematopoietic waves of development. The sexes are similarly affected, but hemocyte number is different in females and males of both mutants and wild type. Additionally, initial tests show that curcumin enhances the dappled melanotic phenotype and establish screening of endogenous and xenobiotic compounds as a route for analysis of cytochrome b5 function. Overall, dappled provides a tractable genetic model for cytochrome b5, which has been difficult to study in higher organisms