Specific ligation to double-stranded RNA for analysis of cellular RNA::RNA interactions

Double-stranded RNA (dsRNA) is formed in cells as intra- and intermolecular RNA interactions and is involved in a range of biological processes including RNA metabolism, RNA interference and translation control mediated by natural antisense RNA and microRNA. Despite this breadth of activities, few molecular tools are available to analyse dsRNA as native hybrids. We describe a two-step ligation method for enzymatic joining of dsRNA adaptors to any dsRNA molecule in its duplex form without a need for prior sequence or termini information. The method is specific for dsRNA and can ligate various adaptors to label, map or amplify dsRNA sequences. When combined with reverse transcription–polymerase chain reaction, the method is sensitive and can detect low nanomolar concentrations of dsRNA in total RNA. As examples, we mapped dsRNA/single-stranded RNA junctions within Escherichia coli hok mRNA and the human immunodeficiency virus TAR element using RNA from bacteria and mammalian cells

Hypoxia causes increased monocyte nitric oxide synthesis which is mediated by changes in Dimethylarginine Dimethylaminohydrolase 2 expression in animal and human models of normobaric hypoxia

Tissue hypoxia is a cardinal feature of inflammatory diseases and modulates monocyte function. Nitric oxide is a crucial component of the immune cell response. This study explored the metabolism of the endogenous inhibitor of nitric oxide production asymmetric dimethylarginine(ADMA) by monocyte dimethylarginine dimethylaminohydrolase 2(DDAH2), and the role of this pathway in the regulation of the cellular response and the local environment during hypoxia.Peritoneal macrophages were isolated from a macrophage-specific DDAH2 knockout mouse that we developed and compared with appropriate controls. Cells were exposed to 3% oxygen followed by reoxygenation at 21%. Healthy volunteers underwent an 8 h exposure to normobaric hypoxia with an inspired oxygen percentage of 12%. Peripheral blood mononuclear cells were isolated from blood samples taken before and at the end of this exposure.Intracellular nitrate plus nitrite(NOx) concentration was higher in wild-type murine monocytes after hypoxia and reoxygenation than in normoxia-treated cells (mean(SD) 13·2(2·4) vs 8·1(1·7) pmols/mg protein, p = 0·009). DDAH2 protein was 4·5-fold (SD 1·3) higher than in control cells (p = 0·03). This increase led to a 24% reduction in ADMA concentration, 0·33(0.04) pmols/mg to 0·24(0·03), p = 0·002). DDAH2-deficient murine monocytes demonstrated no increase in nitric oxide production after hypoxic challenge. These findings were recapitulated in a human observational study. Mean plasma NOx concentration was elevated after hypoxic exposure (3·6(1.8)μM vs 6·4(3·2), p = 0·01), which was associated with a reduction in intracellular ADMA in paired samples from 3·6(0.27) pmols/mg protein to 3·15(0·3) (p < 0·01). This finding was associated with a 1·9-fold(0·6) increase in DDAH2 expression over baseline(p = 0·03).This study shows that in both human and murine models of acute hypoxia, increased DDAH2 expression mediates a reduction in intracellular ADMA concentration which in turn leads to elevated nitric oxide concentrations both within the cell and in the local environment. Cells deficient in DDAH2 were unable to mount this response

An IL-27-Driven Transcriptional Network Identifies Regulators of IL-10 Expression across T Helper Cell Subsets.

Interleukin-27 (IL-27) is an immunoregulatory cytokine that suppresses inflammation through multiple mechanisms, including induction of IL-10, but the transcriptional network mediating its diverse functions remains unclear. Combining temporal RNA profiling with computational algorithms, we predict 79 transcription factors induced by IL-27 in T cells. We validate 11 known and discover 5 positive (Cebpb, Fosl2, Tbx21, Hlx, and Atf3) and 2 negative (Irf9 and Irf8) Il10 regulators, generating an experimentally refined regulatory network for Il10. We report two central regulators, Prdm1 and Maf, that cooperatively drive the expression of signature genes induced by IL-27 in type 1 regulatory T cells, mediate IL-10 expression in all T helper cells, and determine the regulatory phenotype of colonic Foxp3 &lt;sup&gt;+&lt;/sup&gt; regulatory T cells. Prdm1/Maf double-knockout mice develop spontaneous colitis, phenocopying ll10-deficient mice. Our work provides insights into IL-27-driven transcriptional networks and identifies two shared Il10 regulators that orchestrate immunoregulatory programs across T helper cell subsets

Diversity of Murine Norovirus Strains Isolated from Asymptomatic Mice of Different Genetic Backgrounds within a Single U.S. Research Institute

Antibody prevalence studies in laboratory mice indicate that murine norovirus (MNV) infections are common, but the natural history of these viruses has not been fully established. This study examined the extent of genetic diversity of murine noroviruses isolated from healthy laboratory mice housed in multiple animal facilities within a single, large research institute- the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (NIAID-NIH) in Bethesda, Maryland, U.S. Ten distinct murine norovirus strains were isolated from various tissues and feces of asymptomatic wild type sentinel mice as well as asymptomatic immunodeficient (RAG 2−/−) mice. The NIH MNV isolates showed little cytopathic effect in permissive RAW264.7 cells in early passages, but all isolates examined could be adapted to efficient growth in cell culture by serial passage. The viruses, although closely related in genome sequence, were distinguishable from each other according to facility location, likely due to the introduction of new viruses into each facility from separate sources or vendors at different times. Our study indicates that the murine noroviruses are widespread in these animal facilities, despite rigorous guidelines for animal care and maintenance

Turbot reovirus (SMReV) genome encoding a FAST protein with a non-AUG start site

<p>Abstract</p> <p>Background</p> <p>A virus was isolated from diseased turbot <it>Scophthalmus maximus </it>in China. Biophysical and biochemical assays, electron microscopy, and genome electrophoresis revealed that the virus belonged to the genus <it>Aquareovirus</it>, and was named <it>Scophthalmus maximus </it>reovirus (SMReV). To the best of our knowledge, no complete sequence of an aquareovirus from marine fish has been determined. Therefore, the complete characterization and analysis of the genome of this novel aquareovirus will facilitate further understanding of the taxonomic distribution of aquareovirus species and the molecular mechanism of its pathogenesis.</p> <p>Results</p> <p>The full-length genome sequences of SMReV were determined. It comprises eleven dsRNA segments covering 24,042 base pairs and has the largest S4 genome segment in the sequenced aquareoviruses. Sequence analysis showed that all of the segments contained six conserved nucleotides at the 5' end and five conserved nucleotides at the 3' end (5'-GUUUUA ---- UCAUC-3'). The encoded amino acid sequences share the highest sequence identities with the respective proteins of aquareoviruses in species group <it>Aquareovirus </it>A. Phylogenetic analysis based on the major outer capsid protein VP7 and RNA-dependent RNA polymerase were performed. Members in <it>Aquareovirus </it>were clustered in two groups, one from fresh water fish and the other from marine fish. Furthermore, a fusion associated small transmembrane (FAST) protein NS22, which is translated from a non-AUG start site, was identified in the S7 segment.</p> <p>Conclusions</p> <p>This study has provided the complete genome sequence of a novel isolated aquareovirus from marine fish. Amino acids comparison and phylogenetic analysis suggested that SMReV was a new aquareovirus in the species group <it>Aquareovirus </it>A. Phylogenetic analysis among aquareoviruses revealed that VP7 could be used as a reference to divide the aquareovirus from hosts in fresh water or marine. In addition, a FAST protein with a non-AUG start site was identified, which partially contributed to the cytopathic effect caused by the virus infection. These results provide new insights into the virus-host and virus-environment interactions.</p

MDA-5 Recognition of a Murine Norovirus

Noroviruses are important human pathogens responsible for most cases of viral epidemic gastroenteritis worldwide. Murine norovirus-1 (MNV-1) is one of several murine noroviruses isolated from research mouse facilities and has been used as a model of human norovirus infection. MNV-1 infection has been shown to require components of innate and adaptive immunity for clearance; however, the initial host protein that recognizes MNV-1 infection is unknown. Because noroviruses are RNA viruses, we investigated whether MDA5 and TLR3, cellular sensors that recognize dsRNA, are important for the host response to MNV-1. We demonstrate that MDA5−/− dendritic cells(DC) have a defect in cytokine response to MNV-1. In addition, MNV-1 replicates to higher levels in MDA5−/− DCs as well as in MDA5−/− mice in vivo. Interestingly, TLR3−/− DCs do not have a defect in vitro, but TLR3−/− mice have a slight increase in viral titers. This is the first demonstration of an innate immune sensor for norovirus and shows that MDA5 is required for the control of MNV-1 infection. Knowledge of the host response to MNV-1 may provide keys for prevention and treatment of the human disease

Inherent Structural Disorder and Dimerisation of Murine Norovirus NS1-2 Protein

Human noroviruses are highly infectious viruses that cause the majority of acute, non-bacterial epidemic gastroenteritis cases worldwide. The first open reading frame of the norovirus RNA genome encodes for a polyprotein that is cleaved by the viral protease into six non-structural proteins. The first non-structural protein, NS1-2, lacks any significant sequence similarity to other viral or cellular proteins and limited information is available about the function and biophysical characteristics of this protein. Bioinformatic analyses identified an inherently disordered region (residues 1–142) in the highly divergent N-terminal region of the norovirus NS1-2 protein. Expression and purification of the NS1-2 protein of Murine norovirus confirmed these predictions by identifying several features typical of an inherently disordered protein. These were a biased amino acid composition with enrichment in the disorder promoting residues serine and proline, a lack of predicted secondary structure, a hydrophilic nature, an aberrant electrophoretic migration, an increased Stokes radius similar to that predicted for a protein from the pre-molten globule family, a high sensitivity to thermolysin proteolysis and a circular dichroism spectrum typical of an inherently disordered protein. The purification of the NS1-2 protein also identified the presence of an NS1-2 dimer in Escherichia coli and transfected HEK293T cells. Inherent disorder provides significant advantages including structural flexibility and the ability to bind to numerous targets allowing a single protein to have multiple functions. These advantages combined with the potential functional advantages of multimerisation suggest a multi-functional role for the NS1-2 protein

Definitions and pathophysiology of vasoplegic shock.

Vasoplegia is the syndrome of pathological low systemic vascular resistance, the dominant clinical feature of which is reduced blood pressure in the presence of a normal or raised cardiac output. The vasoplegic syndrome is encountered in many clinical scenarios, including septic shock, post-cardiac bypass and after surgery, burns and trauma, but despite this, uniform clinical definitions are lacking, which renders translational research in this area challenging. We discuss the role of vasoplegia in these contexts and the criteria that are used to describe it are discussed. Intrinsic processes which may drive vasoplegia, such as nitric oxide, prostanoids, endothelin-1, hydrogen sulphide and reactive oxygen species production, are reviewed and potential for therapeutic intervention explored. Extrinsic drivers, including those mediated by glucocorticoid, catecholamine and vasopressin responsiveness of the blood vessels, are also discussed. The optimum balance between maintaining adequate systemic vascular resistance against the potentially deleterious effects of treatment with catecholamines is as yet unclear, but development of novel vasoactive agents may facilitate greater understanding of the role of the differing pathways in the development of vasoplegia. In turn, this may provide insights into the best way to care for patients with this common, multifactorial condition

Host Iron Binding Proteins Acting as Niche Indicators for Neisseria meningitidis

Neisseria meningitidis requires iron, and in the absence of iron alters its gene expression to increase iron acquisition and to make the best use of the iron it has. During different stages of colonization and infection available iron sources differ, particularly the host iron-binding proteins haemoglobin, transferrin, and lactoferrin. This study compared the transcriptional responses of N. meningitidis, when grown in the presence of these iron donors and ferric iron, using microarrays