Cytoskeletal dynamics and cell signaling during planar polarity establishment in the Drosophila embryonic denticle

Many epithelial cells are polarized along the plane of the epithelium, a property termed planar cell polarity. Th

The antiviral protein viperin inhibits HCV replication via interaction with NS5A

The interferon-stimulated gene viperin has been shown to have antiviral activity against hepatitis C virus (HCV) in the context of the HCV replicon, although the molecular mechanisms responsible are not well understood. Here we demonstrate that viperin plays an integral part in the ability of interferon to limit replication of cell culture derived HCV (JFH-1) that accurately reflects the complete viral life cycle. Using confocal microscopy and Fluorescence Resonance Energy Transfer (FRET) analysis we demonstrate that viperin localizes and interacts with HCV NS5A at the lipid droplet interface. In addition viperin also associates with NS5A and the pro-viral cellular factor, VAP-A at the HCV replication complex. The ability of viperin to limit HCV replication was dependent on residues within the C-terminus as well as an N-terminal amphipathic helix. Removal of the amphipathic helix redirected viperin from the cytosolic face of the ER and the lipid droplet to a homogenous cytoplasmic distribution, coinciding with a loss of antiviral effect. C-terminal viperin mutants still localized to the lipid droplet interface and replication complexes but did not interact with NS5A proteins as determined by FRET analysis. In conclusion we propose that viperin interacts with NS5A and the host factor VAP-A to limit HCV replication at the replication complex. This highlights the complexity of host control of viral replication by interferon stimulated gene expression

Viperin is induced following dengue virus type-2 (DENV-2) infection and has anti-viral actions requiring the C-terminal end of viperin

The host protein viperin is an interferon stimulated gene (ISG) that is up-regulated during a number of viral infections. In this study we have shown that dengue virus type-2 (DENV-2) infection significantly induced viperin, co-incident with production of viral RNA and via a mechanism requiring retinoic acid-inducible gene I (RIG-I). Viperin did not inhibit DENV-2 entry but DENV-2 RNA and infectious virus release was inhibited in viperin expressing cells. Conversely, DENV-2 replicated to higher tires earlier in viperin shRNA expressing cells. The anti-DENV effect of viperin was mediated by residues within the C-terminal 17 amino acids of viperin and did not require the N-terminal residues, including the helix domain, leucine zipper and S-adenosylmethionine (SAM) motifs known to be involved in viperin intracellular membrane association. Viperin showed co-localisation with lipid droplet markers, and was co-localised and interacted with DENV-2 capsid (CA), NS3 and viral RNA. The ability of viperin to interact with DENV-2 NS3 was associated with its anti-viral activity, while co-localisation of viperin with lipid droplets was not. Thus, DENV-2 infection induces viperin which has anti-viral properties residing in the C-terminal region of the protein that act to restrict early DENV-2 RNA production/accumulation, potentially via interaction of viperin with DENV-2 NS3 and replication complexes. These anti-DENV-2 actions of viperin show both contrasts and similarities with other described anti-viral mechanisms of viperin action and highlight the diverse nature of this unique anti-viral host protein.Karla J. Helbig, Jillian M. Carr, Julie K. Calvert, Satiya Wati, Jennifer N. Clarke, Nicholas S. Eyre, Sumudu K. Narayana, Guillaume N. Fiches, Erin M. McCartney, Michael R. Bear

Cytoskeletal dynamics and cell signaling during planar polarity establishment in the Drosophila embryonic denticle.

Many epithelial cells are polarized along the plane of the epithelium, a property termed planar cell polarity. The Drosophila wing and eye imaginal discs are the premier models of this process. Many proteins required for polarity establishment and its translation into cytoskeletal polarity were identified from studies of those tissues. More recently, several vertebrate tissues have been shown to exhibit planar cell polarity. Striking similarities and differences have been observed when different tissues exhibiting planar cell polarity are compared. Here we describe a new tissue exhibiting planar cell polarity - the denticles, hair-like projections of the Drosophila embryonic epidermis. We describe in real time the changes in the actin cytoskeleton that underlie denticle development, and compare this with the localization of microtubules, revealing new aspects of cytoskeletal dynamics that may have more general applicability. We present an initial characterization of the localization of several actin regulators during denticle development. We find that several core planar cell polarity proteins are asymmetrically localized during the process. Finally, we define roles for the canonical Wingless and Hedgehog pathways and for core planar cell polarity proteins in denticle polarity.</p

Folic acid supplementation in children with sickle cell disease: study protocol for a double-blind randomized cross-over trial

Background: Sickle cell disease (SCD) is a genetic disorder which causes dysfunctional red blood cells (RBC) and is thought to increase requirements for folate, an essential B vitamin, due to increased RBC production and turnover in the disease. High-dose supplementation with 1–5 mg/d folic acid, synthetic folate, has been the standard recommendation for children with SCD. There is concern about whether children with SCD need such high doses of folic acid, following mandatory folic acid fortification of enriched grains in Canada, and advancements in medical therapies which extend the average lifespan of RBCs. In animal and human studies, high folic acid intakes (1 mg/d) have been associated with accelerated growth of some cancers, and the biological effects of circulating unmetabolized folic acid (UMFA), which can occur with doses of folic acid ≥ 0.2 mg/d, are not fully understood. The objective of this study is to determine efficacy of, and alterations in folate metabolism from high-dose folic acid in children with SCD during periods of folic acid supplementation versus no supplementation. Methods: In this double-blind randomized controlled cross-over trial, children with SCD (n = 36, aged 2–19 years) will be randomized to either receive 1 mg/d folic acid, the current standard of care, or a placebo for 12 weeks. After a 12-week washout period, treatments will be reversed. Total folate concentrations (serum and RBC), different folate forms (including UMFA), folate-related metabolites, and clinical outcomes will be measured at baseline and after treatment periods. The sum of the values measured in the two periods will be calculated for each subject and compared across the two sequence groups by means of a test for independent samples for the primary (RBC folate concentrations) and secondary (UMFA) outcomes. Dietary intake will be measured at the beginning of each study period. Discussion: As the first rigorously designed clinical trial in children with SCD, this trial will inform and assess current clinical practice, with the ultimate goal of improving nutritional status of children with SCD. Trial registration: ClinicalTrials.gov NCT04011345 . Registered on July 8, 2019Land and Food Systems, Faculty ofMedicine, Faculty ofOther UBCNon UBCPathology and Laboratory Medicine, Department ofPediatrics, Department ofPopulation and Public Health (SPPH), School ofReviewedFacult

Pliocene-Pleistocene evolution of sea surface and intermediate water temperatures from the southwest Pacific

Over the last 5 million years, the global climate system has evolved toward a colder mean state, marked by large-amplitude oscillations in continental ice volume. Equatorward expansion of polar waters and strengthening temperature gradients have been detected. However, the response of the mid latitudes and high latitudes of the Southern Hemisphere is not well documented, despite the potential importance for climate feedbacks including sea ice distribution and low-high latitude heat transport. Here we reconstruct the Pliocene-Pleistocene history of both sea surface and Antarctic Intermediate Water (AAIW) temperatures on orbital time scales from Deep Sea Drilling Project Site 593 in the Tasman Sea, southwest Pacific. We confirm overall Pliocene-Pleistocene cooling trends in both the surface ocean and AAIW, although the patterns are complex. The Pliocene is warmer than modern, but our data suggest an equatorward displacement of the subtropical front relative to present and a poleward displacement of the subantarctic front of the Antarctic Circumpolar Current (ACC). Two main intervals of cooling, from ~3 Ma and ~1.5 Ma, are coeval with cooling and ice sheet expansion noted elsewhere and suggest that equatorward expansion of polar water masses also characterized the southwest Pacific through the Pliocene-Pleistocene. However, the observed trends in sea surface temperature and AAIW temperature are not identical despite an underlying link to the ACC, and intervals of unusual surface ocean warmth (~2 Ma) and large-amplitude variability in AAIW temperatures (from ~1 Ma) highlight complex interactions between equatorward displacements of fronts associated with the ACC and/or varying poleward heat transport from the subtropics

Alcohol metabolism increases the replication of hepatitis C virus and attenuates the antiviral action of interferon

The interactions between hepatitis C virus (HCV) and alcohol metabolism are not well understood. To determine the effect that alcohol metabolism has on HCV replication and the antiviral action of interferon (IFN), Huh-7 cells that harbor HCV replication and metabolize ethanol via the introduced expression of cytochrome P450 2E1 (Cyp2e1) were treated with ethanol and IFN-alpha. Treatment of these cells with ethanol (0-100 mmol/L) significantly increased HCV replication. This effect was dependent on Cyp2e1 expression and alcohol-metabolized oxidative stress (OS), because the antioxidant N-acetylcysteine blocked this effect. Furthermore, the anti-HCV action of IFN-alpha was attenuated in the presence of ethanol metabolism, most likely via attenuation of Stat1 tyrosine-701 phosphorylation. These in vitro results mimic what is often noted clinically, and further dissection of this model system will aid in our understanding of interactions between HCV and alcohol metabolism.Erin M. McCartney, Ljiljana Semendric, Karla J. Helbig, Susan Hinze, Brett Jones, Steven A. Weinman and Michael R. Bear

Transcriptional gene silencing of HIV-1 through promoter targeted RNA is highly specific

We have previously reported induction of transcriptional gene silencing (TGS) of HIV-1 by short hairpin RNA (shRNA) expressed in MOLT-4 cells. The shRNA (termed shPromA) targets the highly conserved tandem NFκB binding sequences of the HIV-1 promoter. Recent articles have reported that TGS mediated by promoter-targeted siRNAs was exclusively the result of sequence non-specific off-target effects. Specifically, several mismatched siRNAs to the target promoter sequences were reported to also induce significant TGS, suggesting TGS was a consequence of off-target effects. Here, following extensive investigation, we report that shPromA induces sequence specific transcriptional silencing in HIV-1 infection in MOLT-4 cells, while four shRNA variants, mismatched by 2–3 nucleotides, fail to suppress viral replication. We confirm similar levels of shRNA expression from the U6 promoter and the presence of processed/cleaved siRNAs for each construct in transduced MOLT-4 cells. HIV-1 sequence specific shPromA does not suppress HIV-2, which has an alternate NFκB binding sequence. As a result of the unique sequence targeted, shPromA does not induce downregulation of other NFκB driven genes, either at the mRNA or protein level. Furthermore, we confirmed shPromA does not have sequence non-specific off-target effects through unaltered expression of CD4, CXCR4 and CCR5, which are used for viral entry. Additionally, shPromA does not alter PKR, IFN levels, and three downstream mediators of IFNα response genes. Our data clearly shows that shPromA achieved highly specific TGS of HIV-1, demonstrating that effective TGS can be induced with minimal off-target effects

Viperin is anti-viral in primary MDM.

<p>Primary MDM were generated from peripheral blood and transduced with lentiviral particles expressing control td-Tomato or WT viperin. At 24 h post transduction, cells were infected with DENV-2 (MOI = 3). (<b>A</b>) Supernatant was sampled and infectious virus release quantitated by plaque assay. Values represent average ± SEM (n = 3). * p<0.001; (<b>B</b>) Viperin lenti-transduced MDM were DENV-2 or mock infected and at 48 h pi cells were fixed and immunolabelled for viperin and DENV with detection of complexes with Alexa-647 (red) and Alexa-488 (green), respectively. Nuclei were stained with Hoechst (blue) and images collected by confocal microscopy.</p