The Genetics of Life and Death: Virus-Host Interactions Underpinning Resistance to African Swine Fever, a Viral Hemorrhagic Disease

Pathogen transmission from wildlife hosts to genetically distinct species is a major driver of disease emergence. African swine fever virus (ASFV) persists in sub-Saharan Africa through a sylvatic cycle between warthogs and soft ticks that infest their burrows. The virus does not cause disease in these animals, however transmission of the virus to domestic pigs or wild boar causes a hemorrhagic fever that is invariably fatal. ASFV transmits readily between domestic pigs and causes economic hardship in areas where it is endemic. The virus is also a significant transboundary pathogen that has become established in Eastern Europe, and has recently appeared in China increasing the risk of an introduction of the disease to other pig producing centers. Although a DNA genome mitigates against rapid adaptation of the virus to new hosts, extended epidemics of African swine fever (ASF) can lead to the emergence of viruses with reduced virulence. Attenuation in the field leads to large deletions of genetic material encoding genes involved in modulating host immune responses. Therefore resistance to disease and tolerance of ASFV replication can be dependent on both virus and host factors. Here we describe the different virus-host interfaces and discuss progress toward understanding the genetic determinants of disease outcome after infection with ASFV

Autophagy impairment by African swine fever virus

African swine fever is a devastating disease of domestic swine and wild boar caused by a large double-stranded DNA virus that encodes for more than 150 open reading frames. There is no licensed vaccine for the disease and the most promising current candidates are modified live viruses that have been attenuated by deletion of virulence factors. Like many viruses African swine fever virus significantly alters the host cell machinery to benefit its replication and viral genes that modify host pathways represent promising targets for development of gene deleted vaccines. Autophagy is an important cellular pathway that is involved in cellular homeostasis, innate and adaptive immunity and therefore is manipulated by a number of different viruses. Autophagy is regulated by a complex protein cascade and here we show that African swine fever virus can block formation of autophagosomes, a critical functional step of the autophagy pathway through at least two different mechanisms. Interestingly this does not require the A179L gene that has been shown to interact with Beclin-1, an important autophagy regulator

Association of the phosphodiesterase 4D (PDE4D) gene and cardioembolic stroke in an Australian cohort

Background: Large-scale epidemiological studies support an important role for susceptibility genes in the pathogenesis of ischemic stroke, with phosphodiesterase 4D identified as the first gene predisposing to ischemic stroke. Several single nucleotide polymorphisms within the phosphodiesterase 4D gene have been implicated in the pathogenesis of stroke. Aim: Undertake a multivariate analysis of six single nucleotide polymorphisms within the phosphodiesterase 4D gene in a previously defined Australian stroke cohort, to determine whether these single nucleotide polymorphisms have an association with ischemic stroke. Methods: This case–control study was performed using an existing genetic database of 180 ischemic stroke patients and 301 community controls, evaluated previously for cerebrovascular risk factors (hypertension, hypercholesterolemia, diabetes, paroxysmal atrial fibrillation, smoking and history of stroke in a first-degree relative). Based on previously reported associations with large vessel disease, ischemic stroke, cardioembolic stroke or a mixture of these, six single nucleotide polymorphisms in the phosphodiesterase 4D gene were selected for study, these being single nucleotide polymorphisms 13, 19, rs152312, 45, 83 and 87, based on previously utilized DeCODE nomenclature. Single nucleotide polymorphisms were genotyped using a sequence-specific polymerase chain reaction method and gel electrophoresis. Logistic regression was undertaken to determine the relevance of each polymorphism to stroke. Further analysis was undertaken to determine the risk of stroke following stratification for stroke sub-type and etiology. Results: Significant odds ratios were found to be associated with cardioembolic strokes in two single nucleotide polymorphisms: rs152312 and SNP 45 (P<0·05). Conclusions: Our findings demonstrated an association between cardioembolic stroke and phosphodiesterase 4D single nucleotide polymorphisms rs152312 and 45. No significant association was found for the other four single nucleotide polymorphisms investigated within the phosphodiesterase 4D gene. We propose that the results from this Australian population support the concept that a large prospective international study is required to investigate the role of phosphodiesterase 4D in the cardiogenic cause of ischemic stroke.Austin G. Milton, Verna M. Aykanat, M. Anne Hamilton-Bruce, Mark Nezic, Jim Jannes, Simon A. Kobla

Direct Functionalization of Nitrogen Heterocycles via Rh-Catalyzed C−H Bond Activation

Nitrogen heterocycles are present in many compounds of enormous practical importance, ranging from pharmaceutical agents and biological probes to electroactive materials. Direct functionalization of nitrogen heterocycles through C−H bond activation constitutes a powerful means of regioselectively introducing a variety of substituents with diverse functional groups onto the heterocycle scaffold. Working together, our two groups have developed a family of Rh-catalyzed heterocycle alkylation and arylation reactions that are notable for their high level of functional-group compatibility. This Account describes our work in this area, emphasizing the relevant mechanistic insights that enabled synthetic advances and distinguished the resulting transformations from other methods. We initially discovered an intramolecular Rh-catalyzed C-2 alkylation of azoles by alkenyl groups. That reaction provided access to a number of di-, tri-, and tetracyclic azole derivatives. We then developed conditions that exploited microwave heating to expedite these reactions. While investigating the mechanism of this transformation, we discovered that a novel substrate-derived Rh−N-heterocyclic carbene (NHC) complex was involved as an intermediate. We then synthesized analogous Rh−NHC complexes directly by treating precursors to the intermediate [RhCl(PCy3)2] with N-methylbenzimidazole, 3-methyl-3,4-dihydroquinazoline, and 1-methyl-1,4-benzodiazepine-2-one. Extensive kinetic analysis and DFT calculations supported a mechanism for carbene formation in which the catalytically active RhCl(PCy3)2 fragment coordinates to the heterocycle before intramolecular activation of the C−H bond occurs. The resulting Rh−H intermediate ultimately tautomerizes to the observed carbene complex. With this mechanistic information and the discovery that acid cocatalysts accelerate the alkylation, we developed conditions that efficiently and intermolecularly alkylate a variety of heterocycles, including azoles, azolines, dihydroquinazolines, pyridines, and quinolines, with a wide range of functionalized olefins. We demonstrated the utility of this methodology in the synthesis of natural products, drug candidates, and other biologically active molecules. In addition, we developed conditions to directly arylate these heterocycles with aryl halides. Our initial conditions that used PCy3 as a ligand were successful only for aryl iodides. However, efforts designed to avoid catalyst decomposition led to the development of ligands based on 9-phosphabicyclo[4.2.1]nonane (phoban) that also facilitated the coupling of aryl bromides. We then replicated the unique coordination environment, stability, and catalytic activity of this complex using the much simpler tetrahydrophosphepine ligands and developed conditions that coupled aryl bromides bearing diverse functional groups without the use of a glovebox or purified reagents. With further mechanistic inquiry, we anticipate that researchers will better understand the details of the aforementioned Rh-catalyzed C−H bond functionalization reactions, resulting in the design of more efficient and robust catalysts, expanded substrate scope, and new transformations

Suppression of TGFβ-Induced Epithelial-Mesenchymal Transition Like Phenotype by a PIAS1 Regulated Sumoylation Pathway in NMuMG Epithelial Cells

Epithelial-mesenchymal-transition (EMT) is a fundamental cellular process that is critical for normal development and tumor metastasis. The transforming growth factor beta (TGFβ) is a potent inducer of EMT like effects, but the mechanisms that regulate TGFβ-induced EMT remain incompletely understood. Using the widely employed NMuMG mammary epithelial cells as a model to study TGFβ-induced EMT, we report that TGFβ downregulates the levels of the SUMO E3 ligase PIAS1 in cells undergoing EMT. Gain and loss of function analyses indicate that PIAS1 acts in a SUMO ligase dependent manner to suppress the ability of TGFβ to induce EMT in these cells. We also find that TGFβ inhibits sumoylation of the PIAS1 substrate SnoN, a transcriptional regulator that antagonizes TGFβ-induced EMT. Accordingly, loss of function mutations of SnoN sumoylation impair the ability of SnoN to inhibit TGFβ-induced EMT in NMuMG cells. Collectively, our findings suggest that PIAS1 is a novel negative regulator of EMT and reveal that inhibition of the PIAS1-SnoN sumoylation pathway represents a key mechanism by which TGFβ induces EMT, with important implications in normal development and tumor metastasis

Aquatic Birnavirus-Induced ER Stress-Mediated Death Signaling Contribute to Downregulation of Bcl-2 Family Proteins in Salmon Embryo Cells

Aquatic birnavirus induces mitochondria-mediated cell death, but whether connects to endoplasmic reticulum (ER) stress is still unknown. In this present, we characterized that IPNV infection triggers ER stress-mediated cell death via PKR/eIF2α phosphorylation signaling for regulating the Bcl-2 family protein expression in fish cells. The IPNV infection can induce ER stress as follows: (1) ER stress sensor ATF6 cleavaged; (2) ER stress marker GRP78 upregulation, and (3) PERK/eIF2αphosphorylation. Then, the IPNV-induced ER stress signals can induce the CHOP expression at early (6 h p.i.) and middle replication (12 h p.i.) stages. Moreover, IPNV-induced CHOP upregulation dramatically correlates to apparently downregulate the Bcl-2 family proteins, Bcl-2, Mcl-1 and Bcl-xL at middle replication stage (12 h p.i.) and produces mitochondria membrane potential (MMP) loss and cell death. Furthermore, with GRP78 synthesis inhibitor momitoxin (VT) and PKR inhibitor 2-aminopurine (2-AP) treatment for blocking GRP78 expression and eIF2α phosphorylation, PKR/PERK may involve in eIF2α phosphorylation/CHOP upregulation pathway that enhances the downstream regulators Bcl-2 family proteins expression and increased cell survival. Taken together, our results suggest that IPNV infection activates PKR/PERK/eIF2α ER stress signals for regulating downstream molecules CHOP upregulation and Bcl-2 family downregulation that led to induce mitochondria-mediated cell death in fish cells, which may provide new insight into RNA virus pathogenesis and disease

Antiplatelet therapy with aspirin, clopidogrel, and dipyridamole versus clopidogrel alone or aspirin and dipyridamole in patients with acute cerebral ischaemia (TARDIS): a randomised, open-label, phase 3 superiority trial

Background: Intensive antiplatelet therapy with three agents might be more effective than guideline treatment for preventing recurrent events in patients with acute cerebral ischaemia. We aimed to compare the safety and efficacy of intensive antiplatelet therapy (combined aspirin, clopidogrel, and dipyridamole) with that of guideline-based antiplatelet therapy. Methods: We did an international, prospective, randomised, open-label, blinded-endpoint trial in adult participants with ischaemic stroke or transient ischaemic attack (TIA) within 48 h of onset. Participants were assigned in a 1:1 ratio using computer randomisation to receive loading doses and then 30 days of intensive antiplatelet therapy (combined aspirin 75 mg, clopidogrel 75 mg, and dipyridamole 200 mg twice daily) or guideline-based therapy (comprising either clopidogrel alone or combined aspirin and dipyridamole). Randomisation was stratified by country and index event, and minimised with prognostic baseline factors, medication use, time to randomisation, stroke-related factors, and thrombolysis. The ordinal primary outcome was the combined incidence and severity of any recurrent stroke (ischaemic or haemorrhagic; assessed using the modified Rankin Scale) or TIA within 90 days, as assessed by central telephone follow-up with masking to treatment assignment, and analysed by intention to treat. This trial is registered with the ISRCTN registry, number ISRCTN47823388. Findings: 3096 participants (1556 in the intensive antiplatelet therapy group, 1540 in the guideline antiplatelet therapy group) were recruited from 106 hospitals in four countries between April 7, 2009, and March 18, 2016. The trial was stopped early on the recommendation of the data monitoring committee. The incidence and severity of recurrent stroke or TIA did not differ between intensive and guideline therapy (93 [6%] participants vs 105 [7%]; adjusted common odds ratio [cOR] 0·90, 95% CI 0·67–1·20, p=0·47). By contrast, intensive antiplatelet therapy was associated with more, and more severe, bleeding (adjusted cOR 2·54, 95% CI 2·05–3·16, p<0·0001). Interpretation: Among patients with recent cerebral ischaemia, intensive antiplatelet therapy did not reduce the incidence and severity of recurrent stroke or TIA, but did significantly increase the risk of major bleeding. Triple antiplatelet therapy should not be used in routine clinical practice

In vitro inhibition of monkeypox virus production and spread by Interferon-β

<p>Abstract</p> <p>Background</p> <p>The <it>Orthopoxvirus </it>genus contains numerous virus species that are capable of causing disease in humans, including variola virus (the etiological agent of smallpox), monkeypox virus, cowpox virus, and vaccinia virus (the prototypical member of the genus). Monkeypox is a zoonotic disease that is endemic in the Democratic Republic of the Congo and is characterized by systemic lesion development and prominent lymphadenopathy. Like variola virus, monkeypox virus is a high priority pathogen for therapeutic development due to its potential to cause serious disease with significant health impacts after zoonotic, accidental, or deliberate introduction into a naïve population.</p> <p>Results</p> <p>The purpose of this study was to investigate the prophylactic and therapeutic potential of interferon-β (IFN-β) for use against monkeypox virus. We found that treatment with human IFN-β results in a significant decrease in monkeypox virus production and spread <it>in vitro</it>. IFN-β substantially inhibited monkeypox virus when introduced 6-8 h post infection, revealing its potential for use as a therapeutic. IFN-β induced the expression of the antiviral protein MxA in infected cells, and constitutive expression of MxA was shown to inhibit monkeypox virus infection.</p> <p>Conclusions</p> <p>Our results demonstrate the successful inhibition of monkeypox virus using human IFN-β and suggest that IFN-β could potentially serve as a novel safe therapeutic for human monkeypox disease.</p