Introduction Patients with COVID‐19 are known to have a coagulopathy with a thrombosis risk. It is unknown whether this is due to a generalized humoral prothrombotic state or endothelial factors such as inflammation and dysfunction. The aim was to further characterize thrombin generation using a novel analyser (ST Genesia, Diagnostica Stago, Asnières, France) and a panel of haematological analytes in patients with COVID‐19. Methods Platelet poor plasma of 34 patients with noncritical COVID‐19 was compared with 75 patients with critical COVID‐19 (as defined by WHO criteria) in a retrospective study by calibrated automated thrombography and ELISA. Patients were matched for baseline characteristics of age and gender. Results Critical patients had significantly increased fibrinogen, CRP, interleukin‐6 and D‐dimer compared to noncritical patients. Thrombin generation, in critical patients, was right shifted without significant differences in peak, velocity index or endogenous thrombin potential. Tissue plasminogen activator (tPA), tissue factor pathway inhibitor (TFPI) and vascular endothelial growth factor (VEGF) were significantly increased in the critical versus noncritical patients. Critically ill patients were on haemodiafiltration (31%; heparin used in the circuit) or often received escalated prophylactic low‐molecular weight heparin. Conclusion These results confirm increased fibrinogen and D‐dimer in critical COVID‐19‐infected patients. Importantly, disease severity did not increase thrombin generation (including thrombin‐antithrombin complexes and prothrombin fragment 1 + 2) when comparing both cohorts; counter‐intuitively critical patients were hypocoaguable. tPA, TFPI and VEGF were increased in critical patients, which are hypothesized to reflect endothelial dysfunction and/or contribution of heparin (which may cause endothelial TFPI/tPA release).

Hepatitis C virus NS5A has three structural domains, is required for RNA replication and virion assembly, and exists in hypoand hyperphosphorylated forms. Accumulated data suggest that phosphorylation is involved in modulating NS5A functions. We performed a mutational analysis of highly conserved serine residues in the linker region between domains I and II of genotype 2a JFH1 NS5A. As with genotype 1b Con1 NS5A, we found that specific serine residues were important for efficient hyperphosphorylation of JFH1 NS5A. However, in contrast with Con1 replicons, we observed a positive correlation between hyperphosphorylation and JFH1 replicon replication. We previously demonstrated trans-complementation of a hyperphosphorylation-deficient, replication-defective JFH1 replicon. Our results suggested that the defective NS5A encoded by this replicon, while lacking one NS5A function, was capable of performing a separate replication function. In this report, we examined an additional set of replication-defective NS5A mutations in trans-complementation assays. While some behaved similarly to the S232I replicon, others displayed a unique trans-complementation phenotype, suggesting that NS5A trans-complementation can occur by two distinct modes. Moreover, we were able, for the first time, to demonstrate intragenic complementation of replication-defective NS5A alleles. Our results identified three complementation groups: group A, comprising mutations within NS5A domain I; group B, comprising mutations affecting serine residues important for hyperphosphorylation and a subset of the domain I mutations; and group C, comprising a single mutation within the C-terminal region of domain II. We postulate that these complementation groups define three distinct and genetically separable functions of NS5A in RNA replication. H epatitis C virus (HCV) is an enveloped positive-sense singlestranded RNA virus of the genus Hepacivirus in the familyFlaviviridae. The ϳ9.6-kb HCV genome encodes an ϳ3,000-amino-acid (aa) polyprotein that is cleaved by viral and cellular proteases into 10 mature proteins: core, E1, E2, P7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B (reviewed in references 1 and 2). Core is the viral capsid protein, while E1 and E2 are highly glycosylated envelope proteins important for viral entry. P7, a small transmembrane ion channel protein, and NS2, a membrane-associated protease, are required for assembly and release of infectious virions. The remaining nonstructural (NS) proteins are essential components of the HCV RNA replication complex. NS5B is an RNAdependent RNA polymerase that catalyzes synthesis of positiveand negative-strand RNA. NS3 and its cofactor NS4A function as a serine protease responsible for releasing mature NS proteins from the polyprotein precursor. NS3 also possesses RNA helicase and NTPase activities essential for RNA replication. NS4B is an integral membrane protein likely involved in formation of intracellular membranous compartments where viral replication occurs. The final viral component of the replication c...

The NS5A replication complex inhibitor daclatasvir (DCV; BMS-790052) inhibits hybrid replicons containing hepatitis C virus (HCV) genotype 3a (HCV3a) NS5A genes with 50% effective concentrations (EC 50 s) ranging from 120 to 870 pM. Selection studies with a hybrid HCV3a replicon identified NS5A residues 31 and 93 as sites for DCV-selected resistance. Our results support the potential use of DCV as a component in combination therapies for HCV3a chronic infection.

Purpose: Metastatic prostate cancer is a major cause of death of men in the United States. Expression of met, a receptor tyrosine kinase, has been associated with progression of prostate cancer. Experimental Design: To investigate met as a biomarker of disease progression, urinary met was evaluated via ELISA in men with localized (n = 75) and metastatic (n = 81) prostate cancer. Boxplot analysis was used to compare the distribution of met values between each group. We estimated a receiver operating characteristic curve and the associated area under the curve to summarize the diagnostic accuracy of met for distinguishing between localized and metastatic disease. Protein-protein interaction networking via yeast two-hybrid technology supplemented by Ingenuity Pathway Analysis and Human Interactome was used to elucidate proteins and pathways related to met that may contribute to progression of disease. Results: Met distribution was significantly different between the metastatic group and the group with localized prostate cancer and people with no evidence of cancer (P < 0.0001). The area under the curve for localized and metastatic disease was 0.90, with a 95% confidence interval of 0.84 to 0.95. Yeast two-hybrid technology, Ingenuity Pathway Analysis, and Human Interactome identified 89 proteins that interact with met, of which 40 have previously been associated with metastatic prostate cancer. Conclusion: Urinary met may provide a noninvasive biomarker indicative of metastatic prostate cancer and may be a central regulator of multiple pathways involved in prostate cancer progression.

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