Characterization of zebrafish coagulation cofactors Fviii and Fv mutants and modeling hemophilia A and factor V deficiency.

The aim of this study is to characterize zebrafish coagulation cofactors fviii and fv mutant fish and assess if they phenocopy classical hemophilia A and factor V deficiency in humans. The embryos from fviii and fv zebrafish heterozygote mutants generated by ENU mutagenesis were purchased from the ZIRC repository. They were reared to adulthood and genotyped. The heterozygote male and female were crossed to get homozygote, heterozygote, and wild-type fish. Functional kinetic coagulation assays and bleeding assays were performed on normal and mutant adult fish, and venous laser injury assays were performed on the larvae. The DNA from fviii and fv mutants were sequenced to confirm if they have a premature stop codon in exon 19, and in exon 2, respectively, and in both mutants, the amino acid glutamine is replaced with a stop codon. Homozygous and heterozygous 5 days post fertilization (dpf) larvae for fviii and fv deficient mutants exhibited prolonged time to occlusion after venous laser injury compared to wild-type controls. The homozygous and heterozygous fviii adult mutants showed modest bleeding and delayed fibrin formation in the kinetic partial thromboplastin time (kPTT) assay with their plasma. fv homozygous larvae had poor survival beyond 12 dpf. However, heterozygous fv mutants exhibited heavy bleeding and prolonged fibrin formation in the kPTT and kPT assay compared with wild-type siblings. Our characterization showed fviii and fv mutants from ZIRC phenocopied to a considerable extent classical hemophilia A and factor V deficiency in humans, respectively. These models should be useful in studying and developing novel drugs that reverse the phenotype and in generating suppressor mutations to identify novel factors that compensate for these deficiencies.

IFIT1 is rapidly evolving and exhibits disparate antiviral activities across 11 mammalian orders.

Mammalian mRNAs possess an N7-methylguanosine (m7G) cap and 2'O methylation of the initiating nucleotide at their 5' end, whereas certain viral RNAs lack these characteristic features. The human antiviral restriction factor IFIT1 recognizes and binds to specific viral RNAs that lack the 5' features of host mRNAs, resulting in targeted suppression of viral RNA translation. This interaction imposes a significant host-driven evolutionary pressure on viruses, and many viruses have evolved mechanisms to evade the antiviral action of human IFIT1. However, less is known about the virus-driven pressures that may have shaped the antiviral activity of IFIT1 genes across mammals. Here, we take an evolution-guided approach to show that the IFIT1 gene is rapidly evolving in multiple mammalian clades, with positive selection acting upon several residues in distinct regions of the protein. In functional assays with 39 IFIT1s spanning diverse mammals, we demonstrate that IFIT1 exhibits a range of antiviral phenotypes, with many orthologs lacking antiviral activity against viruses that are strongly suppressed by other IFIT1s. We further show that IFIT1s from human and a bat, the black flying fox, inhibit Venezuelan equine encephalitis virus (VEEV) and strongly bind to Cap0 RNAs. Unexpectedly, chimpanzee IFIT1, which differs from human IFIT1 by only 8 amino acids, does not inhibit VEEV infection and exhibits minimal Cap0 RNA-binding. In mutagenesis studies, we determine that amino acids 364 and 366, the latter of which is undergoing positive selection, are sufficient to confer the differential anti-VEEV activity between human and chimpanzee IFIT1. These data suggest that virus-host genetic conflicts have influenced the antiviral specificity of IFIT1 across diverse mammalian orders.

Interferon inhibits a model RNA virus via a limited set of inducible effector genes.

Interferons control viral infection by inducing the expression of antiviral effector proteins encoded by interferon-stimulated genes (ISGs). The field has mostly focused on identifying individual antiviral ISG effectors and defining their mechanisms of action. However, fundamental gaps in knowledge about the interferon response remain. For example, it is not known how many ISGs are required to protect cells from a particular virus, though it is theorized that numerous ISGs act in concert to achieve viral inhibition. Here, we used CRISPR-based loss-of-function screens to identify a markedly limited set of ISGs that confer interferon-mediated suppression of a model alphavirus, Venezuelan equine encephalitis virus (VEEV). We show via combinatorial gene targeting that three antiviral effectors-ZAP, IFIT3, and IFIT1-together constitute the majority of interferon-mediated restriction of VEEV, while accounting for < 0.5% of the interferon-induced transcriptome. Together, our data suggest a refined model of the antiviral interferon response in which a small subset of "dominant" ISGs may confer the bulk of the inhibition of a given virus.

Interferon inhibits a model RNA virus via a limited set of inducible effector genes.

Interferons control viral infection by inducing the expression of antiviral effector proteins encoded by interferon-stimulated genes (ISGs). The field has mostly focused on identifying individual antiviral ISG effectors and defining their mechanisms of action. However, fundamental gaps in knowledge about the interferon response remain. For example, it is not known how many ISGs are required to protect cells from a particular virus, though it is theorized that numerous ISGs act in concert to achieve viral inhibition. Here, we used CRISPR-based loss-of-function screens to identify a markedly limited set of ISGs that confer interferon-mediated suppression of a model alphavirus, Venezuelan equine encephalitis virus (VEEV). We show via combinatorial gene targeting that three antiviral effectors - ZAP, IFIT3, and IFIT1 - together constitute the majority of interferon-mediated restriction of VEEV, while accounting for less than 0.5% of the interferon-induced transcriptome. Together, our data suggests a refined model of the antiviral interferon response in which a small subset of "dominant" ISGs may confer the bulk of the inhibition of a given virus.

Periprocedural myocardial infarction is associated with increased mortality in patients with coronary artery bifurcation lesions after implantation of a drug-eluting stent.

OBJECTIVES: The present study aimed to investigate the association between periprocedural myocardial infarction (PMI), defined by creatine kinase (CK)-MB or troponin I (TNI) level elevations >5 times the 99 th percentile of the upper reference limit (URL) within 48 hr after implantation of a drug-eluting stent (DES), and one-year mortality in patients with coronary bifurcation. BACKGROUND: PMI is reported to be associated with increased one-year mortality after DES implantation. However, the prevalence and association of PMI with mortality after stenting bifurcation lesions remains unclear. METHODS: We prospectively followed 1,971 patients with true coronary bifurcations who underwent DES implantation as part of the multicenter DEFINITION study. These patients were grouped into categories based on PMI outcome: Non-PMI, CKMB-PMI, TNI-PMI, and CKMB/TNI-PMI. The primary endpoint was the rate of all-cause mortality at one year. RESULTS: PMI occurred in 11.4% of patients by CKMB criteria and 41.3% of patients by TNI criteria. At one-year follow-up, the mortality rate was 2.3% in the entire patient population. However, mortality was significantly higher in the CKMB-PMI (6.4%) and CKMB/TNI-PMI (6.1%) groups compared to the Non-PMI (1.7%) and TNI-PMI (2.1%) groups (all P < 0.05). A 10-fold increase in TNI levels resulted in similar PMI rate (5.2%) and mortality risk (adjusted HR 2.7, 95% CI 3.0-5.2) as a fivefold increase in CKMB levels. CONCLUSIONS: PMI, as defined by CKMB elevations following coronary bifurcation lesion stenting, was associated with increased one-year mortality. Additionally, to attain an equal frequency of PMI, the elevation in TNI levels needed to be twice as high as the elevation in CKMB levels.