Major alteration in coxsackievirus B3 genomic RNA structure distinguishes a virulent strain from an a virulent strain

Coxsackievirus B3 (CV-B3) is a cardiovirulent enterovirus that utilizes a 5′ untranslated region (5′UTR) to complete critical viral processes. Here, we directly compared the structure of a 5′UTR from a virulent strain with that of a naturally occurring avirulent strain. Using chemical probing analysis, we identified a structural difference between the two 5′UTRs in the highly substituted stem-loop II region (SLII). For the remainder of the 5′UTR, we observed conserved structure. Comparative sequence analysis of 170 closely related enteroviruses revealed that the SLII region lacks conservation. To investigate independent folding and function, two chimeric CV-B3 strains were created by exchanging nucleotides 104–184 and repeating the 5′UTR structural analysis. Neither the parent SLII nor the remaining domains of the background 5′UTR were structurally altered by the exchange, supporting an independent mechanism of folding and function. We show that the attenuated 5′UTR lacks structure in the SLII cardiovirulence determinant

Damaging Cardiac and Cancer Genetic Variants in the LVAD Population

Background: Next generation sequencing technology, coupled with population genetic databases, have made broad genetic evaluation relatively inexpensive and widely available. Our objective was to assess the prevalence of potentially damaging cancer and cardiac gene variants in advanced non-ischemic cardiomyopathy patients. Methods: Explanted human heart tissue procured at LVAD placement was obtained from the University of Nebraska Medical Center Heart Tissue Bank. Genomic DNA was isolated from tissues and amplified by PCR using targeted ampliseq primer pools from an inherited disease panel. Individual libraries were amplified by emulsion PCR on Ion Sphere particles and sequencing was performed on a PGM sequencer (Ion torrent) using the Ion 316 chip. The Ion Torrent browser suite was used to map the reads and call the variants. The identified single nucleotide polymorphisms, insertions, and deletions were then annotated and characterized with ANNOVAR. Non-synonymous mutations with a population frequency of less than or equal to 1% were identified and analyzed utilizing an open source integrative genomics viewer. Amino acid substitution effects on protein function were determined by a bioinformatics algorithm. Myocardial recovery was defined as an improvement in EF to greater than 45% at three months post implant. Results: Our sample population included 12 males and 2 females with an average age of 49 and an average EF at presentation of 17%. Damaging cardiac gene variants were present in 11/14 patients. Only 1 of the 11 patients with damaging cardiac gene variants improved their ejection fraction to greater than 45% post LVAD. Two of the 2 patients without mutations improved their ejection fraction to greater than 45%, p-value=.04. Nine of the 14 patients in this population had damaging oncogene mutations. Conclusions: Damaging variants in cancer and cardiac genes are common in end-stage non-ischemic cardiomyopathy patients undergoing LVAD placement. Genetic variation likely contributes to disease progression and cancer risk

MOESM1 of A formalin-free method for stabilizing cells for nucleic acid amplification, hybridization and next-generation sequencing

Additional file 1: Table S1. Table of actin PCR primers and probes

Targeted myocardial gene expression in failing hearts by RNA sequencing

Abstract Background Myocardial recovery with left ventricular assist device (LVAD) therapy is highly variable and difficult to predict. Next generation ribonucleic acid (RNA) sequencing is an innovative, rapid, and quantitative approach to gene expression profiling in small amounts of tissue. Our primary goal was to identify baseline transcriptional profiles in non-ischemic cardiomyopathies that predict myocardial recovery in response to LVAD therapy. We also sought to verify transcriptional differences between failing and non-failing human hearts. Methods RNA was isolated from failing (n = 16) and non-failing (n = 8) human hearts. RNA from each patient was reverse transcribed and quantitatively sequenced on the personal genome machine (PGM) sequencer (Ion torrent) for 95 heart failure candidate genes. Coverage analysis as well as mapping the reads and alignment was done using the Ion Torrent Browser Suite™. Differential expression analyses were conducted by empirical analysis of digital gene expression data in R (edgeR) to identify differential expressed genes between failing and non-failing groups, and between responder and non-responder groups respectively. Targeted cardiac gene messenger RNA (mRNA) expression was analyzed in proportion to the total number of reads. Gene expression profiles from the PGM sequencer were validated by performing RNA sequencing (RNAseq) with the Illumina Hiseq2500 sequencing system. Results The failing sample population was 75% male with an average age of 50 and a left ventricular ejection fraction (LVEF) of 16%. Myosin light chain kinase (MYLK) and interleukin (IL)-6 genes expression were significantly higher in LVAD responders compared to non-responders. Thirty-six cardiac genes were expressed differentially between failing and non-failing hearts (23 decreased, 13 elevated). MYLK, Beta-1 adrenergic receptor (ADRB1) and myosin heavy chain (MYH)-6 expression were among those significantly decreased in failing hearts compared to non-failing hearts. Natriuretic peptide B (NPPB) and IL-6 were significantly elevated. Targeted gene expression profiles obtained from the Ion torrent PGM sequencer were consistent with those obtained from Illumina HiSeq2500 sequencing system. Conclusions Heart failure is associated with a network of transcriptional changes involving contractile proteins, metabolism, adrenergic receptors, protein phosphorylation, and signaling factors. Myocardial MYLK and IL-6 expression are positively correlated with ejection fraction (EF) response to LVAD placement. Targeted RNA sequencing of myocardial gene expression can be utilized to predict responders to LVAD therapy and to better characterize transcriptional changes in human heart failure