Summary Hypertrophic obstructive cardiomyopathy is an inherited myocardial disease defined by cardiac hypertrophy (wall thickness ≥15 mm) that is not explained by abnormal loading conditions, and ...left ventricular obstruction greater than or equal to 30 mm Hg. Typical symptoms include dyspnoea, chest pain, palpitations, and syncope. The diagnosis is usually suspected on clinical examination and confirmed by imaging. Some patients are at increased risk of sudden cardiac death, heart failure, and atrial fibrillation. Patients with an increased risk of sudden cardiac death undergo cardioverter-defibrillator implantation; in patients with severe symptoms related to ventricular obstruction, septal reduction therapy (myectomy or alcohol septal ablation) is recommended. Life-long anticoagulation is indicated after the first episode of atrial fibrillation.
Many cellular mRNAs contain the modified base m6A, and recent studies have suggested that various stimuli can lead to changes in m6A. The most common method to map m6A and to predict changes in m6A ...between conditions is methylated RNA immunoprecipitation sequencing (MeRIP-seq), through which methylated regions are detected as peaks in transcript coverage from immunoprecipitated RNA relative to input RNA. Here, we generated replicate controls and reanalyzed published MeRIP-seq data to estimate reproducibility across experiments. We found that m6A peak overlap in mRNAs varies from ~30 to 60% between studies, even in the same cell type. We then assessed statistical methods to detect changes in m6A peaks as distinct from changes in gene expression. However, from these published data sets, we detected few changes under most conditions and were unable to detect consistent changes across studies of similar stimuli. Overall, our work identifies limits to MeRIP-seq reproducibility in the detection both of peaks and of peak changes and proposes improved approaches for analysis of peak changes.
N6-methyladenosine (m⁶A) RNA methylation is the most abundant epitranscriptomic modification of eukaryotic messenger RNAs (mRNAs). Previous reports have found m⁶A on both cellular and viral ...transcripts and defined its role in regulating numerous biological processes, including viral infection. Here, we show that m⁶A and its associated machinery regulate the life cycle of hepatitis B virus (HBV). HBV is a DNA virus that completes its life cycle via an RNA intermediate, termed pregenomic RNA (pgRNA). Silencing of enzymes that catalyze the addition of m⁶A to RNA resulted in increased HBV protein expression, but overall reduced reverse transcription of the pgRNA. We mapped the m⁶A site in the HBV RNA and found that a conserved m⁶A consensus motif situated within the epsilon stem loop structure, is the site for m⁶A modification. The epsilon stem loop is located in the 3′ terminus of all HBV mRNAs and at both the 5′ and 3′ termini of the pgRNA. Mutational analysis of the identified m⁶A site in the 5′ epsilon stem loop of pgRNA revealed that m⁶A at this site is required for efficient reverse transcription of pgRNA, while m⁶A methylation of the 3′ epsilon stem loop results in destabilization of all HBV transcripts, suggesting that m⁶A has dual regulatory function for HBV RNA. Overall, this study reveals molecular insights into how m⁶A regulates HBV gene expression and reverse transcription, leading to an increased level of understanding of the HBV life cycle.
RNA modifications go viral Gokhale, Nandan S; Horner, Stacy M
PLoS pathogens,
03/2017, Letnik:
13, Številka:
3
Journal Article
Recenzirano
Odprti dostop
About the Authors: Nandan S. Gokhale Affiliation: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, United States of America Stacy M. Horner * ...E-mail: stacy.horner@duke.edu Affiliations Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, United States of America, Department of Medicine, Duke University Medical Center, Durham, North Carolina, United States of America ORCID http://orcid.org/0000-0002-9351-7409Citation: Gokhale NS, Horner SM (2017) RNA modifications go viral. Funding: Research in the Horner Lab is supported by the National Institutes of Health (R01AI125416, R21NS100545, and R21AI124100), the Duke University Center for AIDS Research (P30AI064518), and a Duke School of Medicine Whitehead Scholarship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ...the microRNA miR-122 interacts with the viral RNA genome of hepatitis C virus (HCV) and is required for HCV replication 1. The deposition of m6A at the consensus motif DRAmCH (where D = G/A/U, R = G > A, and H = U/C/A) is governed by a cellular methyltransferase complex composed of the “writers” METTL3 and METTL14, and other noncatalytic cofactors. m6A modification can be reversed by the “erasers” FTO and ALKBH5. *We note that FTO has recently been found to have greater specificity for the m6Am modifications present in mRNA cap structures than for m6A 34. The role of m6A and the m6A machinery in RNA function and biological processes is further reviewed in 2. https://doi.org/10.1371/journal.ppat.1006188.g001 A role for m6A in viral...
In recent years, the RNA modification
N6
-methyladenosine (m
6
A) has been found to play a role in the life cycles of numerous viruses and also in the cellular response to viral infection. m
6
A has ...emerged as a regulator of many fundamental aspects of RNA biology. Here, we highlight recent advances in techniques for the study of m
6
A, as well as advances in our understanding of the cellular machinery that controls the addition, removal, recognition, and functions of m
6
A. We then summarize the many newly discovered roles of m
6
A during viral infection, including how it regulates innate and adaptive immune responses to infection. Overall, the goals of this review are to summarize the roles of m
6
A on both cellular and viral RNAs and to describe future directions for uncovering new functions of m
6
A during infection.
Echocardiographic findings vary with shock severity, as defined by the Society for Cardiovascular Angiography and Intervention (SCAI) shock stage. Left ventricular stroke work index (LVSWI) measured ...by transthoracic echocardiography (TTE) can predict mortality in the cardiac intensive care unit (CICU). We sought to determine whether LVSWI could refine mortality risk stratification by the SCAI shock classification in the CICU.
We included consecutive CICU patients from 2007 to 2015 with TTE data available to calculate the LVSWI, specifically the mean arterial pressure, stroke volume index and medial mitral E/e' ratio. In-hospital mortality as a function of LVSWI was evaluated across the SCAI shock stages using logistic regression, before and after multivariable adjustment.
We included 3635 unique CICU patients, with a mean age of 68.1 ± 14.5 years (36.5% females); 61.1% of patients had an acute coronary syndrome. The LVSWI progressively decreased with increasing shock severity, as defined by increasing SCAI shock stage. A total of 203 (5.6%) patients died during hospitalization, with higher in-hospital mortality among patients with lower LVSWI (adjusted OR 0.66 per 10 J/m2 higher) or higher SCAI shock stage (adjusted OR 1.24 per each higher stage). A LVSWI <33 J/m2 was associated with higher adjusted in-hospital mortality, particularly among patients with shock (SCAI stages C, D and E).
The LVSWI by TTE noninvasively characterizes the severity of shock, including both systolic and diastolic parameters, and can identify low-risk and high-risk patients at each level of clinical shock severity.
Hosts have evolved diverse strategies to respond to microbial infections, including the detection of pathogen-encoded proteases by inflammasome-forming sensors such as NLRP1 and CARD8. Here, we find ...that the 3CL protease (3CLpro) encoded by diverse coronaviruses, including Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), cleaves a rapidly evolving region of human CARD8 and activates a robust inflammasome response. CARD8 is required for cell death and the release of pro-inflammatory cytokines during SARS-CoV-2 infection. We further find that natural variation alters CARD8 sensing of 3CLpro, including 3CLpro-mediated antagonism rather than activation of megabat CARD8. Likewise, we find that a single nucleotide polymorphism (SNP) in humans reduces CARD8's ability to sense coronavirus 3CLpros and, instead, enables sensing of 3C proteases (3Cpro) from select picornaviruses. Our findings demonstrate that CARD8 is a broad sensor of viral protease activities and suggests that CARD8 diversity contributes to inter- and intraspecies variation in inflammasome-mediated viral sensing and immunopathology.
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