Publications by authors named "Thomas Tuschl"

198 Publications

RTEL1 influences the abundance and localization of TERRA RNA.

Nat Commun 2021 05 21;12(1):3016. Epub 2021 May 21.

Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.

Telomere repeat containing RNAs (TERRAs) are a family of long non-coding RNAs transcribed from the subtelomeric regions of eukaryotic chromosomes. TERRA transcripts can form R-loops at chromosome ends; however the importance of these structures or the regulation of TERRA expression and retention in telomeric R-loops remain unclear. Here, we show that the RTEL1 (Regulator of Telomere Length 1) helicase influences the abundance and localization of TERRA in human cells. Depletion of RTEL1 leads to increased levels of TERRA RNA while reducing TERRA-containing R loops at telomeres. In vitro, RTEL1 shows a strong preference for binding G-quadruplex structures which form in TERRA. This binding is mediated by the C-terminal region of RTEL1, and is independent of the RTEL1 helicase domain. RTEL1 binding to TERRA appears to be essential for cell viability, underscoring the importance of this function. Degradation of TERRA-containing R-loops by overexpression of RNAse H1 partially recapitulates the increased TERRA levels and telomeric instability associated with RTEL1 deficiency. Collectively, these data suggest that regulation of TERRA is a key function of the RTEL1 helicase, and that loss of that function may contribute to the disease phenotypes of patients with RTEL1 mutations.
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http://dx.doi.org/10.1038/s41467-021-23299-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8140157PMC
May 2021

Serum MicroRNA Transcriptomics and Acute Rejection or Recurrent Hepatitis C Virus in Human Liver Allograft Recipients: A Pilot Study.

Transplantation 2021 May 11. Epub 2021 May 11.

Division of Nephrology and Hypertension, Joan and Sanford I. Weill Department of Medicine and Department of Transplantation Medicine, New York Presbyterian-Weill Cornell Medicine, New York, NY. Laboratory of RNA Molecular Biology, The Rockefeller University, New York, NY. Department of Psychiatry and Behavioral Science, Stony Brook University, Stony Brook, NY. Division of Biostatistics, Department of Public Health Sciences, University of California at Davis, Davis, CA. Rho Federal Systems, Chapel Hill, NC. Division of Transplantation Pathology, Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA.

Background: Acute rejection (AR) and recurrent HCV (R-HCV) are significant complications in liver allograft recipients. Noninvasive diagnosis of intragraft pathologies may improve their management.

Methods: We performed small RNA sequencing and miRNA microarray profiling of RNA from sera matched to liver allograft biopsies from patients with nonimmune, nonviral (NINV) native liver disease. Absolute levels of informative miRNAs in 91 sera matched to 91 liver allograft biopsies were quantified using customized RT-qPCR assays: 30 biopsy-matched sera from 26 unique NINV patients and 61 biopsy-matched sera from 41 unique R-HCV patients. The association between biopsy diagnosis and miRNA abundance was analyzed by logistic regression and calculating the area under the receiver operating characteristic curve.

Results: Nine miRNAs- miR-22, miR-34a, miR-122, miR-148a, miR-192, miR-193b, miR-194, miR-210 and miR-885-5p- were identified by both sRNA-seq and TLDA to be associated with NINV-AR. Logistic regression analysis of absolute levels of miRNAs and goodness-of-fit of predictors identified a linear combination of miR-34a + miR-210 (P<0.0001) as the best statistical model and miR-122 + miR-210 (P<0.0001) as the best model that included miR-122. A different linear combination of miR-34a + miR-210 (P<0.0001) was the best model for discriminating NINV-AR from R-HCV with intragraft inflammation, and miR-34a + miR-122 (P<0.0001) was the best model for discriminating NINV-AR from R-HCV with intragraft fibrosis.

Conclusions: Circulating levels of miRNAs, quantified using customized RT-qPCR assays, may offer a rapid and noninvasive means of diagnosing AR in human liver allografts and for discriminating AR from intragraft inflammation or fibrosis due to recurrent HCV.Supplemental Visual Abstract; http://links.lww.com/TP/C231.
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http://dx.doi.org/10.1097/TP.0000000000003815DOI Listing
May 2021

Convergence of mammalian RQC and C-end rule proteolytic pathways via alanine tailing.

Mol Cell 2021 05 27;81(10):2112-2122.e7. Epub 2021 Apr 27.

Center for Molecular Biology of Heidelberg University (ZMBH), DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany; Department of Molecular Medicine, Scripps Research, Jupiter, FL 33458, USA. Electronic address:

Incompletely synthesized nascent chains obstructing large ribosomal subunits are targeted for degradation by ribosome-associated quality control (RQC). In bacterial RQC, RqcH marks the nascent chains with C-terminal alanine (Ala) tails that are directly recognized by proteasome-like proteases, whereas in eukaryotes, RqcH orthologs (Rqc2/NEMF [nuclear export mediator factor]) assist the Ltn1/Listerin E3 ligase in nascent chain ubiquitylation. Here, we study RQC-mediated proteolytic targeting of ribosome stalling products in mammalian cells. We show that mammalian NEMF has an additional, Listerin-independent proteolytic role, which, as in bacteria, is mediated by tRNA-Ala binding and Ala tailing. However, in mammalian cells Ala tails signal proteolysis indirectly, through a pathway that recognizes C-terminal degrons; we identify the CRL2 E3 ligase complex and the novel C-end rule E3, Pirh2/Rchy1, as bona fide RQC pathway components that directly bind to Ala-tailed ribosome stalling products and target them for degradation. As Listerin mutation causes neurodegeneration in mice, functionally redundant E3s may likewise be implicated in molecular mechanisms of neurodegeneration.
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http://dx.doi.org/10.1016/j.molcel.2021.03.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8141035PMC
May 2021

In vitro antiviral activity of the anti-HCV drugs daclatasvir and sofosbuvir against SARS-CoV-2, the aetiological agent of COVID-19.

J Antimicrob Chemother 2021 Apr 21. Epub 2021 Apr 21.

Instituto D'Or de Pesquisa e Ensino, Rio de Janeiro, RJ, Brazil.

Background: Current approaches of drug repurposing against COVID-19 have not proven overwhelmingly successful and the SARS-CoV-2 pandemic continues to cause major global mortality. SARS-CoV-2 nsp12, its RNA polymerase, shares homology in the nucleotide uptake channel with the HCV orthologue enzyme NS5B. Besides, HCV enzyme NS5A has pleiotropic activities, such as RNA binding, that are shared with various SARS-CoV-2 proteins. Thus, anti-HCV NS5B and NS5A inhibitors, like sofosbuvir and daclatasvir, respectively, could be endowed with anti-SARS-CoV-2 activity.

Methods: SARS-CoV-2-infected Vero cells, HuH-7 cells, Calu-3 cells, neural stem cells and monocytes were used to investigate the effects of daclatasvir and sofosbuvir. In silico and cell-free based assays were performed with SARS-CoV-2 RNA and nsp12 to better comprehend the mechanism of inhibition of the investigated compounds. A physiologically based pharmacokinetic model was generated to estimate daclatasvir's dose and schedule to maximize the probability of success for COVID-19.

Results: Daclatasvir inhibited SARS-CoV-2 replication in Vero, HuH-7 and Calu-3 cells, with potencies of 0.8, 0.6 and 1.1 μM, respectively. Although less potent than daclatasvir, sofosbuvir alone and combined with daclatasvir inhibited replication in Calu-3 cells. Sofosbuvir and daclatasvir prevented virus-induced neuronal apoptosis and release of cytokine storm-related inflammatory mediators, respectively. Sofosbuvir inhibited RNA synthesis by chain termination and daclatasvir targeted the folding of secondary RNA structures in the SARS-CoV-2 genome. Concentrations required for partial daclatasvir in vitro activity are achieved in plasma at Cmax after administration of the approved dose to humans.

Conclusions: Daclatasvir, alone or in combination with sofosbuvir, at higher doses than used against HCV, may be further fostered as an anti-COVID-19 therapy.
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http://dx.doi.org/10.1093/jac/dkab072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8083231PMC
April 2021

Longitudinal profiling of circulating miRNA during cardiac allograft rejection: a proof-of-concept study.

ESC Heart Fail 2021 Jun 13;8(3):1840-1849. Epub 2021 Mar 13.

Department of Medicine I, Division of Cardiology, University Hospital of Friedrich Schiller University Jena, Am Klinikum 1, Jena, 07747, Germany.

Aims: Allograft rejection following heart transplantation (HTx) is a serious complication even in the era of modern immunosuppressive regimens and causes up to a third of early deaths after HTx. Allograft rejection is mediated by a cascade of immune mechanisms leading to acute cellular rejection (ACR) and/or antibody-mediated rejection (AMR). The gold standard for monitoring allograft rejection is invasive endomyocardial biopsy that exposes patients to complications. Little is known about the potential of circulating miRNAs as biomarkers to detect cardiac allograft rejection. We here present a systematic analysis of circulating miRNAs as biomarkers and predictors for allograft rejection after HTx using next-generation small RNA sequencing.

Methods And Results: We used next-generation small RNA sequencing to investigate circulating miRNAs among HTx recipients (10 healthy controls, 10 heart failure patients, 13 ACR, and 10 AMR). MiRNA profiling was performed at different time points before, during, and after resolution of the rejection episode. We found three miRNAs with significantly increased serum levels in patients with biopsy-proven cardiac rejection when compared with patients without rejection: hsa-miR-139-5p, hsa-miR-151a-5p, and hsa-miR-186-5p. We identified miRNAs that may serve as potential predictors for the subsequent development of ACR: hsa-miR-29c-3p (ACR) and hsa-miR-486-5p (AMR). Overall, hsa-miR-486-5p was most strongly associated with acute rejection episodes.

Conclusions: Monitoring cardiac allograft rejection using circulating miRNAs might represent an alternative strategy to invasive endomyocardial biopsy.
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http://dx.doi.org/10.1002/ehf2.13238DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8120386PMC
June 2021

Classifying Lung Neuroendocrine Neoplasms through MicroRNA Sequence Data Mining.

Cancers (Basel) 2020 Sep 17;12(9). Epub 2020 Sep 17.

Laboratory of Translational RNA Biology, Department of Pathology and Molecular Medicine, Queen's University, Kingston, ON K7L 3N6, Canada.

Lung neuroendocrine neoplasms (NENs) can be challenging to classify due to subtle histologic differences between pathological types. MicroRNAs (miRNAs) are small RNA molecules that are valuable markers in many neoplastic diseases. To evaluate miRNAs as classificatory markers for lung NENs, we generated comprehensive miRNA expression profiles from 14 typical carcinoid (TC), 15 atypical carcinoid (AC), 11 small cell lung carcinoma (SCLC), and 15 large cell neuroendocrine carcinoma (LCNEC) samples, through barcoded small RNA sequencing. Following sequence annotation and data preprocessing, we randomly assigned these profiles to discovery and validation sets. Through high expression analyses, we found that miR-21 and -375 are abundant in all lung NENs, and that miR-21/miR-375 expression ratios are significantly lower in carcinoids (TC and AC) than in neuroendocrine carcinomas (NECs; SCLC and LCNEC). Subsequently, we ranked and selected miRNAs for use in miRNA-based classification, to discriminate carcinoids from NECs. Using miR-18a and -155 expression, our classifier discriminated these groups in discovery and validation sets, with 93% and 100% accuracy. We also identified miR-17, -103, and -127, and miR-301a, -106b, and -25, as candidate markers for discriminating TC from AC, and SCLC from LCNEC, respectively. However, these promising findings require external validation due to sample size.
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http://dx.doi.org/10.3390/cancers12092653DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564332PMC
September 2020

Characterizing and classifying neuroendocrine neoplasms through microRNA sequencing and data mining.

NAR Cancer 2020 Sep 15;2(3):zcaa009. Epub 2020 Jul 15.

Laboratory of Translational RNA Biology, Department of Pathology and Molecular Medicine, Queen's University, 88 Stuart Street, Kingston, ON K7L 3N6, Canada.

Neuroendocrine neoplasms (NENs) are clinically diverse and incompletely characterized cancers that are challenging to classify. MicroRNAs (miRNAs) are small regulatory RNAs that can be used to classify cancers. Recently, a morphology-based classification framework for evaluating NENs from different anatomical sites was proposed by experts, with the requirement of improved molecular data integration. Here, we compiled 378 miRNA expression profiles to examine NEN classification through comprehensive miRNA profiling and data mining. Following data preprocessing, our final study cohort included 221 NEN and 114 non-NEN samples, representing 15 NEN pathological types and 5 site-matched non-NEN control groups. Unsupervised hierarchical clustering of miRNA expression profiles clearly separated NENs from non-NENs. Comparative analyses showed that miR-375 and miR-7 expression is substantially higher in NEN cases than non-NEN controls. Correlation analyses showed that NENs from diverse anatomical sites have convergent miRNA expression programs, likely reflecting morphological and functional similarities. Using machine learning approaches, we identified 17 miRNAs to discriminate 15 NEN pathological types and subsequently constructed a multilayer classifier, correctly identifying 217 (98%) of 221 samples and overturning one histological diagnosis. Through our research, we have identified common and type-specific miRNA tissue markers and constructed an accurate miRNA-based classifier, advancing our understanding of NEN diversity.
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http://dx.doi.org/10.1093/narcan/zcaa009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7380486PMC
September 2020

Statistical Assessment of Depth Normalization for Small RNA Sequencing.

JCO Clin Cancer Inform 2020 06;4:567-582

Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY.

Purpose: Methods for depth normalization have been assessed primarily with simulated data or cell-line-mixture data. There is a pressing need for benchmark data enabling a more realistic and objective assessment, especially in the context of small RNA sequencing.

Methods: We collected a unique pair of microRNA sequencing data sets for the same set of tumor samples; one data set was collected with and the other without uniform handling and balanced design. The former provided a benchmark for evaluating evidence of differential expression and the latter served as a test bed for normalization. Next, we developed a data perturbation algorithm to simulate additional data set pairs. Last, we assembled a set of computational tools to visualize and quantify the assessment.

Results: We validated the quality of the benchmark data and showed the need for normalization of the test data. For illustration, we applied the data and tools to assess the performance of 9 existing normalization methods. Among them, trimmed mean of M-values was a better scaling method, whereas the median and the upper quartiles were consistently the worst performers; one variation of remove unwanted variation had the best chance of capturing true positives but at the cost of increased false positives. In general, these methods were, at best, moderately helpful when the level of differential expression was extensive and asymmetric.

Conclusion: Our study (1) provides the much-needed benchmark data and computational tools for assessing depth normalization, (2) shows the dependence of normalization performance on the underlying pattern of differential expression, and (3) calls for continued research efforts to develop more effective normalization methods.
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http://dx.doi.org/10.1200/CCI.19.00118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7330947PMC
June 2020

Comprehensive aptamer-based screening identifies a spectrum of urinary biomarkers of lupus nephritis across ethnicities.

Nat Commun 2020 05 4;11(1):2197. Epub 2020 May 4.

Department Biomedical Engineering, University of Houston, Houston, TX, USA.

Emerging urinary biomarkers continue to show promise in evaluating lupus nephritis (LN). Here, we screen urine from active LN patients for 1129 proteins using an aptamer-based platform, followed by ELISA validation in two independent cohorts comprised of 127 inactive lupus, 107 active LN, 67 active non-renal lupus patients and 74 healthy controls, of three different ethnicities. Urine proteins that best distinguish active LN from inactive disease are ALCAM, PF-4, properdin, and VCAM-1 among African-Americans, sE-selectin, VCAM-1, BFL-1 and Hemopexin among Caucasians, and ALCAM, VCAM-1, TFPI and PF-4 among Asians. Most of these correlate significantly with disease activity indices in the respective ethnic groups, and surpass conventional metrics in identifying active LN, with better sensitivity, and negative/positive predictive values. Several elevated urinary molecules are also expressed within the kidneys in LN, based on single-cell RNAseq analysis. Longitudinal studies are warranted to assess the utility of these biomarkers in tracking lupus nephritis.
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http://dx.doi.org/10.1038/s41467-020-15986-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7198599PMC
May 2020

Chromosome 19 microRNA cluster enhances cell reprogramming by inhibiting epithelial-to-mesenchymal transition.

Sci Rep 2020 02 20;10(1):3029. Epub 2020 Feb 20.

Department of Molecular Pharmacology and Physiology, University of South Florida, Morsani College of Medicine, Tampa, Florida, USA.

During implantation, cytotrophoblasts undergo epithelial-to-mesenchymal transition (EMT) as they differentiate into invasive extravillous trophoblasts (EVTs). The primate-specific microRNA cluster on chromosome 19 (C19MC) is exclusively expressed in the placenta, embryonic stem cells and certain cancers however, its role in EMT gene regulation is unknown. In situ hybridization for miR-517a/c, a C19MC cistron microRNA, in first trimester human placentas displayed strong expression in villous trophoblasts and a gradual decrease from proximal to distal cell columns as cytotrophoblasts differentiate into invasive EVTs. To investigate the role of C19MC in the regulation of EMT genes, we employed the CRISPR/dCas9 Synergistic Activation Mediator (SAM) system, which induced robust transcriptional activation of the entire C19MC cistron and resulted in suppression of EMT associated genes. Exposure of human iPSCs to hypoxia or differentiation of iPSCs into either cytotrophoblast-stem-like cells or EVT-like cells under hypoxia reduced C19MC expression and increased EMT genes. Furthermore, transcriptional activation of the C19MC cistron induced the expression of OCT4 and FGF4 and accelerated cellular reprogramming. This study establishes the CRISPR/dCas9 SAM as a powerful tool that enables activation of the entire C19MC cistron and uncovers its novel role in suppressing EMT genes critical for maintaining the epithelial cytotrophoblasts stem cell phenotype.
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http://dx.doi.org/10.1038/s41598-020-59812-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7033247PMC
February 2020

Single-cell transcriptome analysis of human skin identifies novel fibroblast subpopulation and enrichment of immune subsets in atopic dermatitis.

J Allergy Clin Immunol 2020 06 7;145(6):1615-1628. Epub 2020 Feb 7.

Department of Dermatology, Icahn School of Medicine at Mount Sinai, New York, NY. Electronic address:

Background: Atopic dermatitis (AD) is a prevalent inflammatory skin disease with a complex pathogenesis involving immune cell and epidermal abnormalities. Despite whole tissue biopsy studies that have advanced the mechanistic understanding of AD, single cell-based molecular alterations are largely unknown.

Objective: Our aims were to construct a detailed, high-resolution atlas of cell populations and assess variability in cell composition and cell-specific gene expression in the skin of patients with AD versus in controls.

Methods: We performed single-cell RNA sequencing on skin biopsy specimens from 5 patients with AD (4 lesional samples and 5 nonlesional samples) and 7 healthy control subjects, using 10× Genomics.

Results: We created transcriptomic profiles for 39,042 AD (lesional and nonlesional) and healthy skin cells. Fibroblasts demonstrated a novel COL6A5COL18A1 subpopulation that was unique to lesional AD and expressed CCL2 and CCL19 cytokines. A corresponding LAMP3 dendritic cell (DC) population that expressed the CCL19 receptor CCR7 was also unique to AD lesions, illustrating a potential role for fibroblast signaling to immune cells. The lesional AD samples were characterized by expansion of inflammatory DCs (CD1AFCER1A) and tissue-resident memory T cells (CD69CD103). The frequencies of type 2 (IL13)/type 22 (IL22) T cells were higher than those of type 1 (IFNG) in lesional AD, whereas this ratio was slightly diminished in nonlesional AD and further diminished in controls.

Conclusion: AD lesions were characterized by expanded type 2/type 22 T cells and inflammatory DCs, and by a unique inflammatory fibroblast that may interact with immune cells to regulate lymphoid cell organization and type 2 inflammation.
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http://dx.doi.org/10.1016/j.jaci.2020.01.042DOI Listing
June 2020

The G3BP1-Family-USP10 Deubiquitinase Complex Rescues Ubiquitinated 40S Subunits of Ribosomes Stalled in Translation from Lysosomal Degradation.

Mol Cell 2020 03 24;77(6):1193-1205.e5. Epub 2020 Jan 24.

Laboratory for RNA Molecular Biology, The Rockefeller University, 1230 York Ave, Box 186, New York, NY 10065, USA. Electronic address:

Ribosome-associated quality control (RQC) purges aberrant mRNAs and nascent polypeptides in a multi-step molecular process initiated by the E3 ligase ZNF598 through sensing of ribosomes collided at aberrant mRNAs and monoubiquitination of distinct small ribosomal subunit proteins. We show that G3BP1-family-USP10 complexes are required for deubiquitination of RPS2, RPS3, and RPS10 to rescue modified 40S subunits from programmed degradation. Knockout of USP10 or G3BP1 family proteins increased lysosomal ribosomal degradation and perturbed ribosomal subunit stoichiometry, both of which were rescued by a single K214R substitution of RPS3. While the majority of RPS2 and RPS3 monoubiquitination resulted from ZNF598-dependent sensing of ribosome collisions initiating RQC, another minor pathway contributed to their monoubiquitination. G3BP1 family proteins have long been considered RNA-binding proteins, however, our results identified 40S subunits and associated mRNAs as their predominant targets, a feature shared by stress granules to which G3BP1 family proteins localize under stress.
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http://dx.doi.org/10.1016/j.molcel.2019.12.024DOI Listing
March 2020

Non-reversible tissue fixation retains extracellular vesicles for in situ imaging.

Nat Methods 2019 12 11;16(12):1269-1273. Epub 2019 Nov 11.

Department of Ophthalmology and Margaret M. Dyson Vision Research Institute, Weill Cornell Medicine, New York, NY, USA.

Extracellular vesicles (EVs) are secreted nanosized particles with many biological functions and pathological associations. The inability to image EVs in fixed tissues has been a major limitation to understanding their role in healthy and diseased tissue microenvironments. Here, we show that crosslinking mammalian tissues with formaldehyde results in significant EV loss, which can be prevented by additional fixation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) for visualization of EVs in a range of normal and cancer tissues.
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http://dx.doi.org/10.1038/s41592-019-0623-4DOI Listing
December 2019

The RNA-Binding Protein A1CF Regulates Hepatic Fructose and Glycerol Metabolism via Alternative RNA Splicing.

Cell Rep 2019 10;29(2):283-300.e8

Institute of Molecular Health Sciences, ETH Zurich, Otto-Stern-Weg 7, 8093 Zürich, Switzerland; Medical Faculty, University of Zurich, 8091 Zurich, Switzerland. Electronic address:

The regulation of hepatic gene expression has been extensively studied at the transcriptional level; however, the control of metabolism through posttranscriptional gene regulation by RNA-binding proteins in physiological and disease states is less understood. Here, we report a major role for the hormone-sensitive RNA-binding protein (RBP) APOBEC1 complementation factor (A1CF) in the generation of hepatocyte-specific and alternatively spliced transcripts. Among these transcripts are isoforms for the dominant and high-affinity fructose-metabolizing ketohexokinase C and glycerol kinase, two key metabolic enzymes that are linked to hepatic gluconeogenesis and found to be markedly reduced upon hepatic ablation of A1cf. Consequently, mice lacking A1CF exhibit improved glucose tolerance and are protected from fructose-induced hyperglycemia, hepatic steatosis, and development of obesity. Our results identify a previously unreported function of A1CF as a regulator of alternative splicing of a subset of genes influencing hepatic glucose production through fructose and glycerol metabolism.
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http://dx.doi.org/10.1016/j.celrep.2019.08.100DOI Listing
October 2019

Cell atlas of the foetal human heart and implications for autoimmune-mediated congenital heart block.

Cardiovasc Res 2020 07;116(8):1446-1457

Laboratory of RNA Molecular Biology, The Rockefeller University, 1230 York Avenue, Box 186, New York, NY 10065, USA.

Aims: Investigating human heart development and applying this to deviations resulting in disease is incomplete without molecular characterization of the cell types required for normal functioning. We investigated foetal human heart single-cell transcriptomes from mid-gestational healthy and anti-SSA/Ro associated congenital heart block (CHB) samples.

Methods And Results: Three healthy foetal human hearts (19th to 22nd week of gestation) and one foetal heart affected by autoimmune-associated CHB (21st week of gestation) were subjected to enzymatic dissociation using the Langendorff preparation to obtain single-cell suspensions followed by 10× Genomics- and Illumina-based single-cell RNA-sequencing (scRNA-seq). In addition to the myocytes, fibroblasts, immune cells, and other minor cell types, previously uncharacterized diverse sub-populations of endothelial cells were identified in the human heart. Differential gene expression analysis revealed increased and heterogeneous interferon responses in varied cell types of the CHB heart compared with the healthy controls. In addition, we also identified matrisome transcripts enriched in CHB stromal cells that potentially contribute to extracellular matrix deposition and subsequent fibrosis.

Conclusion: These data provide an information-rich resource to further our understanding of human heart development, which, as illustrated by comparison to a heart exposed to a maternal autoimmune environment, can be leveraged to provide insight into the pathogenesis of disease.
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http://dx.doi.org/10.1093/cvr/cvz257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7314636PMC
July 2020

Single-cell RNA sequencing for the study of lupus nephritis.

Lupus Sci Med 2019 4;6(1):e000329. Epub 2019 Jun 4.

Division of Rheumatology, Albert Einstein College of Medicine, Bronx, New York, USA.

Single-cell RNA sequencing (scRNA-seq) has recently undergone rapid advances in the development of this technology, leading to high throughput and accelerating discovery in many biological systems and diseases. The single-cell resolution of the technique allows for the investigation of heterogeneity in cell populations, and the pinpointing of pathological populations contributing to disease. Here we review the development of scRNA-seq technology and the analysis that has evolved with the ever-increasing throughput. Finally, we highlight recent applications of scRNA-seq to understand the molecular pathogenesis of lupus and lupus nephritis.
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http://dx.doi.org/10.1136/lupus-2019-000329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6560921PMC
June 2019

Human cGAS catalytic domain has an additional DNA-binding interface that enhances enzymatic activity and liquid-phase condensation.

Proc Natl Acad Sci U S A 2019 06 29;116(24):11946-11955. Epub 2019 May 29.

Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065;

The cyclic GMP-AMP synthase (cGAS)-cGAMP-STING pathway plays a key role in innate immunity, with cGAS sensing both pathogenic and mislocalized DNA in the cytoplasm. Human cGAS (h-cGAS) constitutes an important drug target for control of antiinflammatory responses that can contribute to the onset of autoimmune diseases. Recent studies have established that the positively charged N-terminal segment of cGAS contributes to enhancement of cGAS enzymatic activity as a result of DNA-induced liquid-phase condensation. We have identified an additional cGAS-DNA interface (labeled site-C; CD, catalytic domain) in the crystal structure of a human SRY.cGAS-DNA complex, with mutations along this basic site-C cGAS interface disrupting liquid-phase condensation, as monitored by cGAMP formation, gel shift, spin-down, and turbidity assays, as well as time-lapse imaging of liquid droplet formation. We expand on an earlier ladder model of cGAS dimers bound to a pair of parallel-aligned DNAs to propose a multivalent interaction-mediated cluster model to account for DNA-mediated condensation involving both the N-terminal domain of cGAS and the site-C cGAS-DNA interface. We also report the crystal structure of the h-cGAS-DNA complex containing a triple mutant that disrupts the site-C interface, with this complex serving as a future platform for guiding cGAS inhibitor development at the DNA-bound h-cGAS level. Finally, we solved the structure of RU.521 bound in two alternate alignments to apo h-cGAS, thereby occupying more of the catalytic pocket and providing insights into further optimization of active-site-binding inhibitors.
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http://dx.doi.org/10.1073/pnas.1905013116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6575157PMC
June 2019

Development of human cGAS-specific small-molecule inhibitors for repression of dsDNA-triggered interferon expression.

Nat Commun 2019 05 21;10(1):2261. Epub 2019 May 21.

Laboratory for RNA Molecular Biology, The Rockefeller University, 1230 York Ave, Box 186, New York, NY, 10065, USA.

Cyclic GMP-AMP synthase (cGAS) is the primary sensor for aberrant intracellular dsDNA producing the cyclic dinucleotide cGAMP, a second messenger initiating cytokine production in subsets of myeloid lineage cell types. Therefore, inhibition of the enzyme cGAS may act anti-inflammatory. Here we report the discovery of human-cGAS-specific small-molecule inhibitors by high-throughput screening and the targeted medicinal chemistry optimization for two molecular scaffolds. Lead compounds from one scaffold co-crystallize with human cGAS and occupy the ATP- and GTP-binding active site. The specificity and potency of these drug candidates is further documented in human myeloid cells including primary macrophages. These novel cGAS inhibitors with cell-based activity will serve as probes into cGAS-dependent innate immune pathways and warrant future pharmacological studies for treatment of cGAS-dependent inflammatory diseases.
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http://dx.doi.org/10.1038/s41467-019-08620-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6529454PMC
May 2019

Tubular cell and keratinocyte single-cell transcriptomics applied to lupus nephritis reveal type I IFN and fibrosis relevant pathways.

Nat Immunol 2019 07 20;20(7):915-927. Epub 2019 May 20.

Division of Rheumatology and Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA.

The molecular and cellular processes that lead to renal damage and to the heterogeneity of lupus nephritis (LN) are not well understood. We applied single-cell RNA sequencing (scRNA-seq) to renal biopsies from patients with LN and evaluated skin biopsies as a potential source of diagnostic and prognostic markers of renal disease. Type I interferon (IFN)-response signatures in tubular cells and keratinocytes distinguished patients with LN from healthy control subjects. Moreover, a high IFN-response signature and fibrotic signature in tubular cells were each associated with failure to respond to treatment. Analysis of tubular cells from patients with proliferative, membranous and mixed LN indicated pathways relevant to inflammation and fibrosis, which offer insight into their histologic differences. In summary, we applied scRNA-seq to LN to deconstruct its heterogeneity and identify novel targets for personalized approaches to therapy.
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http://dx.doi.org/10.1038/s41590-019-0386-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6584054PMC
July 2019

Detection of circulating extracellular mRNAs by modified small-RNA-sequencing analysis.

JCI Insight 2019 04 11;5. Epub 2019 Apr 11.

Laboratory of RNA Molecular Biology and.

Extracellular mRNAs (ex-mRNAs) potentially supersede extracellular miRNAs (ex-miRNAs) and other RNA classes as biomarkers. We performed conventional small-RNA-sequencing (sRNA-seq) and sRNA-seq with T4 polynucleotide kinase (PNK) end-treatment of total exRNA isolated from serum and platelet-poor EDTA, ACD, and heparin plasma to study the effect on ex-mRNA capture. Compared to conventional sRNA-seq PNK-treatment increased the detection of informative ex-mRNAs reads up to 50-fold. The exRNA pool was dominated by hematopoietic cells and platelets, with additional contribution from the liver. About 60% of the 15- to 42-nt reads originated from the coding sequences, in a pattern reminiscent of ribosome-profiling. Blood sample type had a considerable influence on the exRNA profile. On average approximately 350 to 1,100 distinct ex-mRNA transcripts were detected depending on plasma type. In serum, additional transcripts from neutrophils and hematopoietic cells increased this number to near 2,300. EDTA and ACD plasma showed a destabilizing effect on ex mRNA and non-coding RNA ribonucleoprotein complexes compared to other plasma types. In a proof-of-concept study, we investigated differences between the exRNA profiles of patients with acute coronary syndrome (ACS) and healthy controls. The improved tissue resolution of ex mRNAs after PNK-treatment enabled us to detect a neutrophil-signature in ACS that escaped detection by ex miRNA analysis.
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http://dx.doi.org/10.1172/jci.insight.127317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6538341PMC
April 2019

Single-Cell RNA Profiling of Glomerular Cells Shows Dynamic Changes in Experimental Diabetic Kidney Disease.

J Am Soc Nephrol 2019 04 7;30(4):533-545. Epub 2019 Mar 7.

Division of Nephrology, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York;

Background: Recent single-cell RNA sequencing (scRNA-seq) analyses have offered much insight into cell-specific gene expression profiles in normal kidneys. However, in diseased kidneys, understanding of changes in specific cells, particularly glomerular cells, remains limited.

Methods: To elucidate the glomerular cell-specific gene expression changes in diabetic kidney disease, we performed scRNA-seq analysis of isolated glomerular cells from streptozotocin-induced diabetic endothelial nitric oxide synthase (eNOS)-deficient (eNOS) mice and control eNOS mice.

Results: We identified five distinct cell populations, including glomerular endothelial cells, mesangial cells, podocytes, immune cells, and tubular cells. Using scRNA-seq analysis, we confirmed the expression of glomerular cell-specific markers and also identified several new potential markers of glomerular cells. The number of immune cells was significantly higher in diabetic glomeruli compared with control glomeruli, and further cluster analysis showed that these immune cells were predominantly macrophages. Analysis of differential gene expression in endothelial and mesangial cells of diabetic and control mice showed dynamic changes in the pattern of expressed genes, many of which are known to be involved in diabetic kidney disease. Moreover, gene expression analysis showed variable responses of individual cells to diabetic injury.

Conclusions: Our findings demonstrate the ability of scRNA-seq analysis in isolated glomerular cells from diabetic and control mice to reveal dynamic changes in gene expression in diabetic kidneys, with variable responses of individual cells. Such changes, which might not be apparent in bulk transcriptomic analysis of glomerular cells, may help identify important pathophysiologic factors contributing to the progression of diabetic kidney disease.
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http://dx.doi.org/10.1681/ASN.2018090896DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6442341PMC
April 2019

miR-193b regulates tumorigenesis in liposarcoma cells via PDGFR, TGFβ, and Wnt signaling.

Sci Rep 2019 03 1;9(1):3197. Epub 2019 Mar 1.

Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Liposarcoma is the most common soft tissue sarcoma. Molecularly targeted therapeutics have had limited efficacy in liposarcomas, in part because of inadequate knowledge of the complex molecular alterations in these tumors. Our recent study revealed the tumor suppressive function of miR-193b in liposarcoma. Considering the biological and clinical heterogeneity of liposarcoma, here, we confirmed the under-expression of miR-193b in additional patient liposarcoma samples and cell lines. Based on STRING analysis of protein-protein interactions among the reported putative miR-193b targets, we validated three: PDGFRβ, SMAD4, and YAP1, belonging to strongly interacting pathways (focal adhesion, TGFβ, and Hippo, respectively). We show that all three are directly targeted by miR-193b in liposarcoma. Inhibition of PDGFRβ reduces liposarcoma cell viability and increases adipogenesis. Knockdown of SMAD4 promotes adipogenic differentiation. miR-193b targeting of the Hippo signaling effector YAP1 indirectly inhibits Wnt/β-catenin signaling. Both a PDGFR inhibitor (CP-673451) and a Wnt/ β-catenin inhibitor (ICG-001) had potent inhibitory effects on liposarcoma cells, suggesting their potential application in liposarcoma treatment. In summary, we demonstrate that miR-193b controls cell growth and differentiation in liposarcoma by targeting multiple key components (PDGFRβ, SMAD4, and YAP1) in several oncogenic signaling pathways.
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http://dx.doi.org/10.1038/s41598-019-39560-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6397171PMC
March 2019

Deciphering human ribonucleoprotein regulatory networks.

Nucleic Acids Res 2019 01;47(2):570-581

Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine, Berlin, Germany.

RNA-binding proteins (RBPs) control and coordinate each stage in the life cycle of RNAs. Although in vivo binding sites of RBPs can now be determined genome-wide, most studies typically focused on individual RBPs. Here, we examined a large compendium of 114 high-quality transcriptome-wide in vivo RBP-RNA cross-linking interaction datasets generated by the same protocol in the same cell line and representing 64 distinct RBPs. Comparative analysis of categories of target RNA binding preference, sequence preference, and transcript region specificity was performed, and identified potential posttranscriptional regulatory modules, i.e. specific combinations of RBPs that bind to specific sets of RNAs and targeted regions. These regulatory modules represented functionally related proteins and exhibited distinct differences in RNA metabolism, expression variance, as well as subcellular localization. This integrative investigation of experimental RBP-RNA interaction evidence and RBP regulatory function in a human cell line will be a valuable resource for understanding the complexity of post-transcriptional regulation.
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http://dx.doi.org/10.1093/nar/gky1185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6344852PMC
January 2019

A single-cell survey of the human first-trimester placenta and decidua.

Sci Adv 2018 10 31;4(10):eaau4788. Epub 2018 Oct 31.

Department of Obstetrics and Gynecology, Columbia University Medical Center, 630 W 168th St., New York, NY 10032, USA.

The placenta and decidua interact dynamically to enable embryonic and fetal development. Here, we report single-cell RNA sequencing of 14,341 and 6754 cells from first-trimester human placental villous and decidual tissues, respectively. Bioinformatic analysis identified major cell types, many known and some subtypes previously unknown in placental villi and decidual context. Further detailed analysis revealed proliferating subpopulations, enrichment of cell type-specific transcription factors, and putative intercellular communication in the fetomaternal microenvironment. This study provides a blueprint to further the understanding of the roles of these cells in the placenta and decidua for maintenance of early gestation as well as pathogenesis in pregnancy-related disorders.
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http://dx.doi.org/10.1126/sciadv.aau4788DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6209386PMC
October 2018

Evaluating gastroenteropancreatic neuroendocrine tumors through microRNA sequencing.

Endocr Relat Cancer 2019 01;26(1):47-57

Laboratory of Translational RNA Biology, Department of Pathology and Molecular Medicine, Queen's University, Kingston, Ontario, Canada.

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) can be challenging to evaluate histologically. MicroRNAs (miRNAs) are small RNA molecules that often are excellent biomarkers due to their abundance, cell-type and disease stage specificity and stability. To evaluate miRNAs as adjunct tissue markers for classifying and grading well-differentiated GEP-NETs, we generated and compared miRNA expression profiles from four pathological types of GEP-NETs. Using quantitative barcoded small RNA sequencing and state-of-the-art sequence annotation, we generated comprehensive miRNA expression profiles from archived pancreatic, ileal, appendiceal and rectal NETs. Following data preprocessing, we randomly assigned sample profiles to discovery (80%) and validation (20%) sets prior to data mining using machine-learning techniques. High expression analyses indicated that miR-375 was the most abundant individual miRNA and miRNA cistron in all samples. Leveraging prior knowledge that GEP-NET behavior is influenced by embryonic derivation, we developed a dual-layer hierarchical classifier for differentiating GEP-NET types. In the first layer, our classifier discriminated midgut (ileum, appendix) from non-midgut (rectum, pancreas) NETs based on miR-615 and -92b expression. In the second layer, our classifier discriminated ileal from appendiceal NETs based on miR-125b, -192 and -149 expression, and rectal from pancreatic NETs based on miR-429 and -487b expression. Our classifier achieved overall accuracies of 98.5% and 94.4% in discovery and validation sets, respectively. We also found provisional evidence that low- and intermediate-grade pancreatic NETs can be discriminated based on miR-328 expression. GEP-NETs can be reliably classified and potentially graded using a limited panel of miRNA markers, complementing morphological and immunohistochemistry-based approaches to histologic evaluation.
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http://dx.doi.org/10.1530/ERC-18-0244DOI Listing
January 2019

MicroRNA-206 suppresses TGF-β signalling to limit tumor growth and metastasis in lung adenocarcinoma.

Cell Signal 2018 Oct 20;50:25-36. Epub 2018 Jun 20.

Cancer Biology & Genetics Division, Queen's Cancer Research Institute, Kingston, Canada; Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Canada. Electronic address:

MicroRNA-206 (miR-206) has demonstrated tumor suppressive effects in a variety of cancers. Numerous studies have identified aberrantly expressed targets of miR-206 that contribute to tumor progression and metastasis, however, the broader gene-networks and pathways regulated by miR-206 remain poorly defined. Here, we have ectopically expressed miR-206 in lung adenocarcinoma cell lines and tumors to identify differentially expressed genes, and study the effects on tumor growth and metastasis. In H1299 tumor xenograft assays, stable expression of miR-206 suppressed both tumor growth and metastasis in mice. Profiling of xenograft tumors using small RNA sequencing and a targeted panel of tumor progression and metastasis-related genes revealed a network of genes involved in TGF-β signalling that were regulated by miR-206. Among these were the TGFB1 ligand, as well as direct transcriptional targets of Smad3. Other differentially expressed genes included components of the extracellular matrix involved in TGF-β activation and signalling, including Thrombospondin-1, which is responsible for the activation of latent TGF-β in the stroma. In cultured lung adenocarcinoma cells treated with recombinant TGF-β, ectopic expression of miR-206 impaired canonical signalling, and expression of TGF-β target genes linked to epithelial-mesenchymal transition. This was due at least in part to the suppression of Smad3 protein levels in lung adenocarcinoma cells with ectopic miR-206 expression. Together, these findings indicate that miR-206 can suppress tumor progression and metastasis by limiting autocrine production of TGF-β, and highlight the potential utility of TGF-β inhibitors for the treatment of lung adenocarcinomas.
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http://dx.doi.org/10.1016/j.cellsig.2018.06.008DOI Listing
October 2018

Human plasma and serum extracellular small RNA reference profiles and their clinical utility.

Proc Natl Acad Sci U S A 2018 06 18;115(23):E5334-E5343. Epub 2018 May 18.

Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10065;

Circulating extracellular RNAs (exRNAs) have the potential to serve as biomarkers for a wide range of medical conditions. However, limitations in existing exRNA isolation methods and a lack of knowledge on parameters affecting exRNA variability in human samples may hinder their successful discovery and clinical implementation. Using combinations of denaturants, reducing agents, proteolysis, and revised organic extraction, we developed an automated, high-throughput approach for recovery of exRNAs and exDNA from the same biofluid sample. We applied this method to characterize exRNAs from 312 plasma and serum samples collected from 13 healthy volunteers at 12 time points over a 2-month period. Small RNA cDNA library sequencing identified nearly twofold increased epithelial-, muscle-, and neuroendocrine-cell-specific miRNAs in females, while fasting and hormonal cycle showed little effect. External standardization helped to detect quantitative differences in erythrocyte and platelet-specific miRNA contributions and in miRNA concentrations between biofluids. It also helped to identify a study participant with a unique exRNA phenotype featuring a miRNA signature of up to 20-fold elevated endocrine-cell-specific miRNAs and twofold elevated total miRNA concentrations stable for over 1 year. Collectively, these results demonstrate an efficient and quantitative method to discern exRNA phenotypes and suggest that plasma and serum RNA profiles are stable over months and can be routinely monitored in long-term clinical studies.
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http://dx.doi.org/10.1073/pnas.1714397115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6003356PMC
June 2018

Modulation of LIN28B/Let-7 Signaling by Propranolol Contributes to Infantile Hemangioma Involution.

Arterioscler Thromb Vasc Biol 2018 06 3;38(6):1321-1332. Epub 2018 May 3.

From the Department of Molecular Pharmacology and Physiology (E.F.M., J.C., J.L., J.V., A.M., H.T.-J.)

Objective: Infantile hemangiomas (IHs) are the most common benign vascular neoplasms of infancy, characterized by a rapid growth phase followed by a spontaneous involution, or triggered by propranolol treatment by poorly understood mechanisms. LIN28/let-7 axis plays a central role in the regulation of stem cell self-renewal and tumorigenesis. However, the role of LIN28B/let-7 signaling in IH pathogenesis has not yet been elucidated.

Approach And Results: LIN28B is highly expressed in proliferative IH and is less expressed in involuted and in propranolol-treated IH samples as measured by immunofluorescence staining and quantitative RT-PCR. Small RNA sequencing analysis of IH samples revealed a decrease in microRNAs that target LIN28B, including let-7, and an increase in microRNAs in the mir-498(46) cistron. Overexpression of LIN28B in HEK293 cells induced the expression of miR-516b in the mir-498(46) cistron. Propranolol treatment of induced pluripotent stem cells, which express mir-498(46) endogenously, reduced the expression of both LIN28B and mir-498(46) and increased the expression of let-7. Furthermore, propranolol treatment reduced the proliferation of induced pluripotent stem cells and induced epithelial-mesenchymal transition.

Conclusions: This work uncovers the role of the LIN28B/let-7 switch in IH pathogenesis and provides a novel mechanism by which propranolol induces IH involution. Furthermore, it provides therapeutic implications for cancers in which the LIN28/let-7 pathway is imbalanced.
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http://dx.doi.org/10.1161/ATVBAHA.118.310908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5970092PMC
June 2018