Publications by authors named "Marc K Halushka"

160 Publications

Exercise triggers CAPN1-mediated AIF truncation, inducing myocyte cell death in arrhythmogenic cardiomyopathy.

Sci Transl Med 2021 Feb;13(581)

Division of Cardiology, Department of Medicine, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.

Myocyte death occurs in many inherited and acquired cardiomyopathies, including arrhythmogenic cardiomyopathy (ACM), a genetic heart disease plagued by the prevalence of sudden cardiac death. Individuals with ACM and harboring pathogenic desmosomal variants, such as desmoglein-2 (), often show myocyte necrosis with progression to exercise-associated heart failure. Here, we showed that homozygous mutant mice (), a model of ACM, die prematurely during swimming and display myocardial dysfunction and necrosis. We detected calcium (Ca) overload in hearts, which induced calpain-1 (CAPN1) activation, association of CAPN1 with mitochondria, and CAPN1-induced cleavage of mitochondrial-bound apoptosis-inducing factor (AIF). Cleaved AIF translocated to the myocyte nucleus triggering large-scale DNA fragmentation and cell death, an effect potentiated by mitochondrial-driven AIF oxidation. Posttranslational oxidation of AIF cysteine residues was due, in part, to a depleted mitochondrial thioredoxin-2 redox system. Hearts from exercised mice were depleted of calpastatin (CAST), an endogenous CAPN1 inhibitor, and overexpressing CAST in myocytes protected against Ca overload-induced necrosis. When cardiomyocytes differentiated from embryonic stem cells (ES-CMs) were challenged with β-adrenergic stimulation, CAPN1 inhibition attenuated CAPN1-induced AIF truncation. In addition, pretreatment of ES-CMs with an AIF-mimetic peptide, mirroring the cyclophilin-A (PPIA) binding site of AIF, blocked PPIA-mediated AIF-nuclear translocation, and reduced both apoptosis and necrosis. Thus, preventing CAPN1-induced AIF-truncation or barring binding of AIF to the nuclear chaperone, PPIA, may avert myocyte death and, ultimately, disease progression to heart failure in ACM and likely other forms of cardiomyopathies.
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http://dx.doi.org/10.1126/scitranslmed.abf0891DOI Listing
February 2021

Aortitis masquerading as intramural hematoma: When to observe, when to operate? A case report.

J Card Surg 2021 Jan 27. Epub 2021 Jan 27.

Division of Cardiac Surgery, Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

Radiologic evidence of aortic disease is not always consistent with the diagnosis. With a lack of accompanying symptoms or with an atypical presentation, diagnosis, and management of aortic pathology rely greatly on imaging techniques. We report the case of a 58-year-old female who presented with incidental radiographic findings consistent with a type A aortic intramural hematoma and a vague left-sided chest discomfort. After follow-up, imaging was consistent with disease progression and hematoma expansion; the affected segment was resected and pathology reported lymphoplasmacytic aortitis as the underlying etiology of the imaging findings rather than an intramural hematoma. The patient lacked symptoms or serology consistent with the rheumatologic disease, and the postoperative course was uneventful. The management of a suspected ascending intramural hematoma is controversial, especially when the patient presents with atypical signs and symptoms. Features of disease progression may warrant urgent surgical intervention.
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http://dx.doi.org/10.1111/jocs.15348DOI Listing
January 2021

HPAStainR: a Bioconductor and Shiny app to query protein expression patterns in the Human Protein Atlas.

F1000Res 2020 8;9:1210. Epub 2020 Oct 8.

Department of Pathology, Johns Hopkins University School of Medicine Baltimore, Baltimore, MD, 21205, USA.

The Human Protein Atlas is a website of protein expression in human tissues. It is an excellent resource of tissue and cell type protein localization, but only allows the query of a single protein at a time. We introduce HPAStainR as a new Shiny app and Bioconductor/R package used to query the scored staining patterns in the Human Protein Atlas with multiple proteins/genes of interest. This allows the user to determine if an experimentally-generated protein/gene list associates with a particular cell type. We validated the tool using the Panglao Database cell type specific marker genes and a Genotype Expression (GTEx) tissue deconvolution dataset.  HPAStainR identified 92% of the Panglao cell types in the top quartile of confidence scores limited to tissue type of origin results. It also appropriately identified the correct cell types from the GTEx dataset. HPAStainR fills a gap in available bioinformatics tools to identify cell type protein expression patterns and can assist in establishing ground truths and exploratory analysis. HPAStainR is available from: https://32tim32.shinyapps.io/HPAStainR/.
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http://dx.doi.org/10.12688/f1000research.26771.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7802108PMC
October 2020

Spatial Proteomic Approach to Characterize Skeletal Muscle Myofibers.

J Proteome Res 2021 Jan 30;20(1):888-894. Epub 2020 Nov 30.

Department of Pathology, Johns Hopkins University School of Medicine, Ross Bldg. Rm 632B, 720 Rutland Avenue, Baltimore, Maryland 21205, United States.

Skeletal muscle myofibers have differential protein expression resulting in functionally distinct slow- and fast-twitch types. While certain protein classes are well-characterized, the depth of all proteins involved in this process is unknown. We utilized the Human Protein Atlas (HPA) and the HPASubC tool to classify mosaic expression patterns of staining across 49,600 unique tissue microarray (TMA) images using a visual proteomic approach. We identified 2164 proteins with potential mosaic expression, of which 1605 were categorized as "likely" or "real." This list included both well-known fiber-type-specific and novel proteins. A comparison of the 1605 mosaic proteins with a mass spectrometry (MS)-derived proteomic dataset of single human muscle fibers led to the assignment of 111 proteins to fiber types. We additionally used a multiplexed immunohistochemistry approach, a multiplexed RNA-ISH approach, and STRING v11 to further assign or suggest fiber types of newly characterized mosaic proteins. This visual proteomic analysis of mature skeletal muscle myofibers greatly expands the known repertoire of twitch-type-specific proteins.
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http://dx.doi.org/10.1021/acs.jproteome.0c00673DOI Listing
January 2021

Myocarditis is rare in COVID-19 autopsies: cardiovascular findings across 277 postmortem examinations.

Cardiovasc Pathol 2021 Jan - Feb;50:107300. Epub 2020 Oct 23.

Department of Pathology, Louisiana State University Health Sciences Center, New Orleans, LA.

The COVID-19 pandemic, the result of severe acute respiratory syndrome (SARS)-CoV-2, is a major cause of worldwide mortality with a significant cardiovascular component. While a number of different cardiovascular histopathologies have been reported at postmortem examination, their incidence is unknown, due to limited numbers of cases in any given study. A literature review was performed identifying 277 autopsied hearts across 22 separate publications of COVID-19 positive patients. The median age of the autopsy cohort was 75 and 97.6% had one or more comorbidities. Initial review of the data indicate that myocarditis was present in 20 hearts (7.2%); however, closer examination of additional reported information revealed that most cases were likely not functionally significant and the true prevalence of myocarditis is likely much lower (<2%). At least one acute, potentially COVID-19-related cardiovascular histopathologic finding, such as macro or microvascular thrombi, inflammation, or intraluminal megakaryocytes, was reported in 47.8% of cases. Significant differences in reporting of histopathologic findings occurred between studies indicating strong biases in observations and the need for more consistency in reporting. In conclusion, across 277 cases, COVID-19-related cardiac histopathological findings, are common, while myocarditis is rare.
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http://dx.doi.org/10.1016/j.carpath.2020.107300DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7583586PMC
December 2020

Expression of the Autoantigen Topoisomerase-1 is Enriched in the Lung Tissues of Patients With Autoimmune Interstitial Lung Disease: A Case Control Study.

ACR Open Rheumatol 2020 Nov 29;2(11):657-661. Epub 2020 Oct 29.

Johns Hopkins University School of Medicine, Baltimore, Maryland, United States.

Background: Among the autoimmune rheumatic diseases, it is striking that autoantibodies targeting ubiquitously expressed proteins (eg, topoisomerase-1) associate with specific clinical complications (eg, interstitial lung disease [ILD]). It has been proposed that enriched antigen expression in inflamed target tissue may play a role in focusing the autoimmune response. We sought to determine whether topoisomerase-1 expression is enriched in lungs from patients with autoimmune/inflammatory diseases relative to normal lung.

Methods: We used a 99-core lung tissue microarray (TMA) containing lung tissue from 40 patients with autoimmune inflammatory ILD (cases) and 46 control subjects with normal lungs. We stained the TMA with antibodies to compare topoisomerase-1 and CD8 expression between patients and control subjects and evaluated whether expression is enriched in specific cell types. Staining was analyzed, and statistical comparisons were performed.

Results: Cases were more likely to have global topoisomerase-1 expression (53% vs 21%; P = 0.003), specifically in pneumocytes (47% vs 16%; P = 0.003) and stromal/immune cells (32% vs 5%; P = 0.002) compared with control subjects. CD8 cell density (223 cells/mm vs 102 cells/mm ; P = 0.018) was significantly higher in topoisomerase-1-positive lung tissues compared with topoisomerase-1-negative lung tissues. Interestingly, topoisomerase-1 expression was significantly more common in scleroderma compared with normal lung (67% vs 21%; P = 0.036) and was present more frequently in pneumocytes in these patients (67% vs 16%; P = 0.018).

Conclusions: Pulmonary expression of topoisomerase-1 is increased in the setting of autoimmune ILD relative to normal lung, specifically in pneumocytes. This may contribute to the amplification of pulmonary disease in patients with scleroderma with a loss of tolerance to topoisomerase-1.
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http://dx.doi.org/10.1002/acr2.11191DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7672300PMC
November 2020

An Urgent Need for Studies of the Late Effects of SARS-CoV-2 on the Cardiovascular System.

Circulation 2020 Sep 24. Epub 2020 Sep 24.

Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD.

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http://dx.doi.org/10.1161/CIRCULATIONAHA.120.051362DOI Listing
September 2020

Mycobacterium tuberculosis infection drives mitochondria-biased dysregulation of host tRNA-derived fragments.

J Infect Dis 2020 Sep 22. Epub 2020 Sep 22.

Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Background: Mycobacterium tuberculosis (Mtb), the bacterium that causes tuberculosis (TB), causes 10 million infections and 1.5 million deaths per year worldwide. The success of Mtb as a human pathogen is directly related to its ability to suppress host responses, which are critical for clearing intracellular pathogens. Emerging evidence suggests that key response pathways may be regulated by a novel class of small non-coding RNA, called tRNA-derived fragments (tRFs). tRFs can complex with Argonaute proteins to target and degrade mRNA targets, similarly to miRNAs, but have thus far been overlooked in the context of bacterial infections.

Methods: We generate a novel miRge2.0-based tRF-analysis tool, tRFcluster, and use it to analyze independently-generated and publicly available RNA-sequencing datasets to assess tRF dysregulation in host cells following infection with Mtb and other intracellular bacterial pathogens.

Results: We find that Mtb and Listeria monocytogenes drive dramatic tRF dysregulation, whereas other bacterial pathogens do not. Interestingly, Mtb infection uniquely increased the expression of mitochondria-derived tRFs rather than genomic-derived tRFs, suggesting an association with mitochondrial damage in Mtb infection.

Conclusions: tRFs are dysregulated in some, but not all, bacterial infections. Biased dysregulation of mitochondria-derived tRFs in Mtb infection suggests a link between mitochondrial distress and tRF production.
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http://dx.doi.org/10.1093/infdis/jiaa596DOI Listing
September 2020

Endomyocardial Biopsy Characterization of Heart Failure With Preserved Ejection Fraction and Prevalence of Cardiac Amyloidosis.

JACC Heart Fail 2020 09 8;8(9):712-724. Epub 2020 Jul 8.

Division of Cardiology, The Johns Hopkins School of Medicine, Baltimore, Maryland. Electronic address:

Objectives: This study prospectively evaluated endomyocardial biopsies in patients with heart failure with preserved ejection fraction (HFpEF) to identify histopathologic phenotypes and their association with clinical characteristics.

Background: Myocardial tissue analysis from a prospectively defined HFpEF cohort reflecting contemporary comorbidities is lacking.

Methods: Patients with HFpEF (EF ≥50%) referred to the Johns Hopkins HFpEF Clinic between August 2014 and September 2018 were enrolled for right heart catheterization and endomyocardial biopsy. Clinical features, echocardiography, hemodynamics, and tissue histology were determined and compared with controls (unused donor hearts) and HF with reduced EF (HFrEF).

Results: Of the 108 patients enrolled, median age was 66 years (25th to 75th percentile: 57 to 74 years), 61% were women, 57% were African American, 62% had a previous HF hospitalization, median systolic blood pressure was 141 mm Hg (25th to 75th percentile: 125 to 162 mm Hg), body mass index (BMI) was 37 kg/m (25th to 75th percentile: 32 to 45 kg/m), and 97% were on a loop diuretic. Myocardial fibrosis and myocyte hypertrophy were often present (93% and 88%, respectively); however, mild in 71% with fibrosis and in 52% with hypertrophy. Monocyte infiltration (CD68+ cells/mm) was greater in patients with HFpEF versus controls (60.4 cells/mm [25th to 75th percentile: 36.8 to 97.8] vs. 32.1 cells/mm [25th to 75th percentile: 22.3 to 59.2]; p = 0.02) and correlated with age and renal disease. Cardiac amyloidosis (CA) was diagnosed in 15 (14%) patients (HFpEF-CA: 7 patients with wild-type transthyretin amyloidosis [ATTR], 4 patients with hereditary ATTR, 3 patients with light-chain amyloidosis, and 1 patient with AA (secondary) amyloidosis), of which 7 cases were unsuspected. Patients with HFpEF-CA were older, with lower BMI, higher left ventricular mass index, and higher N-terminal pro-B-type natriuretic peptide and troponin I levels.

Conclusions: In this large, prospective myocardial tissue analysis of HFpEF, myocardial fibrosis and hypertrophy were common, CD68+ inflammation was increased, and CA prevalence was 14%. Tissue analysis in HFpEF might improve precision therapies by identifying relevant myocardial mechanisms.
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http://dx.doi.org/10.1016/j.jchf.2020.04.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7604801PMC
September 2020

Consistent RNA sequencing contamination in GTEx and other data sets.

Nat Commun 2020 04 22;11(1):1933. Epub 2020 Apr 22.

Department of Pathology, Johns Hopkins University SOM, Baltimore, MD, 21205, USA.

A challenge of next generation sequencing is read contamination. We use Genotype-Tissue Expression (GTEx) datasets and technical metadata along with RNA-seq datasets from other studies to understand factors that contribute to contamination. Here we report, of 48 analyzed tissues in GTEx, 26 have variant co-expression clusters of four highly expressed and pancreas-enriched genes (PRSS1, PNLIP, CLPS, and/or CELA3A). Fourteen additional highly expressed genes from other tissues also indicate contamination. Sample contamination is strongly associated with a sample being sequenced on the same day as a tissue that natively expresses those genes. Discrepant SNPs across four contaminating genes validate the contamination. Low-level contamination affects ~40% of samples and leads to numerous eQTL assignments in inappropriate tissues among these 18 genes. This type of contamination occurs widely, impacting bulk and single cell (scRNA-seq) data set analysis. In conclusion, highly expressed, tissue-enriched genes basally contaminate GTEx and other datasets impacting analyses.
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http://dx.doi.org/10.1038/s41467-020-15821-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7176728PMC
April 2020

The effect of tissue composition on gene co-expression.

Brief Bioinform 2021 Jan;22(1):127-139

Department of Biostatistics and Computation Biology, University of Rochester, Rochester, NY, USA.

Variable cellular composition of tissue samples represents a significant challenge for the interpretation of genomic profiling studies. Substantial effort has been devoted to modeling and adjusting for compositional differences when estimating differential expression between sample types. However, relatively little attention has been given to the effect of tissue composition on co-expression estimates. In this study, we illustrate the effect of variable cell-type composition on correlation-based network estimation and provide a mathematical decomposition of the tissue-level correlation. We show that a class of deconvolution methods developed to separate tumor and stromal signatures can be applied to two component cell-type mixtures. In simulated and real data, we identify conditions in which a deconvolution approach would be beneficial. Our results suggest that uncorrelated cell-type-specific markers are ideally suited to deconvolute both the expression and co-expression patterns of an individual cell type. We provide a Shiny application for users to interactively explore the effect of cell-type composition on correlation-based co-expression estimation for any cell types of interest.
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http://dx.doi.org/10.1093/bib/bbz135DOI Listing
January 2021

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

Unification of miRNA and isomiR research: the mirGFF3 format and the mirtop API.

Bioinformatics 2020 02;36(3):698-703

Bioinformatics Core, The Picower Institute for Learning and Memory, Cambridge, MA 02139, USA.

Motivation: MicroRNAs (miRNAs) are small RNA molecules (∼22 nucleotide long) involved in post-transcriptional gene regulation. Advances in high-throughput sequencing technologies led to the discovery of isomiRs, which are miRNA sequence variants. While many miRNA-seq analysis tools exist, the diversity of output formats hinders accurate comparisons between tools and precludes data sharing and the development of common downstream analysis methods.

Results: To overcome this situation, we present here a community-based project, miRNA Transcriptomic Open Project (miRTOP) working towards the optimization of miRNA analyses. The aim of miRTOP is to promote the development of downstream isomiR analysis tools that are compatible with existing detection and quantification tools. Based on the existing GFF3 format, we first created a new standard format, mirGFF3, for the output of miRNA/isomiR detection and quantification results from small RNA-seq data. Additionally, we developed a command line Python tool, mirtop, to create and manage the mirGFF3 format. Currently, mirtop can convert into mirGFF3 the outputs of commonly used pipelines, such as seqbuster, isomiR-SEA, sRNAbench, Prost! as well as BAM files. Some tools have also incorporated the mirGFF3 format directly into their code, such as, miRge2.0, IsoMIRmap and OptimiR. Its open architecture enables any tool or pipeline to output or convert results into mirGFF3. Collectively, this isomiR categorization system, along with the accompanying mirGFF3 and mirtop API, provide a comprehensive solution for the standardization of miRNA and isomiR annotation, enabling data sharing, reporting, comparative analyses and benchmarking, while promoting the development of common miRNA methods focusing on downstream steps of miRNA detection, annotation and quantification.

Availability And Implementation: https://github.com/miRTop/mirGFF3/ and https://github.com/miRTop/mirtop.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btz675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566869PMC
February 2020

Human cardiac myosin light chain 4 (MYL4) mosaic expression patterns vary by sex.

Sci Rep 2019 09 3;9(1):12681. Epub 2019 Sep 3.

Division of Cardiovascular Pathology, Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Sex disparities modulate cardiac function, although the proteins and mechanisms remain to be elucidated. We recently demonstrated a mosaic pattern of protein expression in the heart for over 100 proteins. Here we investigate one of these proteins, myosin light chain 4 (MYL4), which is important for contractile functions by increasing force production. We assayed the expression pattern of MYL4 across 756 ventricular myocardial samples from 668 individuals utilizing a semi-automated Cell Profiler method on five tissue microarrays (TMAs) of cardiac tissues across a diverse set of diseases. The percentage of MYL4 positive cells was significantly higher in male subjects independently across all five TMAs, regardless of disease state (p = 8.66e-15). Higher MYL4 expression was also modestly associated with hypertrophic cardiomyopathy (p = 6.3e-04). MYL4 expression did not associate with sudden cardiac death or other cardiomyopathies. This study demonstrates a new mosaic pattern of protein expression that underlies sex disparities in the human heart.
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http://dx.doi.org/10.1038/s41598-019-49191-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6722118PMC
September 2019

An expanded proteome of cardiac t-tubules.

Cardiovasc Pathol 2019 Sep - Oct;42:15-20. Epub 2019 May 24.

Department of Pathology, Division of Cardiovascular Pathology, Johns Hopkins University SOM, Baltimore, MD, USA. Electronic address:

Background: Transverse tubules (t-tubules) are important structural elements, derived from sarcolemma, found on all striated myocytes. These specialized organelles create a scaffold for many proteins crucial to the effective propagation of signal in cardiac excitation-contraction coupling. The full protein composition of this region is unknown.

Methods: We characterized the t-tubule subproteome using 52,033 immunohistochemical images covering 13,203 proteins from the Human Protein Atlas (HPA) cardiac tissue microarrays. We used HPASubC, a suite of Python tools, to rapidly review and classify each image for a specific t-tubule staining pattern. The tools Gene Cards, String 11, and Gene Ontology Consortium as well as literature searches were used to understand pathways and relationships between the proteins.

Results: There were 96 likely t-tubule proteins identified by HPASubC. Of these, 12 were matrisome proteins and 3 were mitochondrial proteins. A separate literature search identified 50 known t-tubule proteins. A comparison of the 2 lists revealed only 17 proteins in common, including 8 of the matrisome proteins. String11 revealed that 94 of 127 combined t-tubule proteins generated a single interconnected network.

Conclusion: Using HPASubC and the HPA, we identified 78 novel, putative t-tubule proteins and validated 17 within the literature. This expands and improves our knowledge of this important subcellular structure of the cardiac myocyte. This information can be used to identify new structural targets involved in excitation-contraction coupling that may be altered in disease.
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http://dx.doi.org/10.1016/j.carpath.2019.05.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6732025PMC
December 2019

Genetic aetiologies should be considered in paediatric cases of acute heart failure presumed to be myocarditis.

Cardiol Young 2019 Jul 14;29(7):917-921. Epub 2019 Jun 14.

Department of Pediatrics, Pediatric Cardiology, Johns Hopkins University, Baltimore, MD, USA.

There are a variety of causes of acute heart failure in children including myocarditis, genetic/metabolic conditions, and congenital heart defects. In cases with a structurally normal heart and a negative personal and family history, myocarditis is often presumed to be the cause, but we hypothesise that genetic disorders contribute to a significant portion of these cases. We reviewed our cases of children who presented with acute heart failure and underwent genetic testing from 2008 to 2017. Eighty-seven percent of these individuals were found to have either a genetic syndrome or pathogenic or likely pathogenic variant in a cardiac-related gene. None of these individuals had a personal or family history of cardiomyopathy that was suggestive of a genetic aetiology prior to presentation. All of these individuals either passed away or were listed for cardiac transplantation indicating genetic testing may provide important information regarding prognosis in addition to providing information critical to assessment of family members.
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http://dx.doi.org/10.1017/S1047951119001124DOI Listing
July 2019

Lysyl oxidase-like 2 depletion is protective in age-associated vascular stiffening.

Am J Physiol Heart Circ Physiol 2019 07 19;317(1):H49-H59. Epub 2019 Apr 19.

Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University , Baltimore, Maryland.

Vascular stiffening and its sequelae are major causes of morbidity and mortality in the elderly. The increasingly accepted concept of "smooth muscle cell (SMC) stiffness syndrome" along with matrix deposition has emerged in vascular biology to account for the mechanical phenotype of arterial aging, but the molecular targets remain elusive. In this study, using an unbiased proteomic analysis, we identified lysyl oxidase-like 2 (LOXL2) as a critical SMC mediator for age-associated vascular stiffening. We tested the hypothesis that loss of LOXL2 function is protective in aging-associated vascular stiffening. We determined that exogenous and endogenous nitric oxide markedly decreased LOXL2 abundance and activity in the extracellular matrix of isolated SMCs and LOXL2 endothelial cells suppress LOXL2 abundance in the aorta. In a longitudinal study, LOXL2 mice were protected from age-associated increase in pulse-wave velocity, an index of vascular stiffening, as occurred in littermate wild-type mice. Using isolated aortic segments, we found that LOXL2 mediates vascular stiffening in aging by promoting SMC stiffness, augmented SMC contractility, and vascular matrix deposition. Together, these studies establish LOXL2 as a nodal point for a new therapeutic approach to treat age-associated vascular stiffening. Increased central vascular stiffness augments risk of major adverse cardiovascular events. Despite significant advances in understanding the genetic and molecular underpinnings of vascular stiffening, targeted therapy has remained elusive. Here, we show that lysyl oxidase-like 2 (LOXL2) drives vascular stiffening during aging by promoting matrix remodeling and vascular smooth muscle cell stiffening. Reduced LOXL2 expression protects mice from age-associated vascular stiffening and delays the onset of isolated systolic hypertension, a major consequence of stiffening.
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http://dx.doi.org/10.1152/ajpheart.00670.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6692735PMC
July 2019

Integrated Transcriptomic and Proteomic Analysis of Primary Human Umbilical Vein Endothelial Cells.

Proteomics 2019 08 26;19(15):e1800315. Epub 2019 Jun 26.

Center for Molecular Medicine, National Institute of Mental Health and Neurosciences, Hosur Road, Bangalore, 560029, Karnataka, India.

Understanding the molecular profile of every human cell type is essential for understanding its role in normal physiology and disease. Technological advancements in DNA sequencing, mass spectrometry, and computational methods allow us to carry out multiomics analyses although such approaches are not routine yet. Human umbilical vein endothelial cells (HUVECs) are a widely used model system to study pathological and physiological processes associated with the cardiovascular system. In this study, next-generation sequencing and high-resolution mass spectrometry to profile the transcriptome and proteome of primary HUVECs is employed. Analysis of 145 million paired-end reads from next-generation sequencing confirmed expression of 12 186 protein-coding genes (FPKM ≥0.1), 439 novel long non-coding RNAs, and revealed 6089 novel isoforms that were not annotated in GENCODE. Proteomics analysis identifies 6477 proteins including confirmation of N-termini for 1091 proteins, isoforms for 149 proteins, and 1034 phosphosites. A database search to specifically identify other post-translational modifications provide evidence for a number of modification sites on 117 proteins which include ubiquitylation, lysine acetylation, and mono-, di- and tri-methylation events. Evidence for 11 "missing proteins," which are proteins for which there was insufficient or no protein level evidence, is provided. Peptides supporting missing protein and novel events are validated by comparison of MS/MS fragmentation patterns with synthetic peptides. Finally, 245 variant peptides derived from 207 expressed proteins in addition to alternate translational start sites for seven proteins and evidence for novel proteoforms for five proteins resulting from alternative splicing are identified. Overall, it is believed that the integrated approach employed in this study is widely applicable to study any primary cell type for deeper molecular characterization.
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http://dx.doi.org/10.1002/pmic.201800315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6812510PMC
August 2019

Ablation Lesion Characterization in Scarred Substrate Assessed Using Cardiac Magnetic Resonance.

JACC Clin Electrophysiol 2019 01 26;5(1):91-100. Epub 2018 Dec 26.

Division of Cardiology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland; Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland; Department of Radiology, Johns Hopkins University School of Medicine, Baltimore, Maryland.

Objectives: This study examined radiofrequency catheter ablation (RFCA) lesions within and around scar by cardiac magnetic resonance (CMR) imaging and histology.

Background: Substrate modification by RFCA is the cornerstone therapy for ventricular arrhythmias. RFCA in scarred myocardium, however, is not well understood.

Methods: We performed electroanatomic mapping and RFCA in the left ventricles of 8 swine with myocardial infarction. Non-contrast-enhanced T-weighted (T1w) and contrast-enhanced CMR after RFCA were compared with gross pathology and histology.

Results: Of 59 lesions, 17 were in normal myocardium (voltage >1.5 mV), 21 in border zone (0.5 to 1.5 mV), and 21 in scar (<0.5 mV). All RFCA lesions were enhanced in T1w CMR, whereas scar was hypointense, allowing discrimination among normal myocardium, scar, and RFCA lesions. With contrast-enhancement, lesions and scar were similarly enhanced and not distinguishable. Lesion width and depth in T1w CMR correlated with necrosis in pathology (both; r = 0.94, p < 0.001). CMR lesion volume was significantly different in normal myocardium, border zone, and scar (median: 397 [interquartile range (IQR): 301 to 474] mm, 121 [IQR: 87 to 201] mm, 66 [IQR: 33 to 123] mm, respectively). RFCA force-time integral, impedance, and voltage changes did not correlate with lesion volume in border zone or scar. Histology showed that ablation necrosis extended into fibrotic tissue in 26 lesions and beyond in 14 lesions. In 7 lesions, necrosis expansion was blocked and redirected by fat.

Conclusions: T1w CMR can selectively enhance necrotic tissue in and around scar and may allow determination of the completeness of ablation intra- and post-procedure. Lesion formation in scar is affected by tissue characteristics, with fibrosis and fat acting as thermal insulators.
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http://dx.doi.org/10.1016/j.jacep.2018.11.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6355250PMC
January 2019

Opportunities for microRNAs in the Crowded Field of Cardiovascular Biomarkers.

Annu Rev Pathol 2019 01 17;14:211-238. Epub 2018 Oct 17.

Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA; email:

Cardiovascular diseases exist across all developed countries. Biomarkers that can predict or diagnose diseases early in their pathogeneses can reduce their morbidity and mortality in afflicted individuals. microRNAs are small regulatory RNAs that modulate translation and have been identified as potential fluid-based biomarkers across numerous maladies. We describe the current state of cardiovascular disease biomarkers across a range of diseases, including myocardial infarction, acute coronary syndrome, myocarditis, hypertension, heart failure, heart transplantation, aortic stenosis, diabetic cardiomyopathy, atrial fibrillation, and sepsis. We present the current understanding of microRNAs as possible biomarkers in these categories and where their best opportunities exist to enter clinical practice.
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http://dx.doi.org/10.1146/annurev-pathmechdis-012418-012827DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6442682PMC
January 2019

Human and Cow Have Identical miR-21-5p and miR-30a-5p Sequences, Which Are Likely Unsuited to Study Dietary Uptake from Cow Milk.

J Nutr 2018 09;148(9):1506-1507

Molecular and Comparative Pathobiology, and Neurology, The Johns Hopkins University School of Medicine, Baltimore, MD.

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http://dx.doi.org/10.1093/jn/nxy144DOI Listing
September 2018

Seven factors predict a delayed diagnosis of cardiac amyloidosis.

Amyloid 2018 Sep 31;25(3):174-179. Epub 2018 Aug 31.

a Division of Cardiovascular Pathology, Department of Pathology , Johns Hopkins University , Baltimore , MD , USA.

Introduction: Diagnostic delay of cardiac amyloidosis (CAm) continues to challenge clinicians. We investigated features associated with delay and ascertained if a diagnostic delay had negative implications for the patient.

Methods: We performed a retrospective chart review identifying 82 subjects with biopsy-proven and mass-spectrometry-identified CAm with clinical and epidemiologic data including first potential symptom of amyloidosis. Pathology slides were scored for extent of amyloid. Robust statistical analyses including generalized linear and ordered logistic regression analysis were performed.

Results: There was a 22 month (median) delay in diagnosis, more pronounced (34 months) in subjects with transthyretin (ATTR) amyloidosis. Seven factors predict a delayed diagnosis including ATTR amyloid type (ratio =2.17, 95% CI 1.31-3.59), having carpal tunnel syndrome (2.13, CI 1.49-3.03) and age <70 at first symptom (1.85, CI 1.30-2.61). Individuals with delays of 1+ years had higher levels of NT proBNP (4451 vs. 2559 pg/mL, p = .016) and longer PR intervals (225 vs. 162 ms, p < .001) at the time of diagnosis.

Conclusions: Diagnostic delays negatively affect cardiac function. Of the predictive clinical features, carpal tunnel syndrome was frequent and its presence should lead to a more aggressive analysis for CAm in the appropriate clinical settings.
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http://dx.doi.org/10.1080/13506129.2018.1498782DOI Listing
September 2018

A comprehensive evaluation of the genetic architecture of sudden cardiac arrest.

Eur Heart J 2018 11;39(44):3961-3969

Cardiovascular Health Research Unit, Division of Cardiology, Departments of Medicine and Epidemiology, University of Washington, 1730 Minor Ave, Seattle, WA, USA.

Aims: Sudden cardiac arrest (SCA) accounts for 10% of adult mortality in Western populations. We aim to identify potential loci associated with SCA and to identify risk factors causally associated with SCA.

Methods And Results: We carried out a large genome-wide association study (GWAS) for SCA (n = 3939 cases, 25 989 non-cases) to examine common variation genome-wide and in candidate arrhythmia genes. We also exploited Mendelian randomization (MR) methods using cross-trait multi-variant genetic risk score associations (GRSA) to assess causal relationships of 18 risk factors with SCA. No variants were associated with SCA at genome-wide significance, nor were common variants in candidate arrhythmia genes associated with SCA at nominal significance. Using cross-trait GRSA, we established genetic correlation between SCA and (i) coronary artery disease (CAD) and traditional CAD risk factors (blood pressure, lipids, and diabetes), (ii) height and BMI, and (iii) electrical instability traits (QT and atrial fibrillation), suggesting aetiologic roles for these traits in SCA risk.

Conclusions: Our findings show that a comprehensive approach to the genetic architecture of SCA can shed light on the determinants of a complex life-threatening condition with multiple influencing factors in the general population. The results of this genetic analysis, both positive and negative findings, have implications for evaluating the genetic architecture of patients with a family history of SCA, and for efforts to prevent SCA in high-risk populations and the general community.
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http://dx.doi.org/10.1093/eurheartj/ehy474DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6247663PMC
November 2018

miRge 2.0 for comprehensive analysis of microRNA sequencing data.

BMC Bioinformatics 2018 Jul 23;19(1):275. Epub 2018 Jul 23.

Department of Pathology, Johns Hopkins University SOM, 720 Rutland Avenue/Ross Bldg. Rm 632B, Baltimore, MD, 21205, USA.

Background: miRNAs play important roles in the regulation of gene expression. The rapidly developing field of microRNA sequencing (miRNA-seq; small RNA-seq) needs comprehensive, robust, user-friendly and standardized bioinformatics tools to analyze these large datasets. We present miRge 2.0, in which multiple enhancements were made towards these goals.

Results: miRge 2.0 has become more comprehensive with increased functionality including a novel miRNA detection method, A-to-I editing analysis, integrated standardized GFF3 isomiR reporting, and improved alignment to miRNAs. The novel miRNA detection method uniquely uses both miRNA hairpin sequence structure and composition of isomiRs resulting in higher specificity for potential miRNA identification. Using known miRNA data, our support vector machine (SVM) model predicted miRNAs with an average Matthews correlation coefficient (MCC) of 0.939 over 32 human cell datasets and outperformed miRDeep2 and miRAnalyzer regarding phylogenetic conservation. The A-to-I editing detection strongly correlated with a reference dataset with adjusted R = 0.96. miRge 2.0 is the most up-to-date aligner with custom libraries to both miRBase v22 and MirGeneDB v2.0 for 6 species: human, mouse, rat, fruit fly, nematode and zebrafish; and has a tool to create custom libraries. For user-friendliness, miRge 2.0 is incorporated into bcbio-nextgen and implementable through Bioconda.

Conclusions: miRge 2.0 is a redesigned, leading miRNA RNA-seq aligner with several improvements and novel utilities. miRge 2.0 is freely available at: https://github.com/mhalushka/miRge .
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http://dx.doi.org/10.1186/s12859-018-2287-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6112139PMC
July 2018

Human cardiac -regulatory elements, their cognate transcription factors, and regulatory DNA sequence variants.

Genome Res 2018 10 23;28(10):1577-1588. Epub 2018 Aug 23.

McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

-regulatory elements (CRE), short DNA sequences through which transcription factors (TFs) exert regulatory control on gene expression, are postulated to be the major sites of causal sequence variation underlying the genetics of complex traits and diseases. We present integrative analyses, combining high-throughput genomic and epigenomic data with sequence-based computations, to identify the causal transcriptional components in a given tissue. We use data on adult human hearts to demonstrate that (1) sequence-based predictions detect numerous, active, tissue-specific CREs missed by experimental observations, (2) learned sequence features identify the cognate TFs, (3) CRE variants are specifically associated with cardiac gene expression, and (4) a significant fraction of the heritability of exemplar cardiac traits (QT interval, blood pressure, pulse rate) is attributable to these variants. This general systems approach can thus identify candidate causal variants and the components of gene regulatory networks (GRN) to enable understanding of the mechanisms of complex disorders on a tissue- or cell-type basis.
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http://dx.doi.org/10.1101/gr.234633.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6169896PMC
October 2018

Marked disparity of microRNA modulation by cGMP-selective PDE5 versus PDE9 inhibitors in heart disease.

JCI Insight 2018 08 9;3(15). Epub 2018 Aug 9.

Division of Cardiology, Department of Medicine, The Johns Hopkins Medical Institutions, Baltimore, Maryland, USA.

MicroRNAs (miRs) posttranscriptionally regulate mRNA and its translation into protein, and are considered master controllers of genes modulating normal physiology and disease. There is growing interest in how miRs change with drug treatment, and leveraging this for precision guided therapy. Here we contrast 2 closely related therapies, inhibitors of phosphodiesterase type 5 or type 9 (PDE5-I, PDE9-I), given to mice subjected to sustained cardiac pressure overload (PO). Both inhibitors augment cyclic guanosine monophosphate (cGMP) to activate protein kinase G, with PDE5-I regulating nitric oxide (NO) and PDE9-I natriuretic peptide-dependent signaling. While both produced strong phenotypic improvement of PO pathobiology, they surprisingly showed binary differences in miR profiles; PDE5-I broadly reduces more than 120 miRs, including nearly half those increased by PO, whereas PDE9-I has minimal impact on any miR (P < 0.0001). The disparity evolves after pre-miR processing and is organ specific. Lastly, even enhancing NO-coupled cGMP by different methods leads to altered miR regulation. Thus, seemingly similar therapeutic interventions can be barcoded by profound differences in miR signatures, and reversing disease-associated miR changes is not required for therapy success.
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http://dx.doi.org/10.1172/jci.insight.121739DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6129132PMC
August 2018

xMD-miRNA-seq to generate near in vivo miRNA expression estimates in colon epithelial cells.

Sci Rep 2018 06 28;8(1):9783. Epub 2018 Jun 28.

Department of Pathology, Johns Hopkins University SOM, Baltimore, MD, 21205, USA.

Accurate, RNA-seq based, microRNA (miRNA) expression estimates from primary cells have recently been described. However, this in vitro data is mainly obtained from cell culture, which is known to alter cell maturity/differentiation status, significantly changing miRNA levels. What is needed is a robust method to obtain in vivo miRNA expression values directly from cells. We introduce expression microdissection miRNA small RNA sequencing (xMD-miRNA-seq), a method to isolate cells directly from formalin fixed paraffin-embedded (FFPE) tissues. xMD-miRNA-seq is a low-cost, high-throughput, immunohistochemistry-based method to capture any cell type of interest. As a proof-of-concept, we isolated colon epithelial cells from two specimens and performed low-input small RNA-seq. We generated up to 600,000 miRNA reads from the samples. Isolated epithelial cells, had abundant epithelial-enriched miRNA expression (miR-192; miR-194; miR-200b; miR-200c; miR-215; miR-375) and overall similar miRNA expression patterns to other epithelial cell populations (colonic enteroids and flow-isolated colon epithelium). xMD-derived epithelial cells were generally not contaminated by other adjacent cells of the colon as noted by t-SNE analysis. xMD-miRNA-seq allows for simple, economical, and efficient identification of cell-specific miRNA expression estimates. Further development will enhance rapid identification of cell-specific miRNA expression estimates in health and disease for nearly any cell type using archival FFPE material.
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http://dx.doi.org/10.1038/s41598-018-28198-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6023933PMC
June 2018

Cell-type specific expression of oncogenic and tumor suppressive microRNAs in the human prostate and prostate cancer.

Sci Rep 2018 05 8;8(1):7189. Epub 2018 May 8.

The James Buchanan Brady Urologic Institute and Department of Urology, Johns Hopkins School of Medicine, Baltimore, MD, USA.

MiR-1 and miR-143 are frequently reduced in human prostate cancer (PCa), while miR-141 and miR-21 are frequently elevated. Consequently, these miRNAs have been studied as cell-autonomous tumor suppressors and oncogenes. However, the cell-type specificity of these miRNAs is not well defined in prostate tissue. Through two different microdissection techniques, and droplet digital RT-PCR, we quantified these miRNAs in the stroma and epithelium of radical prostatectomy specimens. In contrast to their purported roles as cell-autonomous tumor suppressors, we found miR-1 and miR-143 expression to be predominantly stromal. Conversely, miR-141 was predominantly epithelial. miR-21 was detected in both stroma and epithelium. Strikingly, the levels of miR-1 and miR-143 were significantly reduced in tumor-associated stroma, but not tumor epithelium. Gene expression analyses in human cell lines, tissues, and prostate-derived stromal cultures support the cell-type selective expression of miR-1, miR-141, and miR-143. Analyses of the PCa Genome Atlas (TCGA-PRAD) showed a strong positive correlation between stromal markers and miR-1 and miR-143, and a strong negative correlation between stromal markers and miR-141. In these tumors, loss of miR-1 and gain of miR-21 was highly associated with biochemical recurrence. These data shed new light on stromal and epithelial miRNA expression in the PCa tumor microenvironment.
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http://dx.doi.org/10.1038/s41598-018-25320-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5940660PMC
May 2018

Cardiomyocytes have mosaic patterns of protein expression.

Cardiovasc Pathol 2018 May - Jun;34:50-57. Epub 2018 Mar 24.

Department of Pathology, Johns Hopkins School of Medicine, Baltimore, MD, USA. Electronic address:

Skeletal myocytes have well-established fast and slow twitch fibers with unique gene and protein specific expression patterns. By immunohistochemical staining, these show a mosaic pattern across myocytes. We hypothesized cardiac myocytes may behave similarly where some proteins are differentially expressed between mature cardiomyocytes. We utilized the tool HPASubC on over 52,000 cardiac images of the Human Protein Atlas to identify differential protein expression patterns by immunohistochemistry across the cardiomyocytes. We matched identified proteins to open chromatin and gene expression data. We identified 143 putative proteins with mosaic patterns of expression across the cardiomyocytes. We validated four of these proteins (MYL3, MYL4, PAM, and MYOM1) and demonstrated unique atrial or ventricular patterns of expression for each. Acetylation of histone H3K27 at the promoters of these four genes were consistent with the atrial/ventricular expression patterns. Despite the generally accepted homogeneity of cardiomyocytes, a small subset of proteins varies between cardiomyocytes in a mosaic pattern. This fundamental process has been previously uncharacterized. These changes may inform on different functional and disease-related activities of proteins in individual cardiomyocytes.
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http://dx.doi.org/10.1016/j.carpath.2018.03.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5940500PMC
October 2018

Big Strides in Cellular MicroRNA Expression.

Trends Genet 2018 03 18;34(3):165-167. Epub 2018 Jan 18.

Department of Biostatistics and Computational Biology, University of Rochester Medical Center, Rochester, NY 14642, USA.

A lack of knowledge of the cellular origin of miRNAs has greatly confounded functional and biomarkers studies. Recently, three studies characterized miRNA expression patterns across >78 human cell types. These combined data expand our knowledge of miRNA expression localization and confirm that many miRNAs show cell type-specific expression patterns.
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http://dx.doi.org/10.1016/j.tig.2017.12.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5834366PMC
March 2018