Spatially organized cellular communities form the developing human heart.

The heart, which is the first organ to develop, is highly dependent on its form to function1,2. However, how diverse cardiac cell types spatially coordinate to create the complex morphological structures that are crucial for heart function remains unclear. Here we integrated single-cell RNA-sequencing with high-resolution multiplexed error-robust fluorescence in situ hybridization to resolve the identity of the cardiac cell types that develop the human heart. This approach also provided a spatial mapping of individual cells that enables illumination of their organization into cellular communities that form distinct cardiac structures. We discovered that many of these cardiac cell types further specified into subpopulations exclusive to specific communities, which support their specialization according to the cellular ecosystem and anatomical region. In particular, ventricular cardiomyocyte subpopulations displayed an unexpected complex laminar organization across the ventricular wall and formed, with other cell subpopulations, several cellular communities. Interrogating cell-cell interactions within these communities using in vivo conditional genetic mouse models and in vitro human pluripotent stem cell systems revealed multicellular signalling pathways that orchestrate the spatial organization of cardiac cell subpopulations during ventricular wall morphogenesis. These detailed findings into the cellular social interactions and specialization of cardiac cell types constructing and remodelling the human heart offer new insights into structural heart diseases and the engineering of complex multicellular tissues for human heart repair.

Cell Layers: uncovering clustering structure in unsupervised single-cell transcriptomic analysis.

Motivation: Unsupervised clustering of single-cell transcriptomics is a powerful method for identifying cell populations. Static visualization techniques for single-cell clustering only display results for a single resolution parameter. Analysts will often evaluate more than one resolution parameter but then only report one. Results: We developed Cell Layers, an interactive Sankey tool for the quantitative investigation of gene expression, co-expression, biological processes and cluster integrity across clustering resolutions. Cell Layers enhances the interpretability of single-cell clustering by linking molecular data and cluster evaluation metrics, providing novel insight into cell populations. Availability and implementation: https://github.com/apblair/CellLayers.

Human-gained heart enhancers are associated with species-specific cardiac attributes.

The heart, a vital organ which is first to develop, has adapted its size, structure and function in order to accommodate the circulatory demands for a broad range of animals. Although heart development is controlled by a relatively conserved network of transcriptional/chromatin regulators, how the human heart has evolved species-specific features to maintain adequate cardiac output and function remains to be defined. Here, we show through comparative epigenomic analysis the identification of enhancers and promoters that have gained activity in humans during cardiogenesis. These cis-regulatory elements (CREs) are associated with genes involved in heart development and function, and may account for species-specific differences between human and mouse hearts. Supporting these findings, genetic variants that are associated with human cardiac phenotypic/disease traits, particularly those differing between human and mouse, are enriched in human-gained CREs. During early stages of human cardiogenesis, these CREs are also gained within genomic loci of transcriptional regulators, potentially expanding their role in human heart development. In particular, we discovered that gained enhancers in the locus of the early human developmental regulator ZIC3 are selectively accessible within a subpopulation of mesoderm cells which exhibits cardiogenic potential, thus possibly extending the function of ZIC3 beyond its conserved left-right asymmetry role. Genetic deletion of these enhancers identified a human gained enhancer that was required for not only ZIC3 and early cardiac gene expression at the mesoderm stage but also cardiomyocyte differentiation. Overall, our results illuminate how human gained CREs may contribute to human-specific cardiac attributes, and provide insight into how transcriptional regulators may gain cardiac developmental roles through the evolutionary acquisition of enhancers.

Osteoid Osteoma of the Distal Phalanx: A Rare Condition.

Osteoid osteoma of the distal phalanges in the hand is rare and difficult to diagnose. We report a case of a 37-year-old Caucasian female patient who presented with a mass on the distal phalanx of the index finger. The patient was suffering from intermittent nocturnal pain for more than 18 months along with thickening, localized swelling, and clubbing of the distal phalanx of the right index finger. Radiographs revealed a lytic lesion of the distal phalanx of the right index finger with surrounding sclerosis. An MRI showed an intramedullary lesion with infiltration of the bone marrow, cortex, and surrounding tissue with focal sclerosis and elements of enhancements. A presumptive diagnosis of osteoid osteoma was made and surgical removal of the lesion by curettage and bone grafting was the treatment of choice. The curetted specimen was sent to pathology and the diagnosis of osteoid osteoma was confirmed. The patient was asymptomatic at six months postoperatively. Osteoid osteoma should be included in every differential diagnosis for patients presenting with atypical features of the distal phalanx of the hand.

Cardiac cell type-specific gene regulatory programs and disease risk association.

Misregulated gene expression in human hearts can result in cardiovascular diseases that are leading causes of mortality worldwide. However, the limited information on the genomic location of candidate cis-regulatory elements (cCREs) such as enhancers and promoters in distinct cardiac cell types has restricted the understanding of these diseases. Here, we defined >287,000 cCREs in the four chambers of the human heart at single-cell resolution, which revealed cCREs and candidate transcription factors associated with cardiac cell types in a region-dependent manner and during heart failure. We further found cardiovascular disease-associated genetic variants enriched within these cCREs including 38 candidate causal atrial fibrillation variants localized to cardiomyocyte cCREs. Additional functional studies revealed that two of these variants affect a cCRE controlling KCNH2/HERG expression and action potential repolarization. Overall, this atlas of human cardiac cCREs provides the foundation for illuminating cell type-specific gene regulation in human hearts during health and disease.

Canonical Wnt5b Signaling Directs Outlying Nkx2.5+ Mesoderm into Pacemaker Cardiomyocytes.

Pacemaker cardiomyocytes that create the sinoatrial node are essential for the initiation and maintenance of proper heart rhythm. However, illuminating developmental cues that direct their differentiation has remained particularly challenging due to the unclear cellular origins of these specialized cardiomyocytes. By discovering the origins of pacemaker cardiomyocytes, we reveal an evolutionarily conserved Wnt signaling mechanism that coordinates gene regulatory changes directing mesoderm cell fate decisions, which lead to the differentiation of pacemaker cardiomyocytes. We show that in zebrafish, pacemaker cardiomyocytes derive from a subset of Nkx2.5+ mesoderm that responds to canonical Wnt5b signaling to initiate the cardiac pacemaker program, including activation of pacemaker cell differentiation transcription factors Isl1 and Tbx18 and silencing of Nkx2.5. Moreover, applying these developmental findings to human pluripotent stem cells (hPSCs) notably results in the creation of hPSC-pacemaker cardiomyocytes, which successfully pace three-dimensional bioprinted hPSC-cardiomyocytes, thus providing potential strategies for biological cardiac pacemaker therapy.

Transcriptionally active HERV-H retrotransposons demarcate topologically associating domains in human pluripotent stem cells.

Chromatin architecture has been implicated in cell type-specific gene regulatory programs, yet how chromatin remodels during development remains to be fully elucidated. Here, by interrogating chromatin reorganization during human pluripotent stem cell (hPSC) differentiation, we discover a role for the primate-specific endogenous retrotransposon human endogenous retrovirus subfamily H (HERV-H) in creating topologically associating domains (TADs) in hPSCs. Deleting these HERV-H elements eliminates their corresponding TAD boundaries and reduces the transcription of upstream genes, while de novo insertion of HERV-H elements can introduce new TAD boundaries. The ability of HERV-H to create TAD boundaries depends on high transcription, as transcriptional repression of HERV-H elements prevents the formation of boundaries. This ability is not limited to hPSCs, as these actively transcribed HERV-H elements and their corresponding TAD boundaries also appear in pluripotent stem cells from other hominids but not in more distantly related species lacking HERV-H elements. Overall, our results provide direct evidence for retrotransposons in actively shaping cell type- and species-specific chromatin architecture.

Combinatorial interactions of genetic variants in human cardiomyopathy.

Dilated cardiomyopathy (DCM) is a leading cause of morbidity and mortality worldwide; yet how genetic variation and environmental factors impact DCM heritability remains unclear. Here, we report that compound genetic interactions between DNA sequence variants contribute to the complex heritability of DCM. By using genetic data from a large family with a history of DCM, we discovered that heterozygous sequence variants in the TROPOMYOSIN 1 (TPM1) and VINCULIN (VCL) genes cose-gregate in individuals affected by DCM. In vitro studies of patient-derived and isogenic human-pluripotent-stem-cell-derived cardio-myocytes that were genome-edited via CRISPR to create an allelic series of TPM1 and VCL variants revealed that cardiomyocytes with both TPM1 and VCL variants display reduced contractility and sarcomeres that are less organized. Analyses of mice genetically engineered to harbour these human TPM1 and VCL variants show that stress on the heart may also influence the variable penetrance and expressivity of DCM-associated genetic variants in vivo. We conclude that compound genetic variants can interact combinatorially to induce DCM, particularly when influenced by other disease-provoking stressors.

Mortality Rate and Mid-Term Outcomes of Total Hip Arthroplasty Using Dual Mobility Cups for the Treatment of Femoral Neck Fractures in a Middle Eastern Population.

BACKGROUND: The dual mobility cups (DMCs) were shown to reduce dislocation rate following total hip arthroplasty for any etiology, including femoral neck fractures. No reported studies evaluating DMC results for femoral neck fracture in a Middle Eastern population were found in the literature. METHODS: This study aims to look for mortality rate, clinical, and functional outcomes in a population having specific rituals involving extreme hip positions as part of their daily activities. RESULTS: Of an initial sample of 174 patients (177 operated hips), 18 (10.3%) patients (20 hips) died after a mean of 39.6 ± 13.8 months (ranging from 2 to 49 months) with only 3 (1.7%) during the first post-operative year. Twelve patients (13 hips) were lost to follow-up and 19 patients (19 hips) had their radiological data incomplete. In the final sample of 125 patients (125 hips), no dislocation, aseptic loosening, or infection was encountered. The mean modified Hip Harris Score was of 94.8 ± 8.4. The mean modified Hip Harris Score of 40 patients who used to practice regularly oriental sitting position or prayers was 94.1 ± 3.1. After surgery, 36 of these 40 patients (90%) described their hip as "a forgotten hip." Multivariate analyses found correlation only between mortality and cardiovascular co-morbidities. CONCLUSION: DMC implants showed excellent clinical and functional results. The majority of patients having rituals and customs involving extreme hip positions were able to resume their daily activities. The observed low mortality rate should incite future research to investigate its correlation with the use of DMCs.

Safety of Diagnostic and Therapeutic Cardiac Catheterization in Cancer Patients With Acute Coronary Syndrome and Chronic Thrombocytopenia.

Little data is available on the bleeding risk and outcomes of cancer patients with chronic thrombocytopenia who underwent cardiac catheterization. We sought to assess the safety of coronary angiography, percutaneous coronary intervention, and antiplatelet therapy in cancer patients with acute coronary syndrome (ACS) and chronic thrombocytopenia. We performed a retrospective study of patients with chronic thrombocytopenia who underwent cardiac catheterization for ACS between November 2009 and November 2015. Preprocedural platelet counts were classified into 3 groups: mild thrombocytopenia (50,000 to 100,000/µL), moderate thrombocytopenia (30,000 to 50,000/µL), and severe thrombocytopenia (<30,000/µL). Postprocedural bleeding complications and overall survival (OS) were recorded. A total of 98 patients were included. Mean platelet count on admission was 47.63 ± 29.85 K/µL. Severe thrombocytopenia was identified in 36 patients (36.7%), moderate thrombocytopenia in 20 patients (20.4%), and mild thrombocytopenia in 42 patients (42.9%). Aspirin therapy (alone or in combination with clopidogrel) was used in 66 patients (67.3%), whereas 27 patients (27.6%) were on dual antiplatelet therapy. One procedure-related retroperitoneal hematoma and 3 procedure-related small hematomas were identified. No cerebrovascular events related to the procedure or the antiplatelet therapy were noted. Moderate thrombocytopenia was associated with decreased OS, whereas aspirin, dual antiplatelet therapy, and statin use showed a trend of improved OS. In conclusion, we suggest that coronary angiography and percutaneous coronary intervention can be performed safely in cancer patients with chronic thrombocytopenia. Aspirin therapy and dual antiplatelet therapy should be considered in cancer patients with chronic thrombocytopenia and ACS.

Cell-Surface Marker Signature for Enrichment of Ventricular Cardiomyocytes Derived from Human Embryonic Stem Cells.

To facilitate understanding of human cardiomyocyte (CM) subtype specification, and the study of ventricular CM biology in particular, we developed a broadly applicable strategy for enrichment of ventricular cardiomyocytes (VCMs) derived from human embryonic stem cells (hESCs). A bacterial artificial chromosome transgenic H9 hESC line in which GFP expression was driven by the human ventricular-specific myosin light chain 2 (MYL2) promoter was generated, and screened to identify cell-surface markers specific for MYL2-GFP-expressing VCMs. A CD77+/CD200- cell-surface signature facilitated isolation of >97% cardiac troponin I-positive cells from H9 hESC differentiation cultures, with 65% expressing MYL2-GFP. This study provides a tool for VCM enrichment when using some, but not all, human pluripotent stem cell lines. Tools generated in this study can be utilized toward understanding CM subtype specification, and enriching for VCMs for therapeutic applications.

Progressive and Reversible Conduction Disease With Checkpoint Inhibitors.

Novel antineoplastic therapies are focused on harnessing our own immune system to fight cancer. To that end, cytotoxic T-lymphocyte-associated antigen 4 and programmed death ligand 1 are 2 coinhibitory signals that play central roles in decreasing T-cell response and represent a class of medications termed "checkpoint inhibitors." We present an unusual case of progressive conduction abnormalities induced by checkpoint inhibitors. Prompt medical intervention resulted in full recovery. Despite the anticancer efficacy, the newer antineoplastic agents pose a significant and often life-threatening risk of cardiotoxicity.

Effects of glycaemic variability on cardiac remodelling after reperfused myocardial infarction: Evaluation of streptozotocin-induced diabetic Wistar rats using cardiac magnetic resonance imaging.

AIMS: In addition to hyperglycaemia, glycaemic variability seems to be associated with poor outcomes after acute myocardial infarction. This study explored the impact of glycaemic variability in diabetic Wistar rats subjected to myocardial ischaemia/reperfusion. METHODS: Animals with streptozotocin-induced diabetes received insulin either to maintain stable hyperglycaemia (Dh group) or to generate glycaemic variability (Dv). After experimental myocardial ischaemia/reperfusion was surgically induced, 7T cardiac magnetic resonance imaging (CMR) was performed at weeks 1 (w1) and 3 (w3). RESULTS: Twenty-six rats were randomized [sham group (S): n=5; control group (C): n=7; Dh group: n=6; and Dv group: n=8]. The mean amplitude of glucose reflecting glycaemic variability was higher in the Dv than in the Dh group (9.1±2.7mmol/L vs 5.9±1.9mmol/L; P<0.05). CMR assessment at w3 revealed ventricular enlargement in both Dh and Dv groups compared with the C and S groups (end-diastolic volume: 1.60±0.22 and 1.36±0.30mL/kg compared with 1.11±0.13 and 0.87±0.11mL/kg, respectively; P<0.05). Circumferential strain was altered between w1 and w3 in the remote area only in the Dv group, resulting in a lower value in this group than in the S, C and Dh groups (-0.11±0.01 vs -0.17±0.05, -0.15±0.03 and -0.16±0.03, respectively; P<0.05). In addition, at w3, oedema was also higher in the remote area in the Dv than in the C group (18.3±4.9ms vs 14.5±1.7ms, respectively; P<0.05). CONCLUSION: In the context of experimental myocardial ischaemia/reperfusion, our results suggest that glycaemic variability might have a potentially deleterious impact on myocardial outcomes beyond the classical glucose metrics.

Outcomes of Cancer Patients Undergoing Percutaneous Pericardiocentesis for Pericardial Effusion.

BACKGROUND: Pericardial effusion (PE) is common in cancer patients, but the optimal therapeutic approach is not well defined. Percutaneous pericardiocentesis is less invasive than surgery, but its long-term effectiveness and safety have not been well documented. OBJECTIVES: The goal of this study was to evaluate outcomes of cancer patients undergoing percutaneous pericardiocentesis for PE and assess the procedure's safety in patients with thrombocytopenia. METHODS: Cancer patients who underwent percutaneous pericardiocentesis for PE between November 2009 and October 2014 at the MD Anderson Cancer Center were included. Procedure-related complications, effusion recurrence rate, and overall survival were analyzed. RESULTS: Of 1,645 cancer patients referred for PE, 212 (13%) underwent percutaneous pericardiocentesis. The procedure was successful in 99% of the cases, and there were no procedure-related deaths. Four patients had major procedure-related bleeding that did not vary by platelet count <50,000/µl or ≥50,000/µl (p = 0.1281). Patients with catheter drainage for 3 to 5 days had the lowest recurrence rate (10%). Median overall survival was 143 days; older age (i.e., >65 years), lung cancer, platelet count <20,000/µl, and malignant pericardial fluid were independently associated with poor prognosis. Lung cancer patients with proven malignant effusions had a significantly shorter median 1-year survival compared with those with nonmalignant effusions (16.2% vs. 49.0%, respectively; log-rank test p = 0.0101). A similar difference in 1-year survival was not observed in patients with breast cancer (40.2% vs. 40.0%; log-rank test p = 0.4170). CONCLUSIONS: Percutaneous pericardiocentesis with extended catheter drainage was safe and effective as the primary treatment for PE in cancer patients, including in those with thrombocytopenia. Malignant PE significantly shortened the survival outcome of patients with lung cancer but not those with breast cancer.

Functional organization of excitatory synaptic strength in primary visual cortex.

The strength of synaptic connections fundamentally determines how neurons influence each other's firing. Excitatory connection amplitudes between pairs of cortical neurons vary over two orders of magnitude, comprising only very few strong connections among many weaker ones. Although this highly skewed distribution of connection strengths is observed in diverse cortical areas, its functional significance remains unknown: it is not clear how connection strength relates to neuronal response properties, nor how strong and weak inputs contribute to information processing in local microcircuits. Here we reveal that the strength of connections between layer 2/3 (L2/3) pyramidal neurons in mouse primary visual cortex (V1) obeys a simple rule--the few strong connections occur between neurons with most correlated responses, while only weak connections link neurons with uncorrelated responses. Moreover, we show that strong and reciprocal connections occur between cells with similar spatial receptive field structure. Although weak connections far outnumber strong connections, each neuron receives the majority of its local excitation from a small number of strong inputs provided by the few neurons with similar responses to visual features. By dominating recurrent excitation, these infrequent yet powerful inputs disproportionately contribute to feature preference and selectivity. Therefore, our results show that the apparently complex organization of excitatory connection strength reflects the similarity of neuronal responses, and suggest that rare, strong connections mediate stimulus-specific response amplification in cortical microcircuits.

Ewing sarcoma/primitive neuroectodermal tumor (PNET) of the vulva. Case report and review of the literature.

Ewing sarcoma is a rare highly malignant neoplasm that is mostly encountered as a primary bone tumor of childhood. Extra osseous occurrence is less frequent and cases involving the vulva are very rare with only twelve reported to date. We report the case of a Ewing sarcoma that presented as a vulvar mass in a 28-year-old Lebanese lady and briefly review the differential diagnosis as well as the published literature regarding the clinical presentation and prognosis.

Apical hypertrophic cardiomyopathy.

We describe a patient with asymptomatic apical hypertrophic cardiomyopathy (AHCM) who later developed cardiac arrhythmias, and briefly discuss the diagnostic modalities, differential diagnosis and treatment option for this condition. AHCM is a rare form of hypertrophic cardiomyopathy which classically involves the apex of the left ventricle. AHCM can be an incidental finding, or patients may present with chest pain, palpitations, dyspnea, syncope, atrial fibrillation, myocardial infarction, embolic events, ventricular fibrillation and congestive heart failure. AHCM is frequently sporadic, but autosomal dominant inheritance has been reported in few families. The most frequent and classic electrocardiogram findings are giant negative T-waves in the precordial leads which are found in the majority of the patients followed by left ventricular (LV) hypertrophy. A transthoracic echocardiogram is the initial diagnostic tool in the evaluation of AHCM and shows hypertrophy of the LV apex. AHCM may mimic other conditions such as LV apical cardiac tumors, LV apical thrombus, isolated ventricular non-compaction, endomyocardial fibrosis and coronary artery disease. Other modalities, including left ventriculography, multislice spiral computed tomography, and cardiac magnetic resonance imagings are also valuable tools and are frequently used to differentiate AHCH from other conditions. Medications used to treat symptomatic patients with AHCM include verapamil, beta-blockers and antiarrhythmic agents such as amiodarone and procainamide. An implantable cardioverter defibrillator is recommended for high risk patients.