Spatially resolved multiomics of human cardiac niches.

The function of a cell is defined by its intrinsic characteristics and its niche: the tissue microenvironment in which it dwells. Here we combine single-cell and spatial transcriptomics data to discover cellular niches within eight regions of the human heart. We map cells to microanatomical locations and integrate knowledge-based and unsupervised structural annotations. We also profile the cells of the human cardiac conduction system1. The results revealed their distinctive repertoire of ion channels, G-protein-coupled receptors (GPCRs) and regulatory networks, and implicated FOXP2 in the pacemaker phenotype. We show that the sinoatrial node is compartmentalized, with a core of pacemaker cells, fibroblasts and glial cells supporting glutamatergic signalling. Using a custom CellPhoneDB.org module, we identify trans-synaptic pacemaker cell interactions with glia. We introduce a druggable target prediction tool, drug2cell, which leverages single-cell profiles and drug-target interactions to provide mechanistic insights into the chronotropic effects of drugs, including GLP-1 analogues. In the epicardium, we show enrichment of both IgG+ and IgA+ plasma cells forming immune niches that may contribute to infection defence. Overall, we provide new clarity to cardiac electro-anatomy and immunology, and our suite of computational approaches can be applied to other tissues and organs.

Ultra-high-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight mass spectrometry and molecular networking analysis to investigate the chemodiversity of bioactive extracts of Annona jahnii Saff. fungi from the Brazilian Amazon.

RATIONALE: Annona species are of interest for the isolation of bioactive molecules; however, studies of Annona jahnii Saff. are limited. The exploration of bioactive metabolites of endophytes isolated from this species is unprecedented and allows the preservation of the host plant, in addition to enabling the discovery of compounds with promising biological activities. METHODS: Ethyl acetate extracts from the cultured media of five fungi were obtained. The antioxidant capacity of the extracts was measured using the 1,1-diphenyl-2-picrylhydrazyl free radical method. Antimicrobial activity was determined using the microdilution method in broth in 96-well plates. The exploration of the metabolic profile of the extracts and dereplication of the compounds were performed using ultra-high-performance liquid chromatography/electrospray ionization/tandem mass spectrometry (UHPLC/ESI-MS/MS) combined with analysis using molecular networking (MN). RESULTS: A total of 1818 MS features were detected in the five selected extracts, of which 39 compounds were putatively identified. The secondary metabolites with the highest abundance were alkaloids, naphthopyrons, and cytochalasins. Other secondary metabolites include fumonisins, coumarin, and a meroterpenoid. Most of these compounds are related to specific biological properties such as antioxidant, anti-inflammatory, antimicrobial, antiviral, and antitumor activities. Extracts F398 and F403 showed inhibitory activity of the four pathogens tested. Extracts F475 and F506 did not inhibit the growth of Staphylococcus aureus, and F407 did not inhibit the growth of Escherichia coli in addition to having potent antioxidant activity, with IC50 values of 10 µg/mL or less. CONCLUSIONS: The use of UHPLC/ESI-MS/MS data combined with MN proved useful in the dereplication of bioactive molecules of complex extracts that are still unexplored. These initial investigations should significantly assist in further research and increase the efficiency and speed in the discovery of new sources of secondary metabolites and new natural products.

Achieving the best method to classify Eosinophilic Chronic Rhinosinusitis: a systematic review.

BACKGROUND: Chronic Rhinosinusitis is currently classified into eosinophilic and non-eosinophilic, according to the histologic quantification of the number of eosinophils in nasal mucosa biopsy. There is a lack of unanimous histopathologic criteria and methodology for this classification and no consensus regarding a cut-off point for Eosinophils per High power field. METHODOLOGY: A systematic electronic search was performed on BVS, PUBMED, PUBMED PMC, SCOPUS, WEB OF SCIENCE, EMBASE, COCHRANE and PROQUEST databases looking for studies that reported a cut point for classification of Eosinophilic Chronic Rhinosinusitis (eCRS), and data concerning methodology of classification was extracted. RESULTS: We identified 142 studies that reported 29 different cut-off values for classification of eCRS, and different methods of histologic analysis. Out of these studies 13 reported their own methodology to establish the cut-off point, and used different reference standards as polyp recurrence, asthma and allergy, immunocytochemistry, quality of life index, standard deviation of the control population and cluster analysis. CONCLUSIONS: Further studies are needed to determine a precise cut-off point, especially international multicentered cluster analysis. Moreover, methodologic standardization of biopsy and analysis is needed to certify comparable results. Multiple biopsy sites, densest cellular infiltration area examination and oral steroids restriction at least four weeks before sampling are advisable.

Pedicled Sensate Composite Calcaneal Flap in Children With Congenital Tibial Pseudoarthrosis.

BACKGROUND: The preservation and functionality of a limb affected by a malformation (such as congenital pseudoarthrosis of the tibia) or a severely mangled lower limb in children, despite modern reconstructive techniques, remains challenging, often eventually requiring amputation to achieve a better outcome. The classical Syme and Boyd procedures are functionally better than transtibial (TT) amputation, but are not feasible for congenital tibial pseudoarthrosis. TT amputation delivers an excellent, effective, and functional stump that usually leads, after prosthetization, to a functional gait. Unfortunately, in some situations, particularly when amputation is performed conventionally, the stump is also associated with complications. Future surgical revisions are often needed, particularly in children, because of stump overgrowth. METHODS: Between 2008 and 2010, three patients diagnosed with congenital pseudoarthrosis of the tibia associated with neurofibromatosis who were indicated for TT amputation with calcaneal flap after failure of all previous surgical reconstructive procedures were selected. The chosen method for osteosynthesis was an external fixator of Ilizarov. RESULTS: At 12 weeks of follow-up, the stump had healed in all three patients, and tibiocalcaneal fusion was achieved without complications. All patients were prosthetized and had an asymptomatic gait. After a minimum follow-up of 6 years, all three cases with the pedicled sensate composite calcaneal flap still had a strong, full weight-bearing surface and have adapted easily to the conventional prosthesis, providing a painless stump with excellent functionality. CONCLUSION: With a 0 rate of needed revisions, all 3 cases with the pedicled sensate composite calcaneal flap preserving the hind foot still have a strong, full weight-bearing surface and have easily adapted to the conventional prosthesis, providing a painless and excellent functional stump that could last a lifetime. LEVEL OF EVIDENCE: Level IV.

Biallelic variants in POPDC2 cause a novel autosomal recessive syndrome presenting with cardiac conduction defects and variable hypertrophic cardiomyopathy.

POPDC2 encodes for the Popeye domain-containing protein 2 which has an important role in cardiac pacemaking and conduction, due in part to its cAMP-dependent binding and regulation of TREK-1 potassium channels. Loss of Popdc2 in mice results in sinus pauses and bradycardia and morpholino knockdown of popdc2 in zebrafish results in atrioventricular (AV) block. We identified bi-allelic variants in POPDC2 in 4 families that presented with a phenotypic spectrum consisting of sinus node dysfunction, AV conduction defects and hypertrophic cardiomyopathy. Using homology modelling we show that the identified POPDC2 variants are predicted to diminish the ability of POPDC2 to bind cAMP. In in vitro electrophysiological studies we demonstrated that, while co-expression of wild-type POPDC2 with TREK-1 increased TREK-1 current density, POPDC2 variants found in the patients failed to increase TREK-1 current density. While patient muscle biopsy did not show clear myopathic disease, it showed significant reduction of the expression of both POPDC1 and POPDC2, suggesting that stability and/or membrane trafficking of the POPDC1-POPDC2 complex is impaired by pathogenic variants in any of the two proteins. Single-cell RNA sequencing from human hearts demonstrated that co-expression of POPDC1 and 2 was most prevalent in AV node, AV node pacemaker and AV bundle cells. Sinoatrial node cells expressed POPDC2 abundantly, but expression of POPDC1 was sparse. Together, these results concur with predisposition to AV node disease in humans with loss-of-function variants in POPDC1 and POPDC2 and presence of sinus node disease in POPDC2, but not in POPDC1 related disease in human. Using population-level genetic data of more than 1 million individuals we showed that none of the familial variants were associated with clinical outcomes in heterozygous state, suggesting that heterozygous family members are unlikely to develop clinical manifestations and therefore might not necessitate clinical follow-up. Our findings provide evidence for POPDC2 as the cause of a novel Mendelian autosomal recessive cardiac syndrome, consistent with previous work showing that mice and zebrafish deficient in functional POPDC2 display sinus and AV node dysfunction.

Single-cell transcriptomics for the assessment of cardiac disease.

Cardiovascular disease is the leading cause of death globally. An advanced understanding of cardiovascular disease mechanisms is required to improve therapeutic strategies and patient risk stratification. State-of-the-art, large-scale, single-cell and single-nucleus transcriptomics facilitate the exploration of the cardiac cellular landscape at an unprecedented level, beyond its descriptive features, and can further our understanding of the mechanisms of disease and guide functional studies. In this Review, we provide an overview of the technical challenges in the experimental design of single-cell and single-nucleus transcriptomics studies, as well as a discussion of the type of inferences that can be made from the data derived from these studies. Furthermore, we describe novel findings derived from transcriptomics studies for each major cardiac cell type in both health and disease, and from development to adulthood. This Review also provides a guide to interpreting the exhaustive list of newly identified cardiac cell types and states, and highlights the consensus and discordances in annotation, indicating an urgent need for standardization. We describe advanced applications such as integration of single-cell data with spatial transcriptomics to map genes and cells on tissue and define cellular microenvironments that regulate homeostasis and disease progression. Finally, we discuss current and future translational and clinical implications of novel transcriptomics approaches, and provide an outlook of how these technologies will change the way we diagnose and treat heart disease.

Analysis of the Effect of the TRPC4/TRPC5 Blocker, ML204, in Sucrose-Induced Metabolic Imbalance.

Sugar-induced metabolic imbalances are a major health problem since an excessive consumption of saccharides has been linked to greater obesity rates at a global level. Sucrose, a disaccharide composed of 50% glucose and 50% fructose, is commonly used in the food industry and found in a range of fast, restaurant, and processed foods. Herein, we investigated the effects of a TRPC4/TRPC5 blocker, ML204, in the metabolic imbalances triggered by early exposure to sucrose-enriched diet in mice. TRPC4 and TRPC5 belong to the family of non-selective Ca+2 channels known as transient receptor potential channels. High-sucrose (HS)-fed animals with hyperglycaemia and dyslipidaemia, were accompanied by increased body mass index. mesenteric adipose tissue accumulation with larger diameter cells and hepatic steatosis in comparison to those fed normal diet. HS mice also exhibited enhanced adipose, liver, and pancreas TNFα and VEGF levels. ML204 exacerbated hyperglycaemia, dyslipidaemia, fat tissue deposition, hepatic steatosis, and adipose tissue and liver TNFα in HS-fed mice. Normal mice treated with the blocker had greater hepatic steatosis and adipose tissue cell numbers/diameter than those receiving vehicle, but showed no significant changes in tissue inflammation, glucose, and lipid levels. The results indicate that TRPC4/TRPC5 protect against the metabolic imbalances caused by HS ingestion.

Pathogenic variants damage cell composition and single cell transcription in cardiomyopathies.

Pathogenic variants in genes that cause dilated cardiomyopathy (DCM) and arrhythmogenic cardiomyopathy (ACM) convey high risks for the development of heart failure through unknown mechanisms. Using single-nucleus RNA sequencing, we characterized the transcriptome of 880,000 nuclei from 18 control and 61 failing, nonischemic human hearts with pathogenic variants in DCM and ACM genes or idiopathic disease. We performed genotype-stratified analyses of the ventricular cell lineages and transcriptional states. The resultant DCM and ACM ventricular cell atlas demonstrated distinct right and left ventricular responses, highlighting genotype-associated pathways, intercellular interactions, and differential gene expression at single-cell resolution. Together, these data illuminate both shared and distinct cellular and molecular architectures of human heart failure and suggest candidate therapeutic targets.

Characterization of a common progenitor pool of the epicardium and myocardium.

The mammalian heart is derived from multiple cell lineages; however, our understanding of when and how the diverse cardiac cell types arise is limited. We mapped the origin of the embryonic mouse heart at single-cell resolution using a combination of transcriptomic, imaging, and genetic lineage labeling approaches. This mapping provided a transcriptional and anatomic definition of cardiac progenitor types. Furthermore, it revealed a cardiac progenitor pool that is anatomically and transcriptionally distinct from currently known cardiac progenitors. Besides contributing to cardiomyocytes, these cells also represent the earliest progenitor of the epicardium, a source of trophic factors and cells during cardiac development and injury. This study provides detailed insights into the formation of early cardiac cell types, with particular relevance to the development of cell-based cardiac regenerative therapies.

Spatial protein analysis in developing tissues: a sampling-based image processing approach.

Advances in fluorescence microscopy approaches have made it relatively easy to generate multi-dimensional image volumes and have highlighted the need for flexible image analysis tools for the extraction of quantitative information from such data. Here we demonstrate that by focusing on simplified feature-based nuclear segmentation and probabilistic cytoplasmic detection we can create a tool that is able to extract geometry-based information from diverse mammalian tissue images. Our open-source image analysis platform, called 'SilentMark', can cope with three-dimensional noisy images and with crowded fields of cells to quantify signal intensity in different cellular compartments. Additionally, it provides tissue geometry related information, which allows one to quantify protein distribution with respect to marked regions of interest. The lightweight SilentMark algorithms have the advantage of not requiring multiple processors, graphics cards or training datasets and can be run even with just several hundred megabytes of memory. This makes it possible to use the method as a Web application, effectively eliminating setup hurdles and compatibility issues with operating systems. We test this platform on mouse pre-implantation embryos, embryonic stem cell-derived embryoid bodies and mouse embryonic heart, and relate protein localization to tissue geometry. This article is part of a discussion meeting issue 'Contemporary morphogenesis'.

Human pluripotent stem cell-derived cardiomyocytes as a target platform for paracrine protection by cardiac mesenchymal stromal cells.

Ischemic heart disease remains the foremost cause of death globally, with survivors at risk for subsequent heart failure. Paradoxically, cell therapies to offset cardiomyocyte loss after ischemic injury improve long-term cardiac function despite a lack of durable engraftment. An evolving consensus, inferred preponderantly from non-human models, is that transplanted cells benefit the heart via early paracrine signals. Here, we tested the impact of paracrine signals on human cardiomyocytes, using human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) as the target of mouse and human cardiac mesenchymal stromal cells (cMSC) with progenitor-like features. In co-culture and conditioned medium studies, cMSCs markedly inhibited human cardiomyocyte death. Little or no protection was conferred by mouse tail tip or human skin fibroblasts. Consistent with the results of transcriptomic profiling, functional analyses showed that the cMSC secretome suppressed apoptosis and preserved cardiac mitochondrial transmembrane potential. Protection was independent of exosomes under the conditions tested. In mice, injecting cMSC-conditioned media into the infarct border zone reduced apoptotic cardiomyocytes > 70% locally. Thus, hPSC-CMs provide an auspicious, relevant human platform to investigate extracellular signals for cardiac muscle survival, substantiating human cardioprotection by cMSCs, and suggesting the cMSC secretome or its components as potential cell-free therapeutic products.

Successful accomplishment of educational goals with clinical experience at public primary care facilities.

GOAL: To compare the spectrum of clinical encounters experienced by medical students at the primary level of care in six urban public health units, and to determine the extent to which these educational experiences were sufficient to meet learning objectives proposed for a teaching module. METHOD: During the 4th year of a new six- year curriculum, 113 students cared for adults, the elderly, women and children. They were supervised by faculty and trained supervisors during three 4-hours periods a week, every other week, from January to October at six primary health units. RESULTS: There were 7198 clinical encounters (2493 for adults, 2440 for women, and 2302 for children), during a total of 37 periods, averaging 1.8 cases/student per period. The top five primary diagnoses, similar at all primary health units, included: for adults--hypertension, diabetes, upper respiratory diseases, anxiety/depression, and obesity; for children--first-year follow up, upper respiratory diseases, dermatological, and infectious diseases; for women--antenatal care, vaginal discharge, cervical cancer screening, climacteric symptoms/menstrual disorders, and family planning. CONCLUSIONS: Students were exposed to and cared for the most common conditions observed at the primary level of care, with a sufficient homogeneous clinical spectrum among six primary health units, meeting essential learning objectives related to ambulatory care.

An insight into soil bioremediation through respirometry.

Respirometric tests on a soil contaminated by crude oil were performed. Continuous measurements of oxygen and carbon dioxide concentrations and temperature in the soil atmosphere resulted in a large volume of data. Time series and system identification theories were used to analyze data as a biological signal, allowing us to detect some particularities related to daily cycles of the studied variables as well as its time relationships through autocorrelation and cross-correlation functions. Using system identification techniques, it was possible to build black box models, namely autoregressive moving average models which enable to predict oxygen concentration at the outlet in a good agreement with measured data.

Chemical composition and antimicrobial evaluation of the essential oils of Bocageopsis pleiosperma Maas.

Essential oils from the leaves, twigs and barks of Bocageopsis pleiosperma Maas were obtained by using hydrodistillation and analysed by using gas chromatography coupled to mass spectrometry. Several compounds (51) were detected and identified, being ß-bisabolene the main component in all aerial parts of the plant, with higher concentration in the leaves (55.77%), followed by barks (38.53%) and twigs (34.37%). In order to increase the biological knowledge about the essential oil of Bocageopsis species, antimicrobial activities were evaluated against the microorganisms Escherichia coli, Staphylococcus epidermidis, Enterobacter aerogenes, Candida tropicalis, Candida dubliniensis, Candida glabrata and Candida albicans. The essential oil obtained from the barks exhibited a moderate effect against S. epidermidis ATCC 1228 (MIC = 250 µg/mL), while the other oils did not exhibit antimicrobial activity. These results represent the first report about the chemical composition of B. pleiosperma and the first antimicrobial evaluation with a Bocageopsis species.

Oral anti-inflammatory and anti-ulcerogenic activities of a hydroalcoholic extract and partitioned fractions of Turnera ulmifolia (Turneraceae).

Anti-inflammatory studies were conducted on rats or mice using a crude hydroalcoholic extract of the aerial parts of Turnera ulmifolia and it's partitioned fractions, i.e. the aqueous, ethyl acetate and ethanolic fractions. The hydroalcoholic extract and it's fractions (aqueous and ethanolic) inhibited carrageenan-induced edema. However, only the ethanolic fraction was used in the other experiments due to it's yield. The extract also inhibited the cotton pellet granuloma and the increase of vascular permeability induced by histamine, 5-hydroxytryptamine and prostaglandin E2, but not that produced by bradykinin. The extract or the fraction did not present analgesic activity in the writhing test using acetic acid and did not reduce croton oil-induced ear edema in mice. When the ethanolic fraction and LPS were administered i.p. to Balb/C mice 72 h before blood or peritoneal fluid collection, no changes were observed in the white or total blood cell counts in the peripheral blood. On the other hand, changes were observed in both total and differential cell counts in the peritoneal exudate since all doses of the fraction reduced the number of total leukocytes (mainly lymphocytes) obtained from the peritoneal exudate. In contrast to nonsteroidal anti-inflammatory drugs, the administration of the hydroalcoholic extract or the ethanolic fraction alone did not potentiate gastric mucosal lesions induced by aspirin. The extract and the fraction inhibited the appearance of gastric lesions induced by indomethacin, ethanol and pylorus ligature, but not those induced by stress. As also observed with carbenoxolone, the ethanolic fraction increased the wall mucus in hypothermical-restraint stress-induced gastric lesions. The anti-ulcerogenic effect of the extract and of the ethanolic fraction may be related to an increase of mucosal defensive factors, such as prostaglandin and mucus. The anti-inflammatory actions of the extract and the fraction may be due to an inhibitory effect on histamine and cyclooxygenase II, but not on cyclooxygenase I, because the extract and it's fraction present both anti-inflammatory and anti-ulcerogenic effects. The major substances present in the ethanolic fraction are flavonoids which will be isolated and identified.

Efficacy of a whey protein concentrate on the inhibition of stomach ulcerative lesions caused by ethanol ingestion.

The purpose of this research was to test the ability of a whey protein concentrate (WPC) to inhibit gastric mucosal ulcerative lesions caused by oral administration to rats of absolute ethanol. Acute administration (single doses) of WPC resulted in 41% inhibition of the ulcerative lesion index (ULI), and 73% inhibition was obtained with repetitive doses. In a 10-days subchronic treatment study, the inhibition was 64%, all relative to a saline treatment (negative control). Alkylation of sulfhydryl compounds by subcutaneous injection of N-ethylmaleimide essentially eliminated the WPC protection. Treating the rats with an intraperitoneal injection of butathionine sulfoximine, which inhibits glutathione synthesis, reduced WPC protection to 35% and 52% for single and double doses, respectively. Taken as a whole, the results indicate that WPC does protect gastric mucosa from ethanol damage and that the protection depends on sulfhydryl compounds present in the WPC, including its capacity to stimulate glutathione synthesis.

Protective effect of bovine milk whey protein concentrate on the ulcerative lesions caused by subcutaneous administration of indomethacin.

The protective effect of a whey protein concentrate (WPC) was studied in three models of stomach ulcerative lesions induction: subcutaneous injection of indomethacin, and stress induced by either intraperitoneal injection of reserpine, or immobilization and holding in the cold (4 degrees C, 2 hours). Adult Wistar rats (300-400 g) were used for acute (single-dose), repetitive, or subchronic (10 days) administration of WPC prior to treatment with the ulcerogenic factors. The best protection was achieved in the indomethacin model for repetitive and subchronic experiments, reaching 50.1% and 44%, respectively, inhibition of the ulcerative lesions, which was significant at 1% probability (P <.01). For the immobilization and cold model, maximum inhibition by WPC was 22.1%, and that for the reserpine model was 23.8%. In both models the inhibition was not significant (P >.05) compared with saline (negative control).

Hypotensive and vasorelaxant effects of ethanolic extract from Jatropha gossypiifolia L. in rats.

The present work was carried out to examine the hypotensive effects of ethanolic extract (EE) from Jatropha gossypiifolia L. The oral administration of EE (125 or 250 mg kg(-1) day(-1)) caused a significant and dose-dependent reduction of the systolic blood pressure. The concentration-response curves to norepinephrine (NE) or Ca(2+) were non-parallelly shifted to the right and the maximum contractile responses were concentration dependently depressed by EE (0.1 or 0.5 mg/ml) in endothelium-deprived mesenteric artery. The cumulative additions of EE (0.1-30 mg/ml) caused a concentration-dependent relaxant response in endothelium-deprived mesenteric artery precontracted with NE or Ca(2+). In conclusion, our results have shown that the EE from J. gossypiifolia L. can elicit hypotension, by oral via, in conscious normotensive rats and vasorelaxant activity on rat mesenteric rings precontracted with NE or Ca(2+).

[Growth analysis of 566 children from the fourteen day care centers of Paulínia (São Paulo, Brazil), with ages ranging from 3 months to 3 years]. / Análise do perfil de crescimento de 566 crianças com idade entre 3 meses e 3 anos matriculadas nas 14 creches municipais de Paulínia (SP).

To study the growth of 566 children (273 males and 293 females) from fourteen day care centers of Paulínia (São Paulo, Brazil), with ages ranging from 3 months to 3 years, admitted from March 1st to May 31st, 1993, the authors analyzed the z-score distribution of height for age and weight for height in relation to age group, per capita family income, social class levels, mothers education level and child birth weight. The Kruskal-Wallis test and the Multiple Comparison test were used in the statistical analysis. The children with less than 24 months or with birth weight less than 3000 g as the children with mothers education level less than four years, presented left deviated distribution in the height for age z-score. The weight for height score was less satisfactory in the group with per capita income less than one salary, in the group with birth weight less than 3000 g, and in the group with ages superior to 18 months.Therefore, institutional actions concerning the children and their parents are recommended in order to attenuate these factors.