Brain-derived neurotrophic factor in megakaryocytes

The biosynthesis of endogenous BDNF has thus far been examined in neurons where it is expressed at very low levels, in an activity-dependent fashion. In humans, BDNF has long been known to accumulate in circulating platelets, at levels far higher than in the brain. During the process of blood coagulation, BDNF is released from platelets which has led to its extensive use as a readily accessible biomarker, under the assumption that serum levels may somehow reflect brain levels. To identify the cellular origin of BDNF in platelets, we established primary cultures of megakaryocytes, the progenitors of platelets, and found that human and rat megakaryocytes express the BDNF gene. Surprisingly, the pattern of mRNA transcripts is similar to neurons. In the presence of thapsigargin and of external calcium, the levels of the mRNA species leading to efficient BDNF translation rapidly increase. Under these conditions, pro-BDNF, the obligatory precursor of biologically active BDNF, becomes readily detectable. Megakaryocytes store BDNF in α-granules, with more than 80% of them also containing platelet factor 4. By contrast, BDNF is undetectable in mouse megakaryocytes, in line with the absence of BDNF in mouse serum. These findings suggest that alterations of BDNF levels in human serum as reported in studies dealing with depression or physical exercise may primarily reflect changes occurring in megakaryocytes and platelets, including the ability of the latter to retain and release BDNF

RELEITURA À APLICABILIDADE DO "DANO SOCIAL" NAS RELAÇÕES DE CONSUMO

O presente artigo tem como objeto de estudo o Dano Social, com objetivo de caracterizá-lo como instrumento capaz de sobrepor um dos obstáculos enfrentados para o difícil equilíbrio entre consumidor e fornecedor: a valoração do dano moral e sua destinação; de forma que não ocorra nem o enriquecimento indevido, nem o estímulo à repetição da conduta pela ínfima quantia arbitrada. O relato dos resultados é metodologicamente composto na base lógica-indutiva, enquanto que, para a pesquisa, são utilizadas as técnicas do Referente, da Categoria, do Conceito Operacional e da Pesquisa Bibliográfica

Detection of Leptospira spp. in Water Turtle (Trachemys scripta) Living in Ponds of Urban Parks

Urban parks are green areas of cities where families and children spend hours outside. Turtles often inhabit urban parks. However, even if the animals seem harmless, they may serve as both reservoirs or accidental hosts for different serotypes of Leptospira spp. Leptospira spp. is a waterborne zoonotic bacterium relevant for public health. Reptiles and amphibians may play a role in the epidemiology, transmission, and persistence of Leptospira spp. In the present study, we observed the presence of anti-leptospiral agglutinins in a group of freshwater turtles (Trachemys scripta) captured in three urban ponds of the metropolitan city of Turin, Italy

Modulating hESC-derived cardiomyocyte and endothelial cell function with triple-helical peptides for heart tissue engineering.

In this study, we investigated the role of cardiomyocyte (CM) and endothelial cell (EC) specific interactions with collagen in the assembly of an operational myocardium in vitro. Engineered cardiac patches represent valuable tools for myocardial repair following infarction and are generally constituted of a suitable biomaterial populated by CMs and supportive cell types. Among those, ECs are required for tissue vascularization and positively modulate CM function. To direct the function of human embryonic stem cell (hESC)-derived CM and EC seeded on biomaterials, we replicated cell-collagen interactions, which regulate cellular behaviour in the native myocardium, using triple-helical peptides (THPs) that are ligands for collagen-binding proteins. THPs enhanced proliferation and activity of CMs and ECs separately and in co-culture, drove CM maturation and enabled coordinated cellular contraction on collagen films. These results highlight the importance of collagen interactions on cellular response and establish THP-functionalized biomaterials as novel tools to produce engineered cardiac tissues

Human embryonic stem cell-derived cardiomyocyte platform screens inhibitors of SARS-CoV-2 infection.

Patients with cardiovascular comorbidities are more susceptible to severe infection with SARS-CoV-2, known to directly cause pathological damage to cardiovascular tissue. We outline a screening platform using human embryonic stem cell-derived cardiomyocytes, confirmed to express the protein machinery critical for SARS-CoV-2 infection, and a SARS-CoV-2 spike-pseudotyped virus system. The method has allowed us to identify benztropine and DX600 as novel inhibitors of SARS-CoV-2 infection in a clinically relevant stem cell-derived cardiomyocyte line. Discovery of new medicines will be critical for protecting the heart in patients with SARS-CoV-2, and for individuals where vaccination is contraindicated

SARS-CoV-2 Infects Human Pluripotent Stem Cell-Derived Cardiomyocytes, Impairing Electrical and Mechanical Function

COVID-19 patients often develop severe cardiovascular complications, but it remains unclear if these are caused directly by viral infection or are secondary to a systemic response. Here, we examine the cardiac tropism of SARS-CoV-2 in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) and smooth muscle cells (hPSC-SMCs). We find that that SARS-CoV-2 selectively infects hPSC-CMs through the viral receptor ACE2, whereas in hPSC-SMCs there is minimal viral entry or replication. After entry into cardiomyocytes, SARS-CoV-2 is assembled in lysosome-like vesicles and egresses via bulk exocytosis. The viral transcripts become a large fraction of cellular mRNA while host gene expression shifts from oxidative to glycolytic metabolism and upregulates chromatin modification and RNA splicing pathways. Most importantly, viral infection of hPSC-CMs progressively impairs both their electrophysiological and contractile function, and causes widespread cell death. These data support the hypothesis that COVID-19-related cardiac symptoms can result from a direct cardiotoxic effect of SARS-CoV-2

A highly conserved SOX6 double binding site mediates SOX6 gene downregulation in erythroid cells

The Sox6 transcription factor plays critical roles in various cell types, including erythroid cells. Sox6-deficient mice are anemic due to impaired red cell maturation and show inappropriate globin gene expression in definitive erythrocytes. To identify new Sox6 target genes in erythroid cells, we used the known repressive double Sox6 consensus within the εy-globin promoter to perform a bioinformatic genome-wide search for similar, evolutionarily conserved motifs located within genes whose expression changes during erythropoiesis. We found a highly conserved Sox6 consensus within the Sox6 human gene promoter itself. This sequence is bound by Sox6 in vitro and in vivo, and mediates transcriptional repression in transient transfections in human erythroleukemic K562 cells and in primary erythroblasts. The binding of a lentiviral transduced Sox6FLAG protein to the endogenous Sox6 promoter is accompanied, in erythroid cells, by strong downregulation of the endogenous Sox6 transcript and by decreased in vivo chromatin accessibility of this region to the PstI restriction enzyme. These observations suggest that the negative Sox6 autoregulation, mediated by the double Sox6 binding site within its own promoter, may be relevant to control the Sox6 transcriptional downregulation that we observe in human erythroid cultures and in mouse bone marrow cells in late erythroid maturation

Cardiovascular ACE2 receptor expression in patients undergoing heart transplantation

Abstract: Aims: Membrane‐bound angiotensin‐converting enzyme (ACE)2 is the main cellular access point for SARS‐CoV‐2, but its expression and the effect of ACE inhibition have not been assessed quantitatively in patients with heart failure. The aim of this study was to characterize membrane‐bound ACE2 expression in the myocardium and myocardial vasculature in patients undergoing heart transplantation and to assess the effect of pharmacological ACE inhibition. Methods and results: Left ventricular (LV) tissue was obtained from 36 explanted human hearts from patients undergoing heart transplantation. Immunohistochemical staining with antibodies directed against ACE2 co‐registered with cardiac troponin T (cTnT) and α‐smooth muscle cell actin (SMA) was performed across the entire cohort. ACE2 receptor expression was quantitatively assessed throughout the myocardium and vasculature. ACE2 was consistently expressed throughout the LV myocardium (28.3% ± 22.2% of cardiomyocytes). ACE2 expression was also detected in small calibre blood vessels (range, 2–9 μm), albeit at quantitatively much lower levels (5% ± 9% of blood vessels). There was no significant difference in ACE2 expression between patients receiving ACE inhibitors prior to transplantation and ACE inhibitor‐negative controls (P > 0.05). ACE2 expression did not differ significantly between the different diagnostic groups as the underlying reason for heart transplantation (ANOVA > 0.05). N‐terminal pro‐brain natriuretic peptide (NT‐proBNP) (R2 = 0.37, P = 0.0006) and pulmonary capillary wedge pressure (PCWP) (R2 = 0.13, P = 0.043) assessed by right heart catheterization were significantly correlated with greater ACE2 expression in cardiomyocytes. Conclusions: These data provide a comprehensive characterization of membrane‐bound cardiac ACE2 expression in patients with heart failure with no demonstrable effect exerted by ACE inhibitors

Epitope mapping and characterization of 4-hydroxy-2-nonenal modified-human serum albumin using two different polyclonal antibodies

Lipids are susceptible to damage by reactive oxygen species, and from lipid oxidation reactions many short chain lipid peroxidation products can be formed. 4-Hydroxy-2-nonenal (HNE) is one of the most abundant and cytotoxic lipid oxidation products and is known to form covalent adducts with nucleophilic amino acids of proteins. HNE-modified proteins have value as biomarkers and can be detected by antibody-based techniques, but most commercially available antibodies were raised against HNE-keyhole limpet hemocyanin. We used HNE-treated human serum albumin (HSA) to raise sheep antiserum and report for the first time the use of covalently modified peptide arrays to assess epitope binding of antibodies (Abs). Peptide arrays covering the sequence of HSA and treated post peptide synthesis with HNE were used to compare the different binding patterns of a commercial polyclonal antibody (pAb) raised against HNE-treated KLH and an in-house anti-HNE enriched pAb. The results were correlated with analysis of HNE-modified HSA by high-resolution tandem mass spectrometry. Both anti-HNE pAbs were found to bind strongly to eight common peptides on the HNE-treated HSA membranes, suggesting that HNE adducts per se induced an immune response in both cases even though different immunogens were used. Both antibodies bound with the highest affinity to the peptide 365DPHECYAKVFDEFKPLV381, which contains K378 and was also shown to be modified by the mass spectrometry analysis. Overall, the commercial anti-HNE pAb showed better specificity, recognizing nine out of the eleven adducts found by MS/MS, while the in-house enriched pAb only recognizes six. Nevertheless, the in-house pAb recognized specific peptides that were not recognized by the commercial pAb, which suggests the presence of clones uniquely specific to HNE adducts on HSA

Large-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming.

The production of megakaryocytes (MKs)--the precursors of blood platelets--from human pluripotent stem cells (hPSCs) offers exciting clinical opportunities for transfusion medicine. Here we describe an original approach for the large-scale generation of MKs in chemically defined conditions using a forward programming strategy relying on the concurrent exogenous expression of three transcription factors: GATA1, FLI1 and TAL1. The forward programmed MKs proliferate and differentiate in culture for several months with MK purity over 90% reaching up to 2 × 10(5) mature MKs per input hPSC. Functional platelets are generated throughout the culture allowing the prospective collection of several transfusion units from as few as 1 million starting hPSCs. The high cell purity and yield achieved by MK forward programming, combined with efficient cryopreservation and good manufacturing practice (GMP)-compatible culture, make this approach eminently suitable to both in vitro production of platelets for transfusion and basic research in MK and platelet biology.This work was supported by the Leukemia and Lymphoma Society grant, the UK Medical Research Council (Roger Pedersen), the National Institute for Health Research (NIHR; RP-PG-0310-1002; Willem Ouwehand and Cedric Ghevaert) and a core support grant from the Wellcome Trust and MRC to the Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute. Cedric Ghevaert is supported by the British Heart Foundation (FS/09/039); Marloes Tijssen is supported by the European Hematology Association (Research fellowship) and the British Heart Foundation (PG/13/77/30375). Catherine Hobbs was supported by the National Health Service Blood and Transplant. Matthew Trotter was supported by a Medical Research Council Centre grant (MRC Centre for Stem Cell Biology and Regenerative Medicine); since participation in the work described, Matthew Trotter has become an employee of Celgene Research SLU, part of Celgene Corporation. Nicole Soranzo's research and Sanger Institute affiliates are supported by the Wellcome Trust (WT098051 and WT091310), the EU FP7 (Epigenesys 257082 and Blueprint HEAL TH-F5-2011-282510). The Cambridge Biomedical Centre (BRC) hIPSCs core facility is funded by the NIHR.This is the final version of the article. It first appeared from Nature Publishing Group via https://doi.org/10.1038/ncomms1120