hiPSC and hiPSC-cardiomyocytes are alternative EV biofactories for cardiac regeneration

In cardiac regenerative medicine, there is a growing interest in using EV as cell-mimetic therapeutics due to their potential superior efficacy and overall advantages over cell transplantation: i) absence of oncogenic risk, ii) low immunogenicity [1], iii) easier large-scale manufacturing and iv) consistent product profile. However, most studies have focused particularly on the potential of either mesenchymal or cardiac progenitor cell-derived EV to promote cardiac repair [2]. Here, we study the potential of human induced pluripotent stem cells (hiPSC) and hiPSC-derived cardiomyocytes (hiPSC-CM) as alternative cell factories for the production of a high yield of therapeutic EV for cardiac regeneration. Due to their high self-renewal ability and capacity to differentiate into functional cardiomyocytes, these cells can provide an unlimited source of EV for application in cardiac regeneration. We generated and characterized EV derived from key stages of hiPSC-CM differentiation and maturation, i.e. from hiPSC (hiPSC-EV), cardiac progenitors (CPC-EV), immature (CMi-EV) and mature (CMm-EV) cardiomyocytes, with the goal of studying their potential role as therapeutics, and whether their yield and function was influenced by the state of their parent cell. Two hiPSC lines were differentiated into hiPSC-CM and cultured as 3D spheroids in a fatty acid supplemented medium to improve CM maturation [3,4]. EV isolation was performed based on density separation on an iodixanol discontinuous gradient, and EV were characterized in terms of particle size and particle size distribution, presence of EV-specific markers, and imaging through transmission electron microscopy. Functional studies were performed using human umbilical vein endothelial cells (HUVECs) to evaluate EV-uptake, migration and angiogenesis. EV yield varied along CM differentiation stages, with a minimum for CPC, for both cell lines. Bioactivity assays with HUVECs showed that uptake of PKH26-labelled EV could be blocked by dynasore, an inhibitor of dynamin-2, a GTPase that plays a crucial role in clathrin and caveolin-dependent endocytosis. Increased migration was observed in HUVECs treated with hiPSC, CPC and CM-derived EV (92.25 ± 14.69% wound closure at 24h for hiPSC, 77.13 ± 13.64 % for CPC, 74.71 ± 19.86% for CMi, 69.2 ± 19.12% for CMm versus 45.65 ± 7.26% for control), but angiogenic properties were found only for hiPSC-EV (fold change of 11.2 ± 4.59 in total segment length vs. control, p\u3c0.001). Current efforts towards the characterization of EV small RNA cargo aim at understanding the correlation between cargo composition and in vitro activity, to identify the optimal cell factory for scalable therapeutic EV production. Funding: FCT PhD fellowship PD/BD/139078/2018; IC&TD Projects MetaCardio” (PTDC/BTM-SAL/32566/2017) and NETDIAMOND (SAICTPAC/0047/2015), and iNOVA4Health Research Unit (UIDB/04462/2020). 1. Zhu, X., et al. Comprehensive toxicity and immunogenicity studies reveal minimal effects in mice following sustained dosing of extracellular vesicles derived from HEK293T cells. Journal of Extracellular Vesicles 6, 1, 2017. 2. El Harane, et al. Acellular therapeutic approach for heart failure: in vitro production of extracellular vesicles from human cardiovascular progenitors. European Heart Journal 39, 20, 2018. 3. Correia, C., et al. Distinct carbon sources affect structural and functional maturation of cardiomyocytes derived from human pluripotent stem cells. Scientific reports 7, 1, 2017. 4. Correia, C., et al. 3D aggregate culture improves metabolic maturation of human pluripotent stem cell derived cardiomyocytes. Biotechnology and Bioengineering 115, 3, 2018

Giant congenital melanocytic nevi: 40 years of experience with the serial excision technique

Although giant congenital melanocytic nevus is a rare lesion, it causes significant deformity and carries a risk of malignant degeneration. Different surgical techniques for the lesion removal are described, including serial resection, resection with skin grafting, and resection and coverage with expanded skin flap (skin expanders). The aim of this study is to report the author's 40 years of experience with cases requiring at least 4 serial excisions to complete the treatment. Serial resection is an effective, safe, and simple technique that requires a lot of patience. Treatment often results in a single linear scar, requires no donor sites, nor large flaps. It is not subject to potencial complications of expanders and avoid aesthetic deformities depending on the location.Univ Fed Sao Paulo UNIFESP, Dermatol Dept, Sao Paulo, SP, BrazilUniv Fed Sao Paulo UNIFESP, Dermatol Surg Ctr, Sao Paulo, SP, BrazilUniv Sao Paulo, Sch Med, Sao Paulo, SP, BrazilUniv Fed Sao Paulo UNIFESP, Dermatol Dept, Sao Paulo, SP, BrazilUniv Fed Sao Paulo UNIFESP, Dermatol Surg Ctr, Sao Paulo, SP, BrazilWeb of Scienc

DIÁLOGO EM MARTIN BUBER: POSSIBILIDADES PARA A EDUCAÇÃO

Este artigo problematiza que possibilidades à educação podem ser eleitas a partir das ideias dialógicas expressas nas obras de Martin Buber. Como método, utiliza a dialética e as pesquisas bibliográfica e documental, a partir das obras buberianas e do documentário "Granito de Arena". Constata que o diálogo para Buber se concretiza na relação Eu-Tu e aponta como possibilidade, à educação, o desenvolvimento do conceito comunidade.

Giant atypical lipoma

Liposarcomas correspond to the most common histological subtype of soft tissue sarcomas. They can be subdivided into: well differentiated or atypical lipoma, undifferentiated, myxoid, round, and pleomorphic cells. Atypical lipomas are the most prevalent and usually appear as asymptomatic softened tumors. They are locally aggressive but rarely lead to distant metastases. The diagnosis of this tumor is based on the imaging and histopathologic findings. Treatment consists of excision surgery with complete tumor removal. It has a good prognosis due to the low percentage of distant metastases. We report a rare case of giant atypical lipoma as well as the adopted therapy and evolution.Univ Fed Sao Paulo UNIFESP, Dept Dermatol, Sao Paulo, SP, BrazilUniv Fed Sao Paulo UNIFESP, Dept Pathol, Sao Paulo, SP, BrazilUniv Fed Sao Paulo UNIFESP, Dept Dermatol, Sao Paulo, SP, BrazilUniv Fed Sao Paulo UNIFESP, Dept Pathol, Sao Paulo, SP, BrazilWeb of Scienc

Functionally Conserved Noncoding Regulators of Cardiomyocyte Proliferation and Regeneration in Mouse and Human

BACKGROUND: The adult mammalian heart has little regenerative capacity after myocardial infarction (MI), whereas neonatal mouse heart regenerates without scarring or dysfunction. However, the underlying pathways are poorly defined. We sought to derive insights into the pathways regulating neonatal development of the mouse heart and cardiac regeneration post-MI. METHODS AND RESULTS: Total RNA-seq of mouse heart through the first 10 days of postnatal life (referred to as P3, P5, P10) revealed a previously unobserved transition in microRNA (miRNA) expression between P3 and P5 associated specifically with altered expression of protein-coding genes on the focal adhesion pathway and cessation of cardiomyocyte cell division. We found profound changes in the coding and noncoding transcriptome after neonatal MI, with evidence of essentially complete healing by P10. Over two-thirds of each of the messenger RNAs, long noncoding RNAs, and miRNAs that were differentially expressed in the post-MI heart were differentially expressed during normal postnatal development, suggesting a common regulatory pathway for normal cardiac development and post-MI cardiac regeneration. We selected exemplars of miRNAs implicated in our data set as regulators of cardiomyocyte proliferation. Several of these showed evidence of a functional influence on mouse cardiomyocyte cell division. In addition, a subset of these miRNAs, miR-144-3p, miR-195a-5p, miR- 451a, and miR-6240 showed evidence of functional conservation in human cardiomyocytes. CONCLUSIONS: The sets of messenger RNAs, miRNAs, and long noncoding RNAs that we report here merit further investigation as gatekeepers of cell division in the postnatal heart and as targets for extension of the period of cardiac regeneration beyond the neonatal period.Leducq Foundation funding via the Transatlantic Network of Excellence (Grant 11CVD01), the British Heart Foundation funding via the Imperial College Centre of Research Excellence and the Imperial Cardiovascular Regenerative Medicine Centre RM/13/1/30157

O PERFIL DE SENSIBILIZAÇÃO ACERCA DO DESCARTE E REUTILIZAÇÃO DE RESÍDUOS SÓLIDOS NA CIDADE UNIVERSITÁRIA, UNIVERSIDADE FEDERAL DO MARANHÃO.

An important aspect the still absent in many universities is planning of disposal, storage and reuse of solid waste. The identification ofsituations faced by academic community regarding generation of waste is essential to the elaboration of programs, projects, systems andpolicies for sustainable management of generated waste. This study aimed to understand how aware of this issue is academic communityof the University City, Federal University of Maranhão (UFMA), through a profile of the production of solid waste and its impacts on theenvironment. The methodology was based on the application of 509 questionnaires during the year 2011 in four different campuses centers,including 33 undergraduate and graduate departments from various fields, and six administrative centers. The questionnaires consisted ofquestions about recycling policies and waste sorting. According to our results, 67.97 % of respondents know the 3Rs (reduce, reuse andrecycle) and 92.32 % said they would participate in a program for waste management if the university were to do so. However, over 60 % ofrespondents do not separate their household waste. Thus, it is important to note that, although encouraged by the university administration,an effective campaign should include the individual awareness of the academic body.Identificar situações com as quais a comunidade acadêmica se defronta quanto à geração de resíduos é imprescindível para que haja uma elaboração de programas e políticas de gestão sustentável em universidades. Este trabalho visou compreender como se dá a sensibilização da comunidade acadêmica da Cidade Universitária/ UFMA, acerca da produção de resíduos sólidos e seus devidos impactos ambientais. A metodologia baseou-se na aplicação de 509 questionários, com perguntas acerca de políticas de reciclagem e coleta seletiva, abrangendo 33 cursos de graduação e pós-graduação de diversas áreas e seis instâncias administrativas. De acordo com os resultados, 67,97% dos entrevistados conhecem as políticas de reaproveitamento, reutilização e reciclagem de resíduos e 92,32% afirmou que participaria de um programa interno caso houvesse incentivo para tal. Assim, é importante ressaltar que ainda que incentivado pelas instâncias administrativas, uma campanha efetiva deve incluir a sensibilização dos integrantes da comunidade acadêmica em questão

The impact of surgical delay on resectability of colorectal cancer: An international prospective cohort study

AIM: The SARS-CoV-2 pandemic has provided a unique opportunity to explore the impact of surgical delays on cancer resectability. This study aimed to compare resectability for colorectal cancer patients undergoing delayed versus non-delayed surgery. METHODS: This was an international prospective cohort study of consecutive colorectal cancer patients with a decision for curative surgery (January-April 2020). Surgical delay was defined as an operation taking place more than 4 weeks after treatment decision, in a patient who did not receive neoadjuvant therapy. A subgroup analysis explored the effects of delay in elective patients only. The impact of longer delays was explored in a sensitivity analysis. The primary outcome was complete resection, defined as curative resection with an R0 margin. RESULTS: Overall, 5453 patients from 304 hospitals in 47 countries were included, of whom 6.6% (358/5453) did not receive their planned operation. Of the 4304 operated patients without neoadjuvant therapy, 40.5% (1744/4304) were delayed beyond 4 weeks. Delayed patients were more likely to be older, men, more comorbid, have higher body mass index and have rectal cancer and early stage disease. Delayed patients had higher unadjusted rates of complete resection (93.7% vs. 91.9%, P = 0.032) and lower rates of emergency surgery (4.5% vs. 22.5%, P < 0.001). After adjustment, delay was not associated with a lower rate of complete resection (OR 1.18, 95% CI 0.90-1.55, P = 0.224), which was consistent in elective patients only (OR 0.94, 95% CI 0.69-1.27, P = 0.672). Longer delays were not associated with poorer outcomes. CONCLUSION: One in 15 colorectal cancer patients did not receive their planned operation during the first wave of COVID-19. Surgical delay did not appear to compromise resectability, raising the hypothesis that any reduction in long-term survival attributable to delays is likely to be due to micro-metastatic disease

Impact of Diabetes and its pharmacological treatment on cardiovascular function: studies on ischaemia-reperfusion models

Tese de doutoramento em Biologia, especialidade de Biologia Celular, apresentada ao Departamento de Ciências da Vida da Fac. Ciências e Tecnologia da Universidade de CoimbraCardiovascular diseases are the leading cause of death worldwide of which ischaemic heart disease plays a main role. A diminished supply of oxygen and nutrients is extremely detrimental for the cardiac tissue, which has a high energetic demand. Following ischaemia-reperfusion injury, cell death will occur to great extent. Dead tissue is replaced by scar tissue, resulting in an impairment of cardiac function and heart failure. Importantly, clinical studies have shown that diabetic patients are more prone to suffer from cardiovascular diseases and to have worse clinical outcomes after an ischaemia-reperfusion injury event. The need for pharmacological agents that target both cardiovascular diseases and diabetes is of extreme relevance. Recently, the UKPDS study suggested that diabetic patients treated with metformin would have better clinical outcomes after suffering a cardiovascular event. Metformin is the most prescribed anti-diabetic drug worldwide and is known to target the mitochondria and 5’AMP-activated kinase (AMPK). AMPK is a kinase that functions as the cellular energy sensor. It is activated upon events that reduce energy reserves, such as exercise and ischaemia and coordinates both metabolism and intracellular signalling events towards an effective use of ATP during energy depletion. Activation of AMPK is beneficial if it occurs during myocardial ischaemia, but is role during reperfusion is still controversial. We hypothesised that metformin could be cardioprotective against myocardial infarction when administered at reperfusion via AMPK activation with concomitant improvement of cardiac metabolism and signalling that would prevent the occurrence of mitochondrial permeability transition (MPT) and cell death. To verify this hypothesis, a Langendorff perfused rat heart model of regional ischaemia with myocardial infarction assessed as our primary endpoint. In addition, adult rat ventricular myocytes were exposed to laser induced oxidative stress and imaged using confocal microscopy to follow mitochondrial depolarization and occurence of MPT and Western blotting performed to verify the activation of protective kinases. To study the influence of diabetes upon ischaemia-reperfusion injury, a colony of Goto-Kakizaki (GK) rat was established and characterized as an animal model of type 2 diabetes. Metformin, when administered at the onset of reperfusion was cardioprotective due to a transient activation AMPK and associated enhanced nitric oxide production, stimulation of adenosine receptors and delayed opening of the MPT pore (MPTP), which prevented cell death. In addition, protection of the diabetic heart was obtained by administration of metformin 24 hours prior to the ischaemic insult, concomitant with AMPK activation prior to the ischaemic insult and with nitric oxide production at reperfusion, an effect similar the protection observed in delayed ischaemic preconditioning. Interestingly, the use of an AMPK inhibitor, compound C, either prior to or during ischaemia abolished the reduction of myocardial infarction in the diabetic heart afforded by ischaemic pre- and postconditioning, suggesting a pivotal role for AMPK for the susceptibility of the myocardium to be protected. These results represented a strong background for the second hypothesis investigated that endogenous AMPK activation during myocardial ischaemia above physiological levels would protect against ischaemia-reperfusion injury. The administration during ischaemia of a newly discovered AMPK activator, A-769662, to diabetic and non-diabetic rat hearts, significantly reduced myocardial infarct size in a concentration-dependent manner. The activation of AMPK associated with ischaemia, was significantly increased in the presence of A-769662 and associated with a reduction of infarction after 120 min of reperfusion. Interestingly, there were no changes in the activation of the Reperfusion Injury Salvage Kinase (RISK) pathway, in the Langendorff rat perfused heart model. Our data suggests that enhancing activation of AMPK during ischaemia may be a novel cardioprotective strategy for the ischaemic-reperfused heart. Moreover, A-769662 significantly delayed MPTP opening in adult ventricular myocytes from non-diabetic and diabetic hearts, when administered prior and during the laser-induced oxidative stress, in the same concentrations shown to reduce myocardial infarction. In summary, we have thus demonstrated that metformin can protect the diabetic heart when given prior or after ischaemia, by the activation of the AMPK-eNOS-NO axis that results in a significant delay of MPTP occurrence and cell death. Hence, more details about metformin’s cardioprotective mechanisms have been revealed as this drug seems to be a valuable cardioprotective agent for the diabetic heart. Furthermore, pharmacological activation of AMPK alone in the diabetic heart could constitute a novel cardioprotective strategy that should be further explored and eventually translated from bench side to the clinical setting.As doenças cardiovasculares são a principal causa de morte em todo o mundo, das quais a doença cardíaca isquémica assume um papel principal. Um fornecimento reduzido de oxigénio e nutrientes é deveras deletério para o tecido cardíaco, o qual possui um elevado consumo energético. Após isquémia-reperfusão (IR), a morte celular ocorre de forma extensa. O tecido morto é substituído por tecido fibroso, levando à disfunção e insuficiência cardíacas. Estudos clínicos demonstraram que os diabéticos são mais propensos para sofrer de doenças cadiovasculares e apresentam piores prognósticos após um evento isquémico. A procura de agentes farmacológicos para o tratamento de doenças cardiovasculares e diabetes é de extrema relevância. Recentemente, o estudo UKPDS sugeriu que nos diabéticos tratados com metformina os prognósticos após um evento cardiovascular seriam melhorados. A metformina (MET) é um dos fármacos anti-diabéticos mais receitados em todo o mundo e actua sobre a mitocôndria e a 5’-AMP-activated kinase (AMPK). A AMPK é uma cinase que funciona como sensor de energia da célula, sendo activada durante situações que reduzem as reservas energéticas da célula, como o exercício físico e a isquémia (I). Ela coordena eventos metabólicos e de sinalização celular promovendo um uso eficaz de ATP. A sua activação é benéfica durante a I mas o seu papel durante a reperfusão permaneça controverso. A nossa hipotése é de que a MET é cardioprotectora contra o enfarte do miocárdio quando administrada na reperfusão, por activação da AMPK, devido a uma melhoria do metabolismo e sinalização cardíacos, prevenindo a ocorrência da transição da permeabilidade mitocondrial (MPT) e morte celular. Para verificar esta hipótese, um modelo de perfusão de Langendorff de coração de rato com isquémia regional foi utilizado e o tamanho do enfarte do miocárdio quantificado enquanto principal medida de dano cardíaco. Além disso, cardiomiócitos ventriculares de coração adulto de rato foram expostos a stress oxidativo induzido por laser e suas imagens analizadas por microscopia confocal, monitorizando-se a despolarização mitocondrial e ocorrência de MPT, sendo a activação de cinases protectoras avaliada por Western blotting. Para o estudo da influência da diabetes sobre o danos causados pela IR, uma colónia de ratos Goto-Kakizaki foi estabelecida e caracterizada enquanto modelo experimental de diabetes tipo 2. A MET, quando administrada no início da reperfusão é cardioprotectora devido à activação transiente da AMPK e aumento da produção de óxido nitrico (NO), estimulação de receptores de adenosina e prevenção da abertura do poro associado a MPT (MPTP), reduzindo a morte celular no miocárdio. A presença de MET, 24 horas antes do insulto isquémico, foi suficiente para proteger corações diabéticos, protecção essa devida à activação da AMPK antes da I e da produção de NO durante a reperfusão, um efeito similar aos mecanismos cardioprotectores inerentes ao precondicionamento isquémico de segunda janela. Na presença de composto C, um inibidor da AMPK, antes e durante a I, redução do enfarte de miocárdio no coração diabético derivada do pré e pós-condicionamento isquémicos foi anulada, sugerindo um papel-chave da activação da AMPK na susceptibilidade do miocárdio a estratégias cardioprotectoras. Estes resultados fundamentaram a segunda hipótese de que a activação endógenea da AMPK durante a I miocárdica, acima dos níveis fisiológicos constituiria um mecanismo de protecção contra o dano de IR. A administração de A-769662, um activador da AMPK recentemente identificado, durante a I a corações diabéticos e não-diabéticos, levou a uma redução significativa do tamanho do enfarte do miocárdio, numa forma dependente da concentração de activador usada. A activação da AMPK durante a I, na presença de A-769662 aumentou significativamente sendo concordante com a redução de enfarte no miocárdio. A realçar, a presença de A-769662 não teve efeito sobre os componentes da via de sinalização RISK (Reperfusion Injury Salvage Kinase), num modelo de perfusão de Langendorff de coração de rato. Os resultados aqui apresentados sugerem que a estimulação da activação da AMPK durante a I pode constituir uma nova estratégia cardioprotectora para o coração submetido a IR. Para além destes efeitos, o composto A-769662, adicionado antes e durante o stress oxidativo, aumentou significativamente o tempo necessário para a abertura do MPTP, em cardiomiócitos ventriculares de corações não-diabéticos e diabéticos, em concentrações observadas como cardioprotectoras. Em suma, foi demonstrado que a MET pode proteger o coração diabético quando administrada antes ou depois da I, através da via AMPK-eNOS-NO com consequente inibição do MPTP e morte celular. Para além do mais, alguns mecanismos celulares da cardioprotecção associada à MET foram elucidados, confirmando –a como um valioso agente cardioprotector para o coração diabético. Finalmente, a activação farmocológica da AMPK no coração diabético, pode fazer parte de uma nova estratégia cardioprotectora a ser explorada e eventualmente, transportada do laboratório para a clínica