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

Evaluation of the biological potential of Himanthalia elongata (L.) S.F.Gray and Eisenia bicyclis (Kjellman) setchell subcritical water extracts

Neuroprotection is a need that remains unmet in treating chronic neurodegenerative disorders, despite decades of extensive research. To find new neuroprotective compounds, extracts of Himanthalia elongata (L.) S.F.Gray and of Eisenia bicyclis (Kjellman) Setchell were obtained through subcritical water extraction applying a four-step temperature gradient. The fractions obtained were screened against brain enzymes involved in neurodegenerative etiology, namely in Alzheimer’s and Parkinson’s diseases, and against reactive oxygen and nitrogen species, all contributing factors to the progression of neurodegeneration. Results showed no significant enzyme inhibition but strong radical scavenging activities, particularly in the fourth fraction, extracted at the highest temperature (250 °C), highlighting their ability to retard oxidative and nitrosative stresses. At higher temperatures, fractions were composed of phenolic compounds and Maillard reaction products, a combination that contributed to their antioxidant activity and, consequently, their neuroprotective properties. All fractions were evaluated for the presence of iodine, 14 organochlorine and 7 organophosphorus pesticides, and pharmaceuticals used in Alzheimer’s and Parkinson’s diseases (14), psychiatric drugs (8), and metabolites (8). The fractions studied did not present any of the screened contaminants, and only fraction 1 of E. bicyclis should be used with caution due to iodine content.info:eu-repo/semantics/publishedVersio

Desenvolvimento da competência cultural na formação dos estudantes de enfermagem

The aim was to identify and map the strategies for the development of cultural competence during the training of students in nursing education institutions. For the elaboration of this Integrative Literature Review, the PCC method was used to develop the research question: What are the strategies used by Nursing Education Institutions to develop cultural competence during the training of students? To answer the question asked, a search was carried out, using the EBSCO-host and PubMed search engines, and the respective databases Cumulative Index to Nursing and Allied Health Literature and MedLine. No time horizon limits were applied. The established languages ​​were Portuguese, English and Spanish. For the assessment of methodological quality, we used a critical assessment instrument, specific for each type of study (qualitative and quantitative). Seven studies were included in the selection, which after being analyzed showed that there are strategies through which students can develop CC, but the most used are: Mobility Programs and Cultural Competence Courses. These results provide evidence of the potential use of educational strategies for the development of CC in nursing students.Objetivou-se identificar e mapear as estratégias de desenvolvimento da competência cultural durante a formação dos estudantes nas instituições de ensino de enfermagem. Para a elaboração da presente Revisão Integrativa da Literatura, recorreu-se ao método PCC, de forma a desenvolver a questão de investigação: Quais são as estratégias utilizadas pelas Instituições de Ensino de Enfermagem para Desenvolvimento da competência cultural durante a formação dos estudantes? De modo a dar resposta à questão formulada, realizou-se uma pesquisa, com recurso aos motores de busca EBSCO-host e PubMed, e às respectivas bases de dados Cumulative Index to Nursing and Allied Health Literature e MedLine. Não foram aplicados limites de horizonte temporal. Os idiomas estabelecidos foram, português, inglês e espanhol. Para a avaliação da qualidade metodológica, utilizamos um instrumento de avaliação crítica, específico para cada tipo de estudo (qualitativo e quantitativo). Sete estudos foram incluídos na seleção, que após analisados demonstraram que há estratégias através das quais os estudantes podem desenvolver CC, porém as mais utilizadas são: os Programas de Mobilidade e os Cursos de Competência Cultural. Estes resultados fornecem evidências do potencial da utilização das estratégias educativas relativamente ao desenvolvimento da CC nos estudantes de Enfermagem

Desenvolvimento da competência cultural na formação dos estudantes de enfermagem

The aim was to identify and map the strategies for the development of cultural competence during the training of students in nursing education institutions. For the elaboration of this Integrative Literature Review, the PCC method was used to develop the research question: What are the strategies used by Nursing Education Institutions to develop cultural competence during the training of students? To answer the question asked, a search was carried out, using the EBSCO-host and PubMed search engines, and the respective databases Cumulative Index to Nursing and Allied Health Literature and MedLine. No time horizon limits were applied. The established languages ​​were Portuguese, English and Spanish. For the assessment of methodological quality, we used a critical assessment instrument, specific for each type of study (qualitative and quantitative). Seven studies were included in the selection, which after being analyzed showed that there are strategies through which students can develop CC, but the most used are: Mobility Programs and Cultural Competence Courses. These results provide evidence of the potential use of educational strategies for the development of CC in nursing students.Objetivou-se identificar e mapear as estratégias de desenvolvimento da competência cultural durante a formação dos estudantes nas instituições de ensino de enfermagem. Para a elaboração da presente Revisão Integrativa da Literatura, recorreu-se ao método PCC, de forma a desenvolver a questão de investigação: Quais são as estratégias utilizadas pelas Instituições de Ensino de Enfermagem para Desenvolvimento da competência cultural durante a formação dos estudantes? De modo a dar resposta à questão formulada, realizou-se uma pesquisa, com recurso aos motores de busca EBSCO-host e PubMed, e às respectivas bases de dados Cumulative Index to Nursing and Allied Health Literature e MedLine. Não foram aplicados limites de horizonte temporal. Os idiomas estabelecidos foram, português, inglês e espanhol. Para a avaliação da qualidade metodológica, utilizamos um instrumento de avaliação crítica, específico para cada tipo de estudo (qualitativo e quantitativo). Sete estudos foram incluídos na seleção, que após analisados demonstraram que há estratégias através das quais os estudantes podem desenvolver CC, porém as mais utilizadas são: os Programas de Mobilidade e os Cursos de Competência Cultural. Estes resultados fornecem evidências do potencial da utilização das estratégias educativas relativamente ao desenvolvimento da CC nos estudantes de Enfermagem

Intensifying the manufacture of hiPSC therapy products through metabolic and process understanding

In vitro differentiation of human induced pluripotent stem cells into specific lineages such as cardiomyocytes (hPSC-CM) and hepatocytes (hPCS-Hep) is a crucial process to enable their application in cell therapy and drug discovery. Nevertheless, despite the remarkable efforts over the last decade towards the implementation of protocols for hPSC expansion and differentiation, there are some technological challenges remaining include the low scalability and differentiation yields. Additionally, generated cells are still immature, closely reminiscent of fetal/embryonic cells in what regards phenotype and function. In this study, we aim to overcome this hurdle by devising bioinspired and integrated strategies to improve the generation and functionality of these hiPSC-derivatives. We also applied robust multi-parametric techniques including proteomics, transcriptomics, metabolomics and fluxomics as complementary analytical tools to support bioprocess optimization and product characterization. We cultured hiPSC as 3D aggregates in stirred-tank bioreactors (STB) operated in perfusion and used a capacitance probe for in situ monitoring of cell growth/differentiation. After cell expansion, the hepatic differentiation step was integrated by addition of key soluble factors and controlling the dissolved oxygen concentration at various stages of the process to generate populations enriched for definitive endoderm, hepatocyte progenitors and mature hepatocytes. The analyses of hepatic markers expression throughout the stages of the differentiation confirmed that hepatocyte differentiation was improved in 3D spheroids when compared to 2D culture. Noteworthy, these hiPSC-HLC exhibited functional characteristics typical of hepatocytes (albumin production, glycogen storage and CYP450 activity). We also demonstrate the potential of dielectric spectroscopy to monitor cell expansion and hepatic differentiation in STB. For CM differentiation, we relied on the aggregation of hPSC-derived cardiac progenitors to establish a scalable differentiation protocol capable of generating highly pure CM aggregate cultures. We assessed if alteration of culture medium composition to mimic in vivo substrate usage during cardiac development improved further hPSC-CM maturation in vitro. Our results showed that shifting hPSC-CMs from glucose-containing to galactose- and fatty acid-containing medium promotes their fast maturation into adult-like CMs with higher oxidative metabolism, transcriptional signatures closer to those of adult ventricular tissue, higher myofibril density and alignment, improved calcium handling, enhanced contractility, and more physiological action potential kinetics. “-Omics” analyses showed that addition of galactose to culture medium and culturing the cells under perfusion improves total oxidative capacity of the cells and ameliorates fatty acid oxidation. This study demonstrated that metabolic shifts during differentiation/maturation of hPSC-CM are a cause, rather than a consequence, of the phenotypic and functional alterations observed. The metabolic-based strategy established herein holds technical and economic advantages over the existing protocols due to its scalability, simplicity and ease of application. Funding: This work was supported by FCT-funded projects NETDIAMOND (SAICTPAC/0047/2015), MetaCardio (Ref.032566) and FCT/ERA-Net (ERAdicatPH; Ref. E-Rare3/0002/2015). iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344) is also acknowledged

MIF Participates in Toxoplasma gondii-Induced Pathology Following Oral Infection

BACKGROUND: Macrophage migration inhibitory factor (MIF) is essential for controlling parasite burden and survival in a model of systemic Toxoplasma gondii infection. Peroral T. gondii infection induces small intestine necrosis and death in susceptible hosts, and in many aspects resembles inflammatory bowel disease (IBD). Considering the critical role of MIF in the pathogenesis of IBD, we hypothesized that MIF participates in the inflammatory response induced by oral infection with T. gondii. METHODOLOGY/PRINCIPAL FINDINGS: Mif deficient (Mif(-/-)) and wild-type mice in the C57Bl/6 background were orally infected with T. gondii strain ME49. Mif(-/-) mice had reduced lethality, ileal inflammation and tissue damage despite of an increased intestinal parasite load compared to wt mice. Lack of MIF caused a reduction of TNF-α, IL-12, IFN-γ and IL-23 and an increased expression of IL-22 in ileal mucosa. Moreover, suppressed pro-inflammatory responses at the ileal mucosa observed in Mif(-/-) mice was not due to upregulation of IL-4, IL-10 or TGF-β. MIF also affected the expression of matrix metalloproteinase-9 (MMP-9) but not MMP-2 in the intestine of infected mice. Signs of systemic inflammation including the increased concentrations of inflammatory cytokines in the plasma and liver damage were less pronounced in Mif(-/-) mice compared to wild-type mice. CONCLUSION/SIGNIFICANCE: In conclusion, our data suggested that in susceptible hosts MIF controls T. gondii infection with the cost of increasing local and systemic inflammation, tissue damage and death

Genome of Rhodnius prolixus, an insect vector of Chagas disease, reveals unique adaptations to hematophagy and parasite infection

Rhodnius prolixus not only has served as a model organism for the study of insect physiology, but also is a major vector of Chagas disease, an illness that affects approximately seven million people worldwide. We sequenced the genome of R. prolixus, generated assembled sequences covering 95% of the genome ( approximately 702 Mb), including 15,456 putative protein-coding genes, and completed comprehensive genomic analyses of this obligate blood-feeding insect. Although immune-deficiency (IMD)-mediated immune responses were observed, R. prolixus putatively lacks key components of the IMD pathway, suggesting a reorganization of the canonical immune signaling network. Although both Toll and IMD effectors controlled intestinal microbiota, neither affected Trypanosoma cruzi, the causal agent of Chagas disease, implying the existence of evasion or tolerance mechanisms. R. prolixus has experienced an extensive loss of selenoprotein genes, with its repertoire reduced to only two proteins, one of which is a selenocysteine-based glutathione peroxidase, the first found in insects. The genome contained actively transcribed, horizontally transferred genes from Wolbachia sp., which showed evidence of codon use evolution toward the insect use pattern. Comparative protein analyses revealed many lineage-specific expansions and putative gene absences in R. prolixus, including tandem expansions of genes related to chemoreception, feeding, and digestion that possibly contributed to the evolution of a blood-feeding lifestyle. The genome assembly and these associated analyses provide critical information on the physiology and evolution of this important vector species and should be instrumental for the development of innovative disease control methods

práticas artísticas no ensino básico e secundário

Educação Artística: integrar a inovação. A educação artística apresenta-se como um território a re-cartografar, numa atualização tão rápida quanto aquela que ocorre no campo artístico. As propostas publicadas neste número 11 da Revista Matéria-Prima trazem essa diversidade de abordagens, com novidades conceptuais que estabelecem as devidas relações entre educação e cidadania, participação, sustentabilidade, cultura visual, e também com alguma atenção sobre os debates pós-coloniais e as questões de género. Os 16 artigos reunidos neste 11º número da Revista Matéria-Prima trazem a realidade operativa quer na formação de professores e quer na formulação dos discursos pedagógicos, suas justificações e suas propostas alternativas.As propostas apresentadas devolvem o debate ao terreno, e alargam-no. Provocam as periferias, convocam abordagens diferenciadas sobre o tema da arte e da educação. Em todas elas a proposta de crescimento através da arte, que hoje implica cada vez mais cidadania, crítica, criatividade, interligação, comprometimento, participação.info:eu-repo/semantics/publishedVersio

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