Cerebral Malaria Model Applying Human Brain Organoids.

Neural injuries in cerebral malaria patients are a significant cause of morbidity and mortality. Nevertheless, a comprehensive research approach to study this issue is lacking, so herein we propose an in vitro system to study human cerebral malaria using cellular approaches. Our first goal was to establish a cellular system to identify the molecular alterations in human brain vasculature cells that resemble the blood-brain barrier (BBB) in cerebral malaria (CM). Through transcriptomic analysis, we characterized specific gene expression profiles in human brain microvascular endothelial cells (HBMEC) activated by the Plasmodium falciparum parasites. We also suggest potential new genes related to parasitic activation. Then, we studied its impact at brain level after Plasmodium falciparum endothelial activation to gain a deeper understanding of the physiological mechanisms underlying CM. For that, the impact of HBMEC-P. falciparum-activated secretomes was evaluated in human brain organoids. Our results support the reliability of in vitro cellular models developed to mimic CM in several aspects. These systems can be of extreme importance to investigate the factors (parasitological and host) influencing CM, contributing to a molecular understanding of pathogenesis, brain injury, and dysfunction.

Revolutionizing Disease Modeling: The Emergence of Organoids in Cellular Systems.

Cellular models have created opportunities to explore the characteristics of human diseases through well-established protocols, while avoiding the ethical restrictions associated with post-mortem studies and the costs associated with researching animal models. The capability of cell reprogramming, such as induced pluripotent stem cells (iPSCs) technology, solved the complications associated with human embryonic stem cells (hESC) usage. Moreover, iPSCs made significant contributions for human medicine, such as in diagnosis, therapeutic and regenerative medicine. The two-dimensional (2D) models allowed for monolayer cellular culture in vitro; however, they were surpassed by the three-dimensional (3D) cell culture system. The 3D cell culture provides higher cell-cell contact and a multi-layered cell culture, which more closely respects cellular morphology and polarity. It is more tightly able to resemble conditions in vivo and a closer approach to the architecture of human tissues, such as human organoids. Organoids are 3D cellular structures that mimic the architecture and function of native tissues. They are generated in vitro from stem cells or differentiated cells, such as epithelial or neural cells, and are used to study organ development, disease modeling, and drug discovery. Organoids have become a powerful tool for understanding the cellular and molecular mechanisms underlying human physiology, providing new insights into the pathogenesis of cancer, metabolic diseases, and brain disorders. Although organoid technology is up-and-coming, it also has some limitations that require improvements.

Establishment and characterization of human pluripotent stem cells-derived brain organoids to model cerebellar diseases.

The establishment of robust human brain organoids to model cerebellar diseases is essential to study new therapeutic strategies for cerebellum-associated disorders. Machado-Joseph disease (MJD) is a cerebellar hereditary neurodegenerative disease, without therapeutic options able to prevent the disease progression. In the present work, control and MJD induced-pluripotent stem cells were used to establish human brain organoids. These organoids were characterized regarding brain development, cell type composition, and MJD-associated neuropathology markers, to evaluate their value for cerebellar diseases modeling. Our data indicate that the organoids recapitulated, to some extent, aspects of brain development, such as astroglia emerging after neurons and the presence of ventricular-like zones surrounded by glia and neurons that are found only in primate brains. Moreover, the brain organoids presented markers of neural progenitors proliferation, neuronal differentiation, inhibitory and excitatory synapses, and firing neurons. The established brain organoids also exhibited markers of cerebellar neurons progenitors and mature cerebellar neurons. Finally, MJD brain organoids showed higher ventricular-like zone numbers, an indication of lower maturation, and an increased number of ataxin-3-positive aggregates, compared with control organoids. Altogether, our data indicate that the established organoids recapitulate important characteristics of human brain development and exhibit cerebellar features, constituting a resourceful tool for testing therapeutic approaches for cerebellar diseases.

Rehabilitation Nurse's Perspective on Transitional Care: An Online Focus Group.

The increasing incidence of chronic and dependence leads to the need for hospitalization and adaptation in the process of returning home, as well as transition between care levels to ensure continuity of care. The World Health Organization has been warning about this problem since 2016, and consider reorganizing the care model as one of the solutions. The present study aimed to analyse the nurses' perspective on transitional care for dependent people with rehabilitation care needs after hospital discharge. METHODS: A focus group was developed with the participation of Rehabilitation Nurses from the hospital and community context, and content analysis was defined a posteriori. RESULTS: From the content analysis emerged four related categories: promotion of continuity of care, nurse of advanced practice as a care manager, capacitation of the person and caregiver, and promotion of the care coordination. CONCLUSIONS: The present study allowed the strategies identification that minimize fragmentation risk of care and promote the person participation in transitional care. Ensuring transitional care is imperative to increase the quality of care, the satisfaction of professionals, clients, and the development of a system of sustainable health.

Aprender prática baseada na evidência pelo envolvimento em atividades de investigação - autopercepção dos estudantes / Aprendizaje de prácticas basadas en evidencia por medio del involvimiento en actividades investigativas - la autopercepción de estudiantes / Learning evidence based practice through involvement in investigation activities - the self-perception of students

RESUMO Objetivo: compreender como os estudantes percepcionam o seu envolvimento em atividades de investigação e o contributo para o desenvolvimento de uma prática baseada na evidência. Método: estudo qualitativo, transversal, descritivo e exploratório, realizado em Portugal, em agosto de 2019, que recorreu ao grupo focal, com oito participantes, para responder à questão: "Quais as vantagens da participação dos estudantes de enfermagem em atividades de investigação para o desenvolvimento de conhecimentos, atitudes e competências de utilização da evidência?". A análise de conteúdo foi realizada com o software NVivo. Resultados: da análise qualitativa dos achados, emergiram cinco categorias e subcategorias: autoaprendizagem, integração teórico-prática, trabalho interdisciplinar, tomada de decisão baseada na evidência e literácia científica. Conclusão: a criação de uma experiência eficaz de aprendizagem ajuda na construção do conhecimento e potencialmente contribui para a saúde da comunidade, os resultados de aprendizagem do estágio e o desenvolvimento de competências essenciais para uma Prática Baseada na Evidência.

RESUMEN Objetivo: comprender como estudiantes perciben su envolvimiento con actividades investigativas y su contribución para desarrollar una práctica basada en evidencias. Método: estudio cualitativo, trasversal, descriptivo y exploratorio, hecho en Portugal en agosto de 2019, utilizando un grupo focal con ocho participantes para responder a la cuestión: "Cuales las ventajas de la participación de los estudiantes de enfermería en actividades de investigación para desarrollar conocimientos, actitudes y competencias de utilización de la evidencia?" Se hizo al análisis de contenido con el software NVivo. Resultados: el análisis cuantitativo de los datos generó cinco categorías y subcategorías: autoaprendizaje, integración teórico-práctica, interdisciplinariedad, tomada de decisiones basadas en evidencia, y alfabetización científica. Conclusión: la creación de una experiencia eficaz de aprendizaje ayuda en la construcción del conocimiento y potencialmente contribuye para la salud de la comunidad, para los resultados del aprendizaje en la pasantía y para el desarrollo de competencias esenciales para una Práctica Basada en Evidencias.

ABSTRACT Objective: to understand how students perceive their involvement in investigation activities and how they contribute for the development of evidence-based practices. Method: qualitative, cross-sectional, descriptive, and exploratory study, carried out in Portugal, in August 2019. A focus group of eight participants was formed to answer the question: "What are the advantages of the participation of nursing students in investigations for the development of knowledge, attitudes, and performance in the use of evidence?" The software NVivo was used for a content analysis. Results: five categories and subcategories emerged from the qualitative analysis of findings: self-learning, integration of theory and practice, interdisciplinary work, evidence-based decision making, and scientific literacy. Conclusion: creating an effective learning experience helps building knowledge and can contribute for the health of the community, for improved educational results during the internship, and for the development of the abilities necessary for Evidence Based Practice.

Expansion of a Specific Plasmodium falciparum PfMDR1 Haplotype in Southeast Asia with Increased Substrate Transport.

Artemisinin-based combination therapies (ACTs) have been vital in reducing malaria mortality rates since the 2000s. Their efficacy, however, is threatened by the emergence and spread of artemisinin resistance in Southeast Asia. The Plasmodium falciparum multidrug resistance protein 1 (PfMDR1) transporter plays a central role in parasite resistance to ACT partner drugs through gene copy number variations (CNV) and/or single nucleotide polymorphisms (SNPs). Using genomic epidemiology, we show that multiple pfmdr1 copies encoding the N86 and 184F haplotype are prevalent across Southeast Asia. Applying genome editing tools on the Southeast Asian Dd2 strain and using a surrogate assay to measure transporter activity in infected red blood cells, we demonstrate that parasites harboring multicopy N86/184F PfMDR1 have a higher Fluo-4 transport capacity compared with those expressing the wild-type N86/Y184 haplotype. Multicopy N86/184F PfMDR1 is also associated with decreased parasite susceptibility to lumefantrine. These findings provide evidence of the geographic selection and expansion of specific multicopy PfMDR1 haplotypes associated with multidrug resistance in Southeast Asia.IMPORTANCE Global efforts to eliminate malaria depend on the continued success of artemisinin-based combination therapies (ACTs) that target Plasmodium asexual blood-stage parasites. Resistance to ACTs, however, has emerged, creating the need to define the underlying mechanisms. Mutations in the P. falciparum multidrug resistance protein 1 (PfMDR1) transporter constitute an important determinant of resistance. Applying gene editing tools combined with an analysis of a public database containing thousands of parasite genomes, we show geographic selection and expansion of a pfmdr1 gene amplification encoding the N86/184F haplotype in Southeast Asia. Parasites expressing this PfMDR1 variant possess a higher transport capacity that modulates their responses to antimalarials. These data could help tailor and optimize antimalarial drug usage in different regions where malaria is endemic by taking into account the regional prevalence of pfmdr1 polymorphisms.

Molecular heterogeneity of Rhipicephalus sanguineus sensu lato and screening for Ehrlichia canis in mainland Portugal.

The present study aimed to expand knowledge regarding the molecular characterization of R. sanguineus s.l. in Portugal and to screen for ticks naturally infected with E. canis. A total of 113 R. sanguineus s.l. were collected questing or attached to domestic and wild animals from the 18 administrative regions of mainland Portugal. All the ticks were analyzed for the 16S rRNA and the partial sequences obtained showed high genetic similarities with specimens belonging to the temperate lineage. These sequences revealed eight haplotypes (H1‒H8), with a genetic distance ranging from 0.3% to 1.4%. A convenience sample representing approximately 75% of all the R. sanguineus s.l. ticks collected was tested for the presence of E. canis by qPCR for the dsb gene. No ticks were found to be infected with this pathogen. Accordingly, further studies are required to determine the role of the R. sanguineus s.l. temperate lineage in E. canis maintenance and transmission, as well as to elucidate if a different R. sanguineus s.l. lineage or other tick species act as E. canis vectors for dogs in Portugal.

Comparative efficacy, toxicity and biodistribution of the liposomal amphotericin B formulations Fungisome® and AmBisome® in murine cutaneous leishmaniasis.

Fungisome® (F), a liposomal amphotericin B (AmB) product, is marketed in India as a safe and effective therapeutic for the parasitic infection visceral leishmaniasis. Its potential in the treatment of cutaneous leishmaniasis (CL), a disfiguring form of the disease affecting the skin, is currently unknown. Here, we report the evaluation of the efficacy of F in the Leishmania major BALB/c murine model of CL, including a head-to-head comparison with the standard liposomal AmB formulation AmBisome® (A). Upon intravenous administration at dose levels of 5, 10 and 15 mg/kg of body weight (on days 0, 2, 4, 6 and 8), F showed clear signs of toxicity (at 15 mg/kg), while A did not. After complete treatment (day 10), the tolerated doses of 5 and 10 mg/kg F had significant antileishmanial activity (ED50 = 4.0 and 12.8 mg/kg for qPCR-based parasite load and lesion size, respectively), although less than that of A at identical doses (ED50 = 3.0 and 8.8 mg/kg). The efficacy of F was inferior compared to A because lower levels of the active agent AmB accumulated within the infected lesion. In conclusion, despite possibly being less safe and efficacious than A at equivalent doses, the moderate in vivo activity of F could indicate a role in the systemic pharmacotherapy of CL.