Formation of Giant Unilamellar Proteo-Liposomes by Osmotic Shock.

Giant unilamellar vesicles (GUVs), composed of a phospholipid bilayer, are often used as a model system for cell membranes. However, the study of proteo-membrane interactions in this system is limited as the incorporation of integral and lipid-anchored proteins into GUVs remains challenging. Here, we present a simple generic method to incorporate proteins into GUVs. The basic principle is to break proteo-liposomes with an osmotic shock. They subsequently reseal into larger vesicles which, if necessary, can endure the same to obtain even larger proteo-GUVs. This process does not require specific lipids or reagents, works under physiological conditions with high concentrations of protein, the proteins remains functional after incorporation. The resulting proteo-GUVs can be micromanipulated. Moreover, our protocol is valid for a wide range of protein substrates. We have successfully reconstituted three structurally different proteins, two trans-membrane proteins (TolC and the neuronal t-SNARE), and one lipid-anchored peripheral protein (GABARAP-Like 1 (GL1)). In each case, we verified that the protein remains active after incorporation and in its correctly folded state. We also measured their mobility by performing diffusion measurements via fluorescence recovery after photobleaching (FRAP) experiments on micromanipulated single GUVs. The diffusion coefficients are in agreement with previous data.

Uma Investigação sobre Disfunções Familiares em Estudantes de Medicina / An investigation into Familiar Dysfunctions among Medical Students

RESUMO Introdução As Diretrizes Curriculares Nacionais do Curso de Graduação em Medicina (2014) determinam como perfil do egresso médico uma identidade generalista, humanista, crítica, reflexiva e multicultural. Isto exige habilidades biopsicomotoras qualificadas (competências de cuidar, curar e reabilitar pessoas e suas famílias). Os núcleos psicopedagógicos nacionais encontram no atendimento desses estudantes conflitos pessoais, pedagógicos e familiares traduzidos em transtornos mentais. As disfunções familiares estarão relacionadas ao aumento dessa prevalência na graduação médica (socialização secundária)? Uma socialização primária insatisfatória contribuiria para o adoecer no processo do adultecer? Objetivos Verificar o perfil sociodemográfico, psiquiátrico e familiar de estudantes de Medicina numa instituição federal do Estado do Rio de Janeiro, Brasil. Métodos A pesquisa foi um estudo quali-quantitativo, com intervenção observacional, exploratória, transversal, descritiva e inferencial. A coleta de dados foi feita por meio de dois instrumentos: (1) um questionário construído para esta pesquisa, autoaplicável, com variáveis sociopsicodemográficas e categorias familiares disfuncionais descritas na literatura; (2) o Mini International Neuropsychiatric Interview (Mini), versão 5.0.0, compatível com os critérios do DSM-IV e validado por Patrícia Amorim/2000. Os dados foram colhidos após consentimento livre e esclarecido (TCLE) e organizados em banco de dados, no Excel. A análise foi feita com o software estatístico R. As variáveis quantitativas foram analisadas por desvio padrão, média, mediana e moda. As variáveis qualitativas tiveram análises de frequência. Foram realizados testes estatísticos: exato de Fisher, Wilcoxon bilateral, Qui-Quadrado e Shapiro-Wilk, de acordo com as variáveis e objetivos da análise. A pesquisa foi aprovada pelo Comitê de Ética e Pesquisa. CAAE: 67590317.5.0000.5258, em 25/07/2017. Resultados Foram avaliados 129 alunos, com 18 a 32 anos de idade, do primeiro ao 12º período da graduação, 49% do sexo feminino e 51% do sexo masculino. Prevalência de transtorno de ansiedade generalizada: 39,53%; de depressão (atual ou recorrente): 32,56%; e risco de suicídio: 28,68%. A depressão possuiu forte correlação com história familiar de transtorno psiquiátrico (p-valor: 7.639e-08) e com o relacionamento dos pais. Ela esteve presente naqueles com pais divorciados, viúvos ou sem relacionamento (p-valor: 0.008291). O risco de suicídio foi maior entre acadêmicos do ciclo básico, do primeiro ao quarto período da graduação (p-valor: 0,01), e possuía forte correlação com ter sofrido ou sofrer bullying (p-valor: 0,02), ter uma religião (p-valor: 0,006), e depressão (p-valor: 0,03) ou transtorno de pânico (p-valor: 4.903e-05). Houve correlação entre depressão (p-valor: 1.248 e-09) e risco de suicídio (p valor: 0.0009) nos que tinham problemas de comunicação na família. Transtorno de pânico foi observado em 17,05% dos entrevistados, sexo feminino (p-valor: 0.00583). Isto decorre do relacionamento dos pais. Os alunos com pais casados foram os mais afetados (p-valor: 0.01284) Conclusões: Os dados colhidos demonstraram correlações entre disfunções familiares e transtornos psiquiátricos, como depressão, risco de suicídio, transtorno de pânico e transtorno de ansiedade generalizada. Existe a necessidade de um ambiente de ensino-aprendizagem com espaços e cenários e educadores médicos que identifiquem precocemente o sofrimento psíquico na socialização secundária do futuro médico.

ABSTRACT Introduction The National Curricular Guidelines for Undergraduate Medicine Courses (2014) determine the profile of medical graduates as having a generalist, humanistic, critical, reflective and multicultural identity. This requires qualified biopsychomotor skills (skills of caring, healing and rehabilitating people and their families). The national psycho-pedagogical support centres find in these students personal, pedagogical and family conflicts translated into mental disorders. Will family dysfunctions be related to increased prevalence among medical undergraduate students (secondary socialization)? Would unsatisfactory primary socialization contribute to becoming ill in the ageing process? Objectives To verify the socio-demographic, psychiatric and family profile of medical students in a federal institution in the state of Rio de Janeiro, Brazil. Methods The research was a qualitative-quantitative study, involving an observational intervention, and of an exploratory, transversal, descriptive and inferential nature. Data collection was performed through two strategies: (1) a self-administered questionnaire built for this research, with socio-psycho-demographic variables and dysfunctional family categories described in the literature. (2) the Mini International Neuropsychiatric Interview (Mini), 5.0.0 version, compatible with the DSM-IV criteria; Validated by Patrícia Amorim/2000. Data were collected after an Informed Consent Form (ICF) was signed and a database was created in Excel. The analysis was performed with the statistical software R. Quantitative variables were analysed by standard deviation, mean, median and mode. The qualitative variables underwent frequency analyses. Statistical tests were performed by Fisher exact test, bilateral Wilcoxon, chi-Square and Shapiro-Wilk, according to the variables and objectives of the analysis. The research was approved by the Ethics and Research Committee under number CAAE: 67590317.5.0000.5258, on 25/07/2017. Results 129 students were evaluated, aged 18 to 32 years, ranging from the 1st to 12th semester of the undergraduate course, 49% were female and 51% male. The prevalence of generalized anxiety disorder was 39.53%; depression (current or recurrent), 32.56% and suicide risk, 28.68%. Depression had a strong correlation with a family history of psychiatric disorder (p-value: 7.639 e-08) and the parents' relationship. It was present in those with divorced parents, widowed or without a relationship (P value: 0.008291). The risk of suicide was higher among students of the basic cycle, 1st to 4th semester of the course (p-value: 0.01), that had a strong correlation with suffering or still suffer bullying (p-value: 0.02), having a religion (p-value: 0.006), and depression (p-value: 0.03) or panic disorder (p-value: 4.903 e-05). There was a correlation between depression (p-value: 1.248 and-09) and suicide risk (p-value: 0.0009) in those who had communication problems in the family. Panic disorder was observed in 17.05% of the interviewees, females (p-value: 0.00583). This stems from the parents' relationship. Students with married parents were the most affected. (p-value: 0.01284) Conclusions: The collected data showed correlations between family dysfunctions and psychiatric disorders such as depression, suicide risk, panic disorder, and generalized anxiety disorder. There is a need for a teaching/learning environment, including adequate spaces and scenarios, and also medical educators who can, at an early stage, identify psychological distress in the secondary socialization of the future physician.

Approaches to the study of Atg8-mediated membrane dynamics in vitro.

Macro-autophagy is the intracellular stress-response pathway by which the cell packages portions of the cytosol for delivery into the lysosome. This "packaging" is carried out by the de novo formation of a new organelle called the autophagosome that grows and encapsulates cytosolic material for eventual lysosomal degradation. How autophagosomes form, including especially how the membrane expands and eventually closes upon itself is an area of intense study. One factor implicated in both membrane expansion and membrane fusion is the ubiquitin-like protein, Atg8. During autophagy, Atg8 becomes covalently bound to phosphatidylethanolamine (PE) on the pre-autophagosomal membrane and remains bound through the maturation process of the autophagosome. In this chapter, we discuss two approaches to the in vitro reconstitution of this lipidation reaction. We then describe methods to study Atg8-PE mediated membrane tethering and fusion, two functions implicated in Atg8's role in autophagosome maturation.