Blood pressure responsiveness to resistance training in the hypertensive older adult: a randomized controlled study.

Different lifestyle changes have been employed to improve clinical hypertension. However, there is scarce evidence on the blood pressure responsiveness to resistance training (RT) in hypertensive older adults. Consequently, little is known about some participants clinically reducing blood pressure and others not. Thus, we investigate the effects and responsiveness of RT on blood pressure in hypertensive older adults. We secondarily evaluated the biochemical risk factors for cardiovascular disease and functional performance. Older participants with hypertension were randomly assigned into RT (n = 27) and control group (n = 25). Blood pressure, functional performance (timed up and go, handgrip strength, biceps curl and sit-to-stand), fasting glucose, and lipid profiles were evaluated preintervention and postintervention. The statistic was performed in a single-blind manner, the statistician did not know who was the control and RT. RT was effective in reducing systolic blood pressure (SBP) (pre 135.7 ±â€…14.7; post 124.7 ±â€…11.0; P  < 0.001) and the responses to RT stimuli varied noticeably between hypertensive older adults after 12 weeks. For example, 13 and 1 responders displayed a minimal clinical important difference for SBP attenuation (10.9 mmHg) in the RT and control groups, respectively. RT improved the functional performance of older people with hypertension, while no differences were found in biochemical parameters (triglycerides, HDL, LDL, fasting glucose) after 12 weeks. In conclusion, responses to RT stimuli varied noticeably between hypertensive individuals and RT was effective in reducing SBP.

Severe acute respiratory syndrome coronavirus-2 accessory proteins ORF3a and ORF7a modulate autophagic flux and Ca2+ homeostasis in yeast.

Virus infection involves the manipulation of key host cell functions by specialized virulence proteins. The Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) small accessory proteins ORF3a and ORF7a have been implicated in favoring virus replication and spreading by inhibiting the autophagic flux within the host cell. Here, we apply yeast models to gain insights into the physiological functions of both SARS-CoV-2 small open reading frames (ORFs). ORF3a and ORF7a can be stably overexpressed in yeast cells, producing a decrease in cellular fitness. Both proteins show a distinguishable intracellular localization. ORF3a localizes to the vacuolar membrane, whereas ORF7a targets the endoplasmic reticulum. Overexpression of ORF3a and ORF7a leads to the accumulation of Atg8 specific autophagosomes. However, the underlying mechanism is different for each viral protein as assessed by the quantification of the autophagic degradation of Atg8-GFP fusion proteins, which is inhibited by ORF3a and stimulated by ORF7a. Overexpression of both SARS-CoV-2 ORFs decreases cellular fitness upon starvation conditions, where autophagic processes become essential. These data confirm previous findings on SARS-CoV-2 ORF3a and ORF7a manipulating autophagic flux in mammalian cell models and are in agreement with a model where both small ORFs have synergistic functions in stimulating intracellular autophagosome accumulation, ORF3a by inhibiting autophagosome processing at the vacuole and ORF7a by promoting autophagosome formation at the ER. ORF3a has an additional function in Ca2+ homeostasis. The overexpression of ORF3a confers calcineurin-dependent Ca2+ tolerance and activates a Ca2+ sensitive FKS2-luciferase reporter, suggesting a possible ORF3a-mediated Ca2+ efflux from the vacuole. Taken together, we show that viral accessory proteins can be functionally investigated in yeast cells and that SARS-CoV-2 ORF3a and ORF7a proteins interfere with autophagosome formation and processing as well as with Ca2+ homeostasis from distinct cellular targets.

TNF and IL6/Jak2 signaling pathways are the main contributors of the glia-derived neuroinflammation present in Lafora disease, a fatal form of progressive myoclonus epilepsy.

Lafora disease (LD; OMIM#254780) is a rare form of progressive myoclonus epilepsy (prevalence <1:1,000,000) characterized by the accumulation of insoluble deposits of aberrant glycogen (polyglucosans), named Lafora bodies, in the brain but also in peripheral tissues. LD is the most severe form of the group of progressive myoclonus epilepsies, since patients present a rapid deterioration and dementia with amplification of seizures, leading to death after a decade from the onset of the first symptoms. We have recently described that reactive glia-derived neuroinflammation should be considered a novel hallmark of LD since we observed a florid upregulation of differentially expressed genes in both LD mouse lines, which were mainly related to mediators of inflammatory response. In this work, we define an upregulation of the expression of mediators of the TNF and IL6/JAK2 signaling pathways in LD. In addition, we describe the activation of the non-canonical form of the inflammasome. Furthermore, we describe the infiltration of peripheral immune cells in the brain parenchyma, which could aggravate glia-derived neuroinflammation. Finally, we describe CXCL10 and S100b as blood biomarkers of the disease, which will allow the study of the progression of the disease using serum blood samples. We consider that the identification of these initial inflammatory changes in LD will be very important to implement possible anti-inflammatory therapeutic strategies to prevent the development of the disease.

Breastfeeding by chikungunya virus-infected dams confers resistance to challenge in the offspring.

Vertical transmission of Chikungunya virus (CHIKV) has been reported in humans, but the transmission routes have not been completely understood, and experimental animal models are needed to enable detailed investigation of the transmission and pathogenesis of congenital infections. The intertwining of immune response and virus components at the gestation/breastfeeding interfaces between mother and fetus/newborn may have effects during the offspring development. An experimental model of CHIKV was established by infecting pregnant BALB/c female mice that enabled confirmation that dams inoculated up to the 10th gestational day transmit CHIKV transplacentally to approximately 8.4% of the fetuses, resulting in severe teratogenic effects. CHIKV neutralizing antibodies were detected in sera from adult mice born to healthy females and breastfed by CHIKV-infected dams, while no neutralization was detected in sera from animals born to CHIKV-infected dams. Moreover, adult mice born to healthy dams and cross-fostered for breastfeeding by CHIKV-infected dams were resistant to challenge with CHIKV on the 90th day after birth. The animals also had reduced viral loads in brain and spleen as compared to controls. There was expression of fluorescent CHIKV non-structural protein, and detection of viral RNA by RT-PCR in breast tissue from infected dams. CHIKV RNA and proteins were also detected in breast milk retrieved from the stomachs of recently fed newborns. The experimental results were also complemented by the finding of CHIKV RNA in 6% of colostrum samples from healthy lactating women in a CHIKV-endemic area. Breastfeeding induces immune protection to challenge with CHIKV in mice.

SARS-CoV-2 infects adipose tissue in a fat depot- and viral lineage-dependent manner.

Visceral adiposity is a risk factor for severe COVID-19, and a link between adipose tissue infection and disease progression has been proposed. Here we demonstrate that SARS-CoV-2 infects human adipose tissue and undergoes productive infection in fat cells. However, susceptibility to infection and the cellular response depends on the anatomical origin of the cells and the viral lineage. Visceral fat cells express more ACE2 and are more susceptible to SARS-CoV-2 infection than their subcutaneous counterparts. SARS-CoV-2 infection leads to inhibition of lipolysis in subcutaneous fat cells, while in visceral fat cells, it results in higher expression of pro-inflammatory cytokines. Viral load and cellular response are attenuated when visceral fat cells are infected with the SARS-CoV-2 gamma variant. A similar degree of cell death occurs 4-days after SARS-CoV-2 infection, regardless of the cell origin or viral lineage. Hence, SARS-CoV-2 infects human fat cells, replicating and altering cell function and viability in a depot- and viral lineage-dependent fashion.

Living with an intestinal ostomy and urinary incontinence / Vivir con una estomía intestinal e incontinencia urinaria / Convivendo com estomia intestinal e a incontinência urinária

ABSTRACT Objective: to understand how aged people with intestinal ostomies experience this situation together with urinary incontinence. Method: a qualitative, descriptive and exploratory research study, developed with 77 aged individuals with intestinal ostomies assisted by the Unified Health System, in four municipalities from the Metropolitan Region of Florianópolis. Data collection was conducted using semi-structured interviews from October 2019 to February 2020. The theoretical framework used was Dorothéa Orem's Self-Care Theory and the data were submitted to content analysis, in its thematic modality. Results: the analysis allowed generating three thematic categories: 1) Feelings generated by the intestinal ostomy and urinary incontinence: acceptance, denial, fear, insecurity, constraints experienced due to the ostomy and to the urinary incontinence symptoms; 2) Lifestyle changes; and 3) Deficit in self-image. Conclusion: it was evidenced that, for most of the research participants, it is difficult to accept the experience of living with an intestinal ostomy and urinary incontinence, which generally produce negative feelings. However, the participants proved to be resilient and able to adapt to the changes in lifestyle. Many of these behaviors are due to the health professionals' important contribution in providing them the necessary attention, encouraging self-care strategies in both situations.

RESUMEN Objetivo: comprender de qué manera los ancianos con ostomías intestinales viven esta situación junto con la incontinencia urinaria. Método: investigación cualitativa, descriptiva y exploratoria, desarrollada con 77 ancianos con estomías intestinales atendidos por el Sistema Único de Salud en cuatro municipios de la Región Metropolitana de Florianópolis. La recolección de datos se realizó entre octubre de 2019 y febrero de 2020 por medio de entrevistas semiestructuradas. El marco de referencia teórico empleado fue la Teoría de Autocuidado de Dorothéa Orem; los datos se sometieron a análisis de contenido, en su modalidad temática. Resultados: el análisis permitió generar tres categorías temáticas: 1) Sentimientos generados por la estomía intestinal y por la incontinencia urinaria: aceptación, negación, miedo, inseguridad, restricciones experimentadas a raíz de la ostomía y de los síntomas de la incontinencia urinaria; 2) Cambios en el estilo de vida; y 3) Déficit en la imagen propia. Conclusión: se hizo evidente que a la mayoría de los participantes de la investigación les resulta difícil aceptar la vida con una estomía intestinal e incontinencia urinaria, que generalmente les provocan sentimientos negativos. Sin embargo, los participantes se mostraron resilientes y aptos para adaptarse a los cambios en el estilo de vida. Muchos de estos comportamientos se deben al importante aporte de los profesionales de la salud al brindarles la atención necesaria, estimulando estrategias de autocuidado en ambas situaciones.

RESUMO Objetivo: compreender como o idoso com estomia intestinal vivencia essa situação em conjunto com a incontinência urinária. Método: pesquisa qualitativa, descritiva e exploratória, desenvolvida junto a 77 idosos com estomia intestinal atendidos pelo Sistema Único de Saúde, em quatro municípios da Região Metropolitana de Florianópolis. A coleta de dados foi realizada de outubro/2019 a fevereiro/2020, por meio de entrevista semiestruturada. O referencial teórico utilizado foi a Teoria de Autocuidado de Dorothéa Orem; os dados foram submetidos à análise de conteúdo, na modalidade temática. Resultados: a análise permitiu a geração de três categorias temáticas: 1) sentimentos gerados pela estomia intestinal e pela incontinência urinária: aceitação, negação, medo, insegurança, constrangimentos vivenciados pela estomia e os sintomas da incontinência urinária; 2) alterações do estilo de vida; 3) déficit na autoimagem. Conclusão: evidenciou-se que para a maioria dos participantes da pesquisa é difícil aceitar a vivência com estomia intestinal e incontinência urinária, que geralmente lhes provocam sentimentos negativos. No entanto, os participantes mostraram-se resilientes e aptos a se adaptar às mudanças no estilo de vida. Muitos desses comportamentos se devem à importante contribuição dos profissionais da saúde em dar-lhes a necessária atenção, estimulando estratégias de autocuidado em ambas as situações.

An empirical pipeline for personalized diagnosis of Lafora disease mutations.

Lafora disease (LD) is a fatal childhood dementia characterized by progressive myoclonic epilepsy manifesting in the teenage years, rapid neurological decline, and death typically within ten years of onset. Mutations in either EPM2A, encoding the glycogen phosphatase laforin, or EPM2B, encoding the E3 ligase malin, cause LD. Whole exome sequencing has revealed many EPM2A variants associated with late-onset or slower disease progression. We established an empirical pipeline for characterizing the functional consequences of laforin missense mutations in vitro using complementary biochemical approaches. Analysis of 26 mutations revealed distinct functional classes associated with different outcomes that were supported by clinical cases. For example, F321C and G279C mutations have attenuated functional defects and are associated with slow progression. This pipeline enabled rapid characterization and classification of newly identified EPM2A mutations, providing clinicians and researchers genetic information to guide treatment of LD patients.

Vesicular Stomatitis Virus Chimeras Expressing the Oropouche Virus Glycoproteins Elicit Protective Immune Responses in Mice.

Oropouche virus (OROV) infection of humans is associated with a debilitating febrile illness that can progress to meningitis or encephalitis. First isolated from a forest worker in Trinidad and Tobago in 1955, the arbovirus OROV has since been detected throughout the Amazon basin with an estimated 500,000 human infections over 60 years. Like other members of the family Peribunyaviridae, the viral genome exists as 3 single-stranded negative-sense RNA segments. The medium-sized segment encodes a viral glycoprotein complex (GPC) that is proteolytically processed into two viral envelope proteins, Gn and Gc, responsible for attachment and membrane fusion. There are no therapeutics or vaccines to combat OROV infection, and we have little understanding of protective immunity to infection. Here, we generated a replication competent chimeric vesicular stomatitis virus (VSV), in which the endogenous glycoprotein was replaced by the GPC of OROV. Serum from mice immunized by intramuscular injection with VSV-OROV specifically neutralized wild-type OROV, and using peptide arrays we mapped multiple epitopes within an N-terminal variable region of Gc recognized by the immune sera. VSV-OROV lacking this variable region of Gc was also immunogenic in mice producing neutralizing sera that recognize additional regions of Gc. Challenge of both sets of immunized mice with wild-type OROV shows that the VSV-OROV chimeras reduce wild-type viral infection and suggest that antibodies that recognize the variable N terminus of Gc afford less protection than those that target more conserved regions of Gc. IMPORTANCE Oropouche virus (OROV), an orthobunyavirus found in Central and South America, is an emerging public health challenge that causes debilitating febrile illness. OROV is transmitted by arthropods, and increasing mobilization has the potential to significantly increase the spread of OROV globally. Despite this, no therapeutics or vaccines have been developed to combat infection. Using vesicular stomatitis (VSV) as a backbone, we developed a chimeric virus bearing the OROV glycoproteins (VSV-OROV) and tested its ability to elicit a neutralizing antibody response. Our results demonstrate that VSV-OROV produces a strong neutralizing antibody response that is at least partially targeted to the N-terminal region of Gc. Importantly, vaccination with VSV-OROV reduces viral loads in mice challenged with wild-type virus. These data provide novel evidence that targeting the OROV glycoproteins may be an effective vaccination strategy to combat OROV infection.

Detection of respiratory viruses in primary cholesteatoma tissues.

Cholesteatomas are frequent middle ear benign tumors of unknown etiology. Infectious agents have been considered as possible contributing factors in the pathogenesis of cholesteatomas. Aiming to investigate the presence of respiratory viruses in primary cholesteatoma tissues, 26 formalin-fixed paraffin-embedded primary cholesteatoma tissues obtained from patients seen at the of the Clinical Hospital of the University of São Paulo School of Medicine, in Ribeirão Preto, Brazil were tested by real-time polymerase chain reaction (PCR). Considering the PCR results, 35% of the tissues were positive for human rhinovirus (HRV), 15.3% for human enterovirus (EV), 3.8% for human metapneumovirus (HMPV), and 3.8% for human bocavirus (HBoV). Serial immunohistochemistry for virus antigens and cell surface markers evidenced that the viruses were associated with fibroblasts, dendritic cells, macrophages, B lymphocytes, CD4+ , and CD8+ T lymphocytes. These findings indicate for the first time the presence of active respiratory virus infection in primary cholesteatoma tissues, suggesting that persisting virus infection in the middle could play a role in the pathogenesis and evolution of cholesteatomas.

Human Respiratory Syncytial Virus Infection in a Human T Cell Line Is Hampered at Multiple Steps.

Human respiratory syncytial virus (HRSV) is the most frequent cause of severe respiratory disease in children. The main targets of HRSV infection are epithelial cells of the respiratory tract, and the great majority of the studies regarding HRSV infection are done in respiratory cells. Recently, the interest on respiratory virus infection of lymphoid cells has been growing, but details of the interaction of HRSV with lymphoid cells remain unknown. Therefore, this study was done to assess the relationship of HRSV with A3.01 cells, a human CD4+ T cell line. Using flow cytometry and fluorescent focus assay, we found that A3.01 cells are susceptible but virtually not permissive to HRSV infection. Dequenching experiments revealed that the fusion process of HRSV in A3.01 cells was nearly abolished in comparison to HEp-2 cells, an epithelial cell lineage. Quantification of viral RNA by RT-qPCR showed that the replication of HRSV in A3.01 cells was considerably reduced. Western blot and quantitative flow cytometry analyses demonstrated that the production of HRSV proteins in A3.01 was significantly lower than in HEp-2 cells. Additionally, using fluorescence in situ hybridization, we found that the inclusion body-associated granules (IBAGs) were almost absent in HRSV inclusion bodies in A3.01 cells. We also assessed the intracellular trafficking of HRSV proteins and found that HRSV proteins colocalized partially with the secretory pathway in A3.01 cells, but these HRSV proteins and viral filaments were present only scarcely at the plasma membrane. HRSV infection of A3.01 CD4+ T cells is virtually unproductive as compared to HEp-2 cells, as a result of defects at several steps of the viral cycle: Fusion, genome replication, formation of inclusion bodies, recruitment of cellular proteins, virus assembly, and budding.

O envelhecimento ativo e a promoção da saúde / Active aging and health promotion / Envejecimiento activo y promoción de la salud

Objetivo: identificar a produção científica sobre o envelhecimento ativo. Método: trata-se de um estudo bibliográfico, descritivo, tipo revisão integrativa, sem delimitação temporal, a partir da busca nas bases de dados LILACS, MEDLINE, CINAHL, Scopus e Biblioteca Virtual SciELO. Analisou-se somente trabalhos publicados em periódicos indexados como artigos de pesquisas originais em língua portuguesa, espanhola e inglesa. Observa-se que os procedimentos adotados para a análise da amostra final dos estudos foram descritivos. Resultados: destaca-se que a amostra final foi composta por 66 artigos. Organizaram-se os principais resultados em três categorias: Percepção dos idosos e profissionais sobre o envelhecimento ativo; A promoção da saúde para o envelhecimento ativo e Os fatores determinantes do envelhecimento ativo e os instrumentos de medida. Conclusão: possibilitou-se identificar e analisar uma diversidade de intervenções e desfechos sobre o envelhecimento ativo, notando-se, por meio destes estudos, a fragilidade no cuidado em relação à prevenção de doenças e agravos da população idosa.(AU)

Objetivo: identificar la producción científica sobre envejecimiento activo. Método: se trata de un estudio bibliográfico, descriptivo, tipo revisión integradora, sin demilitación temporal, a partir de la búsqueda em las bases de datos LILACS, MEDLINE, CINAHL, Scopus y Biclioteca Virtual SciELO. Solo los trabajos publicados em revistas indexadas fueron analizados como artículos de investigación orinales en portugués, español e inglés. Se observa que los procedimientos adoptados para el análisis de la muestra final de los estudios fueron descroptivos. Resultados: se destaca que la muestra final estuvo conformada por 66 artículos. Los principales resultados se organizaron en tres categorias: Percepción de personas mayores y profesionales sobre el envejecimiento activo; Promoción de la salud para el envejecimiento activo y Los determinantes del envejecimiento activo e instrumentos de medición. Conclusión: fue posible identificar y analizar una variedad de intervenciones y resultados sobre el envejecimiento activo, notando, através de estos estudios, la debilidad em la atención en relación a la prevención de enfermedades y lesiones de la población anciana.(AU)

Objective: to identify the scientific production on active aging. Method: this is a bibliographic, descriptive, integrative review study, without temporal delimitation, performed through searches in the LILACS, MEDLINE, CINAHL, Scopus, and SciELO Virtual Library databases. Only original articles published in indexed journals and written in Portuguese, Spanish, and English were analyzed. Descriptive procedures were adopted for the analysis of the final sample of studies. Results: the final sample consisted of 66 articles. The main results were organized into the following three categories: Perception of older adults and professionals about active aging, Health promotion for active aging, and Determinants and instruments for active aging assessment. Conclusion: it was possible to identify and analyze various interventions and outcomes on active aging through the analysis of the studies. The fragility of care actions concerning the prevention of diseases and injuries in the elderly population was observed.(AU)

Endocytosis of the glutamate transporter 1 is regulated by laforin and malin: Implications in Lafora disease.

Lafora disease (LD) is a fatal rare type of progressive myoclonus epilepsy that appears during early adolescence. The disease is caused by mutations in EPM2A or EPM2B genes, which encode laforin, a glucan phosphatase, and malin, an E3-ubiquitin ligase, respectively. Although the exact roles of laforin and malin are still not well understood, it is known that they work as a complex in which laforin recruits targets that will be ubiquitinated by malin. Recently, we suggested that the type of epilepsy that accompanies LD could be due to deficiencies in the function of the astrocytic glutamate transporter GLT-1. We described that astrocytes from LD mouse models presented decreased levels of GLT-1 at the plasma membrane, leading to increased levels of glutamate in the brain parenchyma. In this work, we present evidence indicating that in the absence of a functional laforin/malin complex (as in LD cellular models) there is an alteration in the ubiquitination of GLT-1, which could be the cause of the reduction in the levels of GLT-1 at the plasma membrane. On the contrary, overexpression of the laforin/malin complex promotes the retention of GLT-1 at the plasma membrane. This retention may be due to the direct ubiquitination of GLT-1 and/or to an opposite effect of this complex on the dynamics of the Nedd4.2-mediated endocytosis of the transporter. This work, therefore, presents new pieces of evidence on the regulation of GLT-1 by the laforin/malin complex, highlighting its value as a therapeutic target for the amelioration of the type of epilepsy that accompanies LD.

Desafios para enfermeiros e fisioterapeutas assistirem mulheres idosas com incontinência urinária / Challenges for nurses and physiotherapeuts to assist old women with urinary incontinence

estudo qualitativo, do tipo exploratório-descritivo, com análise temática descritiva. Foram realizadas entrevistas semiestruturadas com 10 enfermeiros e 14 fisioterapeutas atuantes em um Distrito Sanitário de Saúde de uma capital do Sul do Brasil. Resultados: os achados ratificam que o cuidado à população idosa fica fragmentado dentro da Atenção Primária de Saúde. Os idosos são atendidos de acordo com a doença crônica que possuem ou a queixa principal que apresentam. O mesmo ocorre com relação à incontinência urinária, que não é investigada nem priorizada no cuidado. Conclusão: é necessária uma educação permanente ou capacitação para que os profissionais possam responder aos desafios do envelhecimento populacional, em todos os aspectos da saúde, com vistas ao autocuidado. (AU)

Objective: To know how nurses and physiotherapists care for elderly women with urinary incontinence in Primary Health Care. Method: qualitative, exploratory-descriptive study, with descriptive thematic analysis. Semi-structured interviews were conducted with 10 nurses and 14 physiotherapists working in a Health District of a capital of southern Brazil. Results: the findings confirm that care for the elderly population is fragmented within Primary Health Care. The elderly are treated according to their chronic disease or their main complaint. The same is true of urinary incontinence, which is neither investigated nor prioritized in care. Conclusion: Continuing education or training is necessary for professionals to respond to the challenges of population aging, in all aspects of health, with a view to self-care. (AU)

Objectivo: conocer cómo las enfermeras y fisioterapeutas atienden a las mujeres mayores con incontinencia urinaria en atención primaria de salud. Metodo: estudio cualitativo, exploratorio-descriptivo, con análisis descriptivo temático. Se realizaron entrevistas semiestructuradas con 10 enfermeras y 14 fisioterapeutas que trabajan en un distrito de salud de una capital del sur de Brasil. Resultados: los hallazgos confirman que la atención a la población anciana está fragmentada dentro de la Atención Primaria de Salud. Los ancianos son tratados de acuerdo con su enfermedad crónica o su queja principal. Lo mismo ocurre con la incontinencia urinaria, que no se investiga ni se prioriza en la atención. Conclusión: la educación o capacitación continua es necesaria para que los profesionales respondan a los desafíos del envejecimiento de la población, en todos los aspectos de la salud, con miras al autocuidado. (AU)

Regulation of the autophagic PI3KC3 complex by laforin/malin E3-ubiquitin ligase, two proteins involved in Lafora disease.

Lafora progressive myoclonus epilepsy is a fatal rare neurodegenerative disorder characterized by the accumulation of insoluble abnormal glycogen deposits in the brain and peripheral tissues. Mutations in at least two genes are responsible for the disease: EPM2A, encoding the glucan phosphatase laforin, and EPM2B, encoding the RING-type E3-ubiquitin ligase malin. Both laforin and malin form a functional complex in which laforin recruits the substrates to be ubiquitinated by malin. We and others have described that, in cellular and animal models of this disease, there is an autophagy impairment which leads to the accumulation of dysfunctional mitochondria. In addition, we established that the autophagic defect occurred at the initial steps of autophagosome formation. In this work, we present evidence that in cellular models of the disease there is a decrease in the amount of phosphatidylinositol-3P. This is probably due to defective regulation of the autophagic PI3KC3 complex, in the absence of a functional laforin/malin complex. In fact, we demonstrate that the laforin/malin complex interacts physically and co-localizes intracellularly with core components of the PI3KC3 complex (Beclin1, Vps34 and Vps15), and that this interaction is specific and results in the polyubiquitination of these proteins. In addition, the laforin/malin complex is also able to polyubiquitinate ATG14L and UVRAG. Finally, we show that overexpression of the laforin/malin complex increases PI3KC3 activity. All these results suggest a new role of the laforin/malin complex in the activation of autophagy via regulation of the PI3KC3 complex and explain the defect in autophagy described in Lafora disease.

A fase inicial do processo da vivência com a doença de Parkinson / The initial phase of the living process with Parkinson's disease / La fase inicial del proceso de la vivencia con la enfermedad de Parkinson

Objetivos: Conhecer a vivência da pessoa com doença de Parkinson desde o surgimento dos sinais e sintomas até a confirmação do diagnóstico. Métodos: Pesquisa qualitativa, utilizando o referencial metodológico da Teoria Fundamentada nos Dados. Participaram da entrevista em profundidade 30 pessoas com doença de Parkinson, cadastradas na Associação Parkinson Santa Catarina, e, posteriormente, cinco pessoas com a doença validaram os resultados. A coleta ocorreu entre setembro de 2013 e abril de 2014. Resultados: Emergiram três categorias: Percebendo que algo está mudando em suas capacidades, Percorrendo os consultórios médicos, e Lidando com o impacto da doença. Conclusão: Foi possível conhecer a vivência das pessoas com a doença em sua fase inicial, mostrando aspectos importantes na compreensão deste processo, e a partir de tal entendimento refletir sobre práticas de cuidados a esse grupo específico, incorporando assistência qualificada a esses usuários dos serviços de saúde (AU).

Objectives: To know the experience of the person with Parkinson's disease from the appearance of the signs and symptoms to the confirmation of the diagnosis. Methods: Qualitative research, using the methodological framework of Data Based Theory. Participants of the in-depth interview were 30 people with Parkinson's disease, enrolled in the Santa Catarina Parkinson's Association, and later five people with the disease validated the results. The collection took place from September 2013 to April 2014. Results: There emerged three categories: Realizing that something is changing in their capacities, Going to physician offices, and Dealing with the impact of the disease. Conclusion: It was possible to know the experience of people with the disease in its initial phase, showing important aspects in the understanding of this process, and from that understanding reflect on care practices to this specific group, incorporating qualified assistance to these users of the services Cheers (AU).

Objetivos: Conocer la vivencia de la persona con enfermedad de Parkinson desde el surgimiento de los signos y síntomas hasta la confirmación del diagnóstico. Métodos: Investigación cualitativa, utilizando el referencial metodológico de la Teoría Fundamentada en los Datos. En la entrevista en profundidad, 30 personas con enfermedad de Parkinson, registradas en la Asociación Parkinson Santa Catarina, y posteriormente cinco personas con la enfermedad validaron los resultados. La recolección ocurrió entre septiembre de 2013 y abril de 2014. Resultados: emergieron tres categorías: Percibiendo que algo está cambiando en sus capacidades, Recorrer los consultorios médicos, y Lidiando con el impacto de la enfermedad. Conclusión: Fue posible conocer la vivencia de las personas con la enfermedad en su fase inicial, mostrando aspectos importantes en la comprensión de este proceso, ya partir de tal entendimiento reflexionar sobre prácticas de cuidados a ese grupo específico, incorporando asistencia calificada a esos usuarios de los servicios de salud salud (AU).

A novel EPM2A mutation yields a slow progression form of Lafora disease.

Lafora disease (LD, OMIM 254780) is a rare disorder characterized by epilepsy and neurodegeneration leading patients to a vegetative state and death, usually within the first decade from the onset of the first symptoms. In the vast majority of cases LD is related to mutations in either the EPM2A gene (encoding the glucan phosphatase laforin) or the EPM2B gene (encoding the E3-ubiquitin ligase malin). In this work, we characterize the mutations present in the EPM2A gene in a patient displaying a slow progression form of the disease. The patient is compound heterozygous with Y112X and N163D mutations in the corresponding alleles. In primary fibroblasts obtained from the patient, we analyzed the expression of the mutated alleles by quantitative real time PCR and found slightly lower levels of expression of the EPM2A gene respect to control cells. However, by Western blotting we were unable to detect endogenous levels of the protein in crude extracts from patient fibroblasts. The Y112X mutation would render a truncated protein lacking the phosphatase domain and likely degraded. Since minute amounts of laforin-N163D might still play a role in cell physiology, we analyzed the biochemical characteristics of the N163D mutation. We found that recombinant laforin N163D protein was as stable as wild type and exhibited near wild type phosphatase activity towards biologically relevant substrates. On the contrary, it showed a severe impairment in the interaction profile with previously identified laforin binding partners. These results lead us to conclude that the slow progression of the disease present in this patient could be either due to the specific biochemical properties of laforin N163D or to the presence of alternative genetic modifying factors separate from pathogenicity.

The daily lives of people with Parkinson's disease / El cotidiano de las personas con la enfermedad de Parkinson / O cotidiano das pessoas com a doença de Parkinson

ABSTRACT Objective: To understand the daily lives of people with Parkinson's disease. Method: Qualitative research, using as methodological and theoretical referential the Grounded Theory and Symbolic Interactionism, respectively. The in-depth interview was conducted with 30 people with Parkinson's disease. Results: From data analysis, three themes were selected: Living with the disease - living with the treatment and changes in lifestyle; Modifying of one's job performance - revealing incapacity for work and the need to anticipate retirement and; Living with the stigma - the feeling of prejudice against the disease and the perceived limitations of the health services. Final considerations: Living with a chronic and non-transferable disease encompasses social, physical and cultural effects, along with the personal experiences of each unique individual. This study assists the improvement of care to people with the disease, because the care practice emerges from the interactions between the subjects.

RESUMEN Objetivo: Comprender el cotidiano de las personas con la enfermedad de Parkinson. Método: Investigación cualitativa, utilizando como referencial metodológico y teórico la Teoría Fundamentada en Datos y el Interaccionismo Simbólico, respectivamente. La entrevista en profundidad fue realizada con 30 personas con la enfermedad de Parkinson. Resultados: Por medio de la codificación de los datos, emergieron tres categorías: Viviendo la rutina de la enfermedad: la convivencia con el tratamiento y las modificaciones en vivir; Modificando su rendimiento en el trabajo: revelando incapacidad para el trabajo y la necesidad de anticipar la jubilación y; Conviviendo con estigmas: el sentimiento de prejuicio por la enfermedad y la percepción de limitaciones de los servicios de salud. Consideraciones finales: La convivencia con una enfermedad crónica no transmisible engloba efectos sociales, físicos, culturales y las experiencias personales de cada uno. Este estudio auxilia la mejora de la asistencia a las personas con la enfermedad, pues la práctica de cuidados emerge de las interacciones entre los sujetos.

RESUMO Objetivo: Compreender o cotidiano das pessoas com a doença de Parkinson. Método: Pesquisa qualitativa, utilizando como referencial metodológico e teórico a Teoria Fundamentada nos Dados e o Interacionismo Simbólico, respectivamente. A entrevista em profundidade foi realizada com 30 pessoas com a doença de Parkinson. Resultados: Por meio da codificação dos dados, emergiram três categorias: Vivendo a rotina da doença - a convivência com o tratamento e as modificações no viver; Modificando seu desempenho no trabalho - revelando incapacidade para o trabalho e a necessidade de antecipar a aposentadoria e; Convivendo com estigmas - o sentimento de preconceito pela doença e a percepção de limitações dos serviços de saúde. Considerações finais: A convivência com uma doença crônica não transmissível engloba efeitos sociais, físicos, culturais e as experiências pessoais de cada um. Este estudo auxilia no aprimoramento da assistência às pessoas com a doença, pois a prática de cuidados emerge das interações entre os sujeitos.

AMPK Protein Interaction Analyses by Yeast Two-Hybrid.

Mammalian AMP-activated protein kinase (AMPK) is a Ser/Thr protein kinase that acts as a crucial energy sensor in the cell. Since AMPK plays a key role in a multitude of different pathways in the cell, major efforts have been concentrated to elucidate its signaling network, mainly by the identification of AMPK downstream targets. In this chapter we describe a yeast two-hybrid method for the direct evaluation of the interaction between an AMPK subunit and putative substrates.

The daily lives of people with Parkinson's disease.

OBJECTIVE: To understand the daily lives of people with Parkinson's disease. METHOD: Qualitative research, using as methodological and theoretical referential the Grounded Theory and Symbolic Interactionism, respectively. The in-depth interview was conducted with 30 people with Parkinson's disease. RESULTS: From data analysis, three themes were selected: Living with the disease - living with the treatment and changes in lifestyle; Modifying of one's job performance - revealing incapacity for work and the need to anticipate retirement and; Living with the stigma - the feeling of prejudice against the disease and the perceived limitations of the health services. FINAL CONSIDERATIONS: Living with a chronic and non-transferable disease encompasses social, physical and cultural effects, along with the personal experiences of each unique individual. This study assists the improvement of care to people with the disease, because the care practice emerges from the interactions between the subjects.

Astrocytes: new players in progressive myoclonus epilepsy of Lafora type.

Lafora disease (LD) is a fatal form of progressive myoclonus epilepsy characterized by the accumulation of insoluble poorly branched glycogen-like inclusions named Lafora bodies (LBs) in the brain and peripheral tissues. In the brain, since its first discovery in 1911, it was assumed that these glycogen inclusions were only present in affected neurons. Mouse models of LD have been obtained recently, and we and others have been able to report the accumulation of glycogen inclusions in the brain of LD animals, what recapitulates the hallmark of the disease. In this work we present evidence indicating that, although in mouse models of LD glycogen inclusions co-localize with neurons, as originally established, most of them co-localize with astrocytic markers such as glial fibrillary acidic protein (GFAP) and glutamine synthase. In addition, we have observed that primary cultures of astrocytes from LD mouse models accumulate higher levels of glycogen than controls. These results suggest that astrocytes may play a crucial role in the pathophysiology of Lafora disease, as the accumulation of glycogen inclusions in these cells may affect their regular functionality leading them to a possible neuronal dysfunction.