Xenotransplantation of Mitochondrial Electron Transfer Enzyme, Ndi1, in Myocardial Reperfusion Injury

A significant consequence of ischemia/reperfusion (I/R) is mitochondrial respiratory dysfunction, leading to energetic deficits and cellular toxicity from reactive oxygen species (ROS). Mammalian complex I, a NADH-quinone oxidoreductase enzyme, is a multiple subunit enzyme that oxidizes NADH and pumps protons across the inner membrane. Damage to complex I leads to superoxide production which further damages complex I as well as other proteins, lipids and mtDNA. The yeast, S. cerevisiae, expresses internal rotenone insensitive NADH-quinone oxidoreductase (Ndi1); a single 56kDa polypeptide which, like the multi-subunit mammalian complex I, serves as the entry site of electrons to the respiratory chain, but without proton pumping. Heterologous expression of Ndi1 in mammalian cells results in protein localization to the inner mitochondrial membrane which can function in parallel with endogenous complex I to oxidize NADH and pass electrons to ubiquinone. Expression of Ndi1 in HL-1 cardiomyocytes and in neonatal rat ventricular myocytes protected the cells from simulated ischemia/reperfusion (sI/R), accompanied by lower ROS production, and preservation of ATP levels and NAD+/NADH ratios. We next generated a fusion protein of Ndi1 and the 11aa protein transduction domain from HIV TAT. TAT-Ndi1 entered cardiomyocytes and localized to mitochondrial membranes. Furthermore, TAT-Ndi1 introduced into Langendorff-perfused rat hearts also localized to mitochondria. Perfusion of TAT-Ndi1 before 30 min no-flow ischemia and up to 2 hr reperfusion suppressed ROS production and preserved ATP stores. Importantly, TAT-Ndi1 infused before ischemia reduced infarct size by 62%; TAT-Ndi1 infused at the onset of reperfusion was equally cardioprotective. These results indicate that restoring NADH oxidation and electron flow at reperfusion can profoundly ameliorate reperfusion injury

The interaction of tropomodulin with tropomyosin stabilizes thin filaments in cardiac myocytes

Actin (thin) filament length regulation and stability are essential for striated muscle function. To determine the role of the actin filament pointed end capping protein, tropomodulin1 (Tmod1), with tropomyosin, we generated monoclonal antibodies (mAb17 and mAb8) against Tmod1 that specifically disrupted its interaction with tropomyosin in vitro. Microinjection of mAb17 or mAb8 into chick cardiac myocytes caused a dramatic loss of the thin filaments, as revealed by immunofluorescence deconvolution microscopy. Real-time imaging of live myocytes expressing green fluorescent protein–α-tropomyosin and microinjected with mAb17 revealed that the thin filaments depolymerized from their pointed ends. In a thin filament reconstitution assay, stabilization of the filaments before the addition of mAb17 prevented the loss of thin filaments. These studies indicate that the interaction of Tmod1 with tropomyosin is critical for thin filament stability. These data, together with previous studies, indicate that Tmod1 is a multifunctional protein: its actin filament capping activity prevents thin filament elongation, whereas its interaction with tropomyosin prevents thin filament depolymerization

Autophagy Induced by Ischemic Preconditioning is Essential for Cardioprotection

Based on growing evidence linking autophagy to preconditioning, we tested the hypothesis that autophagy is necessary for cardioprotection conferred by ischemic preconditioning (IPC). We induced IPC with three cycles of 5 min regional ischemia alternating with 5 min reperfusion and assessed the induction of autophagy in mCherry-LC3 transgenic mice by imaging of fluorescent autophagosomes in cryosections. We found a rapid and significant increase in the number of autophagosomes in the risk zone of the preconditioned hearts. In Langendorff-perfused hearts subjected to an IPC protocol of 3 × 5 min ischemia, we also observed an increase in autophagy within 10 min, as assessed by Western blotting for p62 and cadaverine dye binding. To establish the role of autophagy in IPC cardioprotection, we inhibited autophagy with Tat-ATG5K130R, a dominant negative mutation of the autophagy protein Atg5. Cardioprotection by IPC was reduced in rat hearts perfused with recombinant Tat-ATG5K130R. To extend the potential significance of autophagy in cardioprotection, we also assessed three structurally unrelated cardioprotective agents—UTP, diazoxide, and ranolazine—for their ability to induce autophagy in HL-1 cells. We found that all three agents induced autophagy; inhibition of autophagy abolished their protective effect. Taken together, these findings establish autophagy as an end-effector in ischemic and pharmacologic preconditioning

“Someone’s got to do it” – Primary care providers (PCPs) describe caring for rural women with mental health problems

Objective: Little is known about how primary care providers (PCPs) approach mental health care for low-income rural women. We developed a qualitative research study to explore the attitudes and practices of PCPs regarding the care of mood and anxiety disorders in rural women. Method: We conducted semi-structured interviews with 19 family physicians, internists, and obstetrician-gynecologists (OBGYNs) in office-based practices in rural central Pennsylvania. Using thematic analysis, investigators developed a coding scheme. Questions focused on 1) screening and diagnosis of mental health conditions, 2) barriers to treatment among rural women, 3) management of mental illnesses in rural women, and 4) ideas to improve care for this population. Results: PCP responses reflected these themes: 1) PCPs identify mental illnesses through several mechanisms including routine screening, indicator-based assessment, and self-identification by the patient; 2) Rural culture and social ecology are significant barriers to women in need of mental healthcare; 3) Mental healthcare resource limitations in rural communities lead PCPs to seek creative solutions to care for rural women with mental illnesses; 4) To improve mental healthcare in rural communities, both social norms and resource limitations must be addressed. Conclusion: Our findings can inform future interventions to improve women\u27s mental healthcare in rural communities. Ideas include promoting generalist education in mental healthcare, and expanding access to consultative networks. In addition, community programs to reduce the stigma of mental illnesses in rural communities may promote healthcare seeking and receptiveness to treatment

What Sets Physically Active Rural Communities Apart from Less Active Ones? A Comparative Case Study of Three US Counties

Background: Rural US communities experience health disparities, including a lower prevalence of physical activity (PA). However, “Positive Deviants”—rural communities with greater PA than their peers—exist. The purpose of this study was to identify the factors that help create physically active rural US communities. Methods: Stakeholder interviews, on-site intercept interviews, and in-person observations were used to form a comparative case study of two rural counties with high PA prevalence (HPAs) and one with low PA prevalence (LPA) from a southern US state, selected based on rurality and adult PA prevalence. Interview transcripts were inductively coded by three readers, resulting in a thematic structure that aligned with a Community Capital Framework, which was then used for deductive coding and analysis. Results: Fifteen stakeholder interviews, nine intercept interviews, and on-site observations were conducted. Human and Organizational Capital differed between the HPAs and LPA, manifesting as Social, Built, Financial, and Political Capital differences and a possible “spiraling-up” or cyclical effect through increasing PA and health (Human Capital), highlighting a potential causal model for future study. Conclusions: Multi-organizational PA coalitions may hold promise for rural PA by directly influencing Human and Organizational Capital in the short term and the other forms of capital in the long term

Cadherin 2-Related Arrhythmogenic Cardiomyopathy Prevalence and Clinical Features

Background:Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiac disease characterized by fibrofatty replacement of the right and left ventricle, often causing ventricular dysfunction and life-threatening arrhythmias. Variants in desmosomal genes account for up to 60% of cases. Our objective was to establish the prevalence and clinical features of ACM stemming from pathogenic variants in the nondesmosomal cadherin 2 (CDH2), a novel genetic substrate of ACM.Methods:A cohort of 500 unrelated patients with a definite diagnosis of ACM and no disease-causing variants in the main ACM genes was assembled. Genetic screening of CDH2 was performed through next-generation or Sanger sequencing. Whenever possible, cascade screening was initiated in the families of CDH2-positive probands, and clinical evaluation was performed.Results:Genetic screening of CDH2 led to the identification of 7 rare variants: 5, identified in 6 probands, were classified as pathogenic or likely pathogenic. The previously established p.D407N pathogenic variant was detected in 2 additional probands. Probands and family members with pathogenic/likely pathogenic variants in CDH2 were clinically evaluated, and along with previously published cases, altogether contributed to the identification of gene-specific features (13 cases from this cohort and 11 previously published, for a total of 9 probands and 15 family members). Ventricular arrhythmic events occurred in most CDH2-positive subjects (20/24, 83%), while the occurrence of heart failure was rare (2/24, 8.3%). Among probands, sustained ventricular tachycardia and sudden cardiac death occurred in 5/9 (56%).Conclusions:In this worldwide cohort of previously genotype-negative ACM patients, the prevalence of probands with CDH2 pathogenic/likely pathogenic variants was 1.2% (6/500). Our data show that this cohort of CDH2-ACM patients has a high incidence of ventricular arrhythmias, while evolution toward heart failure is rare.</p

Correction to:The genetic architecture of Plakophilin 2 cardiomyopathy (Genetics in Medicine, (2021), 23, 10, (1961-1968), 10.1038/s41436-021-01233-7)

Due to a processing error Cynthia James, Brittney Murray, and Crystal Tichnell were assigned to the wrong affiliation. Cynthia James, Brittney Murray, and Crystal Tichnell have as their affiliation 5 Division of Cardiology, Department of Medicine, Johns Hopkins University, Baltimore, MD, USA. In addition Hana Zouk, Megan Hawley, and Birgit Funke were assigned only to affiliation 3; they also have affiliation 4 Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA. The original article has been corrected

Organizing risk: organization and management theory for the risk society

Risk has become a crucial part of organizing, affecting a wide range of organizations in all sectors. We identify, review and integrate diverse literatures relevant to organizing risk, building on an existing framework that describes how risk is organized in three ‘modes’ – prospectively, in real-time, and retrospectively. We then identify three critical issues in the existing literature: its fragmented nature; its neglect of the tensions associated with each of the modes; and its tendency to assume that the meaning of an object in relation to risk is singular and stable. We provide a series of new insights with regard to each of these issues. First, we develop the concept of a risk cycle that shows how organizations engage with all three modes and transition between them over time. Second, we explain why the tensions have been largely ignored and show how studies using a risk work perspective can provide further insights into them. Third, we develop the concept of risk translation to highlight the ways in the meanings of risks can be transformed and to identify the political consequences of such translations. We conclude the paper with a research agenda to elaborate these insights and ideas further

No major role for rare plectin variants in arrhythmogenic right ventricular cardiomyopathy

Aims Likely pathogenic/pathogenic variants in genes encoding desmosomal proteins play an important role in the pathophysiology of arrhythmogenic right ventricular cardiomyopathy (ARVC). However, for a substantial proportion of ARVC patients, the genetic substrate remains unknown. We hypothesized that plectin, a cytolinker protein encoded by the PLEC gene, could play a role in ARVC because it has been proposed to link the desmosomal protein desmoplakin to the cytoskeleton and therefore has a potential function in the desmosomal structure. Methods We screened PLEC in 359 ARVC patients and compared the frequency of rare coding PLEC variants (minor allele frequency [MAF] <0.001) between patients and controls. To assess the frequency of rare variants in the control population, we evaluated the rare coding variants (MAF <0.001) found in the European cohort of the Exome Aggregation Database. We further evaluated plectin localization by immunofluorescence in a subset of patients with and without a PLEC variant. Results Forty ARVC patients carried one or more rare PLEC variants (11%, 40/359). However, rare variants also seem to occur frequently in the control population (18%, 4754/26197 individuals). Nor did we find a difference in the prevalence of rare PLEC variants in ARVC patients with or without a desmosomal likely pathogenic/pathogenic variant (14% versus 8%, respectively). However, immunofluorescence analysis did show decreased plectin junctional localization in myocardial tissue from 5 ARVC patients with PLEC variants. Conclusions Although PLEC has been hypothesized as a promising candidate gene for ARVC, our current study did not show an enrichment of rare PLEC variants in ARVC patients compared to controls and therefore does not support a major role for PLEC in this disorder. Although rare PLEC variants were associated with abnormal localization in cardiac tissue, the confluence of data does not support a role for plectin abnormalities in ARVC development

O ensino da leitura e da escrita no 1º ano de escolaridade: os resultados dos alunos em leitura

Tese de Doutoramento em Psicologia Aplicada, Área de especialidade Psicologia EducacionalO presente estudo teve como objetivo caracterizar as práticas de ensino da linguagem escrita em Portugal e perceber o seu impacto na aquisição da leitura no final do 1º ano de escolaridade. A primeira etapa deste estudo correspondeu a um estudo descritivo e comparativo com o realizado por Fijalkow (2003) em França. A partir da resposta a um questionário de 883 professores foi possível verificar que os professores dos dois países se aproximam no que diz respeito aos aspetos metodológicos do ensino da leitura e à avaliação da leitura e da escrita dos alunos. Contudo, os professores portugueses dizem utilizar com maior frequência do que os professores franceses, atividades a partir de livros infantis, outros materiais para além do manual e diferentes propostas de escrita. Na segunda etapa deste estudo, o questionário utilizado foi validado através da sua estrutura fatorial e foram utilizados os fatores revelados para a realização de uma análise hierárquica de clusters. Esta análise revelou três grupos de professores: a) um grupo de professores que centra as suas práticas no ensino do código da linguagem escrita (unidades curtas); b) um grupo que realça a construção de significado na interação com o material escrito (unidades longas); c) e um grupo de professores que reúne nas suas práticas características de ambos os grupos referidos anteriormente (unidades diversificadas). A terceira etapa realizada diz respeito à validação das respostas do questionário através da observação de sala de aula. Foram selecionados 5% dos professores de cada um dos três grupos constituídos (N=42). Cada professor foi observado duas vezes durante 60 minutos em situações escolhidas por si como situações privilegiadas para o ensino da linguagem escrita. Através da utilização de uma grelha de observação que divide as atividades desenvolvidas em atividades que remetem para o código ou para o significado, foi possível confirmar as respostas ao questionário para 55% dos professores participantes. No sentido de perceber o impacto das práticas dos professores na aquisição da leitura dos alunos foram selecionados os professores cujas observações foram coincidentes com as respostas ao questionário e avaliados os seus alunos com recurso a três provas de leitura: leitura de palavras isoladas, decisão lexical e compreensão. Participaram 461 alunos distribuídos pelos três grupos referidos anteriormente. Através da realização de uma MANCOVA, controlando a escolaridade das mães dos alunos, verificou-se que os alunos do grupo unidades diversificadas obtêm melhores resultados em todas as provas do que os alunos dos outros dois grupos. Foi ainda possível constatar que não existiram diferenças de desempenho entre os alunos do grupo unidades curtas e os alunos do grupo unidades longas.ABSTRACT: The present study aimed to characterize the teaching practices of written language and understand its impact on reading acquisition by the end of 1st grade. The first step of this study was a descriptive and comparative study to the one conducted by Fijalkow (2003) in France. Based on questionnaire answers of 883 teachers, we found that teachers from both countries have similar approaches regarding methodological aspects of teaching of reading and students’ assessment of reading and writing abilities. However, Portuguese teachers stated that they use more frequently activities from children's literature, other materials besides the manual and different writing proposals than French teachers. In the second step of the study, the questionnaire was validated through its factorial structure and the factors disclosed were used to perform a hierarchical cluster analysis. This analysis revealed three groups of teachers: a) a group of teachers who focus its practices in teaching the code of written language (short units), b) a group that emphasizes the construction of meaning in interaction with the written material (long units), c) and a group of teachers who gathered in their practices characteristics of both groups mentioned above (diversified units). The third step concerns the validation of the questionnaire answers through classroom observation. 5% of the teachers in each of the three groups were selected (N=42). Each teacher was observed twice during 60 minutes in situations identified by them as privileged situations for teaching written language. Using an observation grid which divides the activities conducted in code-activities or the meaning-activities it was possible to confirm questionnaire answers for 55% of the participating teachers. In order to understand the impact of teachers’ practices in students reading acquisition, teachers whose observations were coincident with the questionnaire answers were selected and their students were evaluated using three reading tests: single words reading, lexical decision task and comprehension task. Participants were 461 students distributed through the three groups previously mentioned. By conducting a MANCOVA, controlling mothers’ education level, we concluded that students in the diversified unit group obtain better results in all reading tests than students from the other groups. It was also possible to confirm that there were no differences in performance between students of the short units group and students of the long units group.Tese de Doutoramento em Psicologia Aplicada, Área de especialidade Psicologia EducacionalApoio financeiro da Fundação para a Ciência e Tecnologia (SFRH/BD/64630/2009