Paired nicking-mediated COL17A1 reframing for junctional epidermolysis bullosa.

Junctional epidermolysis bullosa (JEB) is a debilitating hereditary skin disorder caused by mutations in genes encoding laminin-332, type XVII collagen (C17), and integrin-α6ß4, which maintain stability between the dermis and epidermis. We designed patient-specific Cas9-nuclease- and -nickase-based targeting strategies for reframing a common homozygous deletion in exon 52 of COL17A1 associated with a lack of full-length C17 expression. Subsequent characterization of protein restoration, indel composition, and divergence of DNA and mRNA outcomes after treatment revealed auspicious efficiency, safety, and precision profiles for paired nicking-based COL17A1 editing. Almost 46% of treated primary JEB keratinocytes expressed reframed C17. Reframed COL17A1 transcripts predominantly featured 25- and 37-nt deletions, accounting for >42% of all edits and encoding C17 protein variants that localized accurately to the cell membrane. Furthermore, corrected cells showed accurate shedding of the extracellular 120-kDa C17 domain and improved adhesion capabilities to laminin-332 compared with untreated JEB cells. Three-dimensional (3D) skin equivalents demonstrated accurate and continuous deposition of C17 within the basal membrane zone between epidermis and dermis. Our findings constitute, for the first time, gene-editing-based correction of a COL17A1 mutation and demonstrate the superiority of proximal paired nicking strategies based on Cas9 D10A nickase over wild-type Cas9-based strategies for gene reframing in a clinical context.

An open-source python library for detection of known and novel Kell, Duffy and Kidd variants from exome sequencing.

BACKGROUND AND OBJECTIVES: Next generation sequencing (NGS) has promising applications in transfusion medicine. Exome sequencing (ES) is increasingly used in the clinical setting, and blood group interpretation is an additional value that could be extracted from existing data sets. We provide the first release of an open-source software tailored for this purpose and describe its validation with three blood group systems. MATERIALS AND METHODS: The DTM-Tools algorithm was designed and used to analyse 1018 ES NGS files from the ClinSeq® cohort. Predictions were correlated with serology for 5 antigens in a subset of 108 blood samples. Discrepancies were investigated with alternative phenotyping and genotyping methods, including a long-read NGS platform. RESULTS: Of 116 genomic variants queried, those corresponding to 18 known KEL, FY and JK alleles were identified in this cohort. 596 additional exonic variants were identified KEL, ACKR1 and SLC14A1, including 58 predicted frameshifts. Software predictions were validated by serology in 108 participants; one case in the FY blood group and three cases in the JK blood group were discrepant. Investigation revealed that these discrepancies resulted from (1) clerical error, (2) serologic failure to detect weak antigenic expression and (3) a frameshift variant absent in blood group databases. CONCLUSION: DTM-Tools can be employed for rapid Kell, Duffy and Kidd blood group antigen prediction from existing ES data sets; for discrepancies detected in the validation data set, software predictions proved accurate. DTM-Tools is open-source and in continuous development.

Lung Defense through IL-8 Carries a Cost of Chronic Lung Remodeling and Impaired Function.

IL-8-dependent inflammation is a hallmark of host lung innate immunity to bacterial pathogens, yet in many human lung diseases, including chronic obstructive pulmonary disease, bronchiectasis, and pulmonary fibrosis, there are progressive, irreversible, pathological changes associated with elevated levels of IL-8 in the lung. To better understand the duality of IL-8-dependent host immunity to bacterial infection and lung pathology, we expressed human IL-8 transgenically in murine bronchial epithelium, and investigated the impact of overexpression on lung bacterial clearance, host immunity, and lung pathology and function. Persistent IL-8 expression in bronchial epithelium resulted in neutrophilia, neutrophil maturation and activation, and chemotaxis. There was enhanced protection against challenge with Pseudomonas aeruginosa, and significant changes in baseline expression of innate and adaptive immunity transcripts for Ccl5, Tlr6, IL-2, and Tlr1. There was increased expression of Tbet and Foxp3 in response to the Pseudomonas antigen OprF, indicating a regulatory T-cell phenotype. However, this enhanced bacterial immunity came at a high price of progressive lung remodeling, with increased inflammation, mucus hypersecretion, and fibrosis. There was increased expression of Ccl3 and reduced expression of Claudin 18 and F11r, with damage to epithelial organization leading to leaky tight junctions, all of which resulted in impaired lung function with reduced compliance, increased resistance, and bronchial hyperreactivity as measured by whole-body plethysmography. These results show that IL-8 overexpression in the bronchial epithelium benefits lung immunity to bacterial infection, but specifically drives lung damage through persistent inflammation, lung remodeling, and damaged tight junctions, leading to impaired lung function.

ABCG2/BCRP: Specific and Nonspecific Modulators.

Multidrug resistance (MDR) in cancer cells is the development of resistance to a variety of structurally and functionally nonrelated anticancer drugs. This phenomenon has become a major obstacle to cancer chemotherapy seriously affecting the clinical outcome. MDR is associated with increased drug efflux from cells mediated by an energy-dependent mechanism involving the ATP-binding cassette (ABC) transporters, mainly P-glycoprotein (ABCB1), the MDR-associated protein-1 (ABCC1), and the breast cancer resistance protein (ABCG2). The first two transporters have been widely studied already and reviews summarized the results. The ABCG2 protein has been a subject of intense study since its discovery as its overexpression has been detected in resistant cell lines in numerous types of human cancers. To date, a long list of modulators of ABCG2 exists and continues to increase. However, little is known about the clinical consequences of ABCG2 modulation. This makes the design of novel, potent, and nontoxic inhibitors of this efflux protein a major challenge to reverse MDR and thereby increase the success of chemotherapy. The aim of the present review is to describe and highlight specific and nonspecific modulators of ABCG2 reported to date based on the selectivity of the compounds, as many of them are effective against one or more ABC transport proteins.

[The construction process of managerial profile competencies for nurse coordinators in the hospital field]. / O processo de construção do perfil de competências gerenciais para enfermeiros coordenadores de área hospitalar.

The objective of this study was to construct a profile of managerial competencies, based on the consensus of nurse coordinators in the field. This study was developed in a philanthropic hospital in São Paulo, following the research-action model, and included 13 nurse coordinators as participants. The data collection was performed using the focal group technique. Data analysis was performed using the theoretical frameworks related to the working process and managerial competencies. The results identified the greater emphasis assigned to the competencies related to the mentor, coordinator and director roles. It was, therefore, possible to construct a professional development plan that is based on competencies in the technical, ethical-political, and communicative domains, as well as the development of citizenship. The analysis of the managerial working process and the study of the competencies within the managerial environment were shown to be important, because they highlighted the professionals' need to improve, thus fulfilling personal, professional, and organizational demands.

Lung Nodules in Melanoma Patients: Morphologic Criteria to Differentiate Non-Metastatic and Metastatic Lesions.

Lung nodules are frequent findings in chest computed tomography (CT) in patients with metastatic melanoma. In this study, we assessed the frequency and compared morphologic differences of metastases and benign nodules. We retrospectively evaluated 85 patients with melanoma (AJCC stage III or IV). Inclusion criteria were ≤20 lung nodules and follow-up using CT ≥183 days after baseline. Lung nodules were evaluated for size and morphology. Nodules with significant growth, nodule regression in line with RECIST assessment or histologic confirmation were judged to be metastases. A total of 438 lung nodules were evaluated, of which 68% were metastases. At least one metastasis was found in 78% of patients. A 10 mm diameter cut-off (used for RECIST) showed a specificity of 95% and a sensitivity of 20% for diagnosing metastases. Central location (n = 122) was more common in metastatic nodules (p = 0.009). Subsolid morphology (n = 53) was more frequent (p < 0.001), and calcifications (n = 13) were solely found in non-metastatic lung nodules (p < 0.001). Our data show that lung nodules are prevalent in about two-thirds of melanoma patients (AJCC stage III/IV) and the majority are metastases. Even though we found a few morphologic indicators for metastatic or non-metastatic lung nodules, morphology has limited value to predict the presence of lung metastases.

A non-viral and selection-free COL7A1 HDR approach with improved safety profile for dystrophic epidermolysis bullosa.

Gene editing via homology-directed repair (HDR) currently comprises the best strategy to obtain perfect corrections for pathogenic mutations of monogenic diseases, such as the severe recessive dystrophic form of the blistering skin disease epidermolysis bullosa (RDEB). Limitations of this strategy, in particular low efficiencies and off-target effects, hinder progress toward clinical applications. However, the severity of RDEB necessitates the development of efficient and safe gene-editing therapies based on perfect repair. To this end, we sought to assess the corrective efficiencies following optimal Cas9 nuclease and nickase-based COL7A1-targeting strategies in combination with single- or double-stranded donor templates for HDR at the COL7A1 mutation site. We achieved HDR-mediated correction efficiencies of up to 21% and 10% in primary RDEB keratinocytes and fibroblasts, respectively, as analyzed by next-generation sequencing, leading to full-length type VII collagen restoration and accurate deposition within engineered three-dimensional (3D) skin equivalents (SEs). Extensive on- and off-target analyses confirmed that the combined treatment of paired nicking and single-stranded oligonucleotides constituted a highly efficient COL7A1-editing strategy, associated with a significantly improved safety profile. Our findings, therefore, represent a further advancement in the field of traceless genome editing for genodermatoses.

Planning and building Health Care Networks in Brazil's Federal District. / O Planejamento e a construção das Redes de Atenção à Saúde no DF, Brasil.

This article reports on the experience of implementing Health Care Planning (HCP) in the territories of Itapoã, Paranoá and São Sebastião in the East Region of Brazil's Federal District. HCP began at the end of 2016 with Itapoã and was expanded to the other territories in 2018. The results point to a better organised health care network, specifically as regards care for chronic conditions, hypertension and diabetes. The activities involved a series of thematic theory workshops and tutoring workshops carried out in Primary Health Care (PHC) and Specialised Ambulatory Care (SAC) facilities. In PHC, macro-processes (territorialisation, family registration, risk stratification, family risk classification, local diagnosis, care by block of hours, elimination of waiting times, and others) were organised to support meeting certain of the population's demands. In SAC, an Ambulatory Specialities Clinic was set up using the technology of continuous care provided by a multi-professional team to high- and very high-risk hypertensive and diabetic patients stratified in PHC, and care provision is shared. One of the strong points in the integration of PHC and SAC was matrix support provided by SAC professionals in "laboratory units". HCP has been an important management tool for organising health care in the East Region.

O artigo objetiva descrever o planejamento e a construção das Redes de Atenção à Saúde (RAS), por meio da Planificação da Atenção à Saúde (PAS), na Região Leste, Distrito Federal. Trata-se de um relato de experiência sobre a PAS, realizada de 2016 a 2018. As atividades foram desenvolvidas a partir de um conjunto de oficinas teóricas temáticas, de tutorias realizadas na Atenção Primária à Saúde (APS) e na Atenção Ambulatorial Especializada (AAE). Os resultados apontam uma melhor organização da RAS, especificamente para a linha de cuidado das condições crônicas, hipertensão e diabetes. Na APS foram organizados os macroprocessos: territorialização, cadastramento das famílias, estratificação de risco, classificação de riscos familiares, diagnóstico local, atendimento por bloco de horas, eliminando filas, dentre outros. Na AAE foi implantado o Ambulatório de Especialidades com a tecnologia de atenção continua, realizada por equipe multiprofissional para hipertensos e diabéticos de alto e muito risco, estratificados na APS, compartilhando o cuidado. Uma das potencialidades da integração da APS e AAE foi o matriciamento realizado por profissionais da AAE, nas unidades laboratórios. A PAS configurou-se como um importante instrumento de gestão das RAS.

O Planejamento e a construção das Redes de Atenção à Saúde no DF, Brasil / Planning and building Health Care Networks in Brazil's Federal District

Resumo O artigo objetiva descrever o planejamento e a construção das Redes de Atenção à Saúde (RAS), por meio da Planificação da Atenção à Saúde (PAS), na Região Leste, Distrito Federal. Trata-se de um relato de experiência sobre a PAS, realizada de 2016 a 2018. As atividades foram desenvolvidas a partir de um conjunto de oficinas teóricas temáticas, de tutorias realizadas na Atenção Primária à Saúde (APS) e na Atenção Ambulatorial Especializada (AAE). Os resultados apontam uma melhor organização da RAS, especificamente para a linha de cuidado das condições crônicas, hipertensão e diabetes. Na APS foram organizados os macroprocessos: territorialização, cadastramento das famílias, estratificação de risco, classificação de riscos familiares, diagnóstico local, atendimento por bloco de horas, eliminando filas, dentre outros. Na AAE foi implantado o Ambulatório de Especialidades com a tecnologia de atenção continua, realizada por equipe multiprofissional para hipertensos e diabéticos de alto e muito risco, estratificados na APS, compartilhando o cuidado. Uma das potencialidades da integração da APS e AAE foi o matriciamento realizado por profissionais da AAE, nas unidades laboratórios. A PAS configurou-se como um importante instrumento de gestão das RAS.

Abstract This article reports on the experience of implementing Health Care Planning (HCP) in the territories of Itapoã, Paranoá and São Sebastião in the East Region of Brazil's Federal District. HCP began at the end of 2016 with Itapoã and was expanded to the other territories in 2018. The results point to a better organised health care network, specifically as regards care for chronic conditions, hypertension and diabetes. The activities involved a series of thematic theory workshops and tutoring workshops carried out in Primary Health Care (PHC) and Specialised Ambulatory Care (SAC) facilities. In PHC, macro-processes (territorialisation, family registration, risk stratification, family risk classification, local diagnosis, care by block of hours, elimination of waiting times, and others) were organised to support meeting certain of the population's demands. In SAC, an Ambulatory Specialities Clinic was set up using the technology of continuous care provided by a multi-professional team to high- and very high-risk hypertensive and diabetic patients stratified in PHC, and care provision is shared. One of the strong points in the integration of PHC and SAC was matrix support provided by SAC professionals in "laboratory units". HCP has been an important management tool for organising health care in the East Region.

O processo de construção do perfil de competências gerenciais para enfermeiros coordenadores de área hospitalar / The construction process of managerial profile competencies for nurse coordinators in the hospital field / Proceso de construcción del perfil de competencias gerenciales para enfermeros coordinadores de área hospitalaria

Este estudo objetivou construir o perfil de competências gerenciais, consensuado por enfermeiros coordenadores de área. Desenvolvido em um hospital filantrópico de São Paulo, na modalidade de pesquisa-ação, participaram da pesquisa 13 enfermeiros coordenadores de área. A coleta de dados ocorreu por meio da técnica de grupo focal. Para a análise dos dados utilizaram-se os referenciais teóricos relacionados a processo de trabalho e competências gerenciais. Os resultados identificaram maior ênfase atribuída às competências relacionadas aos papéis de mentor, coordenador e diretor. Foi possível, então, construir um plano de desenvolvimento profissional, pautado em competências que atendam as dimensões técnica, ético-política, comunicativa e de desenvolvimento da cidadania. A análise do processo de trabalho gerencial e o estudo das competências no âmbito gerencial mostraram-se importantes, pois problematizaram necessidades de aprimoramento desses profissionais, assim, respondendo às demandas pessoais, profissionais e organizacionais.

The objective of this study was to construct a profile of managerial competencies, based on the consensus of nurse coordinators in the field. This study was developed in a philanthropic hospital in São Paulo, following the research-action model, and included 13 nurse coordinators as participants. The data collection was performed using the focal group technique. Data analysis was performed using the theoretical frameworks related to the working process and managerial competencies. The results identified the greater emphasis assigned to the competencies related to the mentor, coordinator and director roles. It was, therefore, possible to construct a professional development plan that is based on competencies in the technical, ethical-political, and communicative domains, as well as the development of citizenship. The analysis of the managerial working process and the study of the competencies within the managerial environment were shown to be important, because they highlighted the professionals' need to improve, thus fulfilling personal, professional, and organizational demands.

Estudio que objetivó construir el perfil de competencias gerenciales, consensuado por enfermeros coordinadores de área. Desarrollado en hospital filantrópico de São Paulo. Fue realizado en modalidad investigación-acción. Participaron de la investigación 13 enfermeros coordinadores de área. La recolección de datos se efectuó mediante técnica de grupo focal. Para análisis de datos se utilizaron los referenciales teóricos relacionados al proceso de trabajo y competencias gerenciales. Los resultados mostraron mayor énfasis atribuido a las competencias relacionadas al papel de mentor, coordinador y director. Se pudo efectuar un planeamiento de desarrollo profesional, pautado en competencias que atiendan las dimensiones técnica, ético-política, comunicacional y de desarrollo de la población. El análisis del proceso de trabajo gerencial y el estudio de competencias en el ámbito gerencial mostraron su importancia, pues problematizaron necesidades de mejoramiento de tales profesionales, atendiendo a las demandas personales, profesionales y organizacionales.

O processo de construção do perfil de competências gerenciais para enfermeiros coordenadores de área hospitalar / The construction process of the coordinating nurses' managerial competences profile of hospitable area

As mudanças no perfil do profissional enfermeiro se configuram historicamente em função da dinâmica que ocorre no mercado de trabalho em saúde, que por sua vez, sofre influências do contexto econômico, social e cultural. A crescente competitividade das organizações de saúde demanda a busca por profissionais cada vez mais qualificados e capacitados. A necessidade de incremento de competências assistenciais, gerenciais, de pesquisa e ensino objetivando o exercício da profissão, incorpora-se progressivamente ao perfil do profissional. O presente estudo teve como objetivos: construir o perfil de competências gerenciais, consensuado por enfermeiros coordenadores de área, para subsidiar um plano de desenvolvimento gerencial dos mesmos; identificar como os enfermeiros coordenadores de área percebem o processo de trabalho gerencial que desenvolvem na instituição hospitalar; analisar a percepção dos enfermeiros coordenadores quanto às competências necessárias para atuar no processo de trabalho gerencial e identificar as convergências e divergências entre o perfil de competências traçado pelos enfermeiros coordenadores e o perfil de competências definido pela Gerência de Enfermagem e Serviço de Educação Continuada. Caracterizado como um estudo qualitativo optou-se pela modalidade da pesquisa-ação. Esse método pressupõe a pesquisa voltada para a descrição de situações concretas e para a intervenção orientada em função da resolução de problemas efetivamentedetectados na prática. O cenário da pesquisa foi um hospital filantrópico do município de São Paulo. Participaram da mesma, 13 enfermeiros coordenadores de área. A coleta de dados ocorreu por meio da técnica de grupo focal, com a realização de 04 encontros. A análise dos dados foi realizada com base nos referenciais teóricos relacionados a processo de trabalho e competências gerenciais. Os resultados permitiram construir um ) plano de desenvolvimento profissional para os enfermeiros coordenadores de área, pautado em competências que atendam as dimensões técnica, ético-política, comunicativa e de desenvolvimento da cidadania. Entre as competências essenciais, atribui-se maior ênfase às relacionadas aos papéis de mentor, coordenador e diretor. A análise do processo de trabalho gerencial dos enfermeiros coordenadores de área e o estudo das competências necessárias para um melhor desempenho no âmbito gerencial mostraram-se importantes, pois possibilitam problematizar as necessidades de aprimoramento e desenvolvimento desses profissionais de modo articulado, atendendo as demandas pessoais, profissionais e organizacionais.

The changes in the professional nurse\'s profile set up historically in function of the dynamics occur in the health labor market, which then, suffer influences of the economics, social and cultural context. The increasing competitiveness of the health organizations demands the search of professional nurses more and more qualified and enabled. The necessity of development of the assistance competences, managerial, of research and teaching objectifying the exercise of the profession, it incorporates progressively to the professional\'s profile. The present study had as objectives: build the profile of managerial competences, according to coordinating nurses of area, to subsidize a plan of managerial development of the same; identify how the coordinating nurses of area realize the process of managerial working that develop in the hospitable institution; analyze the perception of the coordinating nurses regarding the necessary competences to act in the process of managerial working and identify the convergences and divergences among competences profile traced by the coordinating nurses and the competences profile defined by the Nursing and Service Management of Continued Education. Characterized as a qualitative study, was opted by the modality of the research-action. That method presupposes the research geared to the description of concrete situations and for the intervention guided regarding the problem solving indeed detected in the practice. The scenery of theresearch was a philanthropically hospital, in São Paulo, Municipal district. They took part in, thirteen coordinating nurses of area. The data collection occurred by means of the technique of focal group, it was made four encounters. The analysis of the data was accomplished based on theoretical references related to the working process and managerial competences. The results allowed to build a plan of professional development for the coordinating nurses of area, ruled in competences that answer the technical dimensions, ethic-politics, talkative and of development of the citizenship. Among essential competences, it assigns larger emphasis to the related to mentor, coordinator and director\'s roles. The analysis of the process of managerial working of the coordinating nurses of area and study of the necessary competences for a better performance in the managerial scope, they showed important, because they enable to create a problematic the needs to refinement and development of these coordinating nurses in an articulated manner, answering the personal, professional institutional demands.