Kinetochore-microtubule interactions "in check" by Bub1, Bub3 and BubR1: the dual task of attaching and signalling

The spindle assembly checkpoint (SAC) prevents anaphase onset until all chromosomes accomplish proper bipolar attachments to the mitotic spindle and come under tension, thereby ensuring the fidelity of chromosome segregation. Despite significant advances in our understanding of SAC signalling, a clear link between checkpoint signalling and the molecular mechanisms underlying chromosome attachment to microtubules has not been established so far. However, independent studies from many groups have interestingly found that the bone-a-fide Bub1, BubR1 and Bub3 SAC proteins are themselves required for proper kinetochoremicrotubule (K-MT) interactions. Here, we review these findings and discuss the specific contribution of each of these proteins in the regulation of K-MT attachment, taking into consideration their interdependencies for kinetochore localization as well as their relationship with other proteins with a known role in chromosome attachment and congression.This work was supported by a grant from FCT—Fundação para a Ciência e Tecnologia (POCTI/BCI/42341/2001) and by CESPU, crl—Cooperativa de Ensino Superior Politécnico e Universitário

Co-silencing of human Bub3 and dynein highlights an antagonistic relationship in regulating kinetochore-microtubule attachments

We previously reported that the spindle assembly checkpoint protein Bub3 is involved in regulating kinetochore-microtubule (KT-MT) attachments. Also, Bub3 was reported to interact with the microtubule motor protein dynein. Here we examined how this interaction contributes to KT-MT attachments. Depletion of Bub3 or dynein induced misaligned chromosomes, consistent with their role in KT-MT attachments. Unexpectedly, co-silencing of both proteins partially suppressed the misalignment phenotype and restored chromosome congression. Consistent with these observations, KT-MT attachments in co-depleted cells were stable, able to drive chromosome congression, and produce inter-and intra-kinetochore stretch, indicating they are functional. We suggest that a mutual antagonism exists between Bub3 and dynein to ensure optimal KT-MT attachments. (C) 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.CESPU [02-GCQF-CICS-2011N]; FCT - Fundacao para a Ciencia e a Tecnologia [CEQUIMED-PEst-OE/SAU/UI4040/2014]; FCT [SFRH/BD/90744/2012]info:eu-repo/semantics/publishedVersio

The Emergent Power of Human Cellular vs Mouse Models in Translational Hair Research

Different animal models have been used for hair research and regeneration studies based on the similarities between animal and human skins. Primary knowledge on hair follicle (HF) biology has arisen from research using mouse models baring spontaneous or genetically engineered mutations. These studies have been crucial for the discovery of genes underlying human hair cycle control and hair loss disorders. Yet, researchers have become increasingly aware that there are distinct architectural and cellular features between the mouse and human HFs, which might limit the translation of findings in the mouse models. Thus, it is enticing to reason that the spotlight on mouse models and the unwillingness to adapt to the human archetype have been hampering the emergence of the long-awaited human hair loss cure. Here, we provide an overview of the major limitations of the mainstream mouse models for human hair loss research, and we underpin a future course of action using human cell bioengineered models and the emergent artificial intelligence.This work was supported by FEDER (Fundo Europeu de Desenvolvimento Regional) funds through COMPETE 2020 (POCI, Programa Operacional Competividade e Internacionalização) and Portugal 2020 in the framework of the project 70201-SI I&DT EMPRESAS EM COPROMOÇÃO. E.L. was supported by CEECIND/00654/2020 grant from FCT—Fundação para a Ciência e a Tecnologia, I.P

A experiência da unidade autónoma de gestão de cirurgia do Centro Hospitalar de São João E.P.E modelos de gestão intermédia hospitalar

As organizações de saúde são muito particulares devido à sua missão, aos recursos que mobilizam, aos processos que dinamizam, à produção que realizam e ainda à envolvente externa onde se inserem (Reis, 2007). Os sucessivos esforços que têm sido utilizados na reforma na saúde, sobretudo a partir de 1988, têm sido uma constante da agenda política na tentativa de aumentar a eficiência dos serviços prestados, a efetividade dos resultados e a responsabilidade dos profissionais. A empresarialização do Hospital de São João operada a partir de 2006, com a publicação do Dec.Lei 233/05 de 29 de Dezembro, tornou como imperativo estratégico a alteração profunda do modelo de gestão até então praticado. Este era caracterizado por uma forte componente administrativa, de cariz burocrática, e sob ponto de vista económico assentava em sucessivos deficits e no permanente aumento e descontrolo da despesa. Tomando como pressuposto que a única via de modificar esse padrão passava entre outras medidas pela efetivação de uma gestão descentralizada, vieram a ser criadas seis estruturas intermédias de gestão designadas por “Unidades Autónomas de Gestão”. Estas tinham como objetivo aumentar o valor em saúde, melhorar a gestão dos serviços clínicos, potenciando desse modo a qualidade e efetividade dos cuidados prestados, bem como a eficiência dos recursos utilizados. Neste sentido, o propósito deste trabalho centra-se em demonstrar que a implementação de um modelo de gestão descentralizado como é o caso da Unidade Autónoma de Gestão de Cirurgia, doravante designada por UAGC, constituiu uma opção gestionária eficaz e altamente promissora na governação clínica, desmistificando o mito da “ingovernabilidade dos hospitais centrais” como era apanágio do Hospital S. João. Cremos que a descentralização da gestão enquanto forma de reengenharia da organização interna dos hospitais constitui um importante instrumento no sentido de orientar e motivar o comportamento dos gestores (sejam eles clínicos ou não) para o cumprimento dos objetivos institucionais, através da implementação de políticas de desconcentração de poderes, competências e responsabilidades. Embora existam outros modelos de organização ao nível da gestão intermédia, na verdade, a implementação destas estruturas descentralizadas traduziu-se numa inegável mais valia organizativa e gestionária do CHSJ. como os indicadores de desempenho mais à frente tentarão demonstrar. Temos consciência que este modelo está longe de ser perfeito, e que por vezes não é corretamente entendido pelos profissionais, que o encaram como uma necessidade de cariz exclusivamente económica. Porém o caminho já percorrido pela UAGC ao longo destes 5 anos permite-nos afirmar que é possível “fazer mais” com “os mesmos recursos”, desde que exista uma clara estratégia de ação suportada em programas concretos e exequíveis, praticados num clima social participado e responsabilizante

Foxm1 modulates cell non-autonomous response in zebrafish skeletal muscle homeostasis

Foxm1 is a master regulator of the cell cycle, contributing to cell proliferation. Recent data have shown that this transcription factor also modulates gene networks associated with other cellular mechanisms, suggesting non-proliferative functions that remain largely unexplored. In this study, we used CRISPR/Cas9 to disrupt foxm1 in the zebrafish terminally differentiated fast-twitching muscle cells. foxm1 genomic disruption increased myofiber death and clearance. Interestingly, this contributed to non-autonomous satellite cell activation and proliferation. Moreover, we observed that Cas9 expression alone was strongly deleterious to muscle cells. Our report shows that foxm1 modulates a muscle non-autonomous response to myofiber death and highlights underreported toxicity to high expression of Cas9 in vivo.This study was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (ERC-2015-StG-680156-ZPR). FEDER (Fundo Europeu de Desenvolvimento Regional) funds through the COMPETE 2020—Operational Programme for Competitiveness and Internationalization (POCI), Portugal 2020 and by Portuguese funds through FCT (Fundação para a Ciência e a Tecnologia) in the framework of the project POCI-01-0145-FEDER-031120 (PTDC/BIA-CEL/31120/2017); and POCI-01-0145-FEDER-007274 i3S framework project co-funded by COMPETE 2020/PORTUGAL 2020 through FEDER. E.L. and J.B. are supported by FCT (J.B.: Grant CEECIND/03482/2018; E.L.: Grants CEECIND/00654/2020 and IF/00916/2014). F.J.F. (PD/BD/105745/2014) is a PhD fellow from FCT

Development and validation of an analytical methodology for the determination of antipsychotic drugs in hospital wastewaters by gas chromatography–tandem mass spectrometry (GC–MS/MS)

The consumption of psychiatric drugs has seen a huge increase during the last years as a consequence of the financial European crisis, and this can lead to psychological health effects causing several psychiatric diseases. These drugs have become pseudo-persistent in the environment due to their large volumes of use, and nowadays they are considered environmental emerging contaminants. Within this main group, the antipsychotic class have experienced an expressive increase in consumption, namely in Portugal, being used for the management of psychotic episodes as well as for other related behavioral symptoms and even other therapeutic indications. The present work describes the development and validation of a highly sensitive analytical method for the simultaneous determination of antipsychotic drugs in influent and effluent hospital wastewaters by GC–MS/MS. The studied compounds were levomepromazine, clozapine, chlorpromazine, haloperidol, quetiapine and ciamemazine using promazine as internal standard. Sample preparation was carried out by solid phase extraction (SPE) using mixed mode-columns (Strata XC – 200 mg) and followed by derivatization of the extracts with MSTFA (with TMCS). Chromatographic separation was achieved on a 5% phenylmethylsiloxane column. All chromatographic conditions and mass spectrometric parameters were previously optimized to enhance the maximum signal. The method was validated following internationally accepted criteria, and the studied parameters included selectivity, linearity, limits of detection (LOD) and quantification (LOQ), instrumental limits, precision and accuracy, stability and recovery. The procedure was linear for concentrations ranging from 0.1 to 10 g/L (0.02–2 g/L for haloperidol), with determination coefficients higher than 0.99 for all analytes. Intra- and inter-day precision was lower than 15% for all analytes at the studied concentrations, while accuracy remained between a ±15% interval. Recoveries ranged from 35% to 80%. Low LODs were achieved, between 2 and 10 pg/mL, allowing a reliable and accurate quantification of the analytes at trace level (low ppb). All studied parameters complied with the defined criteria and the method enabled the successful determination of antipsychotics in hospital wastewater samples

Experiência Profissionalizante na vertente de Farmácia Comunitária, Hospitalar e Investigação

O farmacêutico é, na sociedade atual, um membro importante na área da saúde. Estando ele presente num meio hospitalar, comunitário e de investigação, entre outros, todos os seus atos passam por melhorar a qualidade de vida dos indivíduos. Sendo “fiel aos princípios da honestidade, caridade e da ciência”, é de vital importância na comunidade. Um farmacêutico faz aconselhamento farmacológico, induz alterações ao estilo de vida, melhora o sistema de saúde, sempre com o ponto de partida uma visão orientada para a saúde. A presente dissertação para a obtenção do grau de mestre em Ciências Farmacêuticas é composta por três capítulos diferenciados. O Capítulo I e II diz respeito à experiência profissionalizante em Farmácia Hospitalar e Comunitária, respetivamente, seguindo-se o Capítulo III referente à componente de investigação. O Capítulo I aborda o estágio curricular em Farmácia Hospitalar realizado no Hospital Sousa Martins, na Guarda, entre os dias 3 de fevereiro e 28 de março de 2014. Através do planeamento realizado pelo Dr. Jorge Aperta, foi-me dada a oportunidade de contactar diretamente com o dia-a-dia realizado pelas farmacêuticas responsáveis pelas diferentes áreas numa farmácia de um serviço hospitalar. Através da autonomia e da boa vontade, consegui realizar as tarefas que constavam no plano de atividades proposto. Estas tarefas foram indispensáveis para a aquisição de competências necessárias ao meu futuro profissional. O capítulo em questão, não é mais que a descrição das atividades desenvolvidas no âmbito do estágio seguindo uma lista de objetivos. No Capítulo II é descrita toda a experiência e aprendizagem adquiridas durante o estágio curricular em Farmácia Comunitária, realizado na Farmácia Sena Padez, no Fundão, durante o período compreendido entre os dias 31 de março e 20 de junho de 2014. Este estágio foi realizado sob a orientação da Dra. Teresa Padez, diretora técnica da farmácia supracitada, que através da sua experiência profissional e disponibilidade, possibilitou a minha aprendizagem nas vertentes que um farmacêutico necessita para exercer o seu trabalho. Os conhecimentos adquiridos são imprescindíveis para exercer a profissão num futuro próximo. Este capítulo descreve todas as atividades realizadas no âmbito do estágio, seguindo uma lista de objetivos. O Capítulo III refere-se à componente de investigação que consiste na determinação de antipsicóticos em águas residuais. De modo a tornar isto possível foi desenvolvido um método analítico com recurso à cromatografia gasosa acoplada a um detetor de espectrometria de massa em tandem (GC-MS/MS). Os antipsicóticos em estudo foram a levomepromazina, clozapina, clorpromazina, haloperidol, quetiapina e ciamemazina, usando como padrão interno a promazina. Para o processo de preparação da amostra foi utilizada a extração em fase sólida (SPE) mediante o uso de colunas de modo misto e posterior derivatização dos extratos. O método foi validado seguindo critérios internacionais e os parâmetros estudados incluíram linearidade, seletividade, limites de deteção (LOD) e quantificação (LLOQ), precisão e exatidão. O procedimento foi linear para todos os compostos num intervalo de concentrações de (0,1) 0,5 a 10 ng/mL, exceto para o haloperidol que foi de 0,02 a 2 ng/mL, com coeficientes de determinação (R2) superiores a 0,99 para todos os analitos. Precisões intra- e interdia foram inferiores a 15 % em todos os analitos para a maioria das concentrações estudadas, enquanto a exatidão se situou dentro do intervalo de ± 15 %. As recuperações obtidas variaram entre 30 e 80 %. Todos os parâmetros estudados cumpriram os critérios definidos, pelo que o método foi considerado adequado e aplicado com sucesso na quantificação de antipsicóticos em amostras de águas residuais.The Pharmacist has an important role in healthcare sector in today’s society. Regardless of being in a hospital, community or research environment, among others, all his actions focus on improving the quality of people's lives. Being "faithful to the principles of honesty, charity and science", he is of vital importance in the community. A pharmacist does therapeutic counseling, induces changes to lifestyles and improves the health care system, always with a vision towards health. This thesis to obtain a Master’s degree in Pharmaceutical Sciences is composed of three distinct chapters. Chapters I and II are related with the professional experience in Hospital and Community Pharmacy, respectively, followed by the research component covered in chapter III. Chapter I describes the curricular internship in Hospital Pharmacy that took place in the Hospital Sousa Martins, in the city of Guarda, between the 3rd of February and the 28th of March of 2014. The working plans made by Dr. Jorge Aperta, allowed the opportunity to contact directly with the daily work of the pharmacists in charge of the diverse areas in a Hospital Pharmacy. Through autonomy and the willingness, I was able to carry out the tasks that were assigned to me, serving as basis for the acquisition of skills and experience necessary for my future as a pharmacist. This chapter is a description of the activities carried out under the internship according to a list of goals. Chapter II describes the experience, knowledge and learning acquired during the curricular internship in a Community Pharmacy, conducted at Pharmacy Sena Padez in the city of Fundão between the 31st of March and the 20th of June of 2014. This internship was carried out under the guidance of Dr. Teresa Padez, technical director of the mentioned pharmacy, whose expertise and availability, allowed me to learn the skills that a community pharmacist needs in order to perform his job. The knowledge acquired is vital for a future career in this field. This chapter describes all activities undertaken under the internship, following a list of goals. Chapter III describes the research component that consisted on the determination of antipsychotic drugs in wastewater. In order to make this possible an analytical method was developed using gas chromatography coupled to a tandem mass spectrometry detector (GC–MS/MS). The studied antipsychotics were levomepromazine, clozapine, chlorpromazine, haloperidol, quetiapine and ciamemazine using promazine as internal standard. Samples preparation were carried by solid phase extraction (SPE) using mixed mode columns and followed by derivatization of the extracts. The method was validated following international criteria and the studied parameters included linearity, selectivity, limits of detection (LOD) and quantification (LLOQ), precision and accuracy. The procedure was linear for concentrations ranging from (0.1) 0.5 to 10 ng/mL and, 0.02 to 2 ng/mL for haloperidol, with determination coefficients (R2) higher than 0.99 for all analytes. Intra- and inter-day precisions were lower than 15% for all analytes at most studied concentrations, while the accuracy remained between a ±15% interval. Recoveries ranged from 30% to 80%. All parameters studied complied with the defined criteria and therefore the method was considered adequate and successfully applied for the quantification of antipsychotics in wastewater samples

Exploring direct costs of primary hip and knee arthroplasties healthcare-associated infections: A retrospective study

Hip or knee arthroplasty healthcare-associated infections (HAI) are a public health problem that induces the increase of morbidity and mortality rates and poses an economic problem with significant impact on hospitals budget. The infection rate in primary hip and knee arthroplasties range between 1.5% and 2.5%, and is considered one of the main reasons for surgeries non-effectiveness. A retrospective study was carried out in S. João Hospital Center, EPE (CHSJ) to calculate HAI rate in primary hip and knee arthroplasties, and to analyse their direct costs, for a better understanding of their economic impact. Four hundred and eighty seven arthroplasties were studied and infection was noticed in 11 cases: 3 after hip and 8 after knee arthroplasties. Data collected from infected patients-related costs were compared with the average cost of non-infected patients (standard). An incidence rate of 2.17% for hip arthroplasties and 2.25% for knee arthroplasties was found. Results showed that patients with infection remained in hospital 7.45 times longer than uninfected patients and incurred hospital costs almost 3.8 times higher. This work shows how important is the quantification of additional HAI costs to allow hospital managers to weigh the cost/benefit ratio and better justify investments in HAI prevention and control programmes.info:eu-repo/semantics/publishedVersio

FOXM1 repression increases mitotic death upon antimitotic chemotherapy through BMF upregulation

Inhibition of spindle microtubule (MT) dynamics has been effectively used in cancer treatment. Although the mechanisms by which MT poisons elicit mitotic arrest are fairly understood, efforts are still needed towards elucidating how cancer cells respond to antimitotic drugs owing to cytotoxicity and resistance side effects. Here, we identified the critical G2/M transcription factor Forkhead box M1 (FOXM1) as a molecular determinant of cell response to antimitotics. We found FOXM1 repression to increase death in mitosis (DiM) due to upregulation of the BCL-2 modifying factor (BMF) gene involved in anoikis, an apoptotic process induced upon cell detachment from the extracellular matrix. FOXM1 binds to a BMF intronic cis-regulatory element that interacts with both the BMF and the neighbor gene BUB1B promoter regions, to oppositely regulate their expression. This mechanism ensures that cells treated with antimitotics repress BMF and avoid DiM when FOXM1 levels are high. In addition, we show that this mechanism is partly disrupted in anoikis/antimitotics-resistant tumor cells, with resistance correlating with lower BMF expression but in a FOXM1-independent manner. These findings provide a stratification biomarker for antimitotic chemotherapy response.This work was supported by: FEDER (Fundo Europeu de Desenvolvimento Regional) funds through the COMPETE 2020 - Operational Programme for Competitiveness and Internationalization (POCI), Portugal 2020 and by Portuguese funds through FCT (Fundação para a Ciência e a Tecnologia) in the framework of the project POCI-01-0145-FEDER-031120 (PTDC/BIA-CEL/31120/ 2017); and POCI-01-0145-FEDER-007274 i3S framework project co-funded by COMPETE 2020/ PORTUGAL 2020 through FEDER. S.V. and F.F. were supported by FCT fellowships SFRH/BD/125017/2016 and PD/BD/105745/2014. E.L. was supported by an FCT Investigator Grant (IF/00916/2014). U.B-D. and G.L. were supported by the Azrieli Faculty Fellowship (to U.D.-D.) and the DoD CDMRP Career Development Award (CA191138 to U.B.-D.). J.B. was supported by an FCT Investigator Grant (CEECIND/03482/2018) and the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (ERC-2015-StG-680156-ZPR)

Candidate alzheimer’s disease biomarker mir-483-5p lowers tau phosphorylation by direct erk1/2 repression

MicroRNAs have been demonstrated as key regulators of gene expression in the etiology of a range of diseases including Alzheimer’s disease (AD). Recently, we identified miR-483-5p as the most upregulated miRNA amongst a panel of miRNAs in blood plasma specific to prodromal, early-stage Alzheimer’s disease patients. Here, we investigated the functional role of miR-483-5p in AD pathology. Using TargetScan and miRTarBase, we identified the microtubule-associated protein MAPT, often referred to as TAU, and the extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), known to phosphorylate TAU, as predicted direct targets of miR-483-5p. Employing several functional assays, we found that miR-483-5p regulates ERK1 and ERK2 at both mRNA and protein levels, resulting in lower levels of phosphorylated forms of both kinases. Moreover, miR-483-5p-mediated repression of ERK1/2 resulted in reduced phosphorylation of TAU protein at epitopes associated with TAU neurofibrillary pathology in AD. These results indicate that upregulation of miR-483-5p can decrease phosphorylation of TAU via ERK pathway, representing a compensatory neuroprotective mechanism in AD pathology. This miR-483-5p/ERK1/TAU axis thus represents a novel target for intervention in AD.This work has been supported by the Polish National Science Centre grant OPUS 2018/29 /B/NZ7/02757, by the EU Horizon 2020 FETOPEN grant, agreement no 737390 (ArrestAD), and by the funding from the Polish Ministry of Science and Higher Education within 2016-2020 funds for the implementation of international projects (agreement no 3548/H2020/COFUND/2016/2). A.F. research internship in Portugal was supported by Erasmus fellowship