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1.
Sante Publique ; 33(4): 517-526, 2021.
Artigo em Francês | MEDLINE | ID: mdl-35724134

RESUMO

INTRODUCTION: Unscheduled care (UC) is a current issue, particularly in the context of COVID-19 outbreak. UC is at the center of health policies, mainly in terms of the negative impact they have on hospital emergency services. This occurs in a context of imbalance between increasing health needs and changing healthcare offer. AIM: The aim of this study was to describe the organization of general practitioners in UC management in South-West France. METHOD: Our research was based on the combination of a cross-sectional descriptive study by self-questionnaire, and a collection of qualitative data via semi-structured interviews. RESULTS: 79% of practitioners delegated their hotline to a "medical secretary"; 67% had a specific organization to manage UC, especially by means of dedicated slots; 88% proposed a structured management of UC. The secretary appeared to be a necessary link for managing UC requests, mainly through the role of sorting, regulating and scheduling UC appointments. Group work was highlighted as a means to distribute the response to UC requests more fairly, to carry out unscheduled home visits and to prevent healthcare refusal. Nevertheless, this group management seemed difficult to implement outside of medical centers, especially at the level of health territory. CONCLUSION: the majority of GPs had implemented structured and shared management of UC, but they needed help and support to organize themselves across a territory, for example within the framework of "territorial health professional communities".


Assuntos
COVID-19 , Medicina Geral , Clínicos Gerais , COVID-19/epidemiologia , Estudos Transversais , França , Humanos
3.
Antiviral Res ; 164: 162-175, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30825471

RESUMO

Maturation of human immunodeficiency virus type 1 (HIV-1) particles is a key step for viral infectivity. This process can be blocked using maturation inhibitors (MIs) that affect the cleavage of the capsid-spacer peptide 1 (CA-SP1) junction. Here, we investigated the mechanisms underlying the activity of EP-39, a bevirimat (BVM) derivative with better hydrosolubility. To this aim, we selected in vitro EP-39- and BVM-resistant mutants. We found that EP-39-resistant viruses have four mutations within the CA domain (CA-A194T, CA-T200N, CA-V230I, and CA-V230A) and one in the first residue of SP1 (SP1-A1V). We also identified six mutations that confer BVM resistance (CA-A194T, CA-L231F, CA-L231M, SP1-A1V, SP1-S5N and SP1-V7A). To characterize the EP-39 and BVM-resistant mutants, we studied EP-39 effects on mutant virus replication and performed a biochemical analysis with both MIs. We observed common and distinct characteristics, suggesting that, although EP-39 and BVM share the same chemical skeleton, they could interact in a different way with the Gag polyprotein precursor (Pr55Gag). Using an in silico approach, we observed that EP-39 and BVM present different predicted positions on the hexameric crystal structure of the CACTD-SP1 Gag fragment. To clearly understand the relationship between assembly and maturation, we investigated the impact of all identified mutations on virus assembly by expressing Pr55Gag mutants. Finally, using NMR, we have shown that the interaction of EP-39 with a peptide carrying the SP1-A1V mutation (CA-SP1(A1V)-NC) is almost suppressed in comparison with the wild type peptide. These results suggest that EP-39 and BVM could interact differently with the Pr55Gag lattice and that the mutation of the first SP1 residue induces a loss of interaction between Pr55Gag and EP-39.


Assuntos
Fármacos Anti-HIV/farmacologia , Farmacorresistência Viral/genética , HIV-1/efeitos dos fármacos , HIV-1/genética , Succinatos/química , Succinatos/farmacologia , Triterpenos/química , Triterpenos/farmacologia , HIV-1/fisiologia , Humanos , Células Jurkat , Simulação de Acoplamento Molecular , Mutação , Montagem de Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
4.
BMC Med Educ ; 18(1): 254, 2018 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-30413196

RESUMO

BACKGROUND: There is currently an absence of valid and relevant instruments to evaluate how Evidence-based Practice (EBP) training improves, beyond knowledge, physicians' skills. Our aim was to develop and test a tool to assess physicians' EBP skills. METHODS: The tool we developed includes four parts to assess the necessary skills for applying EBP steps: clinical question formulation; literature search; critical appraisal of literature; synthesis and decision making. We evaluated content and face validity, then tested applicability of the tool and whether external observers could reliably use it to assess acquired skills. We estimated Kappa coefficients to measure concordance between raters. RESULTS: Twelve general practice (GP) residents and eleven GP teachers from the University of Bordeaux, France, were asked to: formulate four clinical questions (diagnostic, prognosis, treatment, and aetiology) from a proposed clinical vignette, find articles or guidelines to answer four relevant provided questions, analyse an original article answering one of these questions, synthesize knowledge from provided synopses, and decide about the four clinical questions. Concordance between two external raters was excellent for their assessment of participants' appraisal of the significance of article results (K = 0.83), and good for assessment of the formulation of a diagnostic question (K = 0.76), PubMed/Medline (K = 0.71) or guideline (K = 0.67) search, and of appraisal of methodological validity of articles (K = 0.68). CONCLUSIONS: Our tool allows an in-depth analysis of EBP skills, thus could supplement existing instruments focused on knowledge or specific EBP step. The actual usefulness of such tools to improve care and population health remains to be evaluated.


Assuntos
Competência Clínica/normas , Prática Clínica Baseada em Evidências , Clínicos Gerais , Avaliação Educacional/métodos , Prática Clínica Baseada em Evidências/educação , França , Clínicos Gerais/educação , Clínicos Gerais/normas , Humanos , Projetos Piloto , Desenvolvimento de Programas , Psicometria/instrumentação , Reprodutibilidade dos Testes , Literatura de Revisão como Assunto
5.
BMC Med Educ ; 16(1): 231, 2016 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-27585603

RESUMO

BACKGROUND: Young French postgraduates in general practice increasingly prefer salaried practice to private practice in spite of the financial incentives offered by the French government or local communities to encourage the latter. This study aimed to explore the determinants of choice between private or salaried practice among young general practitioners. METHODS: A qualitative study was conducted in the South West of France. Semi-structured interviews of young general practitioners were audio-recorded until data saturation. Recordings were transcribed and then analyzed according to Grounded Theory by three researchers working independently. RESULTS: Sixteen general practitioners participated in this study. For salaried and private doctors, the main factors governing their choice were occupational factors: working conditions, need of varied scope of practice, quality of the doctor-patient relationship or career flexibility. Other factors such as postgraduate training, having worked as a locum or self-interest were also determining. Young general practitioners all expected a work-life balance. The fee-for-service scheme or home visits may have discouraged young general practitioners from choosing private practice. CONCLUSIONS: National health policies should increase the attractiveness of ambulatory general practice by promoting the diversification of modes of remuneration and encouraging the organization of group exercises in multidisciplinary medical homes and community health centers.


Assuntos
Atitude do Pessoal de Saúde , Escolha da Profissão , Medicina Geral , Clínicos Gerais/psicologia , Prática Privada , França , Medicina Geral/economia , Medicina Geral/estatística & dados numéricos , Humanos , Motivação , Relações Médico-Paciente , Prática Privada/economia , Prática Privada/estatística & dados numéricos , Pesquisa Qualitativa
6.
BMJ Open ; 6(6): e011488, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27311913

RESUMO

INTRODUCTION: While the relationship between electronic cigarette use and smoking has often been studied, the association between electronic cigarette use and socioeconomic factors has received less attention. This is a study protocol aiming to describe the relationship between the consumption of psychoactive products (in particular: smoking) or some socioeconomic factors and the evolution of the use of electronic cigarette in primary healthcare over 1 year. METHODS AND ANALYSIS: Electronic cigarette, Tobacco, Alcohol and Cannabis (e-TAC) is a prospective multisite cohort study, including 473 patients at baseline and carrying out in general practices in the Aquitaine area (France). The volunteer patients participated in the study regardless of their initial reason for consultation. They filled out a self-administered questionnaire at baseline and will also do so after 12 months by phone, email or letter. The study will focus on the factors that explain the experimentation with or the current use of the electronic cigarette, as well as factors associated with their evolutions over time using multivariate logistic regression modelling or Cox regression modelling. ETHICS AND DISSEMINATION: This study received ethical approval from the University of Bordeaux Committee for the protection of persons. It was also approved by the National Commission for Data Processing and Freedoms. Findings will be submitted for publication in peer-reviewed journals and we will disseminate them by presentations at national or international conferences. TRIAL REGISTRATION NUMBER: RCB: 2015-A00778-41; Pre-results.


Assuntos
Sistemas Eletrônicos de Liberação de Nicotina , Motivação , Fumar/epidemiologia , Fatores Socioeconômicos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , França/epidemiologia , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Atenção Primária à Saúde , Modelos de Riscos Proporcionais , Estudos Prospectivos , Projetos de Pesquisa , Abandono do Hábito de Fumar/métodos , Inquéritos e Questionários , Adulto Jovem
7.
Presse Med ; 44(12 Pt 1): 1219-25, 2015 Dec.
Artigo em Francês | MEDLINE | ID: mdl-26585744

RESUMO

CONTEXT: Statins in primary prevention before 75 years old reduce cardiovascular events from 20 to 30% and mortality from 10% with acceptable side effects. We investigated whether these results persisted for patients aged 75 and older taking statin. METHOD: Methodic review of large randomized clinical trials and meta-analyzes that included patients 75 years and older treated with statins in primary prevention. RESULTS: Since the 1990s, a score of randomized controlled trials studying statins versus placebo in primary prevention were published and studied in meta-analyses. Exclusion criteria, including persons older than 70 years, are often restrictive. The impact on all-cause mortality in the four main studies and meta-analyses in over 75 years has not been demonstrated. On the other hand, a recent meta-analyses of observational studies including subjects between 70 and 89 years treated with statins found that low total cholesterol was associated with a moderate decrease in cardiovascular mortality, with no decrease in all-cause mortality. Moreover, in a common context of comorbidities in this age group, statins may be responsible for many adverse effects, drug interactions and impaired quality of life. CONCLUSION: Given the lack of formal evidence of effectiveness in terms of all-cause mortality and a high level of adverse effects, the benefit/risk of primary prevention with statins is not established in the elderly. The economic weight of statin prescriptions and their possible impact on quality of life justify an economic analysis of discontinuing statin therapy for people 75 years and older.


Assuntos
Doenças Cardiovasculares/prevenção & controle , Inibidores de Hidroximetilglutaril-CoA Redutases/efeitos adversos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Prevenção Primária , Idoso , Doenças Cardiovasculares/mortalidade , Causas de Morte , Interações Medicamentosas , Humanos , Ensaios Clínicos Controlados Aleatórios como Assunto , Medição de Risco , Análise de Sobrevida
8.
Retrovirology ; 10: 157, 2013 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-24344931

RESUMO

BACKGROUND: Host cell proteins, including cellular kinases, are embarked into intact HIV-1 particles. We have previously shown that the Cα catalytic subunit of cAMP-dependent protein kinase is packaged within HIV-1 virions as an enzymatically active form able to phosphorylate a synthetic substrate in vitro (Cartier et al. J. Biol. Chem. 278:35211 (2003)). The present study was conceived to investigate the contribution of HIV-1-associated PKA to the retroviral life cycle. RESULTS: NL4.3 viruses were produced from cells cultured in the presence of PKA inhibitors H89 (H89-NL4.3) or Myr-PKI (PKI-NL4.3) and analyzed for viral replication. Despite being mature and normally assembled, and containing expected levels of genomic RNA and RT enzymatic activity, such viruses showed poor infectivity. Indeed, infection generated reduced amounts of strong-strop minus strand DNA, while incoming RNA levels in target cells were unaffected. Decreased cDNA synthesis was also evidenced in intact H89-NL4.3 and PKI-NL4.3 cell free particles using endogenous reverse transcription (ERT) experiments. Moreover, similar defects were reproduced when wild type NL4.3 particles preincubated with PKA inhibitors were subjected to ERT reactions. CONCLUSIONS: Altogether, our results indicate that HIV-1-associated PKA is required for early reverse transcription of the retroviral genome both in cell free intact viruses and in target cells. Accordingly, virus-associated PKA behaves as a cofactor of an intraviral process required for optimal reverse transcription and for early post-entry events.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Transcrição Reversa , Integração Viral , Linhagem Celular , Humanos
9.
Presse Med ; 42(11): e400-8, 2013 Nov.
Artigo em Francês | MEDLINE | ID: mdl-23958209

RESUMO

INTRODUCTION: To screen and to follow-up the patients with high cardiovascular risk in general practice may amplify the decrease of the cardiovascular morbi-mortality observed since a few years. The objective of this study is to identify the patients with high cardiovascular risk and to describe the management of these patients by general practitioners in Aquitaine. METHODS: Transversal study of a sample of patients from 18 to 70 years old with high cardiovascular risk (combining at least 3 factors), included by voluntary general practitioners (GP). RESULTS: Forty-seven GP included 102 patients, presenting on average 3.7 risk factors among which 2.6 modifiable. The target values were reached for 59 % of patients with high blood pressure, 56 % of patients with diabetes and 53 % of patients with high cholesterol level. The analysis of care pathways identified the cardiologist as the privileged interlocutor. The doctors thought that seven patients out of 10 could change their risk behaviors. For the patients, the scale of declared importance to change was 6.6 on 10 for tobacco, 6.0 for food habits and 6.2 for physical activity. The confidence in their capacity to change was 3.8 on 10 for the tobacco, 5.2 for the food habits and 4.7 for the physical activity. DISCUSSION: Although doctors' sample is not representative, these results give an original overview of the management of patients with high cardiovascular risk and their care pathways. Medical treatments were globally in accordance with guidelines. The difficulty to change risk behaviors illustrates the necessity of patient therapeutic education.


Assuntos
Doenças Cardiovasculares/prevenção & controle , Comportamento de Redução do Risco , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticolesterolemiantes/uso terapêutico , Anti-Hipertensivos/uso terapêutico , Doenças Cardiovasculares/epidemiologia , Doenças Cardiovasculares/etiologia , Procedimentos Clínicos , Estudos Transversais , Diabetes Mellitus/terapia , Exercício Físico , Comportamento Alimentar , Feminino , França/epidemiologia , Medicina Geral , Pesquisas sobre Atenção à Saúde/métodos , Humanos , Hipercolesterolemia/terapia , Hipertensão/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Estilo de Vida , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Fumar/efeitos adversos , Fumar/epidemiologia
10.
Eur J Med Chem ; 62: 453-65, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23399723

RESUMO

Bevirimat (2), the first-in-class HIV-1 maturation inhibitor, shows a low efficacy due essentially to the natural polymorphism of its target, the CA-SP1 junction. Moreover, its low hydrosolubility makes it difficult to study its interaction with the CA-SP1 junction. We have synthesized new derivatives of bevirimat by adding different hydrophilic substituents at the C-28 position to improve their hydrosolubility and perform the structural study of a complex by NMR. Synthesis of the new derivatives, the effect of substituents at the C-28 position and their hydrosolubility are discussed. The ability of these molecules to inhibit viral infection and their cytotoxicity is assessed. Compared to the well-known bevirimat (2), one of our compounds (16) shows a higher hydrosolubility associated with a 2.5 fold increase in activity, a higher selectivity index and a better antiviral profile. Moreover, for the first time a direct interaction between a derivative of bevirimat (16) and the domain CA-SP1-NC is shown by NMR. Information from this study should allow us to decipher the mechanism by which bevirimat inhibits HIV-1 maturation and how the natural polymorphism of the spacer peptide SP1 triggers resistance to inhibitors.


Assuntos
Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , Succinatos/farmacologia , Triterpenos/farmacologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Sítios de Ligação/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Testes de Sensibilidade Microbiana , Relação Estrutura-Atividade , Succinatos/síntese química , Succinatos/química , Triterpenos/síntese química , Triterpenos/química
12.
Infect Genet Evol ; 12(6): 1275-81, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22386853

RESUMO

BACKGROUND: The chikungunya virus (CHIKV) recently caused explosive outbreaks in Indian Ocean islands and India. During these episodes, the virus was mainly spread to humans through the bite of the mosquito Aedes albopictus. Concomitantly to the description of symptoms of an unexpected severity in infants and elderly patients, a viral genome microevolution has been highlighted, in particular consisting in the acquisition of an A226V mutation in the gene encoding envelope glycoprotein E1, which was later found to confer an increased fitness for A. albopictus. We previously decrypted the entry pathway used by CHIKV to infect human epithelial cells and showed that these mechanisms are modulated by the E1-A226V mutation. In this report we investigated the conditions for CHIKV entry into mosquito cells and we assessed the consequence of E1 gene mutation on these parameters. PRINCIPAL FINDINGS: Our main findings indicate that CHIKV infection of A. albopictus cell lines is sensitive to Bafilomycin A1 and chloroquine and to membrane cholesterol depletion. The E1-226V mutated LR-OPY1 isolate collected during the 2005 outbreak in La Réunion replicated more efficiently than the 37997 African reference strain in C6/36 cells. Moreover, the LR-OPY1 strain displayed greater membrane cholesterol dependence and was more sensitive to inhibition of endosomal pH acidification. Finally, using electron microscopy, we imaged CHIKV entry into C6/36 cells. CONCLUSIONS: Our data support that CHIKV is endocyted into A. albopictus cells and requires membrane cholesterol as well as a low-pH environment for entry. These features are modulated in some extent by the A226V mutation in the E1 gene of the LR-OPY1 isolate. Altogether, our data provide information regarding the pathways used by CHIKV to infect A. albopictus cells.


Assuntos
Aedes/virologia , Vírus Chikungunya/fisiologia , Insetos Vetores/virologia , Internalização do Vírus , Animais , Linhagem Celular , Membrana Celular , Vírus Chikungunya/genética , Vírus Chikungunya/metabolismo , Colesterol/metabolismo , Endocitose/fisiologia , Interações Hospedeiro-Patógeno , Concentração de Íons de Hidrogênio , Macrolídeos/farmacologia , Mutação , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo
13.
Retrovirology ; 8: 74, 2011 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-21929758

RESUMO

BACKGROUND: Retroviral gene expression generally depends on a full-length transcript that initiates in the 5' LTR, which is either left unspliced or alternatively spliced. We and others have demonstrated the existence of antisense transcription initiating in the 3' LTR in human lymphotropic retroviruses, including HTLV-1, HTLV-2, and HIV-1. Such transcripts have been postulated to encode antisense proteins important for the establishment of viral infections. The antisense strand of the HIV-1 proviral DNA contains an ORF termed asp, coding for a highly hydrophobic protein. However, although anti-ASP antibodies have been described to be present in HIV-1-infected patients, its in vivo expression requires further support. The objective of this present study was to clearly demonstrate that ASP is effectively expressed in infected T cells and to provide a better characterization of its subcellular localization. RESULTS: We first investigated the subcellular localization of ASP by transfecting Jurkat T cells with vectors expressing ASP tagged with the Flag epitope to its N-terminus. Using immunofluorescence microscopy, we found that ASP localized to the plasma membrane in transfected Jurkat T cells, but with different staining patterns. In addition to an entire distribution to the plasma membrane, ASP showed an asymmetric localization and could also be detected in membrane connections between two cells. We then infected Jurkat T cells with NL4.3 virus coding for ASP tagged with the Flag epitope at its C-terminal end. By this approach, we were capable of showing that ASP is effectively expressed from the HIV-1 3' LTR in infected T cells, with an asymmetric localization of the viral protein at the plasma membrane. CONCLUSION: These results demonstrate for the first time that ASP can be detected when expressed from full-length HIV-1 proviral DNA and that its localization is consistent with Jurkat T cells overexpressing ASP.


Assuntos
Membrana Celular/virologia , Regulação Viral da Expressão Gênica , Infecções por HIV/virologia , HIV-1/genética , RNA Antissenso/genética , RNA Viral/genética , Linfócitos T/virologia , Proteínas Virais/genética , Linhagem Celular , Membrana Celular/metabolismo , Infecções por HIV/metabolismo , HIV-1/metabolismo , Humanos , Mutação , Transporte Proteico , RNA Antissenso/metabolismo , RNA Viral/metabolismo , Linfócitos T/metabolismo , Proteínas Virais/metabolismo
14.
Virol J ; 8: 432, 2011 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-21902836

RESUMO

BACKGROUND: Chikungunya Virus (ChikV) surprised by a massive re-emerging outbreak in Indian Ocean in 2006, reaching Europe in 2007 and exhibited exceptional severe physiopathology in infants and elderly patients. In this context, it is important to analyze the innate immune host responses triggered against ChikV. Autophagy has been shown to be an important component of the innate immune response and is involved in host defense elimination of different pathogens. However, the autophagic process was recently observed to be hijacked by virus for their own replication. Here we provide the first evidence that hallmarks of autophagy are specifically found in HEK.293 infected cells and are involved in ChikV replication. METHODS: To test the capacity of ChikV to mobilize the autophagic machinery, we performed fluorescence microscopy experiments on HEK.GFP.LC3 stable cells, and followed the LC3 distribution during the time course of ChikV infection. To confirm this, we performed electron microscopy on HEK.293 infected cells. To test the effect of ChikV-induced-autophagy on viral replication, we blocked the autophagic process, either by pharmacological (3-MA) or genetic inhibition (siRNA against the transcript of Beclin 1, an autophagic protein), and analyzed the percentage of infected cells and the viral RNA load released in the supernatant. Moreover, the effect of induction of autophagy by Rapamycin on viral replication was tested. RESULTS: The increasing number of GFP-LC3 positive cells with a punctate staining together with the enhanced number of GFP-LC3 dots per cell showed that ChikV triggered an autophagic process in HEK.293 infected cells. Those results were confirmed by electron microscopy analysis since numerous membrane-bound vacuoles characteristic of autophagosomes were observed in infected cells. Moreover, we found that inhibition of autophagy, either by biochemical reagent and RNA interference, dramatically decreases ChikV replication. CONCLUSIONS: Taken together, our results suggest that autophagy may play a promoting role in ChikV replication. Investigating in details the relationship between autophagy and viral replication will greatly improve our knowledge of the pathogenesis of ChikV and provide insight for the design of candidate antiviral therapeutics.


Assuntos
Infecções por Alphavirus/metabolismo , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Autofagia/efeitos dos fármacos , Vírus Chikungunya/efeitos dos fármacos , Proteínas de Membrana/antagonistas & inibidores , Fagossomos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Adenina/análogos & derivados , Adenina/farmacologia , Infecções por Alphavirus/epidemiologia , Infecções por Alphavirus/virologia , Antimetabólitos/farmacologia , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína Beclina-1 , Febre de Chikungunya , Vírus Chikungunya/genética , Vírus Chikungunya/metabolismo , Surtos de Doenças , Europa (Continente) , Inativação Gênica/efeitos dos fármacos , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Imunossupressores/farmacologia , Oceano Índico , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Microscopia de Fluorescência , Fagossomos/virologia , Reação em Cadeia da Polimerase , RNA Interferente Pequeno/farmacologia , Sirolimo/farmacologia , Replicação Viral/fisiologia
15.
PLoS One ; 5(7): e11479, 2010 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-20628602

RESUMO

BACKGROUND: The replicative cycle of chikungunya virus (CHIKV), an alphavirus that recently re-emerged in India and in Indian Ocean area, remains mostly unknown. The aim of the present study was to investigate the intracellular trafficking pathway(s) hijacked by CHIKV to enter mammalian cells. METHODOLOGY/PRINCIPAL FINDINGS: Entry pathways were investigated using a variety of pharmacological inhibitors or overexpression of dominant negative forms of proteins perturbating cellular endocytosis. We found that CHIKV infection of HEK293T mammalian cells is independent of clathrin heavy chain and- dependent of functional Eps15, and requires integrity of Rab5-, but not Rab7-positive endosomal compartment. Cytoskeleton integrity is crucial as cytochalasin D and nocodazole significantly reduced infection of the cells. Finally, both methyl beta-cyclodextrin and lysomotropic agents impaired CHIKV infection, supporting that a cholesterol-, pH-dependent step is required to achieve productive infection. Interestingly, differential sensitivity to lysomotropic agents was observed between the prototypal 37997 African strain of CHIKV and the LR-OPY1 virus isolated from the recent outbreak in Reunion Island. CONCLUSIONS: Together our data indicate that CHIKV entry in its target cells is essentially mediated by clathrin-independent, Eps15-dependent endocytosis. Despite that this property is shared by the prototypal 37997 African strain of CHIKV and the LR-OPY1 virus isolated from the recent outbreak in La Réunion Island, differential sensitivity to lysomotropic agents may support that the LR-OPY1 strain has acquired specific entry mechanisms.


Assuntos
Vírus Chikungunya/metabolismo , Clatrina/metabolismo , Endocitose/fisiologia , Endossomos/metabolismo , Linhagem Celular , Colesterol , Citoesqueleto/genética , Citoesqueleto/metabolismo , Endocitose/efeitos dos fármacos , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Microscopia Confocal , Interferência de RNA , beta-Ciclodextrinas/farmacologia , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7
16.
Proteins ; 78(9): 2144-56, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20455269

RESUMO

Capsid protein (CA) is the major component of the human immunodeficiency virus type 1 (HIV-1) core. Three major phosphorylation sites have been identified at positions S(109), S(149) and S(178) in the amino-acid sequence of CA. Here, we investigated the possible consequences of phosphorylation at these sites on the CA hexamer organization and plasticity using in silico approaches. The biological relevance of molecular modeling was then evaluated by analyzing the in vitro assembly properties of bacterially expressed CA bearing S(109)D, S(149)D, or S(178)D substitutions that mimic constitutive phosphorylation at these sites. We found that a constitutive negative charge at position 109 or 149 impaired the capacity of mature CA to assemble in vitro. In vivo, HIV-1 mutants bearing the corresponding mutation showed dramatic alterations of core morphology. At the level of CA hexamer, S(149) phosphorylation generates inter-monomer repulsions, while phosphorylation at position 109 resulted in cleavage of important bonds required for preserving the stability of the edifice. Addition of a negative charge at position 178 allowed efficient assembly of CA into core-like structures in vitro and in vivo and significantly increased CA hexamer stability when modeled in silico. All mutant viruses studied lacked infectivity since they were unable to produce proviral DNA. Altogether our data indicate that negative charges, that mimic phosphorylation, modulate assembling capacity of CA and affect structural properties of CA hexamers and of HIV-1 cores. In the context of the assembled core, phosphorylation at these sites may be considered as an event interfering with core organization and HIV-1 replicative cycle.


Assuntos
Proteínas do Capsídeo/química , HIV-1/genética , Simulação de Dinâmica Molecular , Multimerização Proteica , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular Tumoral , Humanos , Mutação , Fenótipo , Fosforilação , Estabilidade Proteica , Eletricidade Estática , Vírion , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo
17.
Int J Syst Evol Microbiol ; 60(Pt 6): 1271-1279, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19667386

RESUMO

Three strains of a hitherto unknown, Gram-negative, tiny, anaerobic coccus were collected from human clinical samples originating from skin and soft tissues. The three isolates displayed at least 99.9 % identity in their 16S rRNA gene sequences and more than 99.8 % identity in their dnaK gene sequences. The isolates were affiliated to the family Veillonellaceae, the coccobacillus Dialister micraerophilus being the most closely related species, but there was no more than 91.1 % identity in the 16S rRNA gene sequence between this species and the three isolates. Phylogeny based on the 16S rRNA gene confirmed that the three strains represent a novel and robust lineage within the current family Veillonellaceae. A similar genomic structure was demonstrated for the three isolates by PFGE-based analysis. Morphology and metabolic end products, as well as genotypic and phylogenetic data supported the proposal of the novel genus Negativicoccus gen. nov., with the novel species Negativicoccus succinicivorans sp. nov. [type strain ADV 07/08/06-B-1388(T) (=AIP 149.07(T)=CIP 109806(T)=DSM 21255(T)=CCUG 56017(T)) as type species]. Phylogenetic analyses based on the 16S rRNA gene sequences of members of the phylum Firmicutes and other phyla indicated that the family Veillonellaceae forms a robust lineage clearly separated from those of the classes 'Bacilli', 'Clostridia', Thermolithobacteria and 'Erysipelotrichi' in the phylum Firmicutes. Therefore, we propose that this family is a class-level taxon in the phylum Firmicutes, for which the name Negativicutes classis nov. is proposed, based on the Gram-negative type of cell wall of its members, with the type order Selenomonadales ord. nov. In this order, a novel family, Acidaminococcaceae fam. nov., is proposed and description of the family Veillonellaceae is emended.


Assuntos
Veillonellaceae/genética , Composição de Bases , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/genética , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/genética , Humanos , Dados de Sequência Molecular , Filogenia , RNA Ribossômico 16S/genética , Veillonellaceae/classificação , Veillonellaceae/isolamento & purificação
18.
Virology ; 393(2): 183-97, 2009 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-19732931

RESUMO

Arboviruses (or arthropod-borne viruses), represent a threat for the new century. The 2005-2006 year unprecedented epidemics of chikungunya virus (CHIKV) in the French Reunion Island in the Indian Ocean, followed by several outbreaks in other parts of the world such as India, have attracted the attention of clinicians, scientists, and state authorities about the risks linked to this re-emerging mosquito-borne virus. CHIKV, which belongs to the Alphaviruses genus, was not previously regarded as a highly pathogenic arbovirus. However, this opinion was challenged by the death of several CHIKV-infected persons in Reunion Island. The epidemic episode began in December 2005 and four months later the seroprevalence survey report indicated that 236,000 persons, more than 30% of Reunion Island population, had been infected with CHIKV, among which 0.4-0.5% of cases were fatal. Since the epidemic peak, the infection case number has continued to increase to almost 40% of the population, with a total of more than 250 fatalities. Although information available on CHIKV is growing quite rapidly, we are still far from understanding the strategies required for the ecologic success of this virus, virus replication, its interactions with its vertebrate hosts and arthropod vectors, and its genetic evolution. In this paper, we summarize the current knowledge of CHIKV genomic organization, cell tropism, and the virus replication cycle, and evaluate the possibility to predict its future evolution. Such understanding may be applied in order to anticipate future epidemics and reduce the incidence by development and application of, for example, vaccination and antiviral therapy.


Assuntos
Vírus Chikungunya/fisiologia , Genoma Viral , Replicação Viral , Infecções por Alphavirus/epidemiologia , Infecções por Alphavirus/terapia , Arbovírus/classificação , Arbovírus/genética , Arbovírus/fisiologia , Vírus Chikungunya/classificação , Vírus Chikungunya/genética , Clatrina/metabolismo , Surtos de Doenças , Endocitose , Interações Hospedeiro-Patógeno , Humanos , Filogenia , Reunião/epidemiologia , Estudos Soroepidemiológicos
19.
Can Fam Physician ; 55(8): e14-20, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19675252

RESUMO

OBJECTIVE: To explore the conceptions that family medicine residents from 3 countries have of the roles and responsibilities of family physicians in order to gain a better understanding of challenges that might transcend the specific contexts of different health care systems. DESIGN: Qualitative study using focus groups. SETTING: Resident training programs in France, Belgium, and Canada. PARTICIPANTS: A total of 57 residents in the last year of training. METHOD: Ten focus groups were conducted in 3 countries: 2 in France, 3 in Belgium, and 5 in Canada. All focus groups were held in different cities, with residents registered in different universities in France and Canada and with residents from the same university in Belgium. The study was informed by Abbott's conceptual framework on the system of professions. Each 90-minute focus group was moderated by the same researchers. The transcripts were analyzed according to the immersion-crystallization method. MAIN FINDINGS: Respondents shared common conceptions of the family physician's role: continuity of care and patient advocacy were seen as the foundations of the discipline. Respondents also shared a sense of discomfort about how accessible they were expected to be for patients and about the scope of family practice. They saw family medicine as flexible and reported that they strove for balance between their professional and personal life goals. All respondents strongly believed that their profession was undervalued by the medical schools where they trained. CONCLUSION: This exploratory study suggests that there are more similarities than differences in the understanding that future family physicians from different countries have of their discipline and of their careers. We observed a tension between a desire to develop a "new general practice" and the more traditional vision of the discipline. The culture in academic settings appears to contribute to the persistent low appeal of being a primary care physician.


Assuntos
Medicina de Família e Comunidade/educação , Internato e Residência , Papel do Médico , Médicos de Família/educação , Autoimagem , Adulto , Atitude do Pessoal de Saúde , Bélgica , Canadá , Escolha da Profissão , Feminino , Grupos Focais , França , Humanos , Masculino , Relações Médico-Paciente , Médicos de Família/psicologia , Pesquisa Qualitativa
20.
Med Teach ; 31(1): 39-44, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18825567

RESUMO

INTRODUCTION: Retention of general practitioners (GPs) is crucial to ensure appropriate primary care. However, some recently qualified GPs feel unprepared for practice, which may lead them to leave the profession or restrict their scope of practice. The development of self-efficacy beliefs during vocational training may be an important factor in this phenomenon. METHODS: Five focus groups with a total of 28 GP trainees and recent graduates were conducted in Belgium and France. Initial analysis using the immersion-crystallisation method was followed by analysis using Bandura's self-efficacy framework. RESULTS: Participants described beginning their training with low self-efficacy beliefs. Most participants described how they overcame stressful situations. Some, however, seemed to be developing avoidance strategies. Successfully resolving patient problems, sharing experiences with peers and receiving positive feedback from supervisors, colleagues and patients were conducive to the development of positive self-efficacy beliefs. DISCUSSION: Although low self-efficacy beliefs are natural at the beginning of training, participants seemed to develop in two ways, either overcoming their fears or avoiding them. Identifying the pattern of trainees' responses to allow tailoring of interventions should be investigated by those who run training programs. Interventions could include reassurance, peer interaction and an appropriate degree of autonomy.


Assuntos
Atitude do Pessoal de Saúde , Medicina de Família e Comunidade/educação , Relações Interprofissionais , Médicos de Família/educação , Desenvolvimento de Programas/métodos , Autoeficácia , Adulto , Esgotamento Profissional/prevenção & controle , Escolha da Profissão , Competência Clínica/normas , Feminino , Grupos Focais , França , Humanos , Masculino , Pessoa de Meia-Idade , Padrões de Prática Médica , Meio Social
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