In vitro determination of extracellular proteins from xylella fastidiosa

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)The phytopathogen Xylella fastidiosa causes economic losses in important agricultural crops. Xylem vessel occlusion caused by biofilm formation is the major mechanism underlying the pathogenicity of distinct strains of X. fastidiosa. Here, we provide a detailed in vitro characterization of the extracellular proteins of X. fastidiosa. Based on the results, we performed a comparison with a strain J1a12, which cannot induce citrus variegated chlorosis symptoms when inoculated into citrus plants. We then extend this approach to analyze the extracellular proteins of X. fastidiosa in media supplemented with calcium. We verified increases in extracellular proteins concomitant with the days of growth and, consequently, biofilm development (330 days). Outer membrane vesicles carrying toxins were identified beginning at 10 days of growth in the 9a5c strain. In addition, a decrease in extracellular proteins in media supplemented with calcium was observed in both strains. Using mass spectrometry, 71 different proteins were identified during 30 days of X. fastidiosa biofilm development, including proteases, quorum-sensing proteins, biofilm formation proteins, hypothetical proteins, phage-related proteins, chaperones, toxins, antitoxins, and extracellular vesicle membrane components.The phytopathogen Xylella fastidiosa causes economic losses in important agricultural crops. Xylem vessel occlusion caused by biofilm formation is the major mechanism underlying the pathogenicity of distinct strains of X. fastidiosa. Here, we provide a de7Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2001/07533-7, 2012/51580-4]Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES, Computational Biology Program)CAPESFAPESP [2011/50268-4]CAPES (Computational Biology Program)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Vapd In Xylella Fastidiosa Is A Thermostable Protein With Ribonuclease Activity.

Xylella fastidiosa strain 9a5c is a gram-negative phytopathogen that is the causal agent of citrus variegated chlorosis (CVC), a disease that is responsible for economic losses in Brazilian agriculture. The most well-known mechanism of pathogenicity for this bacterial pathogen is xylem vessel occlusion, which results from bacterial movement and the formation of biofilms. The molecular mechanisms underlying the virulence caused by biofilm formation are unknown. Here, we provide evidence showing that virulence-associated protein D in X. fastidiosa (Xf-VapD) is a thermostable protein with ribonuclease activity. Moreover, protein expression analyses in two X. fastidiosa strains, including virulent (Xf9a5c) and nonpathogenic (XfJ1a12) strains, showed that Xf-VapD was expressed during all phases of development in both strains and that increased expression was observed in Xf9a5c during biofilm growth. This study is an important step toward characterizing and improving our understanding of the biological significance of Xf-VapD and its potential functions in the CVC pathosystem.10e014576

Acute effect of aerobic exercise in different intensities in mucociliary clearance of patients with COPD

Design of the Study: Clinical Trial. Objective (s): To analyze the acute effect of aerobic exercise at different intensities in mucociliary clearance in patients with COPD, and to investigate possible associations of the autonomic nervous system in this response. Methods: 22 COPD patients underwent an initial evaluation for collecting personal data and spirometry to assess lung function. It was performed a progressive treadmill test for aerobic exercise prescription. Finally two randomized sessions of aerobic exercise with intensity of 60 % and 90 % of peak speed reached during the incremental test ( vVO2peack ) were performed with at least 24 hours of rest between them. The mucociliary clerance was assessed before and after the exercise sessions by testing the saccharin transit time (STT). Assessment of autonomic modulation was performed by heart rate variability (HRV) which continued throughout the protocol. Results: The values obtained in the STT test after aerobic exercise at 60 % of vVO2peack (9,08 minutes ± 4,96 ) was lower when compared to the STT before exercise ( 11,96 ± 6,31, p = 0,005 ) . That response also occurred after aerobic exercise at 90% of vVO2peack ( 8,90 ± 4,21 min ) compared to baseline ( 12,94 ± 7,22 , p = 0,023 ). Correlation analysis between the final values of STT test and HRV indexes did not show significant differences. Conclusions: Patients with COPD showed acceleration of mucociliary clerance right after a session of aerobic exercise. It was not possible to observe the association of autonomic modulation in this responseModelo do Estudo: Experimental. Objetivo(s) do estudo: Analisar o efeito agudo do exercício aeróbio em diferentes intensidades no transporte mucociliar de pacientes com DPOC, bem como investigar possíveis associações do sistema nervoso autônomo nesta resposta. Metodologia: Foram analisados 22 pacientes com DPOC que realizaram avaliação inicial para coleta de dados pessoais e espirometria a fim de avaliar a função pulmonar. Realizou-se um teste progressivo em esteira ergométrica para prescrição do exercício aeróbio. Por fim foram realizadas duas sessões de exercício aeróbio randomizadas em esteira ergométrica com intensidade de 60% e 90% do pico da velocidade atingida no teste incremental (vVO2pico) com pelo menos 24 horas de descanso entre elas. O transporte mucociliar foi avaliado antes e após realização do exercício por meio do teste do tempo de trânsito da sacarina (TTS). A avaliação da modulação autonômica foi realizada por meio da variabilidade da frequência cardíaca (VFC) a qual prosseguiu durante todo o protocolo. Resultados: Os valores obtidos no teste de TTS dos pacientes com DPOC após exercício aeróbio a 60% da vVO2pico (9,08 ± 4,96 minutos) foi menor comparado ao TTS antes do exercício (11,96 ± 6,31; p = 0,005). O que também ocorreu após exercício aeróbio a 90% da vVO2pico (8,90 ± 4,21 minutos) quando comparado ao momento basal (12,94 ± 7,22; p = 0,023). As análises de correlação entre os valores finais de TTS e índices da VFC não apontaram diferenças significativas. Conclusões: Pacientes com DPOC apresentaram aceleração da transportabilidade mucociliar frente a uma sessão de exercício aeróbio. Não foi possível observar associação da modulação autonômica nesta resposta após o exercíci

Chronic exposure of diesel exhaust particles induces alveolar enlargement in mice

Background\ud Diesel exhaust particles (DEPs) are deposited into the respiratory tract and are thought to be a risk factor for the development of diseases of the respiratory system. In healthy individuals, the timing and mechanisms of respiratory tract injuries caused by chronic exposure to air pollution remain to be clarified.\ud \ud \ud Methods\ud We evaluated the effects of chronic exposure to DEP at doses below those found in a typical bus corridor in Sao Paulo (150 μg/m3). Male BALB/c mice were divided into mice receiving a nasal instillation: saline (saline; n = 30) and 30 μg/10 μL of DEP (DEP; n = 30). Nasal instillations were performed five days a week, over a period of 90 days. Bronchoalveolar lavage (BAL) was performed, and the concentrations of interleukin (IL)-4, IL-10, IL-13 and interferon-gamma (INF-γ) were determined by ELISA-immunoassay. Assessment of respiratory mechanics was performed. The gene expression of Muc5ac in lung was evaluated by RT-PCR. The presence of IL-13, MAC2+ macrophages, CD3+, CD4+, CD8+ T cells and CD20+ B cells in tissues was analysed by immunohistochemistry. Bronchial thickness and the collagen/elastic fibers density were evaluated by morphometry. We measured the mean linear intercept (Lm), a measure of alveolar distension, and the mean airspace diameter (D0) and statistical distribution (D2).\ud \ud \ud Results\ud DEP decreased IFN-γ levels in BAL (p = 0.03), but did not significantly alter IL-4, IL-10 and IL-13 levels. MAC2+ macrophage, CD4+ T cell and CD20+ B cell numbers were not altered; however, numbers of CD3+ T cells (p ≤ 0.001) and CD8+ T cells (p ≤ 0.001) increased in the parenchyma. Although IL-13 (p = 0.008) expression decreased in the bronchiolar epithelium, Muc5ac gene expression was not altered in the lung of DEP-exposed animals. Although respiratory mechanics, elastic and collagen density were not modified, the mean linear intercept (Lm) was increased in the DEP-exposed animals (p ≤ 0.001), and the index D2 was statistically different (p = 0.038) from the control animals.\ud \ud \ud Conclusion\ud Our data suggest that nasal instillation of low doses of DEP over a period of 90 days results in alveolar enlargement in the pulmonary parenchyma of healthy mice.This work was supported by Coordenação de Aperfeiçoamento de Pessoal\ud de Nível Superior (CAPES) and Fundação de Amparo à Pesquisa do Estado\ud de São Paulo (FAPESP 2010/51377-9 and 2012/16279-1). Dr. Mauad is funded\ud by CNPq (National Research Council), Brazil

p53 Transactivation and the Impact of Mutations, Cofactors and Small Molecules Using a Simplified Yeast-Based Screening System

The p53 tumor suppressor, which is altered in most cancers, is a sequence-specific transcription factor that is able to modulate the expression of many target genes and influence a variety of cellular pathways. Inactivation of the p53 pathway in cancer frequently occurs through the expression of mutant p53 protein. In tumors that retain wild type p53, the pathway can be altered by upstream modulators, particularly the p53 negative regulators MDM2 and MDM4. promoter, ii) single copy, chromosomally located p53-responsive and control luminescence reporters, iii) enhanced chemical uptake using modified ABC-transporters, iv) small-volume formats for treatment and dual-luciferase assays, and v) opportunities to co-express p53 with other cofactor proteins. This robust system can distinguish different levels of expression of WT and mutant p53 as well as interactions with MDM2 or 53BP1.We found that the small molecules Nutlin and RITA could both relieve the MDM2-dependent inhibition of WT p53 transactivation function, while only RITA could impact p53/53BP1 functional interactions. PRIMA-1 was ineffective in modifying the transactivation capacity of WT p53 and missense p53 mutations. This dual-luciferase assay can, therefore, provide a high-throughput assessment tool for investigating a matrix of factors that can influence the p53 network, including the effectiveness of newly developed small molecules, on WT and tumor-associated p53 mutants as well as interacting proteins

Performance of the ALICE experiment at the CERN LHC

ALICE is the heavy-ion experiment at the CERN Large Hadron Collider. The experiment continuously took data during the first physics campaign of the machine from fall 2009 until early 2013, using proton and lead-ion beams. In this paper we describe the running environment and the data handling procedures, and discuss the performance of the ALICE detectors and analysis methods for various physics observables