Analysis of the biofilm proteome of Xylella fastidiosa

<p>Abstract</p> <p>Background</p> <p><it>Xylella fastidiosa </it>is limited to the xylem of the plant host and the foregut of insect vectors (sharpshooters). The mechanism of pathogenicity of this bacterium differs from other plant pathogens, since it does not present typical genes that confer specific interactions between plant and pathogens (avr and/or hrp). The bacterium is injected directly into the xylem vessels where it adheres and colonizes. The whole process leads to the formation of biofilms, which are considered the main mechanism of pathogenicity. Cells in biofilms are metabolically and phenotypically different from their planktonic condition. The mature biofilm stage (phase of higher cell density) presents high virulence and resistance to toxic substances such as antibiotics and detergents. Here we performed proteomic analysis of proteins expressed exclusively in the mature biofilm of <it>X. fastidiosa </it>strain 9a5c, in comparison to planktonic growth condition.</p> <p>Results</p> <p>We found a total of 456 proteins expressed in the biofilm condition, which correspond to approximately 10% of total protein in the genome. The biofilm showed 37% (or 144 proteins) different protein than we found in the planktonic growth condition. The large difference in protein pattern in the biofilm condition may be responsible for the physiological changes of the cells in the biofilm of <it>X. fastidiosa</it>. Mass spectrometry was used to identify these proteins, while real-time quantitative polymerase chain reaction monitored expression of genes encoding them. Most of proteins expressed in the mature biofilm growth were associated with metabolism, adhesion, pathogenicity and stress conditions. Even though the biofilm cells in this work were not submitted to any stress condition, some stress related proteins were expressed only in the biofilm condition, suggesting that the biofilm cells would constitutively express proteins in different adverse environments.</p> <p>Conclusions</p> <p>We observed overexpression of proteins related to quorum sensing, proving the existence of communication between cells, and thus the development of structuring the biofilm (mature biofilm) leading to obstruction of vessels and development of disease. This paper reports a first proteomic analysis of mature biofilm of <it>X. fastidiosa</it>, opening new perspectives for understanding the biochemistry of mature biofilm growth in a plant pathogen.</p

The stress responsive and morphologically regulated hsp90 gene from Paracoccidioides brasiliensis is essential to cell viability

<p>Abstract</p> <p>Background</p> <p><it>Paracoccidioides brasiliensis </it>is a dimorphic fungus that causes the most prevalent systemic mycosis in Latin America. The response to heat shock is involved in pathogenesis, as this pathogen switches from mycelium to yeast forms in a temperature dependent fashion that is essential to establish infection. HSP90 is a molecular chaperone that helps in the folding and stabilization of selected polypeptides. HSP90 family members have been shown to present important roles in fungi, especially in the pathogenic species, as an immunodominant antigen and also as a potential antifungal therapeutic target.</p> <p>Results</p> <p>In this work, we decided to further study the <it>Pbhsp90 </it>gene, its expression and role in cell viability because it plays important roles in fungal physiology and pathogenesis. Thus, we have sequenced a <it>Pbhsp90 </it>cDNA and shown that this gene is present on the genome as a single copy. We have also confirmed its preferential expression in the yeast phase and its overexpression during dimorphic transition and oxidative stress. Treatment of the yeast with the specific HSP90 inhibitors geldanamycin and radicicol inhibited growth at 2 and 10 μM, respectively.</p> <p>Conclusion</p> <p>The data confirm that the <it>Pbhsp90 </it>gene encodes a morphologically regulated and stress-responsive protein whose function is essential to cell viability of this pathogen. This work also enforces the potential of HSP90 as a target for antifungal therapies, since the use of HSP90 inhibitors is lethal to the <it>P. brasiliensis </it>yeast cells in a dose-responsive manner.</p

Anticuerpos antifosfatidilserina en pacientes con síndrome antifosfolipido primario y en individuos sanos

Objective: To investigate the prevalence of IgM, IgG and IgA anti-phosphatidylserine (aPS) antibodies in patients with primary antiphospholipid syndrome (PAPS) and in healthy controls; to analyze sensitivity, and specificity of aPS antibodies for the diagnosis of APS and finally to assess associations between aPS antibodies with specific APS manifestations. Methods: A cross-sectional study was performed in 36 female PAPS patients and in 200 blood donors. IgM, IgG, and IgA antiphosphatidylserine (aPS) antibodies were tested in PAPS patients and controls using an in house technique and a commercial kit. PAPS patients were also tested for lupus anticoagulant (LAC), IgM and IgG anticardiolipin (aCL) antibodies, and for anti-β2 glycoprotein I (anti-β2GPI) antibodies. Results: The prevalence of IgM, IgG, and IgA aPS antibodies in PAPS patients was as follows: 10.8-16.7%, 32.4-35.7%, and 16.1%, respectively. Although a relatively low sensitivity was found for aPS antibodies in PAPS, the specificity of IgM, IgG, and IgA aPS antibodies for PAPS was 94.7-98.9%, 95.3-96.3%, and 97.9%, respectively. All aPS isotypes were significantly associated with obstetric manifestations of APS. IgM aPS antibodies were associated with an increased risk of venous and arterial thrombosis. IgA aPS antibodies were associated with arterial thrombosis whereas IgG aPS antibodies were associated with an increased risk of venous thrombotic events. IgM and IgG aPS antibodies were frequently found in association with anti-β2GPI antibodies. Conclusions: The prevalence of aPS antibodies is low in PAPS but these antibodies are highly specific for PAPS and are associated with specific PAPS manifestations.Objetivo: Investigar la prevalencia de anticuerpos anti-fosfatidilserina (aFS) de tipo IgM, IgG e IgA en pacientes con síndrome antifosfolípido primario (SAFP) y en controles sanos; analizar la sensibilidad y la especificidad de los anticuerpos aFS para el diagnóstico de aFS y finalmente, evaluar las asociaciones entre los anticuerpos específicos aFS y las manifestaciones clínicas del SAF. Métodos: Estudio transversal de 36 pacientes mujeres con SAFP y 200 donantes de sangre. Se determinaron anticuerpos antifosfatidilserina de tipo IgM, IgG e IgA en pacientes y controles con SAFP utilizando una técnica propia y un kit comercial. A los pacientes com SAFP también se les determinó el anticoagulante lúpico (ACL), los anticuerpos anticardiolipina IgM e IgG (aCL), y los anticuerpos anti-β2 glucoproteína I (anti-β2GPI). Resultados: La prevalencia de los anticuerpos AFS IgM, IgG, IgA en pacientes con SAFP fue la siguiente: 10,8-16,7%, 32,4-35,7%, y 16,1%, respectivamente. Aunque se encontro una sensibilidad relativamente baja para los anticuerpos AFS en el SAFP, la especificidad de los anticuerpos AFS IgM, IgG, IgA para el SAFP fue 94,7-98,9%, 95,3-96,3% y 97,9%, respectivamente. Todos los isotipos de AFS se asociaron significativamente con las manifestaciones obstétricas. Los anticuerpos AFS IgM se asociaron con un riesgo aumentado de trombosis venosa y arterial. Los anticuerpos AFS IgA se asociaron con la trombosis arterial mientras que los anticuerpos AFS IgG se asociaron con un mayor riesgo de eventos trombóticos venosos. Los anticuerpos AFS IgM e IgG se encuentran con frecuencia em asociación con anticuerpos anti-β2GPI. Conclusiones: La prevalencia de anticuerpos AFS es baja en SAFS pero estos anticuerpos son altamente específicos para SAFP y se asocian con manifestaciones SAFP específicos

Spontaneously Hypertensive Rats (SHR) Are Resistant to a Reserpine-Induced Progressive Model of Parkinson's Disease: Differences in Motor Behavior, Tyrosine Hydroxylase and alpha-Synuclein Expression

Reserpine is an irreversible inhibitor of vesicular monoamine transporter-2 (VMAT2) used to study Parkinson's disease (PD) and screening for antiparkinsonian treatments in rodents. Recently, the repeated treatment with a low-dose of reserpine was proposed as a progressive model of PD. Rats under this treatment show progressive catalepsy behavior, oral movements and spontaneous motor activity decrement. In parallel, compared to Wistar rats, spontaneously hypertensive rats (SHR) are resistant to acute reserpine-induced oral dyskinesia. We aimed to assess whether SHR would present differential susceptibility to repeated reserpine-induced deficits in the progressive model of PD. Male Wistar and SHR rats were administered 15 subcutaneously (s.c.) injections of reserpine (0.1 mgkg) or vehicle, every other day and motor activity was assessed by the catalepsy, oral movements and open field tests. Only reserpine-treated Wistar rats presented increased latency to step down in the catalepsy test and impaired spontaneous activity in the open field. On the other hand, there was an increase in oral movements in both reserpine-treated strains, although with reduced magnitude and latency to instauration in SHR. After a 15-day withdrawn period, both strains recovered from motor impairment, but SHR animals expressed reduced latencies to reach control levels. Finally, we performed immunohistochemistry for tyrosine hydroxylase (TH) and a-synuclein (alpha-syn) 48 h after the last injection or 15 days after withdrawn. Reserpinetreated animals presented a reduction in TH and an increase in alpha-syn immunoreactivity in the substantia nigra and dorsal striatum (dSTR), which were both recovered after 15 days of withdraw. Furthermore, SHR rats were resistant to reserpine-induced TH decrement in the substantia nigra, and presented reduced immunoreactivity to a-syn inthe dSTR relative to Wistar rats, irrespective of treatment. This effect was accompanied by increase of malondaldhyde (MDA) in the striatum of reserpine-treated Wistar rats, while SHR presented reduced MDA in both control and reserpine conditions relative to Wistar strain. In conclusion, the current results show that SHR are resilient to motor and neurochemical impairments induced by the repeated low-dose reserpine protocol. These findings indicate that the neurochemical, molecular and genetic differences in the SHR strain are potential relevant targets to the study of susceptibility to PD.Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)Fundacao de Apoio a Pesquisa do Estado do Rio Grande do Norte (FAPERN)Pro-reitoria de Pesquisa da Universidade Federal do Rio Grande do Norte (PROPESQ/UFRN)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Univ Fed Rio Grande do Norte, Dept Physiol, Memory Studies Lab, Natal, RN, BrazilUniv Fed Rio Grande do Norte, Brain Inst, Natal, RN, BrazilUniv Fed Sao Paulo, Dept Pharmacol, Behav Neurosci Lab, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Pharmacol, Sao Paulo, BrazilUniv Fed Rio Grande do Norte, Dept Physiol, Neurochem Studies Lab, Natal, RN, BrazilUniv Santa Catarina, Dept Cellular Biol Embryol & Genet, Lab Behav Genet, Florianopolis, SC, BrazilUniv Fed Sao Paulo, Dept Biosci, Santos, BrazilUniv Fed Sao Paulo, Dept Pharmacol, Behav Neurosci Lab, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Pharmacol, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Biosci, Santos, BrazilFAPESP: 2015/12308-5FAPESP: 2015/03354-3Web of Scienc

Contribuições da afetividade na intervenção pedagógica

Muito se fala acerca da importância da afetividade na relação professor-aluno. Porém, existem lacunas a serem preenchidas para a real efetivação dessa relação afetiva em nossas salas de aula. Como pode o professor vincular a afetividade a sua prática cotidiana? Que atividades beneficiam essa prática afetiva? Como articular concepções teóricas sobre afetividade às práticas pedagógicas, ao dia-a-dia em sala de aula? A presente pesquisa norteou-se na busca de respostas a essas questões. Teve como principal objetivo possibilitar um maior conhecimento sobre a relação entre a afetividade e a prática pedagógica, de modo a fornecer aos docentes contribuições de apoio à sua prática pedagógica. A metodologia utilizada foi à pesquisa qualitativa. O instrumento aplicado para levantamento de dados foi à entrevista semiestruturada, pois melhor se adequou às necessidades do trabalho, tendo como participantes quatro psicopedagogas que atuam na área de educação, no Distrito Federal. As categorias selecionadas para organização, análise e discussão dos dados foram as seguintes: O desenvolvimento da afetividade em sala de aula – atividades práticas; Estabelecendo relações afetivas com o chamado “aluno problema”; Contribuições da psicopedagogia nas relações em sala de aula; Estreitando e melhorando laços afetivos; Outras considerações. Os principais resultados da pesquisa foram que: é importante que os professores estabeleçam vínculos afetivos com seus alunos e também se destaca o valor de se trabalhar com atividades atrativas que despertem o interesse dos alunos, como, jogos, músicas, teatro, novelas; é necessário que o aluno confie no professor, sinta-se respeitado e apreciado. O trabalho com a auto-estima dos envolvidos no processo ensino-aprendizagem também é uma importante contribuição; detectou-se a existência do medo e da insegurança, de alguns professores quando precisam buscar ajuda de profissionais como psicopedagogos; percebeu-se que ensinar é mais do que simplesmente transmitir informações. Precisa haver interações entre a docência e a discência, e o diálogo; para se formar cidadãos completos, saudáveis é necessário conhecer o que envolve o aluno e o seu desenvolvimento. Entre as diversas contribuições trazidas por essa pesquisa, destaca-se a necessidade dos docentes ampliarem seus horizontes e compreenderem seus alunos como pessoas, com necessidades e interesses diversos. O papel do docente vai além de transmitir informações; precisa haver uma interação entre os componentes do processo ensino-aprendizagem. A relação afetiva entre professor e aluno exerce papel fundamental, tanto para o desenvolvimento cognitivo como emocional de ambos

Contribuições da afetividade na intervenção pedagógica

Muito se fala acerca da importância da afetividade na relação professor-aluno. Porém, existem lacunas a serem preenchidas para a real efetivação dessa relação afetiva em nossas salas de aula. Como pode o professor vincular a afetividade a sua prática cotidiana? Que atividades beneficiam essa prática afetiva? Como articular concepções teóricas sobre afetividade às práticas pedagógicas, ao dia-a-dia em sala de aula? A presente pesquisa norteou-se na busca de respostas a essas questões. Teve como principal objetivo possibilitar um maior conhecimento sobre a relação entre a afetividade e a prática pedagógica, de modo a fornecer aos docentes contribuições de apoio à sua prática pedagógica. A metodologia utilizada foi à pesquisa qualitativa. O instrumento aplicado para levantamento de dados foi à entrevista semiestruturada, pois melhor se adequou às necessidades do trabalho, tendo como participantes quatro psicopedagogas que atuam na área de educação, no Distrito Federal. As categorias selecionadas para organização, análise e discussão dos dados foram as seguintes: O desenvolvimento da afetividade em sala de aula – atividades práticas; Estabelecendo relações afetivas com o chamado “aluno problema”; Contribuições da psicopedagogia nas relações em sala de aula; Estreitando e melhorando laços afetivos; Outras considerações. Os principais resultados da pesquisa foram que: é importante que os professores estabeleçam vínculos afetivos com seus alunos e também se destaca o valor de se trabalhar com atividades atrativas que despertem o interesse dos alunos, como, jogos, músicas, teatro, novelas; é necessário que o aluno confie no professor, sinta-se respeitado e apreciado. O trabalho com a auto-estima dos envolvidos no processo ensino-aprendizagem também é uma importante contribuição; detectou-se a existência do medo e da insegurança, de alguns professores quando precisam buscar ajuda de profissionais como psicopedagogos; percebeu-se que ensinar é mais do que simplesmente transmitir informações. Precisa haver interações entre a docência e a discência, e o diálogo; para se formar cidadãos completos, saudáveis é necessário conhecer o que envolve o aluno e o seu desenvolvimento. Entre as diversas contribuições trazidas por essa pesquisa, destaca-se a necessidade dos docentes ampliarem seus horizontes e compreenderem seus alunos como pessoas, com necessidades e interesses diversos. O papel do docente vai além de transmitir informações; precisa haver uma interação entre os componentes do processo ensino-aprendizagem. A relação afetiva entre professor e aluno exerce papel fundamental, tanto para o desenvolvimento cognitivo como emocional de ambos

Potential wound healing effect of gel based on chicha gum, chitosan, and mauritia flexuosa oil

Wounds are considered a clinically critical issue, and effective treatment will decrease complications, prevent chronic wound formation, and allow rapid healing. The development of products based on naturally occurring materials is an efficient approach to wound healing. Natural polysaccharides can mimic the extracellular matrix and promote cell growth, thus making them attractive for wound healing. In this context, the aim of this work was to produce a gel based on chicha gum, chitosan, and Mauritia flexuosa oil (CGCHO) for wound treatment. TG and DTG analyzed the thermal behavior of the materials, and SEM investigated the surface roughness. The percentages of total phenolic compounds, flavonoids, and antioxidants were determined, presenting a value of 81.811 ± 7.257 µmol gallic acid/g Mauritia flexuosa oil, 57.915 ± 0.305 µmol quercetin/g Mauritia flexuosa oil, and 0.379 mg/mL, respectively. The anti-inflammatory was determined, presenting a value of 10.35 ± 1.46% chicha gum, 16.86 ± 1.00% Mauritia flexuosa oil, 10.17 ± 1.05% CGCHO, and 15.53 ± 0.65% chitosan, respectively. The materials were tested against Gram-negative (Klebsiella pneumoniae) and Gram-positive (Staphylococcus aureus) bacteria and a fungus (Candida albicans). The CGCHO formulation showed better antimicrobial activity against Gram-positive bacteria. In addition, an in vivo wound healing study was also performed. After 21 days of treatment, the epidermal re-epithelialization process was observed. CGCHO showed good thermal stability and roughness that can help in cell growth and promote the tissue healing process. In addition to the good results observed for the antimicrobial, antioxidant, anti-inflammatory activities and providing wound healing, they provided the necessary support for the healing process, thus representing a new approach to the wound healing process.info:eu-repo/semantics/publishedVersio

Biocompatible gels of chitosan-buriti oil for potential wound healing applications

The buriti oil (Mauritia flexuosa L.) can be associated with polymeric matrices for biomedical applications. This study aimed to evaluate the e ect of chitosan gel (CG) associated with buriti oil (CGB) as a healing agent. The fatty acids and volatile compounds composition of buriti oil were performed and the composite gels were characterized using FTIR and thermal analysis. Biological tests including antimicrobial, antioxidant, anti-inflammatory and healing e ects were also investigated. Buriti oil is composed of oleic and palmitic acids, and the main volatile compounds were identified. The buriti oil did not show antimicrobial activity, on the other hand, the composite gel (chitosan and oil) proved to be e cient against Staphylococcus aureus and Klebsiella pneumonia at the 10 mg/mL. Similar behavior was observed for antioxidant activity, determined by the -carotene bleaching assay, composite gels presenting higher activity and buriti oil showed anti-inflammatory activity, which may be related to the inhibition of the release of free radicals. Regarding wound healing performed using in vivo testing, the composite gel (CGB) was found to promote faster and complete wound retraction. The results indicated that the gel chitosan–buriti oil has a set of properties that improve its antibacterial, antioxidant and healing action, suggesting that this material can be used to treat skin lesions.Maria Onaira Gonçalves Ferreira acknowledges the Coordination for the Improvement of Higher Education Personnel, the Brazilian Ministry of Education, financial support for the scholarship, and other authors acknowledge to National Council for Scientific and Technological Development (CNPQ) and Piauí State Research Support Foundation (FAPEPI).info:eu-repo/semantics/publishedVersio

Determination of Kresoxim-Methyl in Water and in Grapes by High-Performance Liquid Chromatography (HPLC) Using PhotochemicalInduced Fluorescence and Dispersive Liquid-Liquid Microextraction (DLLME)

A high-performance chromatographic method was developed to determine the fungicide kresoxim-methyl. Off-line photochemical derivatization was used to induce the formation of a stable and fluorescent product since the fungicide does not present natural fluorescence. Intense fluorescence at 370/430nm was achieved by treating the analyte in solution at pH 6 to ultraviolet light for 45s. The chromatographic conditions included isocratic elution with 50/ 50% (v/v) acetonitrile/water and the photochemical product appeared at a retention time of 7.2min. The short and long term stabilities of the photoproduct were evaluated and variation of less than 5% was achieved. The limits of detection in water samples and in grapes samples were 0.019mg kg1 and 0.065mg kg1 of kresoxim-methyl residue, respectively. The linear response covered three orders of magnitude up to 10.6mg kg1 of kresoxim-methyl. The robustness was evaluated through a Box–Behnken experimental design showing the insignificance of all factors and their interactions. The potential interference of tebuconazole for the determination of kresoxim-methyl was studied. The use of the dispersive liquid-liquid microextraction (DLLME) allowed recoveries between 80% and 101% depending on concentration with the minimum generation of waste products