Crystallographic studies of proteins involved in the mRNA localization mechanisms in Drosophila melanogaster and amidation of the peptidoglycan residues in Staphylococcus aureus

Part of the work described in this chapter, was the subject of the following publication: D. Vieira, T. a. Figueiredo, A. Verma, R. G. Sobral, A. M. Ludovice, H. de Lencastre, and J. Trincao, “Purification, crystallization and preliminary X-ray diffraction analysis of GatD, a glutamine amidotransferase-like protein from Staphylococcus aureus peptidoglycan,” Acta Crystallogr. Sect. F Struct. Biol. Commun., vol. 70, no. 5, pp. 1–4, Apr. 2014.

Serosurvey of hantavirus infection in humans in the border region between Brazil and Argentina

INTRODUCTION: According to reports by the Ministry of Health, in the far western region of the State of Santa Catarina, there have been no reports of hantavirus pulmonary syndrome, a zoonotic disease transmitted by feces of infected rodents. A seroepidemiological study of residents of this region, was conducted, with the aim of determining the presence of hantavirus infections. A total of 340 volunteers of both genus, from the towns of Belmonte and Paraíso, were studied. METHODS: The serum of these patients was collected and used to detect IgG antibodies against recombinant N protein of Araraquara hantavirus, by ELISA assay. The positive samples were then titrated and confirmed by immunofluorescence assay. RESULTS: This study demonstrated the presence of IgG antibodies against hantavirus N protein in 3.5% of the population. The most frequent occupation was farm worker, 81% had direct and indirect contact with rodents, 91.7% of positive cases were farm workers, indicating that the probable cause of infection occurred during barn cleaning. These antibodies are noteworthy, given that the levels of antibodies were verified in individuals whose contact with hantavirus may have occurred many years ago. CONCLUSIONS: This study shows the circulation of hantavirus in the region, a fact that until now, had not reported. All the serum reagents had contact with the pathogen, but did not develop pulmonary and cardiovascular syndrome. It is important to remain alert, because hantavirus is a serious and emerging disease of some relevance.

A retrospective serologic survey of hantavirus infections in the county of Cássia dos Coqueiros, State of São Paulo, Brazil

INTRODUCTION: In recent years, hantavirus infections producing severe diseases have obtained an increased attention from public health authorities from the countries of Eurasia to the Americas. Brazil has reported 1,300 cases of hantavirus cardiopulmonary syndrome (HCPS) from 1993 to 2010, with about 80 of them occurring in the northeast of the State of São Paulo, with 48% fatality rate. Araraquara virus was the causative agent of HCPS in the region. Considering that hantaviruses causing human disease in the Americas were unknown until 1993, we have looked for hantavirus infections in the population of Cássia dos Coqueiros county, northeast of the State of São Paulo, Brazil, before this time. This county has about 2,800 inhabitants and an economy based on agriculture, including cultivation of Brachiaria decumbens grass. The grass seeds are an important rodent attraction, facilitating transmission of hantavirus to man. Four HCPS cases were reported so far in the county. METHODS: In this study, 1,876 sera collected from 1987 to 1990 were tested for IgG to hantavirus by IgG-ELISA, using the N recombinant protein of Araraquara virus as antigen. RESULTS: Positive results were observed in 89 (4.7%) samples, which were all collected in 1987. The positivity among urban inhabitants was 5.3%, compared with 4.3% among those living in rural areas. CONCLUSIONS: Our results showed that hantavirus infections occurred in Cássia dos Coqueiros, completely unrecognized, even before hantaviruses were described in the Americas.

Production and diagnostic application of recombinant domain III of West Nile envelope protein in Brazil

INTRODUCTION: West Nile virus (WNV) is a flavivirus with a natural cycle involving mosquitoes and birds. Over the last 11 years, WNV has spread throughout the Americas with the imminent risk of its introduction in Brazil. METHODS: Envelope protein domain III of WNV (rDIII) was bacterially expressed and purified. An enzyme-linked immunosorbent assay with WNV rDIII antigen was standardized against mouse immune fluids (MIAFs) of different flavivirus. RESULTS: WNV rDIII reacted strongly with St. Louis encephalitis virus (SLEV) MIAF but not with other flaviviruses. CONCLUSIONS: This antigen may be a potentially useful tool for serologic diagnosis and may contribute in future epidemiological surveillance of WNV infections in Brazil.

Detection of immunogenic proteins from Anopheles sundaicussalivary glands in the human serum

AbstractINTRODUCTION:The saliva of mosquitoes has an important role in the transmission of several diseases, including malaria, and contains substances with vasomodulating and immunomodulating effects to counteract the host physiological mechanisms and enhance pathogen transmission. As immunomodulatory components, salivary gland proteins can induce the generation of specific IgG antibodies in the host, which can be used as specific biomarkers of exposure to Anopheles sundaicus . The objective of this study was to identify immunogenic proteins from the salivary glands of Anopheles sundaicus by reaction with sera from individuals living in malaria-endemic areas who are thus exposed to Anopheles mosquitoes.METHODS:IgG antibodies targeting salivary gland proteins in serum samples from individuals living in malaria-endemic areas were measured by enzyme-linked immunosorbent assay (ELISA). Sera from healthy individuals living in non-endemic areas were used as negative controls. Determination of the presence of salivary gland immunogenic proteins was carried out by western blotting.RESULTS:Sixteen bands appeared in sodium dodecyl sulfate polyacrylamide gel electrophoresis, with molecule weights ranging from 22 to 144kDa. Among the exposed individuals, IgG responses to salivary gland proteins were variable. Protein bands with molecular weights of 46, 41, 33, and 31kDa were the most immunogenic. These immunogenic proteins were consistently recognized by pooled serum and individual samples from people living in malaria-endemic areas but not by negative controls.CONCLUSIONS:These results support the potential use of immunogenic proteins from the salivary glands of Anopheles as candidate markers of bite exposure or in malaria vaccines.

Exploring the relationship between toxin and spore production in the human enteric pathogen Clostridium difficile

RESUMO: Clostridium difficile é presentemente a principal causa de doença gastrointestinal associada à utilização de antibióticos em adultos. C. difficile é uma bactéria Gram-positiva, obrigatoriamente anaeróbica, capaz de formar endósporos. Tem-se verificado um aumento dos casos de doença associada a C. difficile com sintomas mais severos, elevadas taxas de morbilidade, mortalidade e recorrência, em parte, devido à emergência de estirpes mais virulentas, mas também devido à má gestão do uso de antibióticos. C. difficile produz duas toxinas, TcdA e TcdB, que são os principais fatores de virulência e responsáveis pelos sintomas da doença. Estas são codificadas a partir do Locus de Patogenicidade (PaLoc) que codifica ainda para um regulador positivo, TcdR, uma holina, TcdE, e um regulador negativo, TcdC. Os esporos resistentes ao oxigénio são essenciais para a transmissão do organismo e recorrência da doença. A expressão dos genes do PaLoc ocorre em células vegetativas, no final da fase de crescimento exponencial, e em células em esporulação. Neste trabalho construímos dois mutantes de eliminação em fase dos genes tcdR e tcdE. Mostrámos que a auto-regulação do gene tcdR não é significativa. No entanto, tcdR é sempre necessário para a expressão dos genes presentes no PaLoc. Trabalho anterior mostrou que, com a exceção de tcdC, os demais genes do PaLoc são expressos no pré-esporo. Mostrámos aqui que TcdA é detectada à superfície do esporo maduro e que a eliminação do tcdE não influencia a acumulação de TcdA no meio de cultura ou em associação às células ou ao esporo. Estas observações têm consequências para o nosso entendimento do processo infecioso: sugeremque o esporo possa ser também um veículo para a entrega da toxina nos estágios iniciais da infecção, que TcdA possa ser libertada durante a germinação do esporo, e que o esporo possa utilizar o mesmo receptor reconhecido por TcdA para a ligação à mucosa do cólon.---------------------------ABSTRACT: Clostridium difficile is currently the major cause of antibiotic-associated gastrointestinal diseases in adults. This is a Gram-positive bacterium, endospore-forming and an obligate anaerobe that colonizes the gastrointestinal tract. Recent years have seen a rise in C. difficile associated disease (CDAD) cases, associated with more severe disease symptoms, higher rates of morbidity, mortality and recurrence, which were mostly caused due to the emergence of “hypervirulent” strains but also due to changing patterns of antibiotics use. C. difficile produces two potent toxins, TcdA and TcdB, which are the main virulence factors and the responsible for the disease symptoms. These are codified from a Pathogenicity Locus (PaLoc), composed also by the positive regulator, TcdR, the holin-like protein, TcdE, and a negative regulator, TcdC. Besides the toxins, the oxygen-resistant spores are also essential for transmission of the organism through diarrhea; moreover, spores can accumulate in the environment or in the host, which will cause disease recurrence. The expression of the PaLoc genes occurs in vegetative cells, at the end of the exponential growth phase, and in sporulating cells. In this work, we constructed two in-frame deletion mutants of tcdR and tcdE. We showed that the positive auto regulation of tcdR is not significant. However, tcdR is always necessary for the expression of the PaLoc genes. A previous work showed that, except tcdC, all the PaLoc genes are expressed in the forespore. Here, we detected TcdA at the spore surface. Furthermore, we showed that the in-frame deletion of tcdE does not affect the accumulation of TcdA in the culture medium or in association with cells or spores. This data was important for us to conclude about the infeccious process: it suggests that the spore may be the vehicle for the delivery of TcdA in early stages of infection, that TcdA may be released during spores germination and that this spore may use the same receptor recognized by TcdA to bind to the colonic mucosa.

Nutrition for the pediatric surgical patient: approach in the peri-operative period

Nutrition is essential for maintenance of physiologic homeostasis and growth. Hypermetabolic states lead to a depletion of body stores, with decreased immunocompetence and increased morbidity and mortality. The purpose of this paper is to provide an update regarding the provision of appropriate nutrition for the pediatric surgical patient, emphasizing the preoperative and postoperative periods. Modern nutritional support for the surgical patient comprises numerous stages, including assessment of nutritional status, nutritional requirements, and nutritional therapy. Nutritional assessment is performed utilizing the clinical history, clinical examination, anthropometry, and biochemical evaluation. Anthropometric parameters include body weight, height, arm and head circumference, and skinfold thickness measurements. The biochemical evaluation is conducted using determinations of plasma levels of proteins, including album, pre-albumin, transferrin, and retinol-binding protein. These parameters are subject to error and are influenced by the rapid changes in body composition in the peri-operative period. Nutritional therapy includes enteral and/or parenteral nutrition. Enteral feeding is the first choice for nutritional therapy. If enteral feeding is not indicated, parenteral nutrition must be utilized. In all cases, an individualized, adequate diet (enteral formula or parenteral solution) is obligatory to decrease the occurrence of overfeeding and its undesirable consequences.

A Gß protein and the TupA Co-Regulator bind to protein kinase A Tpk2 to act as antagonistic molecular switches of fungal morphological changes

A Gß protein and the TupA Co-Regulator Bind to Protein Kinase A Tpk2 to Act as Antagonistic Molecular Switches of Fungal Morphological Changes

Lower NPAS3 expression during the later stages of abnormal lung development in rat congenital diaphragmatic hernia

Purpose Congenital diaphragmatic hernia (CDH) is characterized by a developmental defect in the diaphragm, pulmonary hypoplasia and pulmonary hypertension. NPAS3 is a PAS domain transcription factor regulating Drosophila tracheogenesis. NPAS3 null mice develop pulmonary hypoplasia in utero and die after birth due to respiratory failure. We aimed to evaluate NPAS3 expres- sion during normal and abnormal lung development due to CDH. Methods CDH was induced by administering 100 mg/ml nitrofen to time-pregnant dams on embryonic day (E) 9 of gestation. Lungs were isolated on E15, E18 and E21 and NPAS3 localization was determined by immunohisto- chemistry and quantified using Western blotting. Results We found that only E21 hypoplastic CDH lungs have reduced expression of NPAS3 in the terminal sac- cules. Western blotting confirmed the down-regulation of NPAS3 protein in the nitrofen-induced hypoplastic lungs. Conclusions We demonstrate for the first time that ni- trofen-induced hypoplastic CDH lungs have reduced NPAS3 expression in the terminal saccules during the later stages of abnormal lung development. Our findings suggest that NPAS3 is associated with pulmonary hypoplasia in CDH.

Micro/nano-structured superhydrophobic surfaces in the biomedical field: part I: basic concepts and biomimetic approaches.

Inspired by natural structures, great attention has been devoted to the study and development of surfaces with extreme wettable properties. The meticulous study of natural systems revealed that the micro/nano-topography of the surface is critical to obtaining unique wettability features, including superhydrophobicity. However, the surface chemistry also has an important role in such surface characteristics. As the interaction of biomaterials with the biological milieu occurs at the surface of the materials, it is expected that synthetic substrates with extreme and controllable wettability ranging from superhydrophilic to superhydrophobic regimes could bring about the possibility of new investigations of cellâ material interactions on nonconventional surfaces and the development of alternative devices with biomedical utility. This first part of the review will describe in detail how proteins and cells interact with micro/nano-structured surfaces exhibiting extreme wettabilities.

Chlamydia pneumoniae and atherosclerosis. Identification of bacterial DNA in the arterial wall

OBJECTIVE: The intracellular Gram-negative bacterium Chlamydia pneumoniae has been associated with atherosclerosis. The presence of Chlamydia pneumoniae has been investigated in fragments of the arterial wall with a technique for DNA identification. METHODS: Arterial fragments obtained from vascular surgical procedures in 58 patients were analyzed. From these patients, 39 were males and the mean age was 65±6 years. The polymerase chain reaction was used to identify the bacterial DNA with a pair of primers that codify the major outer membrane protein (MOMP) of Chlamydia pneumoniae. The amplified product was visualized by electrophoresis in the 2% agarose gel stained with ethidium bromide, and it was considered positive when migrating in the band of molecular weight of the positive controls. RESULTS: Seven (12%) out of the 58 patients showed positive results for Chlamydia pneumoniae. CONCLUSION: DNA from Chlamydia pneumoniae was identified in the arterial wall of a substantial number of patients with atherosclerosis. This association, which has already been described in other countries, corroborates the evidence favoring a role played by Chlamydia pneumoniae in atherogenesis.

Study of the retinoic acid regulated genes in the rodent hypothalamus controlling growth and appetite and the potential for the epigenetic control

Dissertação de mestrado em Bioquímica (área de especialização em Biomedicina)

Correlation between turbidimetric and nephelometric methods of measuring C-reactive protein in patients with unstable angina or non-ST elevation acute myocardial infarction

OBJECTIVE: To evaluate the performance of the turbidimetric method of C-reactive protein (CRP) as a measure of low-grade inflammation in patients admitted with non-ST elevation acute coronary syndromes (ACS). METHODS: Serum samples obtained at hospital arrival from 68 patients (66±11 years, 40 men), admitted with unstable angina or non-ST elevation acute myocardial infarction were used to measure CRP by the methods of nephelometry and turbidimetry. RESULTS: The medians of C-reactive protein by the turbidimetric and nephelometric methods were 0.5 mg/dL and 0.47 mg/dL, respectively. A strong linear association existed between the 2 methods, according to the regression coefficient (b=0.75; 95% C.I.=0.70-0.80) and correlation coefficient (r=0.96; P<0.001). The mean difference between the nephelometric and turbidimetric CRP was 0.02 ± 0.91 mg/dL, and 100% agreement between the methods in the detection of high CRP was observed. CONCLUSION: In patients with non-ST elevation ACS, CRP values obtained by turbidimetry show a strong linear association with the method of nephelometry and perfect agreement in the detection of high CRP.

Rol de la infección bacteriana en el desarrollo y progresión del cáncer prostático. Influence of bacterial infection in the development and progression of prostatitic cancer.

En la patogénesis del cáncer, factores microambientales como la inflamación están estrechamente vinculados al desarrollo y crecimiento tumoral, sustentando la clásica hipótesis de Virchow del origen del cáncer en sitios de inflamación crónica, la cual incitaría a carcinogénesis por múltiples factores. Estudios previos en este laboratorio evidenciaron en un modelo de prostatitis bacteriana agudo con E. coli, profundos cambios estromales semejantes al "estroma reactivo", con predominio de miofibroblasto, que se genera en el cáncer. En correlación, existe abundante evidencia obtenida en modelos experimentales animales confirmando que el microambiente estromal en el cual se desarrollan los tumores epiteliales influencia profundamente la progresión tumoral. El rol protagónico del estroma del huésped en el crecimiento neoplásico, también se ha demostrado inoculando la misma línea tumoral en diferentes tejidos y analizando su comportamiento en comparación con su implantación en el sitio anatómico original del tumor (implante ortotópico); otro factor clave en la repuesta del huésped al tumor está dado por el infiltrado de células inmunes que puede favorecer o limitar el crecimiento tumoral de acuerdo al perfil de citoquinas que secreten. Teniendo en cuenta estos antecedentes, este proyecto tiene como Objetivo General estudiar la influencia de la infección bacteriana crónica en la inducción y evolución del cáncer prostático. Para ello trabajaremos in vivo con dos formas de formas de Tumores Prostáticos, un Tumor Inducido por combinación del carcinógeno N-methyl-N-nitrosourea (MNU) y testosterona; el segundo mediante Transplante Ortotópico de células tumorales prostáticas MAT-LU. En ambos modelos se inducirá previamente una prostatitis bacteriana, a fin de estudiar los efectos de la prostatitis en la inducción del tumor en el primer modelo, y en la implantación de las células tumorales en el segundo. También se inducirá prostatitis después de establecido el tumor por ambos procedimientos, a fin de determinar si la prostatitis bacteriana modula la progresión neoplásica. Finalmente, proponemos un modelo in vitro que permita estudiar la interacción tumor/estroma separado de la influencia del sistema inmune. A tal fin se utilizarán co-cultivos combinando células tumorales con estromales modificadas de diferente modo. La Inducción de Tumores Prostáticos se realizará en ratas de la cepa Wistar adultas, en las cuales se inducirán lesiones displásicas y neoplásicas siguiendo protocolos de carcinogénesis prostática por MNU, para lo cual es necesario el tratamiento previo con acetato de ciprosterona y propionato de testosterona, seguido por administración crónica de testosterona. Los estudios con Transplante Ortotópico de células tumorales se realizarán en ratas Copenhagen. La influencia de la infección bacteriana en el desarrollo tumoral será investigada inyectando E. coli intraprostáticamente: Se realizará Análisis Macroscópico, de Parámetros Morfológicos de las lesiones tumorales, grado de malignidad, extensión e invasión de las lesiones de acuerdo a consensos internacionales, y Bioquímicos mediante análisis de la expresión, por IHQ y WB, de fosfatasa ácida, citoqueratina 8, Prostatic Binding Protein, PTEN (gen supresor tumoral) y el receptor de Andrógenos, todos parámetros de actividad y de transformación celular. También se evaluará apoptosis por TUNEL y proliferación celular. Los cambios del compartimiento estromal en respuesta al implante tumoral y la influencia de la inflamación bacteriana se evaluarán mediante análisis morfológico e inmunocitoquímico, caracterizando el fenotipo de las poblaciones celulares con a-actina, vimentina, calponina y tenascina. Se espera que los resultados aporten evidencias acerca de las interacciones bidireccionales entre células neoplásicas prostáticas y su entorno estromal, que en un futuro puedan servir como base para establecr estrategias para prevenir y/o modificar el crecimiento neoplásico.

Investigación de defectos genéticos en el sistema de la fosforilasa hepática en pacientes argentinos. Definición nosológica mediante una estrategia de análisis molecular. Investigation of genetic defects in the hepatic phosphorylase system in Argentinean patients. Nosological definition by molecular analysis strategy.

Las Enfermedades de Atesoramiento de Glucógeno (EAGs) también llamadas Glucogenosis comprenden un grupo de entidades causadas por una deficiencia enzimática específica relacionada con la vía de síntesis o degradación de esta macromolécula. La heterogeneidad fenotípica de los pacientes afectados dificulta la identificación de las diferentes variantes de EAG y por ende la correcta definición nosológica. En el Centro de Estudio de las Metabolopatías Congénitas, CEMECO, se fueron definiendo los diferentes tipos de Glucogenosis a través de una estrategia multidisciplinaria que integra distintos niveles de investigación clínica y complementaria, laboratorio metabólico especializado, enzimático, histomorfológico y de análisis molecular. Sin embargo, en algunos enfermos, entre los que se encuentran aquellos con defectos en el sistema de la fosforilasa hepática (EAG-VI y EAG-IX), la exacta definición nosológica aún no está resulta. La EAG-VI se refiere a un defecto en la fosforilasa hepática, enzima codificada por el gen PYGL, mientras que la EAG-IX es causada por un defecto genético en una de las subunidades de la fosforilasa b quinasa hepática codicadas por los genes PHKA2, PHKB y PHKG2, respectivamente. El objetivo del presente trabajo es propender a la definición nosológica de pacientes con defectos en el sistema de la fosforilasa mediante una estrategia de análisis molecular investigando los genes PYGL, PHKA2, PHKB y PHKG2. Los pacientes incluidos en este estudio deberán ser compatibles de padecer una EAG-VI o EAG-IX sobre la base de síntomas clínicos y hallazgos bioquímicos. La metodología incluirá la determinación de la enzima fosforilasa b quinasa en glóbulos rojos y dentro del análisis molecular la extracción de DNA genómico a partir de sangre entera para la amplificación por PCR de los exones más las uniones exon/intron de los genes PHKG2 y PYGL y la extracción de RNA total y obtención de cDNA para posterior amplificación de los cDNA PHKA2 y PHKB. Todos los fragmentos amplificados serán sometidos a análisis de secuencia de nucleótidos. Resultados esperados. Este trabajo, primero en Argentina, permitirá establecer las bases moleculares de los defectos del sistema de la fosforilasa hepática (EAG-VI y EAG-IX). El poder lograr este nivel de investigación traerá aparejado, una oferta integrativa en el vasto capítulo de las glucogenosis hepáticas, con extraordinaria significación en la práctica asistencial para el manejo, pronóstico y correspondiente asesoramiento genético. Hepatic glycogen storage diseases (GSDs) are a group of disorders produced by a deficiency in a specific protein involved in the metabolism of glycogen causing different types of GSDs. Phenotypic heterogeneity of affected patients difficult to identify the different GSD variants and therefore the correct definition of the disease. In the “Centro de Estudio de las Metabolopatías Congénitas”, CEMECO, were defined the different GSD types by a protocol which included complex gradual levels of clinical, biochemical, enzymatic and morphological investigation. However, in some patients, like those one with defects in the hepatic phosphorylase system (GSD-VI and GSD-IX) the exact definition of the disease has not yet been resolved. The GSD-VI is produced by a defect in the PYGL gen that encode the liver phosphorylase, while the GSD-IX is caused by a genetic defect in one of the Phosphorylase b kinase subunits, encoded by the PHKA2, PHKB and PHKG2 genes, respectively. The aim of the present study is to define the phosphorylase system defects in argentinian patients through a molecular strategy that involve the investigation of PYGL, PHKA2, PHKB and PHKG2 genes. Patients included in the present study must be compatible with a GSD-VI or GSD-IX on the bases of clinical symptoms and biochemical findings. The phosphorylase b kinase activity will be assay on in blood red cells. The molecular study will include genomic DNA extraction for the amplification of PHKG2 and PYGL genes and the total RNA extraction for amplification of the PHKA2 and PHKB cDNA by PCR. All PCR-amplified fragments will be subjected to direct nucleotide sequencing. This work, first in Argentina, will make possible to establish the molecular basis of the defects on the hepatic phosphorylase system (GSD-VI and GSD IX). To achieve this level of research will entail advance in the study of the hepatic glycogen storage disease, with extraordinary significance in the treatment, prognosis and the genetic counselling.