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Trypanosoma cruzi trypomastigotes adhere to extracellular matrix (ECM) to invade mammalian host cells regulating intracellular signaling pathways. Herein, resin-assisted enrichment of thiols combined with mass spectrometry were employed to map site-specific S-nitrosylated (SNO) proteins from T. cruzi trypomastigotes incubated (MTy) or not (Ty) with ECM. We confirmed the reduction of S-nitrosylation upon incubation with ECM, associated with a rewiring of the subcellular distribution and intracellular signaling pathways. Forty, 248 and 85 SNO-peptides were identified only in MTy, Ty or in both conditions, respectively. SNO proteins were enriched in ribosome, transport, carbohydrate and lipid metabolisms. Nitrosylation of histones H2B and H3 on Cys64 and Cys126, respectively, is described. Protein-protein interaction networks revealed ribosomal proteins, proteins involved in carbon and fatty acid metabolism to be among the enriched protein complexes. Kinases, phosphatases and enzymes involved in the metabolism of carbohydrates, lipids and amino acids were identified as nitrosylated and phosphorylated, suggesting a post-translational modifications crosstalk. In silico mapping of nitric oxide synthase (NOS) genes, previously uncharacterized, matched to four putative T. cruzi proteins expressing C-terminal NOS domain. Our results provide the first site-specific characterization of S-nitrosylated proteins in T. cruzi and their modulation upon ECM incubation before infection of the mammalian hosts. SIGNIFICANCE: Protein S-nitrosylation represents a major molecular mechanism for signal transduction by nitric oxide. We present for the first time a proteomic profile of S-nitrosylated proteins from infective forms of T. cruzi, showing a decrease in SNO proteins after incubation of the parasite with the extracellular matrix, a necessary step for the parasite invasion of the host mammalian cells. We also show for the first time nitrosylation of H2B (Cys64) and H3 (Cys126) histones, sites not conserved in higher eukaryotic cells, and suggest that some specific histone isoforms are sensitive to NO signaling. S-nitrosylation in H2B and H3 histones are more abundant in MTy. Moreover, proteins involved in translation, glycolytic pathway and fatty acid metabolism are enriched in the present dataset. Comparison of the SNO proteome and the phosphoproteome, obtained previously under the same experimental conditions, show that most of the proteins sharing both modifications are involved in metabolic pathways, transport and ribosome function. The data suggest that both PTMs are involved in reprogramming the metabolism of T. cruzi in response to environmental changes. Although NO synthesis was detected in T. cruzi, the identification of NOS remains elusive. Analysis in silico showed two genes similar in domains to NADPH-dependent cytochrome-P450 reductase and two putative oxidoreductases, but no oxygenase domain of NOS was mapped in the T. cruzi genome. It is tempting to speculate that NO synthase-like from T. cruzi and its early NO-mediated pathways triggered in response to host interaction constitute potential diagnostic and therapeutic targets.

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Biofilms can be formed on the surfaces of dairy processing equipment and are a potential source of product contamination. This study evaluated the diversity of multispecies biofilms formed on stainless steel (SS) due to the contaminating microbiota in raw milk. Samples of raw milk were used: one was fresh milk and the other maintained in refrigerated bulk tanks for up to 48 h. The mesophilic aerobic contamination was ∼104 CFU ml-1 in fresh milk and 106 CFU ml-1 in bulk milk. SS coupons were kept immersed in the milk at 7 ±2 °C for 10 days, and every two days, the raw milk was changed for samples of the same origin collected on the current day. After incubation for 10 days, sessile cells in the biofilm reached 105 CFU cm-2 in the presence of fresh milk, and 106 CFU cm-2 in the presence of bulk milk. The genetic diversity analysis showed that Gammaproteobacteria and Bacilli predominated in the biofilms throughout the incubation of both milk samples and these biofilms showed a reduction in diversity over time. The main classes of bacteria found in these biofilms have representatives of great importance since many of them have spoilage potential.

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Since the first assembled genomes, gene sequences alone have not been sufficient to understand complex metabolic processes involving several genes, each playing distinct roles. To identify their roles, a network of interactions, wherein each gene is a node, should be created. Edges connecting nodes are evidence of interaction, for instance, of gene products coexisting in the same cellular component. Such interaction networks are called protein-protein interactions (PPIs). After genome assembling, PPI mapping is used to predict the possibility of proteins interacting with other proteins based on literature evidence and several databases, thus enriching genome annotations. Identifying PPIs involves analyzing each possible protein pair for a set of features, for instance, participation in the same biological process and having the same function and status in a cellular component. Here, we investigated using the three categories of the Gene Ontology (GO) database for efficient PPI prediction, because it provides data about the three features exemplified here. For a broader conclusion, we investigated the genomes of ten different human pathogens, looking for commonality regarding the GO hierarchical relationship-denominated IS_A. The plasmids were examined separately from their main genomes. Protein pairs sharing at least one IS_A value were considered as interacting proteins. STRING results certified the probed interactions as sensitivity (score >0.75) and specificity (score <0.25) analysis. The average areas under the receiver operating characteristic curve for all organisms were 0.66 and 0.53 for their genomes and plasmids, respectively. Thus, GO categories alone could not potentially provide reliable PPI prediction. However, using additional features can improve predictions.

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BACKGROUND: Orthodontic procedures are often limited by the presence of bone defects caused by trauma, periodontal diseases or surgeries, thus requiring the development of materials capable to compensate such deficiencies. Since bone morphogenetic proteins (BMPs) are indicative of bone reconstitution, this study aimed to evaluate histological and immunohistochemically the temporal location of BMP-2 and BMP-4 in osteoblasts of rat alveolar wounds filled with demineralized human dentine matrix (DHDM), used as a graft material. METHODS: After extraction of the upper second molars, the left side alveoli were filled with DHDM and the right side served as the control. The animals were euthanized after 3, 5, 10 and 14 days of surgery. After fixation, demineralization and paraffin embedding, representative samples of each group were stained with H&E and immunohistochemically evaluated. RESULTS: The data showed a statistically significant (p < 0.05) increased number of osteoblasts positively immunostained for BMP-2 and BMP-4 on the experimental side (left) at 10 days. Our results also showed that even when not degraded, dentine matrix was incorporated to new bone formation after 14 days of surgery. CONCLUSIONS: The results suggest that DHDM acts as a scaffold for osteoblast differentiation, actively yielding new bone formation, and it may represent an effective bone implant material.

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Humicola grisea var. thermoidea is a deuteromycete which secretes a large spectrum of hydrolytic enzymes when grown on lignocellulosic residues. This study focused on the heterologous expression and recombinant enzyme analysis of the major secreted cellulase when the fungus is grown on sugarcane bagasse as the sole carbon source. Cellobiohydrolase 1.2 (CBH 1.2) cDNA was cloned in Pichia pastoris under control of the AOX1 promoter. Recombinant protein (rCBH1.2) was efficiently produced and secreted as a functional enzyme, presenting a molecular mass of 47 kDa. Maximum enzyme production was achieved at 96 h, in culture medium supplemented with 1.34 % urea and 1 % yeast extract and upon induction with 1 % methanol. Recombinant enzyme exhibited optimum activity at 60 °C and pH 8, and presented a remarkable thermostability, particularly at alkaline pH. Activity was evaluated on different cellulosic substrates (carboxymethyl cellulose, filter paper, microcrystalline cellulose and 4-para-nitrophenyl ß-D-glucopyranoside). Interestingly, rCBH1.2 presented both exoglucanase and endoglucanase activities and mechanical agitation increased substrate hydrolysis. Results indicate that rCBH1.2 is a potential biocatalyst for applications in the textile industry or detergent formulation.

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A brucelose é uma doença infecto-contagiosa, de caráter zoonótico e de grande importância tanto em produção animal, quanto na saúde pública. É causada por bactérias do gênero Brucella, que provocam orquites e epididimites uni ou bilaterais em touros, podendo levar a sub-fertilidade e a esterilidade. Machos bovinos assintomáticos tornam-se importantes disseminadores desta doença nos rebanhos. Desta forma objetivou-se avaliar o protocolo de extração de DNA de Brucella spp em sêmen in natura bovino, submetendo-os aos testes diagnósticos PCR (protocolo Lise Enzimática por Proteinase e Fenol: Clorofórmio – PK), Antígeno Acidificado Tamponado (AAT) e Sêmem Plasma Aglutinação (SPA), de 33 touros utilizados em monta natural. O protocolo PK foi eficaz na extração de DNA de Brucella spp. Dos 33 touros testados, oito foram reagentes na PCR (24,24%), seis (18,18%) reagentes na SPA, e dos 23 animais com amostra de soro sanguíneo, um (4%) foi reagente para o AAT. Apenas um animal foi reagente na SPA e no AAT, e não reagente na PCR. Por outro lado sete animais apresentaram-se com s&am

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A brucelose é uma doença infecto-contagiosa, de caráter zoonótico e de grande importância tanto em produção animal, quanto na saúde pública. É causada por bactérias do gênero Brucella, que provocam orquites e epididimites uni ou bilaterais em touros, podendo levar a sub-fertilidade e a esterilidade. Machos bovinos assintomáticos tornam-se importantes disseminadores desta doença nos rebanhos. Desta forma objetivou-se avaliar o protocolo de extração de DNA de Brucella spp em sêmen in natura bovino, submetendo-os aos testes diagnósticos PCR (protocolo Lise Enzimática por Proteinase e Fenol: Clorofórmio – PK), Antígeno Acidificado Tamponado (AAT) e Sêmem Plasma Aglutinação (SPA), de 33 touros utilizados em monta natural. O protocolo PK foi eficaz na extração de DNA de Brucella spp. Dos 33 touros testados, oito foram reagentes na PCR (24,24%), seis (18,18%) reagentes na SPA, e dos 23 animais com amostra de soro sanguíneo, um (4%) foi reagente para o AAT. Apenas um animal foi reagente na SPA e no AAT, e não reagente na PCR. Por outro lado sete animais apresentaram-se com s&am

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This paper reports one case, of an ameloblastic fibro-odontosarcoma (AFOS) affecting the mandible, in a 12-year-old girl. This neoplasm is a rare odontogenic neoplasm. To the authors' knowledge this is the fifteenth case of AFOS reported in English. The patient's chief complaint was a swelling in the face for 6 months. An incisional biopsy was performed diagnosing the case as an ameloblastic fibroma. After radiography ameloblastic fibro-odontoma was diagnosed. Computed tomography was performed and a stereolithography model made to plan the surgical procedures. A hemimandibulectomy followed by a vascularized fibular flap was then proposed. The surgery was uneventful. Microscopic features diagnosed an AFOS. After 23 months of close follow-up there is no sign of recurrence or metastasis. Dental implants were recently placed in the fibular flap.

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The present work intended to test the validity of the quantitative datas provided by computerized baropodometry based in three comparisons: between static vertical force on the three regions of the foot and weight, between vertical force on the three regions of the foot during gait and weight, and between peak plantar pressure on the three regions of the foot and weight. It was used body weight because the calibration of the equipment is done in relation to the body weight of the patient. It was selected ten volunteers without foot pain complaints, age between 27-54 years old, 6 women and 4 men. The equipment used was the FSCAN version 1.821 (Teckscan, Boston MA), with new insoles. At the static assessment, the correlation between vertical force and weight was statistically significant only to the midfoot. At the assessment during gait the correlation between vertical force and weight was statistically significant for all regions of the foot and the correlation between peak plantar pressure and weight was statistically significant only to the midfoot. The clinical interpretation of the quantitative data provided by this exam must be done with caution due to uncount variables that are involved.

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