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Rectal cancer is currently the second most common cancer in the large intestine, and represents one-third of the colorectal cancers that are diagnosed. Neoadjuvant chemoradiation is a well-established protocol for rectal cancer treatment and it reduces the risk of local recurrence. However, a pathologic complete response is only achieved in 10–40% of cases and the mechanisms associated with chemoradiation resistance are poorly understood. To identify potential targets for preventing therapy resistance, a proteomic analysis of biopsy specimens collected from stage II and III rectal adenocarcinoma patients before neoadjuvant treatment was performed. These results were then compared with a proteomic analysis of residual rectal adenocarcinoma tissues that were removed by surgery after neoadjuvant therapy. Three proteins, Ku70, Ku80, and Rab5C, exhibited a significant increase in expression after chemoradiation treatment. To better understand the possible role of these proteins in therapy resistance, a rectal adenocarcinoma cell line was irradiated to generate a radiotherapy-resistant lineage. These cells overexpressed the same three proteins that were identified in the tissue samples. Furthermore, radiotherapy resistance in this in vitro model was found to involve modulation of epidermal growth factor receptor (EGFR) internalization by Rab5C in response to irradiation, and this affected expression of the DNA repair proteins, Ku70 and Ku80. Taken together, these findings indicate that EGFR and Rab5C are potential targets for the sensitization of rectal cancer cells to neoadjuvant treatment and they should be further investigated. Supported by FAPESP - Fundação de Amparo à Pesquisa do Estado de São Paulo. Note: This abstract was not presented at the conference. Citation Format: Antuani Rafael Baptistella, Marcos Vinicios Salles Dias, Fernanda Giudice, Bruna Roz Rodrigues, Petrus Paulo Combas Eufrazio Silva, Edson Kuatelela Cassinela, Tonielli Lacerda, Fabio Marchi, Adriana Franco Paes Leme, Maria Dirlei Begnami, Samuel Aguiar Junior, Vilma Regina Martins. Rab5C enhances resistance to ionizing radiation in rectal cancer [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B45.

SummaryHaemorrhage induced by snake venom metalloproteinases (SVMPs) is a complex phenomenon resulting in capillary disruption and extravasation. This study analysed structural elements important for the interaction of four Bothrops jararaca SVMPs of different domain organisation and glycosylation levels with plasma and extracellular matrix proteins: HF3 (P-III class) is highly glycosylated and ~80 times more haemorrhagic than bothropasin (P-III class), which has a minor carbohydrate moiety; BJ-PI (P-I class) is not haemorrhagic and the DC protein is composed of disintegrin-like/cysteine-rich domains of bothropasin. HF3, bothropasin and BJ-PI showed different degradation profiles of fibrinogen, fibronectin, vitronectin, von Willebrand factor, collagens IV and VI, laminin and Matrigel™; however, only bothropasin degraded collagen I. In solid-phase binding assays HF3 and bothropasin interacted with fibrinogen, fibronectin, laminin, collagens I and VI; the DC protein bound only to collagens I and VI; however, no binding of BJ-PI to these proteins was detected. N-deglycosylation caused loss of structural stability of bothropasin and BJ-PI but HF3 remained intact, although its haemorrhagic and fibrinogenolytic activities were partially impaired. Nevertheless, N-deglycosylated HF3 bound with higher affinity to collagens I and VI, although its proteolytic activity upon these collagens was not enhanced. This study demonstrates that features of carbohydrate moieties of haemorrhagic SVMPs may play a role in their interaction with substrates of the extracellular matrix, and the ability of SVMPs to degrade proteins in vitro does not correlate to their ability to cause haemorrhage, suggesting that novel, systemic approaches are necessary for understanding the mechanism of haemorrhage generation by SVMPs.

In situ dental biofilm composition under sugar exposure is well known, but sugar effect on the genotypic diversity of S. mutans in dental biofilm has not been explored. This study evaluated S. mutans genotypic diversity in dental biofilm formed in situ under frequent exposure to sucrose and its monosaccharide constituents (glucose and fructose). Saliva of 7 volunteers was collected for isolation of S. mutans and the same volunteers wore intraoral palatal appliances, containing enamel slabs, which were submitted to the following treatments: distilled and deionized water (negative control), 10% glucose + 10% fructose (fermentable carbohydrates) solution or 20% sucrose (fermentable and EPS inductor) solution, 8x/day. After 3, 7 and 14 days, the biofilms were colleted and S. mutans colonies were isolated. Arbitrarily primed polymerase chain reaction (AP-PCR) of S. mutans showed that salivary genotypes were also detected in almost all biofilm samples, independently of the treatment, and seemed to reflect those genotypes present at higher proportion in biofilms. In addition to the salivary genotypes, others were found in biofilms but in lower proportions and were distinct among treatment. The data suggest that the in situ model seems to be useful to evaluate genotypic diversity of S. mutans, but, under the tested conditions, it was not possible to clearly show that specific genotypes were selected in the biofilm due to the stress induced by sucrose metabolism or simple fermentation of its monosaccharides. A composição do biofilme dental in situ exposto a açúcares é bem conhecida, mas o efeito dos açúcares na diversidade genotípica de S. mutans no biofilme dental ainda não foi explorada. Este estudo avaliou a diversidade genotípica de S. mutans no biofilme dental formado in situ sob frequente exposição à sacarose e seus monossacarídeos constituintes (glicose e frutose). Primeiramente, saliva de voluntários foi coletada para isolamento de S. mutans e os mesmos voluntários usaram um dispositivo intraoral palatino, contendo blocos de esmalte, que foram submetidos 8x/dia aos seguintes tratamentos: água destilada e deionizada (controle negativo), solução de glicose 10% + frutose 10% (carboidratos fermentáveis) e solução de sacarose 20% (fermentável e indutor de PEC). Após 3, 7 e 14 dias, os biofilmes foram coletados e colônias de S. mutans foram isoladas. A técnica de reação em cadeia de polimerase usando primers arbitrários (AP-PCR) demonstrou que o genótipo salivar foi detectado em quase todas as amostras de biofilme, independente do tratamento, e parece refletir aqueles genótipos presentes em maiores proporções no biofilme. Além do genótipo salivar, outros foram encontrados nos biofilmes, mas em uma menor proporção e foram distintos entre os tratamentos. Os dados sugerem que o modelo in situ é útil para a avaliação da diversidade genotípica de S. mutans. Porém, nas condições do presente estudo, não foi possível demonstrar que genótipos específicos foram detectados no biofilme devido ao estresse induzido pelo metabolismo da sacarose ou fermentação de seus monossacarídeos.

Background The composition of the matrix molecules is important in in vitro cell culture experiments of e.g. human cancer invasion and vessel formation. Currently, the mouse Engelbreth-Holm-Swarm (EHS) sarcoma -derived products, such as Matrigel®, are the most commonly used tumor microenvironment (TME) mimicking matrices for experimental studies. However, since Matrigel® is non-human in origin, its molecular composition does not accurately simulate human TME. We have previously described a solid 3D organotypic myoma disc invasion assay, which is derived from human uterus benign leiomyoma tumor. Here, we describe the preparation and analyses of a processed, gelatinous leiomyoma matrix, named Myogel. Methods A total protein extract, Myogel, was formulated from myoma. The protein contents of Myogel were characterized and its composition and properties compared with a commercial mouse Matrigel®. Myogel was tested and compared to Matrigel® in human cell adhesion, migration, invasion, colony formation, spheroid culture and vessel formation experiments, as well as in a 3D hanging drop video image analysis. Results We demonstrated that only 34 % of Myogel’s molecular content was similar to Matrigel®. All test results showed that Myogel was comparable with Matrigel®, and when mixed with low-melting agarose (Myogel-LMA) it was superior to Matrigel® in in vitro Transwell® invasion and capillary formation assays. Conclusions In conclusion, we have developed a novel Myogel TME matrix, which is recommended for in vitro human cell culture experiments since it closely mimics the human tumor microenvironment of solid cancers. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1944-z) contains supplementary material, which is available to authorized users.

The development and progression of oral cavity squamous cell carcinoma (OSCC) involves complex cellular mechanisms that contribute to the low five-year survival rate of approximately 20% among diagnosed patients. However, the biological processes essential to tumor progression are not completely understood. Therefore, detecting alterations in the salivary proteome may assist in elucidating the cellular mechanisms modulated in OSCC and improve the clinical prognosis of the disease. The proteome of whole saliva and salivary extracellular vesicles (EVs) from patients with OSCC and healthy individuals were analyzed by LC-MS/MS and label-free protein quantification. Proteome data analysis was performed using statistical, machine learning and feature selection methods with additional functional annotation. Biological processes related to immune responses, peptidase inhibitor activity, iron coordination and protease binding were overrepresented in the group of differentially expressed proteins. Proteins related to the inflammatory system, transport of metals and cellular growth and proliferation were identified in the proteome of salivary EVs. The proteomics data were robust and could classify OSCC with 90% accuracy. The saliva proteome analysis revealed that immune processes are related to the presence of OSCC and indicate that proteomics data can contribute to determining OSCC prognosis.

Deregulated expression of activin A is reported in several tumors, but its biological functions in oral squamous cell carcinoma (OSCC) are unknown. Here, we investigate whether activin A can play a causal role in OSCCs. Activin A expression was assessed by qPCR and immunohistochemistry in OSCC tissues. Low activin A-expressing cells were treated with recombinant activin A and assessed for apoptosis, proliferation, adhesion, migration, invasion and epithelial-mesenchymal transition (EMT). Those phenotypes were also evaluated in high activin A-expressing cells treated with follistatin (an activin A antagonist) or stably expressing shRNA targeting activin A. Transfections of microRNA mimics were performed to determine whether the overexpression of activin A is regulated by miR-143/miR-145 cluster. Activin A was overexpressed in OSCCs in comparison with normal oral mucosa, and high activin A levels were significantly associated with lymph node metastasis, tumor differentiation and poor survival. High activin A levels promoted multiple properties associated with malignant transformation, including decreased apoptosis and increased proliferation, migration, invasion and EMT. Both miR-143 and miR-145 were markedly downregulated in OSCC cell lines and in clinical specimens, and inversely correlated to activin A levels. Forced expression of miR-143 and miR-145 in OSCC cells significantly decreased the expression of activin A. Overexpression of activin A in OSCCs, which is controlled by downregulation of miR-143/miR-145 cluster, regulates apoptosis, proliferation and invasiveness, and it is clinically correlated with lymph node metastasis and poor survival.

S6Ks are major effectors of the mTOR (mammalian target of rapamycin) pathway, signaling for increased protein synthesis and cell growth in response to insulin, AMP/ATP levels, and amino acids. Deregulation of this pathway has been related to disorders and diseases associated with metabolism, such as obesity, diabetes, and cancer. S6K family is composed of two main members, S6K1 and S6K2, which comprise different isoforms resulted from alternative splicing or alternative start codon use. Although important molecular functions have been associated with p70-S6K1, the most extensively studied isoform, the S6K2 counterpart lacks information. In the present study, we performed immunoprecipitation assays followed by mass spectrometry (MS) analysis of FLAG-tagged p70-S6K1 and p54-S6K2 interactomes, after expression in HEK293 cells. Protein lists were submitted to CRAPome (Contaminant Repository for Affinity Purification) and SAINT (Significance Analysis of INTeractome) analysis, which allowed the identification of high-scoring interactions. By a comparative approach, p70-S6K1 interacting proteins were predominantly related to "cytoskeleton" and "stress response," whereas p54-S6K2 interactome was more associated to "transcription," "splicing," and "ribosome biogenesis." Moreover, we have found evidences for new targets or regulators of the S6K protein family, such as proteins NCL, NPM1, eIF2α, XRCC6, PARP1, and ILF2/ILF3 complex. This study provides new information about the interacting networks of S6Ks, which may contribute for future approaches to a better understanding of the mTOR/S6K pathway.

This study evaluated the effects of pH cycling on fluoride release and bond strength of two self-etching adhesive systems to both enamel and dentin. The ultramorphology of the interfaces produced by the adhesive systems were also analyzed.The buccal surfaces of bovine incisors were flattened to expose enamel and dentin, which were bonded with either Clearfil Protect Bond (CPB) or One-Up Bond F Plus (OBP). The bonded samples were prepared for microtensile bond strength (μTBS) testing, fluoride ion release, and transmission electron microscopy. pH cycling comprised demineralization (8 h/day) and remineralization (16 h/day) cycles for 8 days. The μTBS data were analyzed by two-way ANOVA, while fluoride release was analyzed using the Friedman and Wilcoxon tests.The adhesives presented similar bond strengths to enamel. However, the dentin bond strength of CPB was higher than that of OBP. pH cycling did not influence enamel or dentin μTBS. The amount of fluoride released from the bonded enamel and dentin was low and varied among the groups. The morphological evaluation showed that the thickness of the dentin hybrid layers was similar for both adhesives.The pH-cycling regime did not affect enamel or dentin bond strengths. In enamel, both the self-etching adhesives tested presented similar bond strengths, but in dentin, Clearfil Protect Bond showed higher dentin bonding than One-Up Bond F Plus.

Hepatitis B and C virus (HBV and HCV) infections are an important cause of cirrhosis and hepatocellular carcinoma. The natural history has a prominent latent phase, and infected patients may remain undiagnosed; this situation may lead to the continuing spread of these infections in the community. Compelling reasons exist for using saliva as a diagnostic fluid because it meets the demands of being an inexpensive, noninvasive and easy-to-use diagnostic method. Indeed, comparative analysis of the salivary proteome using mass spectrometry is a promising new strategy for identifying biomarkers. Our goal is to apply an Orbitrap-based quantitative approach to explore the salivary proteome profile in HBV- and HCV-infected patients. In the present study, whole saliva was obtained from 20 healthy, (control) 20 HBV-infected and 20 HCV-infected subjects. Two distinct pools containing saliva from 10 subjects of each group were obtained. The samples were ultracentrifuged and fractionated, and all fractions were hydrolyzed (trypsin) and injected into an LTQ-VELOS ORBITRAP. The identification and analyses of peptides were performed using Proteome Discoverer1.3 and ScaffoldQ + v.3.3.1. From a total of 362 distinct proteins identified, 344 proteins were identified in the HBV, 326 in the HCV and 303 in the control groups. Some blood proteins, such as flavin reductase (which converts biliverdin to bilirubin), were detected only in the HCV group. The data showed a reduced presence of complement C3, ceruloplasmin, alpha(1)-acid glycoprotein and alpha(2)-acid glycoprotein in the hepatitis-infected patients. Peptides of serotransferrin and haptoglobin were less detected in the HCV group. This study provides an integrated perspective of the salivary proteome, which should be further explored in future studies targeting specific disease markers for HBV and HCV infection.

Understanding the molecular mechanisms of oral carcinogenesis will yield important advances in diagnostics, prognostics, effective treatment, and outcome of oral cancer. Hence, in this study we have investigated the proteomic and peptidomic profiles by combining an orthotopic murine model of oral squamous cell carcinoma (OSCC), mass spectrometry-based proteomics and biological network analysis. Our results indicated the up-regulation of proteins involved in actin cytoskeleton organization and cell-cell junction assembly events and their expression was validated in human OSCC tissues. In addition, the functional relevance of talin-1 in OSCC adhesion, migration and invasion was demonstrated. Taken together, this study identified specific processes deregulated in oral cancer and provided novel refined OSCC-targeting molecules.

Analysis of the metaproteome of microbial communities is important to provide an insight of community physiology and pathogenicity. This study evaluated the metaproteome of endodontic infections associated with acute apical abscesses and asymptomatic apical periodontitis lesions. Proteins persisting or expressed after root canal treatment were also evaluated. Finally, human proteins associated with these infections were identified. Samples were taken from root canals of teeth with asymptomatic apical periodontitis before and after chemomechanical treatment using either NaOCl or chlorhexidine as the irrigant. Samples from abscesses were taken by aspiration of the purulent exudate. Clinical samples were processed for analysis of the exoproteome by using two complementary mass spectrometry platforms: nanoflow liquid chromatography coupled with linear ion trap quadrupole Velos Orbitrap and liquid chromatography-quadrupole time-of-flight. A total of 308 proteins of microbial origin were identified. The number of proteins in abscesses was higher than in asymptomatic cases. In canals irrigated with chlorhexidine, the number of identified proteins decreased substantially, while in the NaOCl group the number of proteins increased. The large majority of microbial proteins found in endodontic samples were related to metabolic and housekeeping processes, including protein synthesis, energy metabolism and DNA processes. Moreover, several other proteins related to pathogenicity and resistance/survival were found, including proteins involved with adhesion, biofilm formation and antibiotic resistance, stress proteins, exotoxins, invasins, proteases and endopeptidases (mostly in abscesses), and an archaeal protein linked to methane production. The majority of human proteins detected were related to cellular processes and metabolism, as well as immune defense. Interrogation of the metaproteome of endodontic microbial communities provides information on the physiology and pathogenicity of the community at the time of sampling. There is a growing need for expanded and more curated protein databases that permit more accurate identifications of proteins in metaproteomic studies.

Dental cementum (DC) is a bone-like tissue covering the tooth root and responsible for attaching the tooth to the alveolar bone (AB) via the periodontal ligament (PDL). Studies have unsuccessfully tried to identify factors specific to DC versus AB, in an effort to better understand DC development and regeneration. The present study aimed to use matched human DC and AB samples (n=7) to generate their proteomes for comparative analysis. Bone samples were harvested from tooth extraction sites, whereas DC samples were obtained from the apical root portion of extracted third molars. Samples were denatured, followed by protein extraction reduction, alkylation and digestion for analysis by nanoAcquity HPLC system and LTQ-FT Ultra. Data analysis demonstrated that a total of 318 proteins were identified in AB and DC. In addition to shared proteins between these tissues, 105 and 83 proteins exclusive to AB or DC were identified, respectively. This is the first report analyzing the proteomic composition of human DC matrix and identifying putative unique and enriched proteins in comparison to alveolar bone. These findings may provide novel insights into developmental differences between DC and AB, and identify candidate biomarkers that may lead to more efficient and predictable therapies for periodontal regeneration.Periodontal disease is a highly prevalent disease affecting the world population, which involves breakdown of the tooth supporting tissues, the periodontal ligament, alveolar bone, and dental cementum. The lack of knowledge on specific factors that differentiate alveolar bone and dental cementum limits the development of more efficient and predictable reconstructive therapies. In order to better understand cementum development and potentially identify factors to improve therapeutic outcomes, we took the unique approach of using matched patient samples of dental cementum and alveolar bone to generate and compare a proteome list for each tissue. A potential biomarker for dental cementum was identified, superoxide dismutase 3 (SOD3), which is found in cementum and cementum-associated cells in mouse, pig, and human tissues. These findings may provide novel insights into developmental differences between alveolar bone and dental cementum, and represent the basis for improved and more predictable therapies.

The phosphate-dependent transition between enzymatically inert dimers into catalytically capable tetramers has long been the accepted mechanism for the glutaminase activation. Here, we demonstrate that activated glutaminase C (GAC) self-assembles into a helical, fiber-like double-stranded oligomer and propose a molecular model consisting of seven tetramer copies per turn per strand interacting via the N-terminal domains. The loop 321LRFNKL326 is projected as the major regulating element for self-assembly and enzyme activation. Furthermore, the previously identified in vivo lysine acetylation (Lys311 in humans, Lys316 in mouse) is here proposed as an important down-regulator of superoligomer assembly and protein activation. Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide, a known glutaminase inhibitor, completely disrupted the higher order oligomer, explaining its allosteric mechanism of inhibition via tetramer stabilization. A direct correlation between the tendency to self-assemble and the activity levels of the three mammalian glutaminase isozymes was established, with GAC being the most active enzyme while forming the longest structures. Lastly, the ectopic expression of a fiber-prone superactive GAC mutant in MDA-MB 231 cancer cells provided considerable proliferative advantages to transformed cells. These findings yield unique implications for the development of GAC-oriented therapeutics targeting tumor metabolism. Background: GAC supplies for increased metabolic needs of tumors because of exclusive localization and kinetic properties. Results: Higher than tetramer oligomers are the active form in in vitro and in cellular assays. Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide disrupts oligomers. Conclusion: A novel molecular mechanism for GAC activation is proposed. Significance: The data affect the development of therapies targeting GAC in tumors, with emphasis on allosteric inhibitors.

Oral cancer disease represents a significant fraction of all human cancer types and its poor early diagnosis contributes to reduced individual survival rate. The identification of proteins modulated in tumorigenic cells and its post-translational modifications may improve our understanding of tumor development in epithelial cells. We have analyzed the phosphoproteome of tumorigenic (SCC-9) and non-tumorigenic (HaCaT) cell lines using MS-based approach in order to identify phosphopeptides with differing patterns of modifications and/or abundance. Our results revealed the identity of 4,206 protein phosphorylation sites with sixty-two sites showing to be significantly modulated between the two cell lines. The phosphoproteome data showed an overrepresentation of proteins with a possible role in nuclear regulatory functions. Pathway analysis was further performed on the phosphoproteome dataset and differences and commonalities of the functional pathways present in tumorigenic and non-tumorigenic cells were identified. Phosphopeptides that belong to the proteins lamina-associated polypeptide 2 isoform alpha and serine–arginine repetitive matrix protein 2 were identified with differential abundance and they appear as promising tumor-related phosphopeptides. These two proteins may be related to the structural alterations generally found in the nucleus of tumorigenic cells. The identification of phosphorylation sites in tumorigenic cells may contribute to disclose novel signaling mechanisms associated with OSCC. Significance Oral Squamous Cell Carcinoma (OSCC) is an important cancer disease affecting thousands of people worldwide. Many cellular processes related to the development of oral cancer remain unknown; however, the studies performed in vitro with cancer cells have contributed to guide more specific research which may be further performed by using in vivo approaches or clinical samples. To our knowledge, only few studies have been published showing the results of phosphoproteome profiling of squamous cell carcinoma models, and many signaling proteins must be identified and functionally characterized in order to increase the knowledge available about the complexity of the signaling networks responsible for oral cancer development and its progression. Furthermore, our knowledge regarding proteins exclusive or very low abundant in cancer cells remains limited. A better understanding of the differences between signaling pathways present in epithelial cell lines may contribute to reveal the processes underlying the OSCC.

Background Snake Venom Metalloproteinases (SVMPs) are amongst the key enzymes that contribute to the high toxicity of snake venom. We have recently shown that snake venoms from the Bothrops genus activate the Complement system (C) by promoting direct cleavage of C-components and generating anaphylatoxins, thereby contributing to the pathology and spread of the venom. The aim of the present study was to isolate and characterize the C-activating protease from Bothrops pirajai venom. Results Using two gel-filtration chromatography steps, a metalloproteinase of 23 kDa that activates Complement was isolated from Bothrops pirajai venom. The mass spectrometric identification of this protein, named here as C-SVMP, revealed peptides that matched sequences from the P-I class of SVMPs. C-SVMP activated the alternative, classical and lectin C-pathways by cleaving the α-chain of C3, C4 and C5, thereby generating anaphylatoxins C3a, C4a and C5a. In vivo, C-SVMP induced consumption of murine complement components, most likely by activation of the pathways and/or by direct cleavage of C3, leading to a reduction of serum lytic activity. Conclusion We show here that a P-I metalloproteinase from Bothrops pirajai snake venom activated the Complement system by direct cleavage of the central C-components, i.e., C3, C4 and C5, thereby generating biologically active fragments, such as anaphylatoxins, and by cleaving the C1-Inhibitor, which may affect Complement activation control. These results suggest that direct complement activation by SVMPs may play a role in the progression of symptoms that follow envenomation., Author Summary The genus Bothrops inflicts the vast majority of snakebites in Central and South America and is responsible for 90% of snake envenomations in Brazil. Envenomations are characterized by prominent local effects, including edema and necrosis, and by systemic manifestations such as hemorrhage, coagulopathy and acute renal failure. Several components have been isolated from Bothrops venoms, and the snake venom metalloproteinases (SVMPs) are key enzymes contributing to the high toxicity of the venoms. Previously, we analyzed the pro-inflammatory properties of snake venoms from the genus Bothrops and demonstrated that several of them were potent activators of the Complement (C) system. C3a, C4a and C5a were generated in venom-treated sera not only through C-activation but also by direct cleavage of C-components. In the present study, we have isolated and characterized a metalloproteinase from Bothrops pirajai snake venom, named here as C-SVMP, which interferes with all three complement pathways, generating potent pro-inflammatory fragments, such as C3a, C4a and C5a. Our data suggest that C-activation by Bothrops pirajai venom is due to activity of an SVMP, which may play a role in the progression of symptoms that follow envenomation.

In eukaryotes, ribosome biogenesis involves excision of transcribed spacer sequences from the preribosomal RNA, base and ribose covalent modification at specific sites, assembly of ribosomal proteins, and transport of subunits from the nucleolus to the cytoplasm where mature ribosomes engage in mRNA translation. The biochemical reactions throughout ribosome synthesis are mediated by factors that associate transiently to the preribosomal complexes. In this work, we describe the complexes containing the human protein FTSJ3. This protein functions in association with NIP7 in ribosome synthesis and contains a putative RNA-methyl-transferase domain (FtsJ) in the N-terminal region and two uncharacterized domains in the central (DUF3381) and C-terminal (Spb1_C) regions. FLAG-tagged FTSJ3 coimmunoprecipitates both RPS and RPL proteins, ribosome synthesis factors, and proteins whose function in ribosome synthesis has not been demonstrated yet. A similar set of proteins coimmunoprecipitates with the Spb1_C domain, suggesting that FTSJ3 interaction with the preribosome complexes is mediated by the Spb1_C domain. Approximately 50% of the components of FTSJ3 complexes are shared by complexes described for RPS19, Par14, nucleolin, and NOP56. A significant number of factors are also found in complexes described for nucleophosmin, SBDS, ISG20L2, and NIP7. These findings provide information on the dynamics of preribosome complexes in human cells.

Impaired activity of the lysosomal enzyme glucocerebrosidase (GCR) results in the inherited metabolic disorder known as Gaucher disease. Current treatment consists of enzyme replacement therapy by administration of exogenous GCR. Although effective, it is exceptionally expensive, and patients worldwide have a limited access to this medicine. In Brazil, the public healthcare system provides the drug free of charge for all Gaucher’s patients, which reaches the order of $ 84 million per year. However, the production of GCR by public institutions in Brazil would reduce significantly the therapy costs. Here, we describe a robust protocol for the generation of a cell line producing recombinant human GCR. The protein was expressed in CHO-DXB11 (dhfr−) cells after stable transfection and gene amplification with methotrexate. As expected, glycosylated GCR was detected by immunoblotting assay both as cell-associated (~64 and 59 kDa) and secreted (63–69 kDa) form. Analysis of subclones allowed the selection of stable CHO cells producing a secreted functional enzyme, with a calculated productivity of 5.14 pg/cell/day for the highest producer. Although being laborious, traditional methods of screening high-producing recombinant cells may represent a valuable alternative to generate expensive biopharmaceuticals in countries with limited resources.

Class III peroxidases are present as large multigene families in all land plants. This large number of genes together with the diversity of processes catalyzed by peroxidases suggests possible functional specialization of each isoform. However, assigning a precise role for each individual peroxidase gene has continued to be a major bottleneck. Here we investigated the enzyme activity and translational profile of class III peroxidases during stem development of sugarcane as a first step in the estimation of physiological functions of individual isoenzymes. Internodes at three different developmental stages (young, developing and mature) were divided into pith (inner tissue) and rind (outer tissue) fractions. The rind of mature internodes presented the highest enzymatic activity and thus could be considered the ideal tissue for the discovery of peroxidase gene function. In addition, activity staining of 2DE gels revealed different isoperoxidase profiles and protein expression regulation among different tissue fractions. In-gel tryptic digestion of excised spots followed by peptide sequencing by LC-MS/MS positively matched uncharacterized peroxidases in the sugarcane database SUCEST. Multiple spots matching the same peroxidase gene were found, which reflects the generation of more than one isoform from a particular gene by post-translational modifications. The identified sugarcane peroxidases appear to be monocot-specific sequences with no clear ortholog in dicot model plant Arabidopsis thaliana.

Plant pathogenic bacteria utilize an array of effector proteins to cause disease. Among them, transcriptional activator-like (TAL) effectors are unusual in the sense that they modulate transcription in the host. Although target genes and DNA specificity of TAL effectors have been elucidated, how TAL proteins control host transcription is poorly understood. Previously, we showed that the Xanthomonas citri TAL effectors, PthAs 2 and 3, preferentially targeted a citrus protein complex associated with transcription control and DNA repair. To extend our knowledge on the mode of action of PthAs, we have identified new protein targets of the PthA4 variant, required to elicit canker on citrus. Here we show that all the PthA4-interacting proteins are DNA and/or RNA-binding factors implicated in chromatin remodeling and repair, gene regulation and mRNA stabilization/modification. The majority of these proteins, including a structural maintenance of chromosomes protein (CsSMC), a translin-associated factor X (CsTRAX), a VirE2-interacting protein (CsVIP2), a high mobility group (CsHMG) and two poly(A)-binding proteins (CsPABP1 and 2), interacted with each other, suggesting that they assemble into a multiprotein complex. CsHMG was shown to bind DNA and to interact with the invariable leucine-rich repeat region of PthAs. Surprisingly, both CsHMG and PthA4 interacted with PABP1 and 2 and showed selective binding to poly(U) RNA, a property that is novel among HMGs and TAL effectors. Given that homologs of CsHMG, CsPABP1, CsPABP2, CsSMC and CsTRAX in other organisms assemble into protein complexes to regulate mRNA stability and translation, we suggest a novel role of TAL effectors in mRNA processing and translational control.

Rear-fanged and aglyphous snakes are usually considered not dangerous to humans because of their limited capacity of injecting venom. Therefore, only a few studies have been dedicated to characterizing the venom of the largest parcel of snake fauna. Here, we investigated the venom proteome of the rear-fanged snake Thamnodynastes strigatus , in combination with a transcriptomic evaluation of the venom gland. About 60% of all transcripts code for putative venom components. A striking finding is that the most abundant type of transcript (∼47%) and also the major protein type in the venom correspond to a new kind of matrix metalloproteinase (MMP) that is unrelated to the classical snake venom metalloproteinases found in all snake families. These enzymes were recently suggested as possible venom components, and we show here that they are proteolytically active and probably recruited to venom from a MMP-9 ancestor. Other unusual proteins were suggested to be venom components: a protein related to lactadherin and an EGF repeat-containing transcript. Despite these unusual molecules, seven toxin classes commonly found in typical venomous snakes are also present in the venom. These results support the evidence that the arsenals of these snakes are very diverse and harbor new types of biologically important molecules.

Made available in DSpace on 2013-08-28T14:12:10Z (GMT). No. of bitstreams: 1 WOS000300718400001.pdf: 1303731 bytes, checksum: 558800e48e75bb2935d3f06e9cda50f0 (MD5) Made available in DSpace on 2013-09-30T18:47:00Z (GMT). No. of bitstreams: 2 WOS000300718400001.pdf: 1303731 bytes, checksum: 558800e48e75bb2935d3f06e9cda50f0 (MD5) WOS000300718400001.pdf.txt: 52377 bytes, checksum: 530737fc5207627abc77dde30fd6b8d5 (MD5) Previous issue date: 2011-11-14 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:55:54Z No. of bitstreams: 2 WOS000300718400001.pdf: 1303731 bytes, checksum: 558800e48e75bb2935d3f06e9cda50f0 (MD5) WOS000300718400001.pdf.txt: 52377 bytes, checksum: 530737fc5207627abc77dde30fd6b8d5 (MD5) Made available in DSpace on 2014-05-20T13:55:54Z (GMT). No. of bitstreams: 2 WOS000300718400001.pdf: 1303731 bytes, checksum: 558800e48e75bb2935d3f06e9cda50f0 (MD5) WOS000300718400001.pdf.txt: 52377 bytes, checksum: 530737fc5207627abc77dde30fd6b8d5 (MD5) Previous issue date: 2011-11-14 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Background: Lignocellulosic materials have been moved towards the forefront of the biofuel industry as a sustainable resource. However, saccharification and the production of bioproducts derived from plant cell wall biomass are complex and lengthy processes. The understanding of termite gut biology and feeding strategies may improve the current state of biomass conversion technology and bioproduct production.Results: The study herein shows comprehensive functional characterization of crude body extracts from Coptotermes gestroi along with global proteomic analysis of the termite's digestome, targeting the identification of glycoside hydrolases and accessory proteins responsible for plant biomass conversion. The crude protein extract from C. gestroi was enzymatically efficient over a broad pH range on a series of natural polysaccharides, formed by glucose-, xylose-, mannan- and/or arabinose-containing polymers, linked by various types of glycosidic bonds, as well as ramification types. Our proteomic approach successfully identified a large number of relevant polypeptides in the C. gestroi digestome. A total of 55 different proteins were identified and classified into 29 CAZy families. Based on the total number of peptides identified, the majority of components found in the C. gestroi digestome were cellulose-degrading enzymes. Xylanolytic enzymes, mannan-hydrolytic enzymes, pectinases and starch-degrading and debranching enzymes were also identified. Our strategy enabled validation of liquid chromatography with tandem mass spectrometry recognized proteins, by enzymatic functional assays and by following the degradation products of specific 8-amino-1,3,6-pyrenetrisulfonic acid labeled oligosaccharides through capillary zone electrophoresis.Conclusions: Here we describe the first global study on the enzymatic repertoire involved in plant polysaccharide degradation by the lower termite C. gestroi. The biochemical characterization of whole body termite extracts evidenced their ability to cleave all types of glycosidic bonds present in plant polysaccharides. The comprehensive proteomic analysis, revealed a complete collection of hydrolytic enzymes including cellulases (GH1, GH3, GH5, GH7, GH9 and CBM 6), hemicellulases (GH2, GH10, GH11, GH16, GH43 and CBM 27) and pectinases (GH28 and GH29). Univ Estadual Campinas, Genet Evolut & Bioagents Dept, LGE, Campinas, SP, Brazil CNPEM, Lab Nacl Cencia & Tecnol Bioetanol CTBE, Campinas, SP, Brazil Univ Estadual Campinas, Hematol & Hemotherapy Ctr, Campinas, SP, Brazil Univ Estadual Paulista UNESP, Inst Biocencias, Dept Biol, Rio Claro, Brazil CNPEM, Lab Nacl Biociencias LNBio, Campinas, SP, Brazil Univ Estadual Paulista UNESP, Inst Biocencias, Dept Biol, Rio Claro, Brazil FAPESP: 08/50114-4 FAPESP: 08/58037-9 FAPESP: 06/59086-8 FAPESP: 10/11469-1

The pharmacological activities displayed by Bothrops jararaca venom undergo a significant ontogenetic shift. Similarly, the diet of this species changes from ectothermic prey in early life to endothermic prey in adulthood. In this study we used large and representative newborn and adult venom samples consisting of pools from 694 and 110 specimens, respectively, and demonstrate a significant ontogenetic shift in the venom proteome complexity of B. jararaca. 2-DE coupled to MS protein identification showed a clear rearrangement of the toxin arsenal both in terms of the total proteome, as of the glycoproteome. N-glycosylation seems to play a key role in venom protein variability between newborn and adult specimens. Upon the snake development, the subproteome of metalloproteinases undergoes a shift from a P-III-rich to a P-I-rich profile while the serine proteinase profile does not vary significantly. We also used isobaric tag labeling (iTRAQ) of venom tryptic peptides for the first time to examine the quantitative changes in the venom toxins of B. jararaca upon neonate to adult transition. The iTRAQ analysis showed changes in various toxin classes, especially the proteinases. Our study expands the in-depth understanding of venom complexity variation particularly with regard to toxin families that have been associated with envenomation pathogenesis.

The oral cavity harbors several Streptococcus mutans genotypes, which could present distinct virulence properties. However, little is known about the diversity and virulence traits of S. mutans genotypes isolated in vivo under controlled conditions of high cariogenic challenge. This study evaluated the genotypic diversity of S. mutans isolated from dental biofilms formed in vivo under sucrose exposure, as well as their acidogenicity and aciduricity. To form biofilms, subjects rinsed their mouths with distilled water or sucrose solution 8 times/day for 3 days. S. mutans collected from saliva and biofilms were genotyped by arbitrarily-primed PCR. Genotypes identified in the biofilms were evaluated regarding their ability to lower the suspension pH through glycolysis and their acid susceptibility and F-ATPase activity. Most subjects harbored only one genotype in saliva, which was detected in almost all biofilm samples at high proportions. Genotypes isolated only in the presence of sucrose had higher acidogenicity than those isolated only in the presence of water. Genotypes from biofilms formed with sucrose were more aciduric after 30 and 60 min of incubation at pH 2.8 and 5.0, respectively. The present results suggest that biofilms formed under high cariogenic conditions may harbor more aciduric and acidogenic S. mutans genotypes. A cavidade oral apresenta vários genótipos de Streptococcus mutans, que podem possuir diferentes capacidades de virulência. Entretanto, pouco se sabe sobre a diversidade e virulência de genótipos de S. mutans isolados in vivo sob uma condição controlada de alto desafio cariogênico. Este estudo avaliou a diversidade genotípica de S. mutans identificados no biofilme dental formado in vivo na presença de sacarose, assim como a acidogenicidade e aciduricidade desses genótipos. Para possibilitar formação de biofilme, voluntários bochecharam com água destilada ou solução de sacarose 8x/dia durante 3 dias. S. mutans isolados da saliva e do biofilme dental foram genotipados por PCR com primers-arbitrários. Genótipos isolados do biofilme foram avaliados em relação à habilidade de reduzir o pH da suspensão devido à glicólise, em relação à susceptibilidade a ácidos e também atividade F-ATPase. A maioria dos voluntários apresentou apenas 1 genótipo na saliva, que foram detectados em quase todas as amostras de biofilme em altas proporções. Genótipos isolados somente na presença de sacarose apresentaram maior acidogenicidade do que aqueles genótipos isolados apenas na presença de água. Genótipos de biofilmes formados na presença de sacarose foram mais acidúricos após 30 e 60 min de incubação em pH 2,8 e 5,0, respectivamente. Os resultados do presente estudo sugerem que biofilmes formados sob condição de alto desafio cariogênico podem apresentar genótipos de S. mutans mais acidúricos e mais acidogênicos.

Made available in DSpace on 2019-09-12T16:53:48Z (GMT). No. of bitstreams: 0 Previous issue date: 2011 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) Objective: The present study aimed to determine if the addition of fluoride (F) and calcium (Ca) to home-applied 10% carbamide peroxide (CP) bleaching agents reduces mineral loss on the enamel surface and subsurface. Background: Home-applied 10% carbamide peroxide effectively whitens teeth. However, bleaching possibly promotes adverse side effects such as tooth hypersensitivity and morphological and chemical alterations, such reductions in calcium and fluoride, that affect the enamel surface. Methods: Sixty human enamel slabs were selected based on evaluation of their surface microhardness (n = 10) and submitted to the application of either a placebo agent, Whiteness (TM), Opalescence (TM), Pola Night,(TM) or one of two experimental carbamide peroxide gels: the first with 0.2% fluoride added and the second with 0.2% calcium added. The gel was applied for 6 h/day for 14 days. Samples were submitted to FT-Raman spectroscopy to evaluate surface mineral content before and after treatment. Additionally, cross-sectional microhardness and polarized light microscopy were carried out, to determine enamel subsurface inorganic loss and lesion depth. Results: A significant decrease in mineral content was observed after carbamide peroxide treatment, which resulted in increased lesion depth (p < 0.05). Cross-sectional microhardness assessment and polarized light microscopy demonstrated that all groups (except the placebo) exhibited enamel subsurface mineral loss; however, the inorganic deficit could be effectively controlled by the addition of F and Ca to the experimental bleaching agents. Conclusions: The addition of F and Ca to home-applied bleaching agents may reduce enamel mineral loss. [Cavalli, Vanessa] Universidade de Taubaté (Unitau), Dept Dent [Azevedo Rodrigues, Lidiany Karla] Univ Fed Ceara, Fac Pharm Dent & Nursing, Dept Restorat Dent, Fortaleza, Ceara, Brazil [Paes-Leme, Adriana Franco] Brazilian Biosci Natl Lab, Campinas, SP, Brazil [Silva Soares, Luis Eduardo; Martin, Airton Abrahao] Univ Vale do Paraiba, Inst Res & Dev IP&D, Lab Biomed Vibrat Spect, Sao Jose Dos Campos, SP, Brazil [Berger, Sandrine Bittencourt; Giannini, Marcelo] Univ Estadual Campinas, Piracicaba Sch Dent, Dept Restorat Dent, Campinas, SP, Brazil

Both serine and metalloproteinases have been shown to play the role of toxins in the venoms of many snakes. Determination of the natural protein substrates of these toxins is an important feature in the toxinological characterization of these proteinases. Furthermore, characterization of their peptide bond specificity is of value for understanding active site preference of the proteinase associated with effective proteolysis as well as of use in the design of peptide substrates and inhibitor lead compounds. Typically the determination of peptide bond cleavage specificity of snake venom serine proteinases (SVSPs) and snake venom metalloproteinases (SVMPs) has been performed using limited sets of peptides or small oligopeptides as experimental substrates. Although this approach has yielded valuable data it is generally limited in scope due to the relatively small sets of substrates used to generate the consensus specificity sequences for these proteinases. In this study we use a large, plasma based, proteome-derived peptide library as substrates along with mass spectrometry to explore the peptide bond specificity of three PI SVMPs and one PIII SVMP to determine their individual peptide cleavage consensus sequences. All of the proteinases assayed displayed a clear preference for a leucine residue in the P1′ site. Careful analysis of the specificity profiles of the SVMPs examined showed interesting differences in the preferences at the other P and P′ sites suggesting functional differences between these proteinases. The PI SVMPs, leucurolysin-a, atrolysin C, and BaP1, showed preferences across the full P4 to P4′ range whereas the PIII SVMP bothropasin showed a narrower range of preferences across the sites. In silico docking experiments with the experimentally derived consensus sequences as well as with comparison of the results to those in the literature regarding peptide bond specificity based on both peptide and protein substrates give rise to a fresh understanding of the specificity of these SVMPS and may serve as a foundation for future experiments to better elucidate their mechanism of action in the complex pathophysiology of snakebite envenomation. UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

Physical protein-protein interactions are fundamental to all biological processes and are organized in complex networks. One branch of the kinome network is the evolutionarily conserved NIMA-related serine/threonine kinases (Neks). Most of the 11 mammalian Neks studied so far are related to cell cycle regulation, and due to association with diverse human pathologies, Neks are promising chemotherapeutic targets. Human Nek6 was associated to carcinogenesis, but its interacting partners and signaling pathways remain elusive. Here we introduce hNek6 as a highly connected member in the human kinase interactome. In a more global context, we performed a broad data bank comparison based on degree distribution analysis and found that the human kinome is enriched in hubs. Our networks include a broad set of novel hNek6 interactors as identified by our yeast two-hybrid screens classified into 18 functional categories. All of the tested interactions were confirmed, and the majority of tested substrates were phosphorylated in vitro by hNek6. Notably, we found that hNek6 N-terminal is important to mediate the interactions with its partners. Some novel interactors also colocalized with hNek6 and γ-tubulin in human cells, pointing to a possible centrosomal interaction. The interacting proteins link hNek6 to novel pathways, for example, Notch signaling and actin cytoskeleton regulation, or give new insights on how hNek6 may regulate previously proposed pathways such as cell cycle regulation, DNA repair response, and NF-κB signaling. Our findings open new perspectives in the study of hNek6 role in cancer by analyzing its novel interactions in specific pathways in tumor cells, which may provide important implications for drug design and cancer therapy.

Since little is known about the effects of carbamide peroxide (CP) containing fluoride (F) or calcium (Ca), this study evaluated the effects of experimental and commercially available bleaching agents, with or without F and Ca, on enamel.Sound enamel slabs were randomly divided into six groups (n = 10): placebo gel (PLA); Whiteness (WHI-10% CP; FGM); Opalescence F (OPA-10% CP+F; Ultradent); Pola Night F (PN-10% CP+F; SDI); and experimental gels: 10% CP + F (CPF) and 10% CP + Ca (CPCa). The samples were submitted to 6-hour gel applications daily for 14 days and stored in remineralizing solution after treatment. Enamel microhardness measurements were performed at baseline and after bleaching. In addition, the analytical concentrations of F and Ca and the pH of the water used to rinse the bleached surface were analyzed by means of ion-selective electrode, atomic absorption spectroscopy, and pH meter, respectively.Enamel surface microhardness significantly decreased after bleaching with nonenhanced 10% CP (WHI). The chemical analyses suggest F uptake promoted by high-concentrate F bleaching gels (CPF, OPA, and PN) and a F loss with nonenhanced 10% CP bleaching gels (PLA, WHI). CP agent enhanced with Ca (CPCa) also caused Ca enamel uptake.Enamel was susceptible to mineral changes during bleaching treatment, but mineral loss was minimized by the addition of F and Ca to bleaching agents.

Little is known about the biochemical properties of the non-catalytic domains of snake venom metalloproteinases (SVMPs). The ECD sequence of the disintegrin-like domain (D-domain) has been assigned as the disintegrin motif and, recently, the hyper-variable region (HVR) of the cysteine-rich domain (C-domain) was suggested to constitute a potential protein-protein adhesive interface. Here we show that the recombinant C-domain of HF3, a hemorrhagic SVMP from Bothrops jararaca, as well as three peptides resembling its HVR, inhibit collagen-induced platelet aggregation, which indicates a role for the C-domain and its HVR in targeting HF3 to platelets. Site-directed mutagenesis was used for the first time to identify charged residues essential for the functionality of the disintegrin-like/cysteine-rich domains (DC-domains). Residues of the disintegrin loop (E467 and D469), and of the HVR (K568, K569 and K575) of HF3 were individually mutated to Ala. Interestingly, only the mutant D469A was obtained in soluble form in Escherichia coli and this single mutation caused loss of two functional activities of the DC-domains: inhibition of platelet aggregation and increase of leukocyte rolling in the microcirculation. In summary we demonstrate that the C-domain and its HVR are critical for HF3 to affect platelets and leukocytes, however, the disintegrin loop may be important for the functionality of the D-domain in the context of the C-domain.

To evaluate the effect of a novel antibacterial and fluoride-releasing adhesive formulation on enamel demineralization under sucrose exposure in situ (simulating high caries risk conditions).This crossover, blind study was performed in two phases of 14 days. Volunteers (n = 14) wore an intraoral appliance containing four bovine enamel blocks with cavity preparations restored using self-etching primers/composite resin (Clearfil SE Bond or Clearfil Protect Bond/Clearfil AP-X). The volunteers dropped 20% sucrose solution 8x/day and used fluoridated dentifrice 3x/day. After 14 days, enamel mineral loss was assessed by cross-sectional microhardness (CSMH), and the demineralization areas in enamel adjacent to the restoration were examined by scanning electron microscopy (SEM) and polarizing light microscopy (PLM). The CSMH data were analyzed using ANOVA and Tukey's test (p0.05).Enamel blocks restored with Clearfil Protect Bond showed higher mineral content (%vol) adjacent to restorations than Clearfil SE Bond only for the first site of microhardness measurement and close to enamel surface (20 microm). SEM and PLM analysis suggested that the Clearfil Protect Bond adhesive promoted less enamel demineralization around restorations.After 14 days of cariogenic challenge, the findings suggested that Clearfil Protect Bond might help to control the demineralization around restorations in cases of high caries risk.

This study evaluated the effect of 10% carbamide peroxide (CP) bleaching on Knoop surface microhardness (KHN) and morphology of sound enamel and enamel with early artificial caries lesions (CL) after pH-cycling model (pHcm). Human dental enamel blocks were randomly divided into 6 groups (n=10): 1 - sound enamel bleached (S) with CP (Rembrandt/Den-Mat); 2 - S and submitted to pHcm; 3 - CL bleached with CP; 4 - CL stored in artificial saliva and submitted to pHcm; 5 - CL treated with placebo gel and submitted to pHcm; 6 - CL bleached with CP and submitted to pHcm. Enamel blocks with known initial KHN values were demineralized (groups 3 to 6) and submitted to 12 day pHcm (groups 2, 4, 5 and 6). After demineralization and treatments, KHN was determined and the specimens were examined using scanning electron microscopy (SEM). Data were analyzed statistically by ANOVA and Tukey's test at 5% significance level. The results showed that among CL groups (3 to 6) only the group 3 presented remineralization after treatments. S groups (1 and 2) showed higher KHN and presented less formation of porosities on enamel surface than CL groups after treatments. In conclusion, bleaching procedures on enamel with CL did not exacerbate the demineralization, but should be indicated with caution. Este estudo analisou o efeito do peróxido de carbamida a 10% (PC) na microdureza Knoop de superfície (KHN) e morfologia do esmalte hígido e com lesões iniciais de cárie artificial (EC), após modelo de ciclagem de pH (cpH). Blocos de esmalte dental humano foram divididos aleatoriamente em 6 grupos (n=10): 1- esmalte hígido clareado (EHC) com PC (Rembrandt/Den-Mat); 2- EHC e submetido a cpH; 3- EC clareado com PC; 4- EC armazenado em saliva artificial e submetido a cpH; 5- EC tratado com gel placebo e submetido a cpH; 6- EC clareado com PC e submetido a cpH. Blocos de esmalte com a KHN conhecida eram desmineralizados (grupos 3 a 6) e submetidos a cpH (grupos 2, 4, 5 e 6). KHN foi determinada após a desmineralização e os tratamentos. Os espécimes foram examinados através de Microscopia Eletrônica de Varredura (MEV). Os dados foram analisados através de ANOVA e teste de Tukey (p

Enzymes of the pyrophosphatase/phosphodiesterase family have multiple roles in extracellular nucleotide metabolism and in the regulation of nucleotide-based intercellular signaling. Snake venoms contain enzymes that hydrolyze nucleic acids and nucleotides, but their function is poorly understood. Here we describe for the first time the isolation and functional characterization of a soluble phosphodiesterase from Bothrops jararaca venom, which shows amino acid sequence similarity to mammalian nucleotide pyrophosphatase/phosphodiesterase 3 (NPP3), and inhibits ADP-induced platelet aggregation. The enzyme, named NPP-BJ, showed an apparent molecular mass of 228 kDa by size exclusion chromatography. NPP-BJ exhibited nuclease activity as well as pyrophosphatase and phosphatase activities, preferentially hydrolyzing nucleoside 5'-triphosphates over nucleoside 5'-diphosphates, but was not active upon nucleoside 5'-monophosphates. Depending on the substrate used, dithiothreitol and EDTA differently inhibited the catalytic activity of NPP-BJ. Platelet aggregation induced by ADP was also abrogated by NPP-BJ, whereas thrombin-induced platelet aggregation was only slightly attenuated. However, polyclonal antibodies raised against NPP-BJ could not abolish the lethal activity of B. jararaca venom. Altogether, these results show that NPP-BJ has a minor contribution to the lethal activity of this venom, but interferes with mechanisms of ADP-induced platelet aggregation.

Made available in DSpace on 2013-08-28T14:12:29Z (GMT). No. of bitstreams: 1 WOS000263769500010.pdf: 652672 bytes, checksum: c1c4851735e3848359eb1380128ef732 (MD5) Made available in DSpace on 2013-09-30T18:31:32Z (GMT). No. of bitstreams: 2 WOS000263769500010.pdf: 652672 bytes, checksum: c1c4851735e3848359eb1380128ef732 (MD5) WOS000263769500010.pdf.txt: 26359 bytes, checksum: 647ccd1fb3cd9fccf0b3c20958c38fb2 (MD5) Previous issue date: 2009-02-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:44:58Z No. of bitstreams: 2 WOS000263769500010.pdf: 652672 bytes, checksum: c1c4851735e3848359eb1380128ef732 (MD5) WOS000263769500010.pdf.txt: 26359 bytes, checksum: 647ccd1fb3cd9fccf0b3c20958c38fb2 (MD5) Made available in DSpace on 2014-05-20T13:44:58Z (GMT). No. of bitstreams: 2 WOS000263769500010.pdf: 652672 bytes, checksum: c1c4851735e3848359eb1380128ef732 (MD5) WOS000263769500010.pdf.txt: 26359 bytes, checksum: 647ccd1fb3cd9fccf0b3c20958c38fb2 (MD5) Previous issue date: 2009-02-01 Objective: The purpose of this study was to evaluate the efficacy of the laser fluorescence (LF) device in detecting in vitro demineralization and remineralization of smooth surface caries-like lesions. Background Data: The early detection of smooth surface caries-like lesions is important to provide proper management of carious lesions, and allows monitoring of them over time. Also, some authors suggest that LF could be useful in monitoring the caries pathological process. Materials and Methods: Seventy-eight blocks of bovine teeth were obtained, and then submitted to artificial caries lesion induction and to a pH-cycling process. Superficial microhardness (SMH) and laser fluorescence analysis were performed at baseline, after induction of caries-like lesions, and after the pH-cycling regimen to promote remineralization. Results: Friedman's and multiple comparison tests were performed for all variables. SMH analysis showed significant differences (p < 0.05) between baseline (286.77 +/- 1.49 Vickers hardness number [VHN] units), before (38.48 +/- 0.85 VHN), and after remineralization (131.93 +/- 2.63 VHN). Baseline values for LF were extremely low (2.71 +/- 0.05), and a statistically significant difference was observed only after remineralization (3.61 +/- 0.08), as demonstrated by the increase in LF values. Conclusion: The LF device did not show efficacy for monitoring in vitro demineralization and remineralization of smooth enamel surfaces. UNESP, Sch Dent Araraquara, Dept Pediat Dent, Araraquara, SP, Brazil Univ Estadual Campinas, Sch Dent Piracicaba, Dept Physiol Sci, Piracicaba, Brazil UNESP, Sch Dent Araraquara, Dept Pediat Dent, Araraquara, SP, Brazil

HF3 and bothropasin are P-III hemorrhagic snake venom metalloproteinases (SVMPs) of Bothrops jararaca. The DC protein is composed of the disintegrin-like/cysteine-rich domains derived from the autolysis of P-III SVMPs. Here we describe simplified procedures for the isolation of HF3, bothropasin, the DC protein, and BJ-PI, a novel P-I SVMP. The isolated proteins were identified by mass spectrometry. BJ-PI is a potent caseinolytic enzyme devoid of hemorrhagic activity. HF3, bothropasin and BJ-PI show distinct fibrinogenolytic activities.

Since the currently available pH-cycling models do not differentiate the anti-caries potential of dentifrices with low fluoride (F) concentration, two models were developed and tested in the present. Bovine enamel blocks were subjected to the models and treated with F solutions containing from 70 to 280 mug F/mL in order to validate them in terms of dose-response effect. The models were also tested by evaluating the dentifrices Colgate Baby (500 mug F/g, as a low fluoride dentifrice), Tandy (1,100 mug F/g, as an active F-dentifrice) and Crest (1,100 mug F/g, as positive control). Enamel mineral loss or gain was assessed by surface and cross-sectional microhardness, and lesion depth was analyzed by polarized light microscopy. The pH-cycling models showed F dose-response effect either reducing enamel demineralization or enhancing remineralization. The low F dentifrice presented anti-caries potential, but it was not equivalent to the dentifrices containing 1,100 mug F/g. These data suggest that the models developed in this study were able to evaluate the anti-caries potential of low F dentifrice either on resistance to demineralization or on enhancement of remineralization. Tendo em vista que os modelos atuais de ciclagens de pH não diferenciam o potencial anti-cárie de dentifrícios com baixa concentração de fluoreto (F), dois modelos foram desenvolvidos e testados. Blocos de esmalte bovino foram submetidos aos modelos e tratados com soluções de concentrações crescentes de F (70 a 280 mig F/mL) para validar os modelos em termos de dose-resposta. A seguir, os modelos foram testados avaliando o potencial anti-cárie dos dentifrícios Colgate Baby (500 mig F/g, dentifrício de baixa concentração), Tandy (1.100 mig F/g, como controle ativo) e Crest (1.100 mig F/g, como controle positivo). Perda ou ganho de mineral pelo esmalte foi avaliada por microdureza e profundidade de lesão de cárie foi avaliada por microscopia de luz polarizada. Os modelos de ciclagens de pH desenvolvidos mostraram efeito do F dose-resposta quer seja na redução da desmineralização como na remineralização do esmalte. O dentifrício de baixa concentração de F mostrou ter potencial anti-cárie, o qual não foi equivalente aos dentifrícios contendo 1.100 mig F/g. Os resultados sugerem que os modelos desenvolvidos são capazes de avaliar o potencial anti-cárie de dentifrício de concentração reduzida de F, quer seja na sua capacidade de aumentar a resistência do esmalte a desmineralização como na ativação da remineralização.

Solid tumors must develop direct and indirect ways to induce angiogenesis in order to continue progression and expansion. The expression of angiogenic factors in the tumor microenvironment is a complex process involving interactions among different cell types. Previous studies demonstrated activin A, a member of the TGF-β superfamily, participates in the development and progression of oral squamous cell carcinomas (OSCC) via regulation of the tumor microenvironment, but its effects in the modulation of angiogenesis are unknown. We examined whether activin A, recombinant or derivate from OSCC cells, promotes angiogenesis of the human umbilical vein endothelial cells (HUVECs). Activin A-treated cells increased tubulogenesis activity concomitantly with high cellular proliferation and low apoptosis. Conversely, follistatin, an activin A antagonist, and activin A knock down in HUVECs significantly inhibited proliferation, induced apoptosis and decreases tube formation. Similarly, conditioned media harvested from OSCC cells expressing high activin A levels increased the proliferative rate and the tubulogenic activity of HUVECs more than those obtained from OSCC cells expressing a shRNA to neutralize activin A expression. In conclusion, our results show that activin A derived from OSCC cells promotes endothelial cell proliferation and tumor angiogenesis, suggesting that activin A signaling could be an important target for tumor vascular disruption in oral cancer. Financial support: FAPESP #2013/19856-2 and #2013/01607-6. Citation Format: Carine Ervolino de Oliveira, Nilva de Karla Cervigne, Carolina Carneiro Souza Macedo, Adriana Franco Paes Leme, Edgard Graner, Ricardo Della Coletta. Activin A induces vascular endothelial cell proliferation and angiogenesis in oral cancer. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr B27.

OBJECTIVE: The aim of this study was to evaluate the effects of carbamide peroxide-based bleaching agents (CPG) containing fluoride (CF) or calcium (CCa) on the ultimate tensile strength of enamel (UTS). METHOD: A "cube-like" resin composite structure was built-up on the occlusal surface of twenty-two sound third molars to facilitate specimen preparation for the micro-tensile test. The restored teeth were serially sectioned in buccal-lingual direction in slices with approximate 0.7 mm thickness. Each slice was trimmed with a fine diamond bur to reduce the buccal, internal slope enamel of the cusps to a dumb-bell shape with a cross-sectional area at the "neck" of less than 0.5 mm². The samples were randomly divided into 12 groups (n=11). The control groups were not submitted to the bleaching regimen. Specimens were treated with 10% CPG gel or with 10% CPG formulations containing CF (0.2% and 0.5%) or CCa (0.05% and 0.2%). Bleached groups received the application of the 10% CPGs for 6 hours/day at 37º C, during 14 consecutive days and were stored in artificial saliva (AS) or 100% relative humidity (RH) among each application. After bleaching, specimens were tested with the microtensile method at 0.5 mm/min. Data were analyzed by two-way ANOVA and Tukey test (5%). RESULTS: No significant difference was observed between groups stored in AS or RH. Specimens treated with CF or CCa presented similar UTS as unbleached control groups. CONCLUSION: Either 10% CPG formulations containing CF or CCa can preserve the UTS after bleaching regimen. OBJETIVO: O propósito deste estudo foi avaliar os efeitos de agents clareadores à base de peróxido de carbamida (CPG) contendo fluoreto (CF) e cálcio (CCa) na resistência à tração do esmalte (UTS). MÉTODO: Um bloco de resina composta foi confeccionada na superfície oclusal de vinte e dois terceiros molars hígidos para facilitar a preparação dos espécimes para o teste de micro-tração. Os dentes restaurados foram seccionados com disco diamantado no sentido vestíbulo-lingual em fatias de aproximadamente 0,7 mm de espessura. Com uma ponta diamantada, foi realizada uma constrição na região de esmalte da vertente oclusal interna. Os espécimes apresentaram aproximadamente 0,5 mm² de área na secção transversal da região de constrição e foram divididos em 12 grupos (n=11). Os grupos controles não foram submetidos ao regime clareador e os experimentais foram tratados com gel de CPG 10% ou com formulações de CPG 10% contendo CF (0,2% e 0,5%) ou CCa (0,05% e 0,2%). Os grupos clareadores receberam a aplicação dos CPGs por 6 horas/dia a 37ºC, durante 14 dias consecutivos e foram armazenados em saliva artificial (AS) ou em umidade relativa 100% (RH), entre as aplicações do gel clareador. Após o clareamento, os espécimes foram testados através do método de micro-tração (0,5 mm/min). Os dados foram analisados pela ANOVA (2 fatores) e teste Tukey (5%). RESULTADOS: Nenhuma diferença foi observada entre os grupos armazenados em AS ou RH. Os espécimes tratados com CPG com CF ou Cca apresentaram similar UTS aos grupos controles não clareados. CONCLUSÃO: Ambos CPGs 10% CF or CCa não alteraram a UTS após o tratamento clareador.

This in vitro study evaluated the surface microhardness of human enamel submitted to bleaching with 10% carbamide peroxide (CP) containing calcium or fluoride. Ninety-eight dental blocks (5 x 5 mm²) with polished enamel surfaces were randomly assigned to 7 treatment groups (n=14), as follows: without bleaching and storage in artificial saliva (control); 10% CP; 10% CP + 0.05% calcium; 10% CP + 0.1% calcium; 10% CP + 0.2% calcium; 10% CP + 0.2% fluoride; and 10% CP + 0.5% fluoride. During 14 days, enamel surfaces were daily exposed to a 6-h bleaching regimen followed by storage in artificial saliva. Surface microhardness was measured before (baseline), during (7th day), immediately after bleaching (14th day) and 1 week post bleaching. Data were analyzed by two-way ANOVA and Tukey's test (p

To evaluate the occluding effect of five desensitizing agents on human dentin tubules.30 buccal and lingual surfaces were prepared from 15 extracted intact third molars. Each surface was polished with aluminum oxide abrasive papers to remove enamel and to expose the underlying dentin in cervical area. The flat dentin surfaces were treated with 0.5 M EDTA for 2 minutes to expose dentin tubule orifices. The samples were randomly divided into six groups: AS - immersed in artificial saliva at 37 degrees C for 2 weeks (control); OX - Oxagel (monopotassium oxalate), DU - Duraphat (sodium fluoride), DE - Desensibilize (strontium chloride), OD - Odahcam (acidulated phosphate fluoride) and SE - Sensodyne (strontium chloride + calcium carbonate abrasive). Dentin desensitizers were applied during 2 weeks and after each application the samples were kept in artificial saliva at 37 degrees C. The samples were prepared according to the scanning electron microscope procedures and were examined at x2000 magnification.The results were expressed in percent (%) of tubule occlusion and analyzed by ANOVA and Duncan's multiple range test (P0.05): AS- 45.41 +/- 11.65a; OX- 42.65 +/- 11.79a; DU- 47.25 +/- 8.59ab; DE- 49.36 +/- 18.27ab; OD- 64.43 +/- 15.55b and SE- 65.44 +/- 10.93b. Results suggest that the dentin surfaces treated with OD and SE showed higher tubule occlusion when compared to AS and OX, but were not different compared to DU and DE treatments.

Similar to that which occurred in most developed countries, dental caries have shown a significant decline in Brazil over the last two decades. Water fluoridation, expansion of preventive programs at schools, and especially, the widespread use of fluoride dentifrice are discussed as factors related to this reduction in caries. Data from epidemiological surveys and historical facts are presented to support the importance of fluoride dentifrices to the current caries prevalence in Brazil. Assim como observado na maioria dos países desenvolvidos, a prevalência de cárie dental no Brasil apresentou um declínio significativo nas últimas duas décadas. A fluoretação das águas de abastecimento público, a expansão de programas preventivos nas escolas e principalmente o uso abrangente de dentifrícios fluoretados no país são apresentados como fatores relacionados a essa redução de cárie. Dados de levantamentos epidemiológicos e fatos históricos são apresentados para corroborar a importância dos dentifrícios fluoretados na atual prevalência de cárie no Brasil

Since the efficacy of topical fluoride products is related to the fluoride (F) availability and its reactivity with enamel, this study was conducted. The F concentration of the following materials was verified: I- acidulated phosphate fluoride (APF) gel (1.23% F), II- APF foam (1.23% F) and III- Varnish (2.26% F). Forty blocks of bovine enamel were divided into 4 groups and treated according to the materials described, being one of them used as control. Loosely bound fluoride (''CaF2'') was determined on enamel after extraction with 1.0M KOH and analyzed by ion-selective electrode. Total F concentration found in gel was 12,642, in foam 12,755 and in varnish 23,183 mg F/g. All products formed statistically higher amounts of ''CaF2'' on enamel compared to the control group (p < 0.05), but the difference between them was not significant (p > 0.05). Thus, ''CaF2'' formation was not proportional to the total F content in the products, suggesting that the pH and the vehicle used are more important. Considerando que a eficácia dos produtos para aplicação tópica profissional de flúor (géis, espumas e vernizes) está relacionada com a reatividade do flúor (F) com o esmalte e sendo esta dependente da disponibilidade do F em cada produto, este estudo foi conduzido. A concentração de F nos seguintes produtos foi estudada: I - Flúor Fosfato Acidulado (FFA) gel (1,23% F), II - FFA espuma (1,23% F) e III- Verniz fluoretado (2,26% F). Foram confeccionados 40 blocos de esmalte bovino, tratados de acordo com os grupos descritos, sendo um deles utilizado como controle. O F fracamente ligado (''CaF2'') ao esmalte foi determinado após a extração com 1.0 M KOH e analisado em eletrodo específico. A concentração de F encontrada no gel foi de 12.642, na espuma 12.755 e no verniz 23.183 mg F/g. Todos os produtos formaram uma quantidade significantemente maior de ''CaF2'' na superfície do esmalte, comparado ao grupo controle (p < 0,05), mas entre eles, esta diferença não foi significante (p > 0,05). Assim, a formação de ''CaF2'' na superfície do esmalte não foi proporcional ao conteúdo de F nos produtos, sugerindo que o pH e o veículo utilizado são mais importantes.

To evaluate the effect of a combination of fluoride dentifrice and acidulated phosphate fluoride (APF) on enamel with incipient caries lesions.Bovine dental enamel blocks with artificial caries lesions were divided into four treatment groups and submitted to a pH-cycling model: (1) Placebo non-fluoridated dentifrice (PD); (2) Fluoridated dentifrice (FD), 1,100 ppm F; (3) APF (12,300 ppm F, pH 3.5) + PD; 4) APF+FD. APF was applied to enamel blocks of groups APF+PD and APF+FD before the pH-cycling regimen and all of them were exposed to dentifrice during the cycling.The results showed that 61-81% of the fluoride previously deposited in enamel by APF was released to the media during the pH-cycling. The final concentration of fluoride in enamel in the group APF+PD was lower than that before the cycling (P0.05), but in the group APF+FD the reduction was not significant (P0.05). FD treatment was significantly more efficient than PD in rehardening the enamel surface and increasing the hardness of the caries lesions. APF+PD treatment was not more efficient than PD in increasing enamel hardness and an additive effect of APF+FD was not observed.

Orientador : Jaime Aparecido Cury Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba Resumo: A associação de métodos de uso de flúor tem sido indicada para indivíduos ou grupos considerados de alto risco de cárie, mas o efeito somatório dessa associação não está claramente estabelecido. Assim, a combinação de métodos de uso de flúor foi avaliada in situ quando o desafio cariogênico foi aumentado pela fteqüência de exposição à sacarose. Dezesseis voluntários utilizando dispositivos palatinos contendo quatro blocos de esmalte bovino participaram desse estudo cruzado, dividido em quatro fases de 14 dias. Os voluntários foram submetidos aos tratamentos: dentifrício placebo de flúor (DP), dentifrício fluoretado (DF), aplicação tópica profissional de flúor (ATF) + DP e ATF + DF. A ATF foi utilizada como pré-tratamento nos blocos de esmalte e dentifiicio, fluoretado ou não, (1:3) foi gotejado sobre os blocos 3x/dia. Solução de sacarose a 20% foi gotejada 4x/dia em dois dos quatro blocos e 8x/dia, nos outros dois blocos para simular o desafio cariogênico. Ao final dos 14 dias, a microdureza de superficie e do esmalte seccionado longitudinalmente e concentração de flúor foram avaliadas no esmalte. As concentrações de F, Ca, P, polissacarídeo insolúvel (PI) e contagens de estreptococos do grupo mutans foram avaliadas na placa dental. Os resultados mostraram efeito estatisticamente significante da freqüência de exposição à sacarose na desmineralização do esmalte e na concentração de F, Ca e PI na placa dental (pO,O5). Conclui-se que a combinação de métodos (ATF+DF) não diminui a cariogenicidade da placa dental e não apresenta efeito adicional na redução da desmineralização do esmalte, sugerindo que em adição ao uso de flúor, outras medidas para interferir nos fatores responsáveis pela doença devem ser simultaneamente utilizadas para seu controle completo Abstract: The combination of methods of fluoride use has been suggested for high caries risk individuaIs or groups, but its additive effect on dental caries is not clearly established. Thus, this combination was evaluated in situ under high cariogenic challenge induced by the ftequency of sucrose exposure. Sixteen volunteers, using a palatal acrylic appliance containing four bovine dental enamel blocks, took part in this crossover study done in four phases of 14 days each. The volunteers were subjected to the following groups of treatments: 1 - Non-fluoridateddentiftice (PD); 2 - Fluoridated dentiftice (FD); 3 - Acidulated phosphate fluoride gel (APF) and PD (APF+PD); 4 - APF+FD. APF was applied to the enamel before the experimentalperiod and the dentiftices (a slurry 1:3) were dripped onto the blocks 3x/day. A 20% sucrose solution was dripped onto 2 of the blocks 4x/day and 8x/day onto the other two, simulating an increase in the cariogenic challenge. Surface and cross-sectional microhardness and F uptake were evaluated in the enamel. F, Ca, P, insoluble polysaccharide (IP) and mutans streptococci in dental plaque were determined after 14 days. The results showed effect statistically significant of the ftequency of sucrose exposure on enamel demineralization and F, Ca, IP concentration in dental plaque (p0.05). In conclusion, the combination of methods (APF+FD) neither decreases the cariogenicity of dental plaque nor has an additional efIect on reducing enamel demineralization, suggesting that in addition to fluoride use, other measures to interfere with the factors responsible for the disease must be simultaneouslyused to its complete control Mestrado Cariologia Mestre em Odontologia

Oral squamous cell carcinoma is the most common type of cancer in the oral cavity, representing more than 90% of all oral cancers. The characterization of altered molecules in oral cancer is essential to understand molecular mechanisms underlying tumor progression as well as to contribute to cancer biomarker and therapeutic target discovery. Proteoglycans are key molecular effectors of cell surface and pericellular microenvironments, performing multiple functions in cancer. Two of the major basement membrane proteoglycans, agrin and perlecan, were investigated in this study regarding their role in oral cancer. Using real time quantitative PCR (qRT-PCR), we showed that agrin and perlecan are highly expressed in oral squamous cell carcinoma. Interestingly, cell lines originated from distinct sites showed different expression of agrin and perlecan. Enzymatically targeting chondroitin sulfate modification by chondroitinase, oral squamous carcinoma cell line had a reduced ability to adhere to extracellular matrix proteins and increased sensibility to cisplatin. Additionally, knockdown of agrin and perlecan promoted a decrease on cell migration and adhesion, and on resistance of cells to cisplatin. Our study showed, for the first time, a negative regulation on oral cancer-associated events by either targeting chondroitin sulfate content or agrin and perlecan levels.

Oral squamous cell carcinoma (OSCC) is the eighth most prevalent malignant neoplasm and accounts for 2% of all deaths by cancer worldwide. OSCCs have a highly variable clinical course, and because it is often diagnosed only after it has reached an advanced stage, the overall survival rate is less than 50% in 5 years. A better knowledge of the mechanisms that influence OSCC development and its progression is still needed. Particularly, most of the attention has been driven to mechanisms associated with tumor cell invasion and how the surrounding tumoral stroma may influence it. The myoma organotypic model is a novel fully human organotypic invasion model that mimics the tumor microenvironment (TME) (Nurmenniemi et al. 2009). Aiming to investigate the molecular mechanisms related to OSCC development and search for prognostic markers for this tumor, we used the myoma organotypic assay, followed by MS-ESI technique of laser microdissected invaded tissue, to analyze several features related to OSCC invasive vs. non-invasive cells. Our results showed that the HSC-3, an aggressive and invasive oral squamous cell carcinoma cell line, invaded as small islands and cell chords and penetrated deeply in the tissue. Unlike, the less aggressive LN2 cells, formed a well structured epithelium layer, but they invaded less in myoma. The proteomic analysis was able to characterize the differential expressed proteins of these, and revealed a group of proteins exclusively expressed in the cells that deeply invade the myoma, such as HMGA2 (high mobility group), JUP (junction plakoglobin), PLEC (plectin), HIST1H2B (Histone H2B), IQGAP1 (Ras GTPase-activating-like protein), EEF1A1 (elongation factor 1-alpha), HSPA9 (Stress-70 protein, mitochondrial), HADHA (Trifunctional enzyme subunit alpha). Additionally, the TME is a unique ambient created and shaped by the tumor, which orchestrates molecular and cellular events taking place in surrounding tissues. Interestingly, we found a group of high abundant proteins expressed in the TME surrounding the invasive island. Among them are PALLD (palladin), TNC (tenascin C), LMNA (laminin), VIM (vimentin), and COL1A (collagen-type I-alpha I), which have been described to be involved in cell migration and proliferation. Our preliminary data suggests that the myoma organotypic tumor invasion assay is successful in determining new molecules and potential pathways associated with OSCC tumor invasion. We also present here a new insight for head and neck cancer biomarkers research that can, ultimately, contribute to more individualized treatment of patients affected by OSCC. However, validation of such results in clinical sample cohorts is important for a better understanding of the biological events associate with tumor invasion and disease spread (metastasis) in those patients. Citation Format: Nilva K. Cervigne, Carolina Bitu, Bianca A. Pauletti, Adriana F. Paes Leme, Tuula Salo, Ricardo Della Coletta. In-depth analysis of myoma organotypic model can determine potential markers of oral cancer invasion. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4998. doi:10.1158/1538-7445.AM2014-4998

Cytoskeleton and protein trafficking processes, including vesicle transport to synapses, are key processes in neuronal differentiation and axon outgrowth. The human protein FEZ1 (fasciculation and elongation protein zeta 1 / UNC-76, in C. elegans), SCOCO (short coiled-coil protein / UNC-69) and kinesins (e.g. kinesin heavy chain / UNC116) are involved in these processes. Exploiting the feature of FEZ1 protein as a bivalent adapter of transport mediated by kinesins and FEZ1 protein interaction with SCOCO (proteins involved in the same path of axonal growth), we investigated the structural aspects of intermolecular interactions involved in this complex formation by NMR (Nuclear Magnetic Resonance), cross-linking coupled with mass spectrometry (MS), SAXS (Small Angle X-ray Scattering) and molecular modelling. The topology of homodimerization was accessed through NMR (Nuclear Magnetic Resonance) studies of the region involved in this process, corresponding to FEZ1 (92-194). Through studies involving the protein in its monomeric configuration (reduced) and dimeric state, we propose that homodimerization occurs with FEZ1 chains oriented in an anti-parallel topology. We demonstrate that the interaction interface of FEZ1 and SCOCO defined by MS and computational modelling is in accordance with that previously demonstrated for UNC-76 and UNC-69. SAXS and literature data support a heterotetrameric complex model. These data provide details about the interaction interfaces probably involved in the transport machinery assembly and open perspectives to understand and interfere in this assembly and its involvement in neuronal differentiation and axon outgrowth.

The functionality of the disintegrin-like/cysteine-rich domains of snake venom metalloproteinases (SVMPs) has been shown to reside in the cysteine-rich region, which can interact with VWA-containing proteins. Recently, the hyper-variable region (HVR) of the cysteine-rich domain was suggested to constitute a potential protein–protein adhesive interface. Here we show that recombinant proteins of HF3, a hemorrhagic P-III SVMP, containing the cysteine-rich domain (disintegrin-like/cysteine-rich and cysteine-rich proteins) but not the disintegrin-like protein were able to significantly increase leukocyte rolling in the microcirculation. Peptides from the HVR also promoted leukocyte rolling and this activity was inhibited by anti-alpha M /beta 2 antibodies. These results show, for the first time, that the cysteine-rich domain and its HVR play a role in triggering pro-inflammatory effects mediated by integrins.

We investigated the putative toxins of Philodryas olfersii (Colubridae), a representative of a family of snakes neglected in venom studies despite their growing medical importance. Transcriptomic data of the venom gland complemented by proteomic analysis of the gland secretion revealed the presence of major toxin classes from the Viperidae family, including serine proteases, metalloproteases, C-type lectins, Crisps, and a C-type natriuretic peptide (CNP). Interestingly, the phylogenetic analysis of the CNP precursor showed it as a linker between two related precursors found in Viperidae and Elapidae snakes. We suggest that these precursors constitute a monophyletic group derived from the vertebrate CNPs.