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Calorie restriction (CR) is a type of dietary intervention that is essential in weight loss through modulation of critical metabolic control pathways, is well established and understood in cases of systemic arterial hypertension, however, its role in renovascular hypertension is still unclear.Rats were divided into three groups: SHAM, and two groups that underwent surgery to clip the left renal artery and induce renovascular hypertension (OH and OHR). The SHAM diet was as follows: 14 weeks normolipidic diet; OH: 2 weeks normolipidic diet + 12 weeks hyperlipidic diet, both ad libitum; OHR, 2 weeks normolipidic diet + 8 weeks ad libitum high-fat diet + 4 weeks 40% calorie-restricted high-fat diet.Rats in the OHR group had decreased blood pressure, body weight, and glucose levels. Reductions in insulinemia and in lipid and islet fibrotic areas in the OHR group were observed, along with increased insulin sensitivity and normalization of insulin-degrading enzyme levels. The expression of nicotinamide phosphoribosyltransferase (NAMPT), insulin receptor (IR), sirtuin 1 (SIRT1), and complex II proteins were increased in the liver tissue of the OHR group. Strong correlations, whether positive or negative, were evaluated via Spearman's model between SIRT1, AMPK, NAMPT, PGC-1α, and NNMT expressions with the restoration of normal blood pressure, weight loss, glycemic and lipid panel, and mitochondrial adaptation.CR provided short-term beneficial effects to recover the physiological parameters induced by a high-fat diet and renal artery stenosis in obese and hypertensive animals. These benefits, even in the short term, can provide physiological benefits in the long term.

This study aimed to investigate the application of low-intensity electrostimulation (ES) and electromagnetic stimulation (EM) associated with bioactive glass (BG) or allogeneic grafts (BB) in bone regeneration. A cell viability test on osteoblasts (UMR-106) was performed in the presence of BB and BG grafts associated with ES (10 μA/5 min) and EM (500 Hz/2 min). Critical defects (25 mm2 ) in calvaria were generated in male Wistar rats, and bone regeneration was evaluated on the 30th, 60th, and 120th days after surgery. Cell proliferation increased with the application of ES in both grafts and after EM with BG. Bone remodeling was more effective using the allogeneic graft in both therapies, with increased angiogenesis, osteoblast proliferation, and OPN expression in the BB + EM group. A higher number of osteoblasts and osteoclasts, and an increase in bone sialoprotein, Runx-2, and Opn gene expression were found in the BB + ES group. The BG graft associated with EM therapy had an increased proliferation of osteoblasts and increased expression of Runx-2 and Opn. Groups that had BG and ES therapy had increased numbers of osteoblasts, osteoclasts, and increased OPN expression. The expression of voltage-gated calcium channels increased in groups with ES, while calmodulin expression increased in therapies without grafting. ES and EM therapies favored the repair of bone defects upon grafting by improving angiogenesis, osteogenic gene expression, and tissue reorganization. Despite activating different pathways, both therapies increased the intracellular concentrations of calmodulin, leading to cell proliferation and bone regeneration.

The literature has shown the beneficial effects of microcurrent (MC) therapy on tissue repair. We investigated if the application of MC at 10 μA/90 s could modulate the expression of remodeling genes transforming growth factor beta (Tgfb), connective tissue growth factor (Ctgf), insulin-like growth factor 1 (Igf1), tenascin C (Tnc), Fibronectin (Fn1), Scleraxis (Scx), Fibromodulin (Fmod) and tenomodulin in NIH/3T3 fibroblasts in a wound healing assay. The cell migration was analyzed between days 0 and 4 in both fibroblasts (F) and fibroblasts + MC (F+MC) groups. On the 4th day, cell viability and gene expression were also analyzed after daily MC application. Higher expression of Ctgf and lower expression of Tnc and Fmod, respectively, were observed in the F+MC group in relation to F group (p

Angiotensin II induced increase in hypertension enhances oxidative stress and compromises insulin action and pancreatic function. Quercetin-rich foods are beneficial for hypertensive and diabetic animals owing to their antioxidant function. The aim of this study was to evaluate the antioxidant effects of quercetin in hypertensive rats on insulin action, signaling, and secretion. Wistar rats were randomly divided into three groups: sham, hypertensive rats (H), and hypertensive rats supplemented with quercetin (HQ). After three months of initial hypertension, quercetin was administered at 50 mg/kg/day for 30 days. Our results indicate that hypertension and serum lipid peroxidation levels were reduced by quercetin supplementation. We observed increased insulin sensitivity in adipose tissue, corroborating the insulin tolerance test, HOMA index, and improvements in lipid profile. Despite normal insulin secretion at 2.8 and 20 mM of glucose, animals treated with quercetin exhibited increased number of islets per section; increased protein expression of muscarinic receptor type 3, VEGF, and catalase in islets; and hepatic mRNA levels of Ide were normalized. In conclusion, supplementation with quercetin improved insulin action and prevented pancreatic and metabolic dysfunction.

Background: Caloric restriction (CR) is a type of dietary intervention that is essential in weight loss through modulation of critical pathways of metabolic control; however, it is not yet clear what this intervention results to in association with renovascular hypertension.Methods: Rats were divided into three groups: SHAM, and two groups that underwent surgery to clip the left renal artery and induce renovascular hypertension (OH and OHR). The SHAM diet was as follows: 14 weeks normolipidic diet; OH: 2 weeks normolipidic diet + 12 weeks hyperlipidic diet, both ad libitum; OHR, 2 weeks normolipidic diet + 8 weeks ad libitum high-fat diet + 4 weeks 40% calorie-restricted high-fat diet. Results: Rats in the OHR group had decreased blood pressure, body weight, and glucose levels. Reductions in insulinemia and in lipid and islet fibrotic areas in the OHR group were observed, along with increased insulin sensitivity and normalization of insulin-degrading enzyme levels. The expression of nicotinamide phosphoribosyltransferase (NAMPT), insulin receptor (IR), sirtuin 1 (SIRT1), and complex II proteins were increased in the liver tissue of the OHR group. Strong correlations, whether positive or negative, were evaluated via Spearman’s model between SIRT1, AMPK, NAMPT, PGC-1α, and NNMT expressions with the restoration of normal blood pressure, weight loss, glycemic and lipid panel, and mitochondrial adaptation. Conclusion: CR provided short-term beneficial effects to recover the physiological parameters induced by a high-fat diet and renal artery stenosis in obese and hypertensive animals. These benefits, even in the short term, can provide physiological benefits in the long term.

Epithelial-to-mesenchymal transition (EMT) and endothelial-to-mesenchymal transition are processes that can occur under different biological conditions, including tissue healing due to hypertension and oxidative stress. The purpose of the present study was to evaluate the differences in gene expression of epithelial/endothelial and mesenchymal markers in different tissues. A two-kidney, one-clip (2K1C) renovascular hypertension rat model was used. Hypertension was induced by the clipping of the left renal artery; the rats were randomized into sham and 2K1C groups and monitored for up to 4 weeks. The gene expressions of E-cadherin (E-cad), N-cadherin (N-cad), α-smooth muscle actin (α-SMA), collagen I (COL1A1), collagen III (COL3A1) and hepatocyte growth factor (HGF) were determined by reverse transcription-PCR. The levels of the cytokines transforming growth factor-β1, tumor necrosis factor-α, interleukin (IL)-4, IL-6 and IL-10 were evaluated using ELISAs. The levels of thiobarbituric acid reactive substances and thiol groups were measured to evaluate oxidative stress. All analyses were performed on the liver, heart and kidneys tissues of sham and model rats. The 2K1C animals exhibited a higher systolic blood pressure, as well as cardiac hypertrophy and atrophy of the left kidney. Fibrotic alterations in the heart and kidneys were observed, as was an increase in the collagen fiber areas, and higher levels of inflammatory cytokines, which are associated with the increased expression of fibroproliferative and anti-fibrotic genes. Renovascular hypertension regulated epithelial/endothelial and mesenchymal markers, including E-cad, N-cad, α-SMA and COL1A1 in the kidneys and heart. EMT in the kidneys was mediated by an increased level of inflammatory and profibrotic cytokines, as well as by oxidative stress. The data in the present study suggested that the expression of epithelial/endothelial and mesenchymal markers are differentially regulated by hypertension in the liver, heart and kidneys.

BackgroundCaloric restriction (CR) is a type of dietary intervention enjoyed as an essential tool in weight loss by modulating critical pathways of metabolic control, although it is not yet clear what repercussions this intervention model results when associated with renovascular hypertension. Here we demonstrate that CR can be beneficial in obese and hypertensive animals.MethodsRats were divided into groups: SHAM, and two groups underwent surgery to clip the left renal artery, to induce renovascular hypertension (OH and OHR). The SHAM diet was performed: 14 weeks normolipidic diet; OH: 2 weeks normolipidic diet + 12 weeks hyperlipidic diet, both ad libitum; OHR: 2 weeks normolipidic diet + 8 weeks ad libitum high fat diet + 4 weeks restricted 40% high fat diet.Resultsthe OHR group dissipated blood pressure, body weight and glucose homeostasis. Reductions in insulinemia, lipids, islets fibrotic areas in the OHR group were observed along with increased insulin sensitivity and normalization of the insulin-degrading enzyme. Nicotinamide phosphoribosyltransferase, insulin receptor, Sirtuin 1 and complex II protein were modulated in liver tissue in the OHR group. Strong correlations, direct or indirect, were evaluated by Spearman’s model between SIRT1, AMPK, NAMPT, PGC-1α and NNMT with the reestablishment of blood pressure, weight loss, glycidic and lipid panel and mitochondrial adaptation.ConclusionCR provided short-term beneficial effects to recover physiological parameters induced by a high-fat diet and renal artery stenosis in obese and hypertensive animals. These benefits, even in the short term, can bring physiological benefits in the long run.

The objective of this study was to evaluate the effects of red Light Emiting Diode (red LED) irradiation on fibroblasts in adipose-derived mesenchymal stem cells (ASC) co-culture on the scratch assay. We hypothesized that red LED irradiation could stimulate paracrine secretion of ASC, contributing to the activation of genes and molecules involved in cell migration and tissue repair. ASC were co-cultured with NIH/3T3 fibroblasts through direct contact and subjected to red LED irradiation (1.45 J/cm2/5min6s) after the scratch assay, during 4 days. Four groups were established: fibroblasts (F), fibroblasts + LED (FL), fibroblasts + ASC (FC) and fibroblasts + LED + ASC (FLC). The analyzes were based on Ctgf and Reck expression, quantification of collagen types I and III, tenomodulin, VEGF, TGF-β1, MMP-2 and MMP-9, as well as viability analysis and cell migration. Higher Ctgf expression was observed in FC compared to F. Group FC presented higher amount of tenomodulin and VEGF in relation to the other groups. In the cell migration analysis, a higher number of cells was observed in the scratched area of the FC group on the 4th day. There were no differences between groups considering cell viability, Reck expression, amount of collagen types I and III, MMP-2 and TGF-β1, whereas TGF-β1 was not detected in the FC group and the MMP-9 in none of the groups. Our hypothesis was not supported by the results because the red LED irradiation decreased the healing response of ASC. An inhibitory effect of the LED irradiation associated with ASC co-culture was observed with reduction of the amount of TGF-β1, VEGF and tenomodulin, possibly involved in the reduced cell migration. In turn, the ASC alone seem to have modulated fibroblast behavior by increasing Ctgf, VEGF and tenomodulin, leading to greater cell migration. In conclusion, red LED and ASC therapy can have independent effects on fibroblast wound healing, but the combination of both does not have a synergistic effect. Therefore, future studies with other parameters of red LED associated with ASC should be tested aiming clinical application for tissue repair., Biological sciences; Cell biology; Proteins; Biomedical engineering; Molecular biology; Regenerative medicine; Scratch assay; Ctgf; TGF-β1; Tenomodulin; Repair; Cell migration

Vitamin E and caloric restriction have antioxidant effects in mammals. The aim of this study was to evaluate effects of vitamin E supplementation and caloric restriction upon insulin secretion and glucose homeostasis in rats. Male Wistar rats were distributed among the following groups: C, control group fed ad libitum; R, food quantity reduction of 40%; CV, control group supplemented with vitamin E [30 mg·kg–1·day–1]; and RV, food-restricted group supplemented with vitamin E. The experiments ran for 21 days. Glucose tolerance and insulin sensitivity was higher in the CV, R, and RV groups. Insulin secretion stimulated with different glucose concentrations was lower in the R and RV groups, compared with C and CV. In the presence of glucose and secretagogues, insulin secretion was higher in the CV group and was lower in the R and RV groups. An increase in insulin receptor occurred in the fat pad and muscle tissue of groups CV, R, and RV. Levels of hepatic insulin receptor and phospho-Akt protein were higher in groups R and RV, compared with C and CV, while muscle phospho-Akt was increased in the CV group. There was a reduction in hepatic RNA levels of the hepatocyte growth factor gene and insulin degrading enzyme in the R group, and increased levels of insulin degrading enzyme in the CV and RV groups. Thus, vitamin E supplementation and caloric restriction modulate insulin secretion by different mechanisms to maintain glucose homeostasis.

Connexins (Cx) and cadherins are responsible for cell homeostasis. The Cx activity is directly related to cholesterol. The present work investigates whether vitamin E, with or without caloric restriction (CR), alters the mRNA expression of Cx26, Cx32, Cx43, N-cadherins (N-cads), E-cadherins (E-cads) and alpha-smooth muscle actin (α-SMA), and evaluates their relation to cholesterol metabolism in rat liver. Animals were divided into different groups: control with ad libitum diet (C), control+vitamin E (CV), aloric restriction with intake to 60% of group C (CR), and the intake of group CR+vitamin E (RV). There were increases of manganese superoxide dismutase (Mn-SOD) and glutathione S-transferase mu 1, indicating antioxidant effects of CR and vitamin E. An increase of nitric oxide in the CR group was in agreement with the Mn-SOD data. Supplementation with vitamin E, with or without CR, upregulated the expression of Cx26 mRNA and increased low-density lipoprotein cholesterol (LDL-c) in the CV group. Reductions of Cx32 and Cx43 were associated with lower LDL-c. Increases in Hmgcr and low-density lipoprotein receptor (LDLr) in the CV and RV groups could be explained by the effect of vitamin E. A reduction of LDLr in the CR group was due to the reduced dietary intake. Increases in cadherins in the CV, CR and RV groups were indicative of tissue maintenance, which was also supported by increases of α-SMA in groups CV and RV. Finally, vitamin E, with or without CR, increased Cx26, probably modulated by expression of the Hmgcr and LDLr genes. This suggests important relationship of Cxs and cholesterol metabolism genes.

Objective Complexes like conjugated linoleic acid (CLA) reduce the percentage of body fat by increasing energy expenditure, fat oxidation, or both. The aim of this study was to verify if CLA is able to mimic caloric restriction (CR), and determine the effects of CLA on liver metabolic profile of young adult male Wistar rats. Materials and methods We divided 36 animals into the following groups: 1) Control; 2) CLA (1% of daily food intake, 21 days, orogastric intubation); 3) Restr (fed 60% of the diet offered to controls); and 4) CLA Restr. Liver tissues were processed for biochemical and molecular or mitochondrial isolation (differential centrifugation) and blood samples were collected for biochemical analyses. Results Treatment of the animals for 21 days with 1% CLA alone or combined with CR increased liver weight and respiration rates of liver mitochondria suggesting significant mitochondrial uncoupling. We observed a decrease in adipose tissue leading to insulin resistance, hyperinsulinemia, and hepatic steatosis due to increased liver cholesterol and triacylglycerol levels, but no significant effects on body mass. The expression of hepatic cellular connexins (43 and 26) was significantly higher in the CLA group compared with the Control or Restr groups. Conclusion CLA does not seem to be a safe compound to induce mass loss because it upregulates the mRNA expression of connexins and induces hepatic mitochondrial changes and lipids disorders.

Made available in DSpace on 2019-10-04T12:32:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-01-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Herminio Ometto Foundation Introduction: Periodontitis is characterized by inflammatory mediators beyond T lymphocyte function and phenotype (Thl/Th2/Th17). The clinical diversity in periodontitis makes it difficult to characterize the immune response in patients. This study evaluated the profile of the adaptive immune response in the periodontal disease model. Methods: 72 rats (Wistar) were divided into a control group (CTL/day 0) and periodontitis (PD15/15 days and PD60/60 days). In the PD15 and PD60 groups, periodontal disease was induced by ligature with a silk thread placed in the cervical region of the upper first molar. After euthanasia, the periodontal tissue was analyzed by flow cytometry (CD4, CD8, CD25, CD44), semi-quantitative RT-PCR (T-bet, GATA-3, ROR gamma t), semi-quantitative RT-PCR and ELISA IFN-gamma, TNF-alpha, IFN-gamma, IL-4, IL-6, IL-10, IL-17) and by Western blotting (Caspase-9, PCNA). Results: The number of CD4(+) CD25(+), CD4(+) CD44(+), CD8(+) CD25(+) and CD8(+) CD44(+) cells and expression levels of T-bet and GATA-3 are increased in the PD60 group compared to PD15 and CTL. The ROR gamma-t gene transcript increased in the PD15 group in relation to PD60 and CTL. The cytokines IFN-gamma, TNF-alpha and IL-17 increased in the PD60 group in relation to PD15. The expression of Caspase-9 was higher in the PD60 group than in PD15. Conclusions: The results suggest that the evolution of gingivitis to periodontitis is related to the accumulation of activated Thl cells (IFN-gamma and TNF-alpha) associated with the presence of increased IL-17. Studies with inhibitors of these cytokines in periodontal disease may lead to therapy directed at blocking the inflammatory process in this pathology, interrupting bone loss. Ctr Univ Herminio Ometto UNIARARAS, Grad Program Biomed Sci, Araras, SP, Brazil Ctr Univ Herminio Ometto UNIARARAS, Grad Program Odontol, Araras, SP, Brazil Sao Paulo State Univ, Dept Diag & Surg, Sch Dent, Araraquara, SP, Brazil Univ Estadual Campinas, Inst Biol, Dept Genet Evolut & Bioagents, Campinas, SP, Brazil Sao Paulo State Univ, Dept Diag & Surg, Sch Dent, Araraquara, SP, Brazil FAPESP: 2012-51110-8

Single nucleotide polymorphisms (SNPs) are important markers in many studies that link DNA sequence variations to phenotypic changes; such studies are expected to advance the understanding of human physiology and elucidate the molecular basis of diseases. TheDFNB1locus, which contains theGJB2andGJB6genes, plays a key role in nonsyndromic hearing loss. Previous studies have identified important mutations in this locus, but the contribution of SNPs in the genes has not yet been much investigated. The aim of this study was to investigate the association of nine polymorphisms located within theDFNB1locus with the occurrence of autosomal recessive nonsyndromic hearing loss (ARNSHL). The SNPs rs3751385 (C/T), rs7994748 (C/T), rs7329857 (C/T), rs7987302 (G/A), rs7322538 (G/A), rs9315400 (C/T), rs877098 (C/T), rs945369 (A/C), and rs7333214 (T/G) were genotyped in 122 deaf patients and 132 healthy controls using allele-specific PCR. There were statistically significant differences between patients and controls, in terms of allelic frequencies in the SNPs rs3751385, rs7994748, rs7329857, rs7987302, rs945369, and rs7333214 (P<0.05). No significant differences between the two groups were observed for rs7322538, rs9315400, and rs877098. Our results suggest that SNPs present in theGJB2andGJB6genes may have an influence on ARNSHL in humans.

Made available in DSpace on 2018-12-11T16:45:38Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-02-25 Studies focusing on possible genotoxic effects of excess fluoride are contradictory and inconclusive. Currently, studies have reported a probable link to oxidative stress, DNA damage and apoptosis induced by fluoride in rat hepatocytes. We developed an in vivo study administering three doses of fluoride by gavage given to rats for 60 day. Micronucleus test was applied to investigate genotoxic potential of fluoride. The TUNEL method determined DNA fragmentation and apoptosis. Biochemical parameters to investigate mitochondrial swelling and oxidative stress. Semi-quantitative RT-PCR and immunostaining to determine mRNA and protein expression of antioxidant enzymes. Analyses of the hepatic function and morphology were performed. Our results revealed the genotoxic potential of fluoride but did not confirm mitochondrial swelling nor an increase of positive TUNEL labelling induced by fluoride, indicating absence of apoptosis. Oxidative stress induction was confirmed and is probably associated to DNA damage. Cell death events such as empty nuclear spaces, cytoplasm degeneration, nuclear pyknosis, karyorrhexis and karyorrhexis followed by karyolysis were observed. Hepatic function did not appear to be significantly modified makes no evidence of necrosis and suggesting other cell death pathway, the autophagic. In conclusion, prolonged fluoride intake at chosen concentrations caused imbalance of the cellular oxidative state, affected DNA and disrupted cellular homeostasis. It is recommended that fluoride supplementation requires a fresh consideration in light of the current study. São Paulo State University (UNESP) Department of Biology Institute of Biosciences, Avenida 24-A, nº 1515, Bela Vista Heath Sciences Nucleus Hermínio Ometto University Center UNIARARAS Jd. Universitário, Avenida Dr. Maximiliano Barutto, nº 500 Structural and Functional Biology Laboratory (LABEF) Federal University of São Carlos – UFSCAR, Rodovia João Leme dos Santos, Km 110 - SP-264 Graduate Program in Biomedical Sciences Hermínio Ometto University Center UNIARARAS Jd. Universitário, Avenida Dr. Maximiliano Barutto, nº 500 Reproductive Biology Laboratory Structural and Functional Biology Department State University of Campinas – UNICAMP, CP. 6109 São Paulo State University (UNESP) Department of Biology Institute of Biosciences, Avenida 24-A, nº 1515, Bela Vista

The aim of this study was to evaluate markers of bone loss and immune response present in evolution of periodontal disease. One hundred and two Wistar rats were divided into three animals groups: PD0, without ligation and PD15 days and PD60 days, submitted to ligation placement with a sterile 3-0 silk cord in the cervical region of the upper first molar on both sides. Samples were obtained from the gingival tissue for histomorphometric analysis, immunohistochemical analysis of RANK, RANKL, OPG, characterization of the inflammatory infiltrate, quantification of nitric oxide, MCP-1, RANTES, IP10 chemokines, and expression of the TGF-b1, VEG, and bFGF. The number of inflammatory cells in gingival tissue was higher in PD60 samples. The collagen content and the area occupied by birefringent collagen fibers were lower for PD60. Differential leukocyte counting showed that there was a significantly higher polymorphonuclear influx in group PD15, while PD60 showed a greater number of lymphocytes. PD60 showed higher RANTES, IP-10, MCP-1 gene transcripts, as well as a higher nitric oxide concentration. Clinical evaluation revealed that the PD60 group presented an increase in furcal area. In conclusion, in this animal model the increase of RANK/RANKL and HGF markers is related to a specific immune response, and probably contributed to the evolution of periodontal disease. Investigating the effect of these biomarkers can help in targeted therapy for bone resorption, since blocking these can inhibit bone loss.

Tendo em vista a complexidade do mecanismo da audição, não é difícil compreender que a deficiência auditiva possa resultar de ampla variedade de anomalias geneticamente determinadas, bem como de diversos fatores ambientais. Mutações específicas no gene 12S rRNA em DNA mitocondrial são responsáveis por perda da audição não-sindrômica de herança materna, e pelo aumento da susceptibilidade ototóxica dos antibióticos aminoglicosídeos. OBJETIVO: Neste trabalho, nós avaliamos a presença da mutação C1494T entre os indivíduos ouvintes e com deficiência auditiva que utilizaram aminoglicosídeos e os que não tiveram contato com o antibiótico. MATERIAL E MÉTODO: Foram estudados 20 pacientes com deficiência auditiva neurossensorial não-sindrômica sem histórico de sensibilização aos aminoglicosídeos e 40 recém-nascidos, prematuros e de alto-risco que utilizaram a droga ototóxica, dos quais 20 eram ouvintes e 20 com perda auditiva. As amostras foram analisadas por PCR-RFLP com a enzima de restrição Hph I. FORMA DE ESTUDO: Experimental. RESULTADOS: A mutação mitocondrial C1494T no gene 12S rRNA não foi detectada em nenhuma das amostras analisadas. CONCLUSÃO: Nossos dados sugerem que a deficiência auditiva dos indivíduos analisados não está relacionada com a ototoxicidade da mutação C1494T, demonstrando que esta mutação não é frequente em nossa população.

Hereditary hearing loss is a complex disorder that involves a large number of genes. In developed countries, 1 in 1,000 children is born with deafness severe enough to require special education services, and about 60% of the cases of isolated deafness have a genetic origin. Although more than 100 genes for hearing loss are known currently, only a few are routinely tested in the clinical practice. In this study, we present our findings from the molecular diagnostic screening of the GJB2 and GJB3 genes, del(GJB6-D13S1830) and del(GJB6-D13S1854) deletions in the GJB6 gene, Q829X mutation in the otoferlin gene (OTOF) and, the A1555G and A7445G mutations in the mitochondrial genome over an 8-year period. Mutations analysis in the previously mentioned genes and mutations was performed on 645 unrelated Brazilian patients with hearing loss who fell into two different testing groups. Different mutations in the GJB2 gene were responsible for most of cases studied, but deletions in the GJB6 gene as well as mitochondrial mutations were also found. While most cases of hearing loss in this country are due to environmental factors, the genetic etiology of deafness will increasingly be determined as more genetic tests become available. © 2007 Wiley-Liss, Inc.

Sao Paulo state, Brazil, is one of the main areas of sugar cane planting in the world. Extensive use of ametryn, a triazine herbicide, in sugar cane agriculture and the properties of this herbicide suggest it could be present in the environment as a potential contaminant of soil, surface water, groundwater, and river sediment. In order to clarify the mechanism through which ametryn could be toxic, an in vivo study with Wistar rats was conducted using hematological, biochemical, molecular, morphological and genotoxic approaches. For this purpose, two sub-lethal ametryn concentrations (15 mg and 30 mg/kg/day) were administered to 42 rats divided into three groups (n=12) by gavage during 56 days, whereupon blood, liver and bone marrow were collected. The results showed ametryn genotoxic activity by in vivo micronuclei testing. This event probably occurred as consequence of oxidative stress induction demonstrated by GSTM1 transcript levels increase (indicating complexation between ametryn and/or metabolites with GSH) and by SOD activity decrease. Also, Mn-SOD transcripts were increased, probably avoiding mtDNA damage caused by EROS. These mechanisms displayed hepatic stellate cell (HSCs) activation because two major biomarkers were regulated, connexin and cadherin. N-cad transcripts were increased on both exposed groups while E-cad decreased in the T1 group, indicating epithelial-to-mesenchymal transition. In addition, Cx43 transcripts were decreased suggesting an increase in collagen content. Volumetric proportion of sinusoids was significantly decreased in T1 group and no significant alteration in hepatocyte volume was observed, indicating an increase in the space of Disse, due to fibrosis. Hepatocyte nuclei showed significant decrease in diameter and volume. Few hematological alterations were found. We emphasize the importance of other approaches, such as cell death and proliferation assays, so that ametryn toxicity can better be understood.

Aproximadamente 1/1000 recém-nascidos apresentam deficiência auditiva congênita, sendo 60% dessas de etiologia genética. Na maioria dos casos, a deficiência auditiva é uma doença multifatorial causada por ambos os fatores, genéticos e ambientais. A genética molecular da deficiência auditiva tem apresentado grandes avanços na última década, pois os genes responsáveis pela deficiência auditiva hereditária vêm sendo progressivamente mapeados e clonados. Esta revisão enfatiza a deficiência auditiva não-sindrômica, uma vez que, os genes envolvidos nesse tipo de deficiência foram identificados recentemente.

OBJETIVO: Investigar a prevalencia da mutacao 35delG em amostra de recem-nascidos, com teste molecular especifico; avaliar as perspectivas para a triagem neonatal genetica para a deficiencia auditiva. CASUISTICA E METODO: Foram avaliados 223 recem-nascidos no Hospital de Base de Sao Jose do Rio Preto, em Sao Paulo, para analise molecular da mutacao 35delG, no gene da conexina 26, com a tecnica da reacao em cadeia da polimerase alelo-especifico, apos extracao do DNA genomico de sangue de cordao umbilical. RESULTADOS: Foram identificados cinco heterozigotos, obtendo-se prevalencia de 2,24% de portadores da mutacao 35delG, na populacao do estudo. CONCLUSAO: O uso do teste molecular permitiu a identificacao da mutacao 35delG na populacao do estudo, podendo ser utilizado como complemento aos rastreamentos audiometricos neonatais por ser simples, rapido, de facil execucao e de baixo custo.

Aproximadamente 1/1000 recém-nascidos apresentam deficiência auditiva congênita, sendo 60% dessas de etiologia genética. Na maioria dos casos, a deficiência auditiva é uma doença multifatorial causada por ambos os fatores, genéticos e ambientais. A genética molecular da deficiência auditiva tem apresentado grandes avanços na última década, pois os genes responsáveis pela deficiência auditiva hereditária vêm sendo progressivamente mapeados e clonados. Esta revisão enfatiza a deficiência auditiva não-sindrômica, uma vez que, os genes envolvidos nesse tipo de deficiência foram identificados recentemente. One in every 1,000 newborn suffers from congenital hearing impairment. More than 60% of the congenital cases are caused by genetic factors. In most cases, hearing loss is a multifactorial disorder caused by both genetic and environmental factors. Molecular genetics of deafness has experienced remarkable progress in the last decade. Genes responsible for hereditary hearing impairment are being mapped and cloned progressively. This review focuses on non-syndromic hearing loss, since the gene involved in this type of hearing loss have only recently begun to be identified.

Mutations in the GJB2 gene are a major cause of congenital deafness. One specific mutation, the 35delG mutation, has accounted for most of the GJB2 mutations detected in European populations and is one of the most frequent disease mutations identified so far. We evaluated the frequency of the 35delG mutation in DNA samples from Brazilians of European, Asian, and African ancestry. All DNA samples were screened for the 35delG mutation using an allele-specific PCR. This study shows that the frequency of a common mutation (35delG) is significantly lower in non-European populations.

Congenital deafness occurs in approximately 1 in 1000 live births. In developed countries about 60% of hearing loss is genetic. However, in Brazil most cases of hearing loss are due to environmental factors, such as congenital infections (mainly rubella), perinatal anoxia, kernicterus and meningitis. Recently, it has been demonstrated that the GJB2 gene is a major gene underlying congenital sensorial deafness. Mutations in this gene cause 10-20% of all genetic sensory hearing loss. One specific mutation, 35delG, accounts for the majority of mutant alleles. The extent of the hearing impairment varies from mild/moderate to profound, even within the patients homozygous for the common 35delG mutation. There may also be progression with age. Mutation analysis in the GJB2 gene was performed on 36 families (group A) presenting with at least one individual with non-syndromic deafness (NSD). An unselected series of 26 deaf individuals referred by other services where the environmental factors were not completely excluded was also part of the study (group B). Mutations in the GJB2 gene were found in 22% (eight patients) of the families tested in group A, and 11.5% (three patients) of individuals within group B. This finding should facilitate diagnosis of congenital deafness and allow early treatment of the affected subjects.

Background: Human and animal evidence suggests that environmental toxicants may have an adverse impact on male reproductive health, reducing the population's reproductive output. Owing to the renewed attraction for natural products, some of them constitute effective alternatives to mitigate these effects. Propolis is a candidate for this use because of its intrinsic properties. In many situations, it improved the testicular damage and alleviated the toxic effects induced by environmental contaminant exposure. Objective: The aim of this study was to investigate possible alterations of testicular parameters and certify if its use is really advantageous to the testis, since this could affect rat reproductive function. Materials and Methods: Forty-eight adult male Wistar rats were divided into four groups (Co = control, T1 = 3 mg propolis/kg/day, T2 = 6 mg/kg/day, T3 = 10 mg/kg/day) and were exposed during 56 days. The testes were assessed with morphometrical, stereological, and ultrastructural analyses. Cell proliferation and death were diagnosed, respectively, by immunocytochemistry. Connexin 43 (Cx43) and N-cadherin transcript levels were determined by reverse transcription-polymerase chain reaction. Results: Increased cell proliferation and Leydig cell volume were observed in T2, and in contrast, Cx43 upregulation and cell death were observed in T3. Both T2 and T3 showed ultrastructural abnormalities in testicular parenchyma. Conclusion: We recommend a cautious intake of propolis to avoid deleterious effects. SUMMARY Chronic intake of Brazilian green propolis induced N.-cadherin downregulation and decreased on seminiferous tubule volumeIncrease on connexin 43 expression and cell death and decrease in Leydig cell.(LC) number/testis with the concentration of 10 mg/kg/day were observedIncrease on cell proliferation, cytoplasmic proportion, and volume of LC with the concentration of 6 mg/kg/day was detectedThe presence of empty spaces between spermatids and malformed spermatozoa in the lumen of seminiferous tubule was showedThis male reproductive disruption can be linked to phenolic compounds present in Brazilian green propolis. Abbreviation Used: AEC: 3-amino-9-ethylcarbazole; AJ: Adherens junction; AME: Aromadendrin-40-methyl ether; CAPE: Caffeic acid phenethyl ester; Co: Control group; C×43: Connexin 43; DAB: Diaminobenzidine; dNTP: Deoxyribonucleotide phosphate; DSP: Daily sperm production; FA: Ferulic acid; FSH: Follicle-stimulating hormone; GJ: Gap junction; GJIC: Gap junction intercellular communication; HPLC: High-performance liquid chromatography; LC: Leydig cell; LH: Luteinizing hormone; N-cad: N-cadherin; PCNA: Proliferating cell nuclear antigen; PCR: Polymerase chain reaction; RT-PCR: Reverse transcription-polymerase chain reaction; SDM: Standard deviation of mean; T1: Group exposed to 3 mg of propolis/kg/day; T2: Group exposed to 6 mg of propolis/kg/day; T3: Group exposed to 10 mg of propolis/kg/day; TUNEL: Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; WB-ras 2 cells: Ras-transformed rat liver epithelial cell line.

The tetra-primer amplification refractory mutation system-polymerase chain (ARMS-PCR) reaction is a simple and economical method to genotype single-nucleotide polymorphisms (SNPs). It uses four primers in a single PCR and is followed just by gel electrophoresis. However, the optimization step can be very hardworking and time-consuming. Hence, we propose to demonstrate and discuss critical steps for its development, in a way to provide useful information. Two SNPs that provided different amplification conditions were selected. DNA extraction methods, annealing temperatures, PCR cycles protocols, reagents, and primers concentration were also analyzed. The use of tetra-primer ARMS-PCR could be impaired for SNPs in DNA regions rich in cytosine and guanine and for samples with DNA not purified. The melting temperature was considered the factor of greater interference. However, small changes in the reagents concentration significantly affect the PCR, especially MgCl2. Balancing the inner primers band is also a key step. So, in order to balance the inner primers band, intensity is important to observe which one has the weakest band and promote its band by increasing its concentration. The use of tetra-primer ARMS-PCR attends the expectations of modern genomic research and allows the study of SNPs in a fast, reliable, and low-cost way.

Oncidium flexuosum Sims. is an orchidwhose leaves are frequently used for therapeutic applications although its action mechanism is not exactly understood. So the aim of this study was investigate its effects in Wistar rat liver, kidney and bone marrow. For this purpose, twenty-five adult male Wistar rats were divided into five groups of 5 animals each: a control group receiving only water, a positive control receiving a mutagen compound and three groups treated for 15 days with 100, 250 or 500 mg/kg/day of a crude hydroalcoholic leaf extract of O. flexusosum Sims. No significant alterations were observed in –SH groups, MDA or catalase, showing that the extract does not induce hepatic oxidative damage. There was no evidence of kidney injury and hepatic function alteration (ALT). However, significant mitochondrial swelling was observed in the treated groups and is probably related to cell death induction observed in cells that displayed acidophilia, pyknosis and positive labeling with the TUNEL technique at higher concentrations of the extract. We demonstrated that the hepatotoxicity of the extract resulted from its ability to induce DNA and mitochondrial damage that arrests cells, leading to cell death, probably caused by the flavonoidsand tannins present in this extract.

Pertencente à família Papillomaviridae, o papilomavírus humano (HPV) é formado por um capsídeo e uma única fita dupla de DNA. Sua infecção ocorre principalmente por forma sexual, apresentando grande tropismo por células cutâneas e mucosas. OBJETIVO: Avaliar a presença do HPV em mucosa oral de pacientes assintomáticos e em paralelo correlacionar os hábitos sociais comportamentais com a presença viral. MATERIAL E MÉTODO: Estudo de coorte contemporânea com corte transversal. O diagnóstico da presença viral foi realizado por PCR, utilizando os primers gerais MY09/11 em 125 amostras de mucosa oral, submetidas à extração de DNA e PCR para a pesquisa do gene da beta-globina para avaliação da qualidade do DNA extraído. Em paralelo, foi realizado um estudo de questões comportamentais dos pacientes. RESULTADOS: Todas as amostras apresentaram o diagnóstico positivo para o gene da betaglobina. O HPV foi diagnosticado em 23,2% das amostras analisadas. CONCLUSÃO: O vírus esteve presente em 29 dos 125 pacientes, sem que estes apresentassem qualquer manifestação clinicopatológica relacionada com o HPV. Quanto ao comportamento social dos pacientes, concluiu-se que a prática de sexo oral está correlacionada de forma estatisticamente significante com a presença viral, além de o HPV ter sido estatisticamente mais prevalente em pacientes do sexo feminino. The Human Papillomavirus (HPV) belongs to the Papillomaviridae family and has a capsid and a single DNA strand. Its infection occurs mainly through sexual intercourse, having an important tropism for skin and mucosal cells. AIM: To evaluate the HPV presence in normal oral mucosa of asymptomatic subjects and; in parallel, to correlate social behavioral habits with the virus. MATERIALS AND METHODS: Contemporary cohort cross-sectional study. The HPV was found by PCR, using general primers MY09/11 in 125 oral mucosa samples submitted to DNA extraction and PCR to search for the beta-globin gene in order to assess the quality of the extracted DNA. In parallel, we carried out a study of behavioral issues associated with the patients. RESULTS: All the samples had a positive diagnosis of the beta-hemoglobin gene. HPV was diagnosed in 23.2% of the samples analyzed. CONCLUSION: The virus was present in 29 of the 125 patients, without them having any clinical-pathological manifestation associated with the HPV. As to the social behavior of the patients, we concluded that oral sex is statistically correlated to the virus, and besides the HPV has been statistically more present in female patients.

Risk assessments suggest that intermediate and long-term exposure to triazine herbicides and its metabolites through water can cause severe damage to human health. The objective of this study was to investigate the possible effects of atrazine on Wistar rats submitted to subacute treatment. For this purpose, the activity of catalase and alanine aminotransferase was quantified, and the effect of the herbicide on cell membranes was examined based on the measurement of lipid peroxidation and consequent formation of malondialdehyde and on the mRNA expression of antioxidant enzymes (Mn-superoxide dismutase [SOD] and GSTM1) and connexins. In addition, we evaluated histopathological alterations in the liver, cellular expression of SOD and glutathione (GST), activation of heat shock proteins (HSPs) by immunohistochemistry, and the induction of apoptosis. The genotoxic potential of the herbicide was investigated by the micronucleus test in bone marrow smears. Adult male Wistar rats were treated with an aqueous solution of atrazine at a concentration of 400mg/kg/day, by gavage, for 14 consecutive days. Control groups were also included. The results showed an increase of catalase levels and maintenance of the expression of antioxidant enzymes (SOD and GST). In addition, lipid peroxidation, hepatic tissue degeneration, activation of HSP90, increased levels of connexin mRNA, and genotoxicity were observed. In conclusion, atrazine induced early hepatic oxidative stress that triggered defense mechanisms to maintain the morphophysiological integrity of the liver. Further studies are needed to better understand the effects of this herbicide on human health.

O processo de morte e danos em células ciliadas devido à exposição ao ruído e ototoxinas parece ser mediado por espécies reativas de oxigênio. OBJETIVO: Investigar a relação entre polimorfismos gênicos na Glutationa S-transferase e a susceptibilidade à deficiência auditiva induzida pelos aminoglicosídeos. CASUÍSTICA E MÉTODO: Genótipos nulos foram analisados por PCR-multiplex em amostras de DNA de 50 pacientes e 72 controles. Os pacientes foram divididos em três grupos, sendo 10 com deficiência auditiva e uso de aminoglicosídeos (grupo A), 20 com deficiência auditiva sem exposição à droga (grupo B), e 20 ouvintes que utilizaram o antibiótico (grupo C). FORMA DE ESTUDO: Experimental. RESULTADOS: Polimorfismos nos genes GSTT1 e GSTM1 foram encontrados em 16% e 42% dos pacientes e em 18% e 53% do grupo controle, respectivamente. Após a análise estatística nenhuma diferença significativa foi observada entre os grupos controle e A (p=0,86) e (p=0,41), controle e B (p=0,27) e (p=0,24), controle e C (p=0,07) e (p=0,47), controle e A+C (p=0,09) e (p=0,47), C e A (p=0,32) e (p=0,75), GSTT1 e GSTM1, respectivamente. CONCLUSÃO: Nossos dados demonstram que polimorfismos na GSTT1 e GSTM1 não exercem influência sobre a ototoxicidade dos aminoglicosídeos. The process of hair cell damage and death as a result of exposure to noise and ototoxins seems to be mediated by reactive oxygen species. AIM: To investigate the relationship between genetic polymorphisms in the Glutathione S-transferase and the susceptibility to hearing loss induced by aminoglycosides. MATERIALS AND METHODS: Null genotypes were analyzed by multiplex-PCR in the DNA samples from 50 patients and 72 controls. The patients were divided into 3 groups, 10 with hearing loss using aminoglycosides (group A), 20 with hearing loss without exposure to the drug (group B) and 20 hearing individuals who used the antibiotic (group C). STUDY DESIGN: Experimental. RESULTS: Polymorphisms in the GSTM1 and GSTT1 genes were found in 16% and 42% of patients and in 18% and 53% of the control group, respectively. After statistical analysis no significant difference was observed between the control groups and A (p=0.86) and (p=0.41), controls and B (p=0.27) and (p=0.24), controls and C (p=0.07) and (p=0.47), controls and A + C (p=0.09) and (p=0.47), C and A (p=0.32) and (p=0.75), GSTT1 and GSTM1, respectively. CONCLUSION: Our data show that polymorphisms in GSTM1 and GSTT1 genes have no influence on the ototoxicity of aminoglycosides.

Objective: An early diagnosis has been a priority in the audiological practice. Identifying hearing loss until 3 months old through Universal Newborn Hearing Screening and intervention before 6 months old, minimize the impact of auditory loss in the health and communication development of these children. However, in the clinical practice, despite the help of the risk indicators in the audiological and etiological diagnosis, the integrated services have come up against the challenge of determining the causes of auditory loss, bearing in mind that approximately 50% of the subjects who have congenital loss do not show risk factors in their clinical history. The current research aims introduce together etiologic and audiological diagnosis of newborns. Methods: We eluted dried blood spots from paper and performed genetic testing for 35delG mutation in 8974 newborns that were also screened for transient otoacoustic emissions (TOAE). In addition, the A1555G and A827G mutations in the MTRNR1 mitochondrial gene were screened in all newborns. Results: We have found 17 individuals who failed in TOAE. Among them, we detected 4 homozygous newborns for 35delG mutation and 3 individuals with A827G mutation in the MTRNR1 mitochondrial gene. The frequency of 35delG carriers was 0.94% [84/8974]. In all 17 individuals who failed in OAE no other mutation besides those mentioned above was found. Conclusions: The results greatly contribute to the public health area indicating the etiologic diagnosis, allowing family counseling as well as the early rehabilitation treatment or surgical intervention. Over time that will help to reduce the costs of rehabilitation considerably.

The connexins are a family of proteins whose major function is as part of the gap junctions of cell-to-cell channels. They are expressed in several tissues including brain, skin, and cochlea. Mutations in connexin genes play a major role in non-syndromic sensorineural deafness, but have been also described in individuals with variable dermatological features. In recent years, many genes responsible for hereditary skin diseases have been discovered. These genes may encode different proteins that participate in the terminal differentiation of the epidermis. Therefore alteration or absence of these proteins causes a keratinization disorder. It has been demonstrated that distinct germline mutations within six connexin (Cx) genes GJB2 (Cx26), GJB6 (Cx30), GJB3 (Cx31), GJA1 (Cx43), GJB4 (Cx30.3), and GJB5 (Cx31.1), may cause sensorineural hearing loss and various skin disease phenotypes. The crucial functional importance of each of these connexins in the mentioned ectodermic tissues is reflected by the finding that genetic defects in their genes produce a wide spectrum of genetic disorders comprising sensorineural hearing loss, disorders of cornification of the skin, hair, and nails, and keratitis. Here, we report on different mutations in the connexin genes in individuals with or without hearing loss and different skin disorders illustrating the clinical and genetic heterogeneity of the condition.

The process of hair cell damage and death as a result of exposure to noise and ototoxins seems to be mediated by reactive oxygen species.To investigate the relationship between genetic polymorphisms in the Glutathione S-transferase and the susceptibility to hearing loss induced by aminoglycosides.Null genotypes were analyzed by multiplex-PCR in the DNA samples from 50 patients and 72 controls. The patients were divided into 3 groups, 10 with hearing loss using aminoglycosides (group A), 20 with hearing loss without exposure to the drug (group B) and 20 hearing individuals who used the antibiotic (group C).Experimental.Polymorphisms in the GSTM1 and GSTT1 genes were found in 16% and 42% of patients and in 18% and 53% of the control group, respectively. After statistical analysis no significant difference was observed between the control groups and A (p=0.86) and (p=0.41), controls and B (p=0.27) and (p=0.24), controls and C (p=0.07) and (p=0.47), controls and A + C (p=0.09) and (p=0.47), C and A (p=0.32) and (p=0.75), GSTT1 and GSTM1, respectively.Our data show that polymorphisms in GSTM1 and GSTT1 genes have no influence on the ototoxicity of aminoglycosides.

Tendo em vista a complexidade do mecanismo da audição, não é difícil compreender que a deficiência auditiva possa resultar de ampla variedade de anomalias geneticamente determinadas, bem como de diversos fatores ambientais. Mutações específicas no gene 12S rRNA em DNA mitocondrial são responsáveis por perda da audição não-sindrômica de herança materna, e pelo aumento da susceptibilidade ototóxica dos antibióticos aminoglicosídeos. OBJETIVO: Neste trabalho, nós avaliamos a presença da mutação C1494T entre os indivíduos ouvintes e com deficiência auditiva que utilizaram aminoglicosídeos e os que não tiveram contato com o antibiótico. MATERIAL E MÉTODO: Foram estudados 20 pacientes com deficiência auditiva neurossensorial não-sindrômica sem histórico de sensibilização aos aminoglicosídeos e 40 recém-nascidos, prematuros e de alto-risco que utilizaram a droga ototóxica, dos quais 20 eram ouvintes e 20 com perda auditiva. As amostras foram analisadas por PCR-RFLP com a enzima de restrição Hph I. FORMA DE ESTUDO: Experimental. RESULTADOS: A mutação mitocondrial C1494T no gene 12S rRNA não foi detectada em nenhuma das amostras analisadas. CONCLUSÃO: Nossos dados sugerem que a deficiência auditiva dos indivíduos analisados não está relacionada com a ototoxicidade da mutação C1494T, demonstrando que esta mutação não é frequente em nossa população. In view of the complex mechanism of hearing, it is not difficult to understand that hearing impairment may result from a wide variety of genetically determined anomalies and various environmental factors. Specific mutations in the mitochondrial DNA 12S rRNA gene are responsible for maternally inherited non-syndromic hearing loss, and for increased susceptibility to the ototoxicity of aminoglycoside antibiotics. AIM: To asses the presence of C1494T mutation among individuals with normal hearing and hearing impairment who used aminoglycosides and those who had not had contact with the antibiotic. MATERIAL AND METHOD: The study was composed of 20 patients with nonsyndromic sensorineural hearing loss without prior use of aminoglycosides and 40 premature and high-risk newborns who used ototoxic drugs, of whom 20 had good hearing and 20 had hearing loss. The samples were analyzed by PCR-RFLP with the restriction enzyme Hph I. STUDY DESIGN: Experimental. RESULTS: The mitochondrial 12S rRNA C1494T mutation was not detected in any of the samples analyzed. CONCLUSION: Our data suggest that the hearing loss of the individuals we analyzed was not related to the ototoxicity of mutation C1494T, showing that this mutation is not frequent in our population.

The most common form of non-syndromic autosomal recessive deafness (NSRD) is caused by mutations in the GJB2 gene. Recently, a deletion truncating the GJB6 gene, called del(GJB6-D13S1,830) has also been described normally accompanying mutations in another allele of the GJB2 gene. Among all the mutations described to date, 35delG in the GJB2 gene is the most common. Preliminary data suggest that pathologic changes due to GJB2 mutations do not affect the spiral ganglion cells, which are the site of stimulation of the cochlear implant. Besides, the survival of the spiral ganglion cells is believed to be an important determinant of the outcome after surgery. Therefore, we have studied 49 non-syndromic deaf patients with unknown etiologies in order to determine the prevalence of GJB2 and GJB6 gene mutations in patients undergoing cochlear implantation surgery. Also, the molecular studies were performed using polymerase chain reaction amplification and direct sequencing. As a result, we found 19 individuals with GJB2 mutation including one new mutation (K168R), one patient homozygous for the del(GJB6-D13S1,830). These results establish that genetic screening can provide an etiologic diagnosis, and may help with prognosis after cochlear implantation, as has been hypothesized in previous studies.

OBJETIVO: Investigar a prevalência da mutação 35delG em amostra de recém-nascidos, com teste molecular específico; avaliar as perspectivas para a triagem neonatal genética para a deficiência auditiva. CASUÍSTICA E MÉTODO: Foram avaliados 223 recém-nascidos no Hospital de Base de São José do Rio Preto, em São Paulo, para análise molecular da mutação 35delG, no gene da conexina 26, com a técnica da reação em cadeia da polimerase alelo-específico, após extração do DNA genômico de sangue de cordão umbilical. RESULTADOS: Foram identificados cinco heterozigotos, obtendo-se prevalência de 2,24% de portadores da mutação 35delG, na população do estudo. CONCLUSÃO: O uso do teste molecular permitiu a identificação da mutação 35delG na população do estudo, podendo ser utilizado como complemento aos rastreamentos audiométricos neonatais por ser simples, rápido, de fácil execução e de baixo custo. OBJECTIVES: To investigate the prevalence of the 35delG mutation in a newborn population, with specific molecular testing, and to evaluate the prospects for genetic neonatal screening for hearing impairment. POPULATION AND METHOD: 233 newborn were evaluated at the Hospital de Base de São José do Rio Preto, SP, for molecular analysis of the 35delG mutation in the connexin 26 gene, with the reaction technique in allele-specific polymerase chain reaction, after genomic DNA extraction from umbilical cord blood. RESULTS: Five heterozygotes were identified, obtaining a prevalence of 2.24% of 35delG mutation carriers in the study population. CONCLUSION: Using the molecular test allowed for the identification of the 35delG mutation in the study population with the possibility of being used as a complement to neonatal audiometric screening as being simple, fast, and easily to perform with low costs.

A crescente prevalência de hipertensão arterial na população mundial e os riscos por ela apresentados nas doenças coronarianas eleva a importância de seu controle. Sendo sua causa, frequentemente multifatorial, o tratamento da patologia é dificultado. Fatores ambientais associados à predisposição genética levam o indivíduo a apresentar índices pressóricos elevados de pressão arterial quando comparados a indivíduos que não apresentam tal predisposição. Identificar a predisposição genética seria ideal para amenizar ou, até mesmo, evitar o desenvolvimento da patologia. As nanopartículas estão cada vez mais associadas com biomoléculas, uma vez que suas propriedades associadas às questões médicas podem criar novos métodos potencialmente eficientes, tanto no diagnóstico como na terapêutica. O presente trabalho teve como objetivo a conjugação de nanopartículas de ouro, estabilizadas com dendrímero poli(amidoamina) de geração 4, com oligonucleotídeo para obtenção de genossensores capazes de detectar o polimorfismo de inserção e deleção do gene da enzima conversora de angiotensina I, o qual está intimamente relacionado com a predisposição à hipertensão arterial sistêmica. As nanopartículas foram caracterizadas por Microscopia Eletrônica de Transmissão (TEM), potencial zeta e Espectroscopia no Ultravioleta-Visível (UV-VIS). A formação do conjugado entre a nanopartícula e o oligonucleotídeo foi confirmada por UV-VIS, Espalhamento Dinâmico de Luz (DLS) e Espectroscopia no Infravermelho com Transformada de Fourier (FTIR). Foram construídos três sistemas de detecção diferentes, nos quais as técnicas empregadas foram Espectroscopia de Impedância Elétrica, Espectroscopia de Impedância Eletroquímica e Transistor de Efeito de Campo de Porta Estendida e Separada (SEGFET). O polimorfismo foi detectado em concentrações da ordem de 1 nM. Com destaque para aqueles em que o emprego do conjugado amplificou o sinal pelas propriedades das nanopartículas de ouro. Os genossensores propostos são promissores e futuramente poderão contribuir com a medicina preventiva. The increasing prevalence of hypertension in the world population and the risks presented by it in coronary heart disease reveals the importance of their control. Due to its multifactorial causes, the treatment of this disease is difficult. Environmental factors associated with genetic predisposition lead the individual to present high indexes of blood pressure when compared to individuals who do not have a predisposition. To identify the genetic predisposition would be ideal to minimize or even to prevent the pathology development. Nanoparticles are increasingly associated with biomolecules, their properties added with medical questions, can create new methods potentially efficient, both in diagnosis and therapy. This study aims at developing of poly(amidoamine) dendrimer-stabilized gold nanoparticles, conjugated with oligonucleotides to obtain genosensors able to detect the polymorphism of insertion and deletion of angiotensin I converting enzyme (ACE) gene, which is closely related with the predisposition to systemic blood hypertension. The nanoparticles were characterized by Transmission Electronic Microscopy (TEM), Zeta potential and Ultraviolet-Visible Spectroscopy (UV-VIS). The formation of the conjugate formed by the nanoparticle and the oligonucleotide was confirmed by UV-VIS, Dynamic Light Scattering (DLS) and Fourier Transform Infrared Spectroscopy (FTIR). Three different detection systems were built, in which the following techniques were applied: Electrical Impedance Spectroscopy, Electrochemical Impedance Spectroscopy and Separative Extended Gate Field Effect Transitor (SEGFET). For all systems polymorphism - related sequences were detected at concentrations down to nanomolar. The use of the conjugate amplified the signal of the genosensor due to the nanoparticles. The proposed genosensors may contribute to preventative medicine.