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The aim of this study was to investigate socioeconomic stressors predictive of depressive symptoms and possible epigenetic changes in the glucocorticoid receptor - NR3C1-1F - an encoding gene involved in depressive symptoms. A total of 321 adult volunteers from southeastern Brazil were recruited to evaluate depressive symptoms, socio-demographic and economic factors, including food and nutritional security (FNS) or insecurity (FNiS) status, and NR3C1-1F region methylation by pyrosequencing. Depressive symptom determinants were investigated using a Poisson regression model with robust variance. Mann-Whitney tests and structural mediation equation models were used to evaluate the relationship between NR3C1 DNA methylation, FNiS, and depressive symptoms. Multivariate Poisson regression with robust variance adjusted for sex and FNiS and NR3C1-1F region methylation predicted risk factors for depressive symptoms. Mediation analysis revealed that NR3C1-1F region methylation mediated the relationship between FNiS exposure and depressive symptoms as an outcome, and depressive volunteers and FNiS individuals exhibited a significant increase in NR3C1 methylation when compared to healthy individuals and FNS volunteers, respectively. Therefore, we suggest that stress caused by FNiS may lead to depressive symptoms and that NR3C1-1F DNA methylation can act as a mediator of both FNiS and depressive symptoms.
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Depressão , Insegurança Alimentar , Estresse Psicológico , Adulto , Metilação de DNA , Depressão/genética , Epigênese Genética , Humanos , Regiões Promotoras Genéticas , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Estresse Psicológico/genéticaRESUMO
BACKGROUND: Epidermal growth factor receptor (EGFR) is frequently activated in head and neck squamous cell carcinoma (HNSCC) and serves as a valuable target for therapy. Despite the availability of the EGFR inhibitors Cetuximab, Afatinib, and Allitinib, there are limited predictive markers for their response. Understanding molecular aberrations in HNSCC could facilitate the identification of new strategies for patient clinical and biological classification, offering novel therapeutic avenues. METHODS: We assessed CCNA1, DCC, MGMT, CDKN2A/p16, and DAPK methylation status in HNSCC cell lines and their association with anti-EGFR treatment response. RESULTS: MGMT methylation status displayed high sensitivity and specificity in distinguishing sensitive and resistant HNSCC cell lines to Afatinib (AUC = 0.955) and Allitinib (AUC = 0.935). Moreover, DAPK methylation status predicted response to Allitinib with high accuracy (AUC = 0.852), indicating their putative predictive biomarker roles. CONCLUSION: These findings hold promise for the development of more personalized and effective treatment approaches for HNSCC patients.
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Acrilamidas , Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Quinazolinas , Humanos , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Afatinib , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/genética , Cetuximab/farmacologia , Cetuximab/uso terapêutico , Receptores ErbB/metabolismo , Linhagem Celular Tumoral , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/uso terapêutico , Proteínas Supressoras de Tumor , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/uso terapêuticoRESUMO
BACKGROUND: HOXA1 is a prognostic marker and a potential predictive biomarker for radioresistance in head and neck tumors. Its overexpression has been associated with promoter methylation and a worse prognosis in oral squamous cell carcinoma (OSCC) patients. However, opposite outcomes are also described. The effect of the methylation of this gene on different gene regions, other than the promoter, remains uncertain. We investigated the methylation profile at different genomic regions of HOXA1 in OSCC and correlated differentially methylated CpG sites with clinicopathological data. METHODS: The HOXA1 DNA methylation status was evaluated by analyzing data from The Cancer Genome Atlas and three Gene Expression Omnibus datasets. Significant differentially methylated CpG sites were considered with a |∆ß| ≥ 0.10 and a Bonferroni-corrected p-value < 0.01. Differentially methylated CpGs were validated by pyrosequencing using two independent cohorts of 15 and 47 OSCC patients, respectively. RESULTS: Compared to normal tissues, we found significantly higher DNA methylation levels in the 3'UTR region of HOXA1 in OSCC. Higher methylation levels in tumor samples were positively correlated with smoking habits and patients' overall survival. CONCLUSIONS: Our findings suggest that HOXA1 gene body methylation is a promising prognostic biomarker for OSCC with potential clinical applications in patient monitoring.
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METHODS: One-hundred-six patients diagnosed with non-muscle invasive bladder cancer and treated with intravesical BCG were included and divided into two groups, BCG-responsive (n = 47) and -unresponsive (n = 59). Immunohistochemistry was used to evaluate PD-L1 expression and MSI was assessed by a commercial multiplex PCR kit. The mRNA expression profile of 15 immune checkpoints was performed using the nCounter technology. For in silico validation, two distinct cohorts sourced from the Gene Expression Omnibus (GEO) database were used. RESULTS: Among the 106 patients, only one (<1 %) exhibited MSI instability. PD-L1 expression was present in 9.4 % of cases, and no association was found with BCG-responsive status. We found low gene expression of canonic actionable immune checkpoints PDCD1 (PD-1), CD274 (PD-L1), and CTLA4, while high expression was observed for CD276 (B7-H3), CD47, TNFRSF14, IDO1 and PVR (CD155) genes. High IDO1 expression levels was associated with worst overall survival. The PDCD1, CTLA4 and TNFRSF14 expression levels were associated with BCG responsiveness, whereas TIGIT and CD276 were associated with unresponsiveness. Finally, CD276 was validated in silico cohorts. CONCLUSION: In NMIBC, MSI is rare and PD-L1 expression is present in a small subset of cases. Expression levels of PDCD1, CTLA4, TNFRSF14, TIGIT and CD276 could constitute predictive biomarkers of BCG responsiveness.
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In light of advancements in the field of immuno-oncology, the demand for obtaining mononuclear cells for in vitro assays has surged. However, obtaining these cells from healthy donors remains a challenging task due to difficulties in donor recruitment and the requirement for substantial blood volumes. Here, we present a protocol for isolating peripheral blood mononuclear cells (PBMCs) from leukodepletion filters used in whole blood and erythrocytes by apheresis donations at the Hemonucleus of the Barretos Cancer Hospital, Brazil. The method involves rinsing the leukodepletion filters and subsequent centrifugation using a Ficoll-Paque concentration gradient. The isolated PBMCs were analyzed by flow cytometry, which allowed the identification of various subpopulations, including CD4+ T lymphocytes (CD45+CD4+), CD8+ T lymphocytes (CD45+CD8+), B lymphocytes (CD45+CD20+CD19+), non-classical monocytes (CD45+CD64+CD14-), classical monocytes (CD45+CD64+CD14+), and granulocytes (CD45+CD15+CD14-). In our comparative analysis of filters, we observed a higher yield of PBMCs from whole blood filters than those obtained from erythrocytes through apheresis. Additionally, fresh samples exhibited superior viability when compared to cryopreserved ones. Given this, leukodepletion filters provide a practical and cost-effective means to isolate large quantities of pure PBMCs, making it a feasible source for obtaining mononuclear cells for in vitro experiments. SUMMARY: Here, we provide a detailed protocol for the isolation of mononuclear cells from leukodepletion filters, which are routinely discarded at the Barretos Cancer Hospital's Hemonucleus.
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Leucócitos Mononucleares , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/citologia , Citometria de Fluxo , Separação Celular/métodos , Separação Celular/instrumentação , Leucaférese/instrumentação , Leucaférese/métodos , Brasil , Criopreservação/métodosRESUMO
Background: Dietary composition can modify gene expression, favoring the development of chronic diseases via epigenetic mechanisms. Objective: Our study aimed to investigate the relationship between dietary patterns and NR3C1 gene methylation in users of the Brazilian Public Unified Health System (SUS). Methods: We recruited 250 adult volunteers and evaluated their socioeconomic status, psychosocial characteristics, lifestyle, and anthropometrics. Peripheral blood was collected and evaluated for cortisol levels, glycemia, lipid profile, and insulin resistance; methylation of CpGs 40-47 of the 1F region of the NR3C1 gene was also measured. Factors associated with degree of methylation were evaluated using generalized linear models (p < 0.05). Lifestyle variables and health variables were included as confounding factors. Results: The findings of our cross-sectional study indicated an association between NR3C1 DNA methylation and intake of processed foods. We also observed relevant associations of average NR3C1 DNA across the segment analyzed, methylation in component 1 (40-43), and methylation in component 2 (44-47) with a pattern of consumption of industrialized products in relation to BMI, serum cortisol levels, and lipid profile. These results may indicate a relationship between methylation and metabolic changes related to the stress response. Conclusion: These findings suggest an association of methylation and metabolic alterations with stress response. In addition, the present study highlights the significant role of diet quality as a stress-inducing factor that influences NR3C1 methylation. This relationship is further linked to changes in psychosocial factors, lifestyle choices, and cardiometabolic variables, including glucose levels, insulin resistance, and hyperlipidemia.
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Immune checkpoint blockade (ICB) agents are prominent immunotherapies for the treatment of advanced melanoma. However, they fail to promote any durable clinical benefit in a large cohort of patients. This study assessed clinical and molecular predictors of ICB response and survival in advanced melanoma. A retrospective analysis was performed on 210 patients treated with PD-1 or CTLA-4 inhibitors at Barretos Cancer Hospital, Brazil. PD-L1 expression was assessed by immunohistochemistry using formalin-fixed paraffin-embedded tumor tissues collected prior to ICB therapy. Patients were divided into responders (complete and partial response and stable disease for more than 6 months) and non-responders (stable disease for less than 6 months and progressive disease). Among them, about 82% underwent anti-PD-1 immunotherapy, and 60.5% progressed after the ICB treatment. Patients that received ICB as first-line therapy showed higher response rates than previously treated patients. Higher response rates were further associated with superficial spreading melanomas and positive PD-L1 expression (>1%). Likewise, PD-L1 positive expression and BRAF V600 mutations were associated with a higher overall survival after ICB therapy. Since ICBs are expensive therapies, evaluation of PD-L1 tumor expression in melanoma patients should be routinely assessed to select patients that are most likely to respond.
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BACKGROUND: Metastatic lymph node involvement influences therapy decisions and serves as a prognostic indicator in oral squamous cell carcinoma (OSCC). However, many early-stage patients with clinically negative lymph nodes exhibit no metastasis upon surgical staging. This study aimed to identify differentially expressed miRNAs capable of distinguishing pathologically positive (pN+) from negative (pN0) nodes in OSCC patients without clinical evidence of lymph node metastases (cN0). METHODS: Expression levels of 798 miRNAs were assessed in tumor samples from 10 pN+ and 10 pN0 patients using the Nanostring nCounter platform. Validation was performed in an independent cohort of 15 pN+ and 24 pN0 patients through RT-qPCR. RESULTS: Eight miRNAs exhibited differential expression between pN0 and pN+ patients. Notably, hsa-miR-99a-5p demonstrated high sensitivity and specificity in predicting patients at higher risk of positive lymph nodes. CONCLUSIONS: These findings highlight hsa-miR-99a-5p as a potential biomarker for detecting lymph node metastasis in primary OSCC tumors.
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Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , MicroRNAs , Neoplasias Bucais , Humanos , Carcinoma de Células Escamosas de Cabeça e Pescoço , Carcinoma de Células Escamosas/patologia , Metástase Linfática , Neoplasias Bucais/patologia , Biomarcadores Tumorais/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Regulação Neoplásica da Expressão GênicaRESUMO
This study aimed to investigate BDNF gene methylation in individuals with depression based on tobacco use. Therefore, 384 adults from southeastern Brazil were recruited to assess depression, socioeconomic status, lifestyle, and methylation by pyrosequencing exon IV promoter region of the BDNF gene. The Generalized Linear Model (GzLM) was used to check the effect of depression, tobacco, and the interaction between depression and tobacco use in methylation levels. In addition, the Kruskal-Wallis test, followed by Dunn's post hoc test, was used to compare methylation levels. Interaction between depression and tobacco use was significant at levels of BDNF methylation in the CpG 5 (p = 0.045), 8 (p = 0.016), 9 (p = 0.042), 10 (p = 0.026) and mean 5-11 (p < 0.001). Dunn's post hoc test showed that individuals with depression and tobacco use compared to those with or without depression who did not use tobacco had lower levels of BDNF methylation in CpG 5, 6, 7, 8, 9, 11, and mean 5-11. Therefore, we suggest that tobacco use appears to interfere with BDNF gene methylation in depressed individuals.
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Fator Neurotrófico Derivado do Encéfalo , Metilação de DNA , Adulto , Humanos , Fator Neurotrófico Derivado do Encéfalo/genética , Depressão/genética , Éxons , Uso de TabacoRESUMO
BACKGROUND: Epigenetic mechanisms may affect the ideal and non-ideal kidneys selected for transplantation and their inflammatory gene expression profile differently and may contribute to poor clinical outcomes. OBJECTIVE: Study the Global DNA methylation and the expression profiles of the DNA methyltransferases (DNMTs) and nuclear factor kappa B (NF-κB) in preimplantation kidney biopsies from ideal and non-ideal kidneys (expanded criteria donor (ECD) and with KDPI > 85%). METHODS: In a sample consisting of 45 consecutive pre-implantation biopsies, global DNA methylation levels were detected by LINE-1 repeated elements using bisulfite pyrosequencing. DNMT gene expression was assessed by real-time quantitative polymerase chain reaction, and NF-κB protein expression by immunofluorescence. RESULTS: ECD kidneys displayed increased methylation levels in LINE-1, and DNMT1 and DNMT3B expression was upregulated when comparing ECD to standard criteria donor kidneys. Similarly, kidneys with KDPI > 85% exhibited increased LINE-1 methylation and DNMT1 upregulation when compared to a KDPI ≤ 85%. NF-κB protein expression levels were greatly increased in both types of non-ideal kidneys compared to ideal kidneys. Moreover, hypermethylation of LINE-1 was associated with cold ischemia time > 20 h and ECD kidney classification. CONCLUSIONS: This study shows that global DNA hypermethylation and high expression of NF-κB occurred in both types of non-ideal kidneys and were associated with prolonged cold ischemia time. Global DNA methylation can be a useful tool to assess non-ideal kidneys and hence, could be used to expand the pool of kidneys donors.
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Transplante de Rim , NF-kappa B , Biópsia , DNA , Metilação de DNA , Humanos , Rim/patologia , NF-kappa B/genéticaRESUMO
AIMS: the present study aimed to investigate how glucose and insulin levels may be associated with changes in NR3C1 gene methylation levels in adults. MAIN METHODS: 375 volunteers users of the Brazilian Public Unified Health System (SUS) were recruited to assess socioeconomic status, lifestyle, anthropometric data, blood glucose and serum cortisol levels, insulin resistance, and NR3C1 gene methylation assessment. Factors associated with glucose levels and insulin resistance were investigated using multivariate analysis GLzM at 5% significance (p<0.05). KEY FINDINGS: our results verified that glucose levels and insulin resistance were directly related to NR3C1 gene methylation and age, while not being overweight and obese and no tobacco consumption were indirectly related to glucose levels and insulin resistance. SIGNIFICANCE: habits and lifestyle may influence NR3C1 gene regulation, revealing the complexity of environmental impacts on NR3C1 methylation. Furthermore, associated risk factors must be taken into account in epigenetic studies as they directly interfere with blood glucose levels and insulin resistance.
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Resistência à Insulina , Insulinas , Adulto , Humanos , Metilação de DNA , Hidrocortisona , Receptores de Glucocorticoides/metabolismo , Resistência à Insulina/genética , Glicemia , Éxons , Estilo de Vida , Insulinas/genéticaRESUMO
Evaluate the biological action of valproic acid in the acetylation of histones and in the methylation of tumor suppressor genes via oral rinse in patients with a previous history of head and neck squamous cell carcinoma (HNSCC). Forty-two active or former smokers were included in this randomized, double-blind, placebo-controlled trial. Oral rinse samples were collected prior to treatment with valproic acid or placebo and after 90 days of treatment. The methylation status of five tumor suppressor genes and histone acetylation were evaluated by pyrosequencing and ELISA techniques, respectively. Differences between the 90-day and baseline oral rinse acetylation and methylation results were analyzed by comparing groups. Thirty-four patients were considered for analysis. The mean percentage adherence in the valproic and placebo groups was 93.4 and 93.0, respectively (p = 0.718). There was no statistically significant difference between groups when comparing the medians of the histone acetylation ratio and the methylation ratio for most of the studied genes. A significant reduction in the DCC methylation pattern was observed in the valproic group (p = 0.023). The use of valproic acid was safe and accompanied by good therapeutic adherence. DCC methylation was lower in the valproic acid group than in the placebo group.
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Acetilação/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Ácido Valproico/uso terapêutico , Método Duplo-Cego , Feminino , Neoplasias de Cabeça e Pescoço/metabolismo , Histonas/metabolismo , Humanos , Masculino , Estudos Prospectivos , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismoRESUMO
The identification of molecular markers in negative surgical margins of oral squamous cell carcinoma (OSCC) might help in identifying residual molecular aberrations, and potentially improve the prediction of prognosis. We performed an Infinium MethylationEPIC BeadChip array on 32 negative surgical margins stratified based on the status of tumor recurrence in order to identify recurrence-specific aberrant DNA methylation (DNAme) markers. We identified 2512 recurrence-associated Differentially Methylated Positions (DMPs) and 392 Differentially Methylated Regions (DMRs) which were enriched in cell signaling and cancer-related pathways. A set of 14-CpG markers was able to discriminate recurrent and non-recurrent cases with high specificity and sensitivity rates (AUC 0.98, p = 3 × 10-6; CI: 0.95-1). A risk score based on the 14-CpG marker panel was applied, with cases classified within higher risk scores exhibiting poorer survival. The results were replicated using tumor-adjacent normal HNSCC samples from The Cancer Genome Atlas (TCGA). We identified residual DNAme aberrations in the negative surgical margins of OSCC patients, which could be informative for patient management by improving therapeutic intervention. This study proposes a novel DNAme-based 14-CpG marker panel as a promising predictor for tumor recurrence, which might contribute to improved decision-making for the personalized treatment of OSCC cases.
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The NR3C1 glucocorticoid receptor (GR) gene is a component of the stress response system, which can be regulated by epigenetic mechanisms. NR3C1 methylation has been associated with trauma and mental issues, including depression, post-traumatic stress, anxiety, and personality disorders. Previous studies have reported that stressful events are involved in NR3C1 gene methylation, suggesting that its regulation under environmental effects is complex. The present study aimed to analyze associations involving stressors such as socioeconomic status, health conditions, and lifestyle in relation to NR3C1 methylation in adults. This study included 386 individual users of the Brazilian Public Unified Health System (SUS), and evaluated socioeconomic and health conditions, body mass index, cortisol levels, and lifestyle. Data were correlated with NR3C1 methylation, determined using DNA pyrosequencing. The results showed that alcohol consumption, overweight, and high cortisol levels were related to NR3C1 demethylation, while depression was related to its methylation. Habits, lifestyle, and health status may influence NR3C1 gene regulation via methylation, revealing the complexity of environmental impacts on NR3C1 methylation.
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Consumo de Bebidas Alcoólicas/genética , Cortisona/sangue , Metilação de DNA , Depressão/genética , Sobrepeso/genética , Receptores de Glucocorticoides/genética , Adulto , Biomarcadores , Ilhas de CpG , Estudos Transversais , Depressão/metabolismo , Suscetibilidade a Doenças , Feminino , Estudos de Associação Genética , Humanos , Masculino , Pessoa de Meia-Idade , Sobrepeso/metabolismo , Receptores de Glucocorticoides/metabolismo , Fatores Socioeconômicos , Adulto JovemRESUMO
Epigenetic mechanisms such as aberrant DNA methylation (DNAme) are known to drive esophageal squamous cell carcinoma (ESCC), yet they remain poorly understood. Here, we studied tumor-specific DNAme in ESCC cases from nine high-incidence countries of Africa, Asia, and South America. Infinium MethylationEPIC array was performed on 108 tumors and 51 normal tissues adjacent to the tumors (NAT) in the discovery phase, and targeted pyrosequencing was performed on 132 tumors and 36 NAT in the replication phase. Top genes for replication were prioritized by weighting methylation results using RNA-sequencing data from The Cancer Genome Atlas and GTEx and validated by qPCR. Methylome analysis comparing tumor and NAT identified 6,796 differentially methylated positions (DMP) and 866 differential methylated regions (DMR), with a 30% methylation (Δß) difference. The majority of identified DMPs and DMRs were hypermethylated in tumors, particularly in promoters and gene-body regions of genes involved in transcription activation. The top three prioritized genes for replication, PAX9, SIM2, and THSD4, had similar methylation differences in the discovery and replication sets. These genes were exclusively expressed in normal esophageal tissues in GTEx and downregulated in tumors. The specificity and sensitivity of these DNAme events in discriminating tumors from NAT were assessed. Our study identified novel, robust, and crucial tumor-specific DNAme events in ESCC tumors across several high-incidence populations of the world. Methylome changes identified in this study may serve as potential targets for biomarker discovery and warrant further functional characterization. SIGNIFICANCE: This largest genome-wide DNA methylation study on ESCC from high-incidence populations of the world identifies functionally relevant and robust DNAme events that could serve as potential tumor-specific markers. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/10/2612/F1.large.jpg.
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Biomarcadores Tumorais/genética , Metilação de DNA , DNA de Neoplasias/genética , Epigênese Genética , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/patologia , Genoma Humano , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , DNA de Neoplasias/análise , Neoplasias Esofágicas/epidemiologia , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas do Esôfago/epidemiologia , Carcinoma de Células Escamosas do Esôfago/genética , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Saúde Global , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , PrognósticoRESUMO
Background: Head and neck squamous cell carcinoma (HNSCC) is usually associated to tobacco and alcohol consumption. Increased telomerase activity has been consistently detected in 80-90% of malignant tumors, including HNSCC. Mutations within the promoter region of telomerase reverse transcriptase (TERT) that confer enhanced TERT promoter activity have been reported in two major hotspots, designated C228T and C250T. Objectives: To evaluate TERT promoter mutations C228T and C250T in HNSCC patients from Brazil and correlate with patients' outcome. Materials and Methods: Formalin-fixed paraffin-embedded tissues were obtained from 88 HNSCC patients and analyzed for TERT promoter mutations C228T and C250T by pyrosequencing. Results: The overall prevalence of hotspot TERT mutations in HNSCC samples was of 27.3%, with 6.8% at locus C228T and 20.5% at C250T. The majority (92%) of mutated cases were located in oral cavity, mainly at the tongue. We observed that 94.4% of the patients harboring TERT promoter mutation C250T were alcohol consumers (p = 0.032) and 66.7% of the patients harboring TERT promoter mutation C228T were not alcohol consumers (p = 0.035). The presence of C228T mutation impacted patient outcome, with a significant decrease in disease-free survival (20.0 vs. 63.0%, p =0.017) and in overall survival (16.7 vs. 45.1%, p = 0.017). Conclusion: This is the first report of a TERT promoter mutations in HNSCC patients from South America. The high prevalence of TERT mutation, as well as its association with poor disease-free survival and overall survival, particular at C228T locus might serve as a prognostic biomarker in HNSCC to help clinicians in the management of treatment.
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BACKGROUND: Lack of complete genomic data of Bothrops jararaca impedes molecular biology research focusing on biotechnological applications of venom gland components. Identification of full-length coding regions of genes is crucial for the correct molecular cloning design. METHODS: RNA was extracted from the venom gland of one adult female specimen of Bothrops jararaca. Deep sequencing of the mRNA library was performed using Illumina NextSeq 500 platform. De novo assembly of B. jararaca transcriptome was done using Trinity. Annotation was performed using Blast2GO. All predicted proteins after clustering step were blasted against non-redundant protein database of NCBI using BLASTP. Metabolic pathways present in the transcriptome were annotated using the KAAS-KEGG Automatic Annotation Server. Toxins were identified in the B. jararaca predicted proteome using BLASTP against all protein sequences obtained from Animal Toxin Annotation Project from Uniprot KB/Swiss-Pro database. Figures and data visualization were performed using ggplot2 package in R language environment. RESULTS: We described the in-depth transcriptome analysis of B. jararaca venom gland, in which 76,765 de novo assembled isoforms, 96,044 transcribed genes and 41,196 unique proteins were identified. The most abundant transcript was the zinc metalloproteinase-disintegrin-like jararhagin. Moreover, we identified 78 distinct functional classes of proteins, including toxins, inhibitors and tumor suppressors. Other venom proteins identified were the hemolytic lethal factors stonustoxin and verrucotoxin. CONCLUSION: It is believed that the application of deep sequencing to the analysis of snake venom transcriptomes may represent invaluable insight on their biotechnological potential focusing on candidate molecules.
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DNA methylation is involved in the oncogenesis of head and neck squamous cell carcinoma and could be used for early detection of cancer to increase the chances of cure, but unfortunately diagnosis is usually made at late stages of the disease. In this work we developed genosensors to detect DNA methylation of the MGMT gene in head and neck cancer cell lines. The probe for MGMT promoter methylation was immobilized on gold electrodes modified with 11-mercaptoundecanoic acid (11-MUA) self-assembled monolayers (SAM). Detection was performed with electrochemical impedance spectroscopy, with clear distinction between methylated and non-methylated DNA from head and neck cell lines. The genosensor is sensitive with a low detection limit of 0.24 × 10-12 mol L-1. In addition, the cell lines FaDu, JHU28 and SCC25 for the MGMT gene, could be distinguished from the HN13 cell line which has a high degree of MGMT methylation (97%), thus confirming the selectivity. Samples with different percentages of MGMT DNA methylation could be separated in multidimensional projections using the visualization technique interactive document mapping (IDMAP). The genosensor matrix and the immobilization procedures are generic, and can be extended to other DNA methylation biomarkers.
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Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Técnicas Eletroquímicas , Ácidos Graxos/química , Neoplasias de Cabeça e Pescoço/genética , Compostos de Sulfidrila/química , Tioglicolatos/química , Proteínas Supressoras de Tumor/genética , Linhagem Celular Tumoral , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Eletrodos , Ouro/química , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Metilação , Regiões Promotoras Genéticas/genética , Espectrofotometria Infravermelho , Proteínas Supressoras de Tumor/metabolismoRESUMO
OBJECTIVE: The aim of this study was to verify determinant factors for depression and analyze the relationship between possible changes in HPA axis and depression, in this case NR3C1 DNA methylation and serum cortisol levels. METHODS: 349 adult volunteers were recruited to evaluate depression, socio-demographic, economic and lifestyle factors, serum cortisol levels and NR3C1 DNA methylation by pyrosequencing. Depression determinant factors were investigated using a Poisson regression model with robust variance (pâ¯<â¯0.05). RESULTS: Poisson regression with robust variance adjusted by gender, tobacco use, self-perceived stress, leisure activity, suicidal ideation, low cortisol levels and NR3C1 DNA methylation was performed and predicted risk factors for depression. Furthermore, depressive volunteers showed a significant increase in NR3C1 DNA methylation when compared to healthy volunteers. CONCLUSIONS: This findings provide a basis for understanding the role of HPA axis in depression, especially its regulation by NR3C1 DNA methylation. Furthermore, it emphasizes the stressful lifestyle risk factors (female, tobacco uso, self perceived stress, leisure activities absence and suicidal ideation) that can contribute to future research and the search for public health policies to improve quality of live, mental and general health.
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Metilação de DNA , Depressão/metabolismo , Epigênese Genética , Hidrocortisona/sangue , Sistema Hipotálamo-Hipofisário/metabolismo , Estilo de Vida , Receptores de Glucocorticoides/metabolismo , Estresse Psicológico/metabolismo , Ideação Suicida , Adulto , Brasil/epidemiologia , Depressão/sangue , Depressão/epidemiologia , Feminino , Humanos , Atividades de Lazer , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Fatores Sexuais , Fumar/epidemiologia , Fumar/metabolismo , Estresse Psicológico/epidemiologia , Adulto JovemRESUMO
Tobacco- or human papillomavirus- driven oropharyngeal squamous cell carcinomas (OpSCC) represent distinct clinical, biological and epidemiological entities. The aim of this study was to identify genetic variants based on somatic alterations in OpSCC samples from an admixed population, and to test for association with clinical features. The entire coding region of 15 OpSCC driver genes was sequenced by next-generation sequencing in 51 OpSCC FFPE samples. Thirty-five percent of the patients (18/51) were HPV-positive and current or past tobacco consumption was reported in 86.3% (44/51). The mutation profile identified an average of 2.67 variants per sample. Sixty-three percent of patients (32/51; 62.7%) were mutated for at least one of the genes tested and TP53 was the most frequently mutated gene. The presence of mutation in NOTCH1 and PTEN, significantly decreased patient's recurrence-free survival, but only NOTCH1 mutation remained significant after stepwise selection, with a risk of recurrence of 4.5 (HR 95% CI = 1.11-14.57; Cox Regression p = 0.034). These results show that Brazilian OpSCC patients exhibit a similar clinical and genetic profile in comparison to other populations. Molecular characterization is a promising tool for the definition of clinical subgroups, aiding in a more precise tailoring of treatment and prognostication.