Development of an electrochemical DNA-based biosensor for the detection of the cardiovascular pharmacogenetic-altering SNP CYP2C9*3.

Cardiovascular diseases are among the major causes of mortality and morbidity. Warfarin is often prescribed for these disorders, an anticoagulant with inter and intra-dosage variability dose required to achieve the target international normalized ratio. Warfarin presents a narrow therapeutic index, and due to its variability, it can often be associated with the risk of hemorrhage, or in other patients, thromboembolism. Single-nucleotide polymorphisms are included in the causes that contribute to this variability. The Cytochrome P450 (CYP) 2C9*3 genetic polymorphism modifies its enzymatic activity, and hence warfarin's plasmatic concentration. Thus, the need for a selective, rapid, low-cost, and real-time detection device is crucial before prescribing warfarin. In this work, a disposable electrochemical DNA-based biosensor capable of detecting CYP2C9*3 polymorphism was developed. By analyzing genomic databases, two specific 78 base pairs DNA probes; one with the wild-type adenine (Target-A) and another with the cytosine (Target-C) single-nucleotide genetic variation were designed. The biosensor implied the immobilization on screen-printed gold electrodes of a self-assembled monolayer composed by mercaptohexanol and a linear CYP2C9*3 DNA-capture probe. To improve the selectivity and avoid secondary structures a sandwich format of the CYP2C9*3 allele was designed using complementary fluorescein isothiocyanate-labeled signaling DNA probe and enzymatic amplification of the electrochemical signal. Chronoamperometric measurements were performed at a range of 0.015-1.00 nM for both DNA targets achieving limit of detection of 42 p.m. The developed DNA-based biosensor was able to discriminate between the two synthetic target DNA targets, as well as the targeted denatured genomic DNA, extracted from volunteers genotyped as non-variant homozygous (A/A) and heterozygous (A/C) of the CYP2C9*3 polymorphism.

Telomere length and telomerase activity of leukocytes as biomarkers of selective serotonin reuptake inhibitor responses in patients with major depressive disorder.

We analyze the leukocyte telomere length (LTL) and telomerase activity in patients with major depressive disorder (MDD) before and after treatment with selective serotonin reuptake inhibitors (SSRIs). Before treatment, there was a reduction in the LTLs and expression levels of the human telomerase reverse transcriptase (hTERT) in the patients with MDD compared with controls. However, after 24 weeks of treatment with SSRIs, there was a significant increase in the LTLs and the expression levels of hTERT, with values approaching those observed in the controls. We conclude that SSRI antidepressant therapy can directly influence the increased expression levels of hTERT in patients.

Mechanism of preservation of the intestinal mucosa architecture and NF-κB/PGE2 reduction by hydrogen sulfide on cholera toxin-induced diarrhea in mice.

AIMS: Investigate the involvement of Hydrogen sulfide (H2S) in inflammatory parameters and intestinal morphology caused by cholera toxin (CT) in mice. MAIN METHODS: Mice were subjected to the procedure of inducing diarrhea by CT in the isolated intestinal loop model. The intestinal loops were inoculated with H2S donor molecules (NaHS and GYY 4137) or saline and CT. To study the role of EP2 and EP4 prostaglandin E2 (PGE2) receptors in the H2S antisecretory effect, PAG (DL-propargylglycine - inhibitor of cystathionine-γ-lyase (CSE)), PF-04418948 (EP2 antagonist) and ONO-AE3-208 (EP4 antagonist) were used. The intestinal loops were evaluated for intestinal secretion, relation of the depth of villi and intestinal crypts, and real-time PCR for the mRNA of the CXCL2, IL-6, NOS-2, IL-17, NF-κB1, NF-κBIA, SLC6A4 and IFN-γ genes. KEY FINDINGS: H2S restored the villus/crypt depth ratio caused by CT. NaHS and GYY 4137 increased the expression of NF-κB1 and for the NF-κBIA gene, only GYY 4137 increased the expression of this gene. The increased expression of NF-κB inhibitors, NF-κB1 and NF-κBIA by H2S indicates a possible decrease in NF-κB activity. The pretreatment with PAG reversed the protective effect of PF-04418948 and ONO-AE3-208, indicating that H2S probably decreases PGE2 because in the presence of antagonists of this pathway, PAG promotes intestinal secretion. SIGNIFICANCE: Our results point to a protective activity of H2S against CT for promoting a protection of villus and crypt intestine morphology and also that its mechanism occurs at least in part due to decreasing the activity of NF-κB and PGE2.

New prognostic markers revealed by RNA-Seq transcriptome analysis after MYC silencing in a metastatic gastric cancer cell line.

MYC overexpression is considered a driver event in gastric cancer (GC), and is frequently correlated with poor prognosis and metastasis. In this study, we evaluated the prognostic value of genes upregulated by MYC in patients with GC. Metastatic GC cells (AGP01) characterized by MYC amplification, were transfected with siRNAs targeting MYC. RNA-seq was performed in silenced and non-silenced AGP01 cells. Among the differentially expressed genes, CIAPIN1, MTA2, and UXT were validated using qRT-PCR, western blot, and immunohistochemistry in gastric tissues of 213 patients with GC; and their expressions were correlated with clinicopathological and survival data. High mRNA and protein levels of CIAPIN1, MTA2, and UXT were strongly associated with advanced GC stages (P < 0.0001). However, only CIAPIN1 and UXT gene expressions were able to predict distant metastases in patients with early-stage GC (P < 0.0001), with high sensitivity (> 92%) and specificity (> 90%). Overall survival rate of patients with overexpressed CIAPIN1 or UXT was significantly lower (P < 0.0001). In conclusion, CIAPIN1 and UXT may serve as potential molecular markers for GC prognosis.

Topical application of cashew gum or chlorhexidine gel reduces overexpression of proinflammatory genes in experimental periodontitis.

This study aimed to explore the effect of topically administering an orabase gel containing cashew gum (CG), a complex polysaccharide from Anacardium occidentale L., on the transcription of important proinflammatory (COX-2, NOS-2, INF-γ, OSCAR, and MYD88) and anti-inflammatory genes (IL-10, IL-4, and TGFß1) in the gingival tissues of rats with ligature-induced periodontitis, compared to the effect observed upon topically applying a well-known antibiofilm agent (chlorhexidine) under the same experimental conditions. The gene expression profile in the gingival tissues of rats with periodontitis treated with CG did not statistically significantly differ from that observed in the group of animals treated with chlorhexidine. Results showed that CG is able to attenuate general inflammation in the periodontium by reducing the transcription of proinflammatory mediators in a MYD88-independent manner, and not by inducing the expression of anti-inflammatory factors. In conclusion, this study demonstrated that CG and chlorhexidine treatment reduced significantly the gene overexpression (COX-2, NOS-2, INF-γ, OSCAR, and TGFß1) in the model of ligature-induced periodontitis.

The SLC6A3 3'-UTR VNTR and intron 8 VNTR polymorphisms association in the time estimation.

OBJECTIVE: The present study investigated the association of 3'-UTR VNTR and intron 8 VNTR polymorphisms with a time estimation task performance. MATERIALS AND METHODS: One hundred and eight men in a Brazilian Northeast population (18-32 years old) participated in the experiment. The 3'-UTR VNTR and intron 8 VNTR polymorphisms were associated alone and combined to absolute error (AE) and relative error (RE) in a time estimation task (target duration: 1 s, 4 s, 7 s and 9 s). RESULTS: We found an association of the behavioral variable with intron 8 VNTR for the time intervals of 1 s and 9 s (p < 0.001) and polymorphisms combinatorial effect for 1 s (p ≤ 0.05). CONCLUSION: The intron 8 VNTR polymorphism and the combinatorial effect can modulate the time estimate in the domain of supra seconds, and thus our study indicates a role of the dopamine transporter in the neurobiological areas related to the time intervals judgment.

Lack of detection of human papillomavirus DNA in prostate carcinomas in patients from northeastern Brazil.

Prostate cancer is the second most common cancer among men in western populations, and despite its high mortality, its etiology remains unknown. Inflammatory processes are related to the etiology of various types of tumors, and prostate inflammation, in particular, has been associated with prostate cancer carcinogenesis and progression. Human papillomavirus (HPV) is associated with benign and malignant lesions in the anogenital tract of both females and males. The possible role of HPV in prostate carcinogenesis is a subject of great controversy. In this study, we aimed to examine the prevalence of HPV infections in prostate carcinomas of patients from northeastern Brazil. This study included 104 tissue samples from primary prostate carcinoma cases. HPV DNA was purified and then amplified using MY09/11 and GP5+/GP6+ degenerate primer sets that detect a wide range of HPV types, and with specific PCR primers sets for E6 and E7 HPV regions to detect HPV 16. None of the samples showed amplification products of HPV DNA for primer sets MY09/11 and GP5+/GP6+, or the specific primer set for the E6 and E7 HPV regions. HPV infection, thus, does not seem to be one of the causes of prostate cancer in the population studied.

A Case of Severe Carotid Stenosis in a Patient with Familial Hypercholesterolemia without Significant Coronary Artery Disease.

Familial hypercholesterolemia (FH) is an inherited metabolic disorder characterized by elevated low-density lipoprotein cholesterol levels in the blood. In its heterozygous form, it occurs in 1 in 500 individuals in the general population. It is an important contributor to the early onset of coronary artery disease (CAD), accounting for 5-10% of cases of cardiovascular events in people younger than 50 years. Atherogenesis triggered by hypercholesterolemia generally progresses faster in the coronary arteries, followed by the subsequent involvement of other arteries such as the carotids. Thus, symptoms of CAD commonly appear before the onset of significant carotid stenosis. Herein, we report the case of a patient with untreated FH who had severe carotid atherosclerosis at the age of 46 years but had no evidence of significant CAD.

Prevalência do polimorfismo C677T do gene MTHFR em uma população de idosos / Prevalence of MTHFR C677T polymorphism in an elderly population

Objetivo: determinar a prevalência do polimorfismo MTHFR C677T em uma amostra de 200 idosos da cidade de Parnaíba-PI e comparar suas freqüências genotípicas e alélicas com as observadas em outras populações. Método: a presença dopolimorfismo MTHFR C677T foi determinada pela técnica de reação em cadeia da polimerase seguida por tratamentocom a enzima de restrição HinfI (PCR-RFLP), acompanhado de eletroforese em gel de poliacrilamida 8%, corado comnitrato de prata. Resultados: dos 200 indivíduos estudados, 120 (60%) apresentaram genótipo homozigoto normal (CC);63 (31,5%) foram heterozigotos (CT) e 17 (8,5%) mostraram-se homozigotos TT. A frequência do alelo polimórfico T foide 23,4%. As frequências genotípicas mostraram-se sob equilíbrio de Hardy-Weinberg e não houve diferenças estatisticamentesignificantes quanto à distribuição do alelo T por sexo ou faixa etária. Conclusão: os resultados apresentadosneste estudo representam o primeiro relato indicativo da frequência deste polimorfismo em uma população piauiense. Afrequência do alelo T foi consideravelmente elevada (24,3%) comparada com a população geral e, portanto, estudos sãonecessários para investigar a contribuição desse polimorfismo na etiologia e/ou gravidade a determinadas doenças, nessapopulação.

Objective: to determine the prevalence of MTHFR C677T polymorphism in a sample of 200 elderly individuals fromParnaíba, Piauí, Brazil and to compare its genotypic and allelic frequencies with those observed in other populations.Method: the presence of MTHFR C677T polymorphism was evaluated by polymerase chain reaction followed by restrictionenzyme analysis with HinfI endonuclease (PCR-RFLP). After cleavage, the genotypes were evaluated by 8% silverstained polyacrylamide gel. Results: of the 200 individuals studied, 120 (60%) were homozygous normal (CC), 63 (31.5%) were heterozygous (CT) and 17 (8.5%) were homozygous TT. The frequency of the polymorphic allele T was23.4%. The genotypic frequencies were found to be in Hardy-Weinberg equilibrium and there was no statistically significantdifferences regarding the distribution of T-allele by sex or age. Conclusion: the results presented in this studyrepresent the first report of the MTHFR C677T polymorphism frequency in a population of Piauí. The T-allele frequencywas significantly higher (24.3%) compared to the general population and therefore studies are needed to investigate thecontribution of this polymorphism in the etiology and/or severity of certain diseases in this population