Frequency of the toxic shock syndrome toxin-1 gene in methicillin-susceptible and -resistant Staphylococcus aureus isolates from teaching hospitals in Shiraz, Iran

INTRODUCTION: Staphylococcus aureus produces a range of virulence factors such as toxic shock syndrome toxin-1. METHODS: In this cross-sectional study of 345 clinical S. aureus isolates, the presence of the tst gene was assessed by polymerase chain reaction (PCR). RESULTS: The study revealed 53/345 (15.4%) isolates were positive for the tst gene. The tst gene was present in 18.1% of methicillin-susceptible S. aureus (MSSA) isolates and 11.6% of methicillin-resistant S. aureus (MRSA) isolates (p = 0.136). CONCLUSIONS: These results reveal the remarkable risk of S. aureus infections in hospitals, regardless of methicillin-resistance status.

Toll-like receptor 3 gene polymorphisms are not associated with the risk of hepatitis B and hepatitis C virus infection

INTRODUCTION: The present study investigated the prevalence of two single-nucleotide polymorphisms (SNPs) in the Toll-like receptor 3 (TLR3) gene in patients infected with hepatitis B virus (HBV) and hepatitis C virus (HCV). METHODS: Samples collected from HCV (n = 74) and HBV (n = 35) carriers were subjected to quantitative real-time PCR (qPCR) to detect the presence of the SNPs rs5743305 and rs3775291 in TLR3 and to measure the following biomarkers: alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT), and prothrombin time (PT). A healthy control group was investigated and consisted of 299 HCV- and HBV-seronegative individuals. RESULTS: No significant differences in allele, genotype and haplotype frequencies were observed between the investigated groups, and no association was observed between the polymorphisms and histopathological results. Nevertheless, genotypes TA/AA (rs5743305) and GG (rs3775291) appear to be associated with higher levels of ALT (p<0.01), AST (p<0.05) and PT (p<0.05). In addition, genotypes TT (rs5743305; p<0.05) and GG (rs3775291; p<0.05) were associated with higher GGT levels. CONCLUSIONS: This genetic analysis revealed the absence of an association between the polymorphisms investigated and susceptibility to HBV and HCV infection; however, these polymorphisms might be associated with a greater degree of biliary damage during the course of HCV infection.

Improving silicon probe performance through layer-by-layer coating

Fully comprehending brain function, as the scale of neural networks, will only be possi-ble with the development of tools by micro and nanofabrication. Regarding specifically silicon microelectrodes arrays, a significant improvement in long-term performance of these implants is essential. This project aims to create a silicon microelectrode coating that provides high-quality electrical recordings, while limiting the inflammatory response of chronic implants. To this purpose, a combined chitosan and gold nanoparticles coating was produced allied with electrodes modification by electrodeposition with PEDOT/PSS in order to reduce the im-pedance at 1kHz. Using a dip-coating mechanism, the silicon probe was coated and then charac-terized both morphologically and electrochemically, with focus on the stability of post-surgery performance in anesthetized rodents. Since not only the inflammatory response analysis is vital, the electrodes recording degradation over time was also studied. The produced film presented a thickness of approximately 50 μm that led to an increase of impedance of less than 20 kΩ in average. On a 3 week chronic implant, the impedance in-crease on the coated probe was of 641 kΩ, compared with 2.4 MΩ obtained for the uncoated probe. The inflammatory response was also significantly reduced due to the biocompatible film as proved by histological tests.

P53 and Rb tumor suppressor gene alterations in gastric cancer

Inactivation of tumor suppressor genes has been frequently observed in gastric carcinogenesis. Our purpose was to study the involvement of p53, APC, DCC, and Rb genes in gastric carcinoma. METHOD: Loss of heterozygosity of the p53, APC, DCC and Rb genes was studied in 22 gastric cancer tissues using polymerase chain reaction; single-strand conformation polymorphism of the p53 gene exons 5-6 and exons 7-8 was studied using 35S-dATP, and p53 expression was detected using a histological immunoperoxidase method with an anti-p53 clone. RESULTS AND DISCUSSION: No loss of heterozygosity was observed in any of these tumor suppressor genes; homozygous deletion was detected in the Rb gene in 23% (3/13) of the cases of intestinal-type gastric carcinoma. Eighteen (81.8%) cases showed band mobility shifts in exons 5-6 and/or 7-8 of the p53 gene. The presence of the p53 protein was positive in gastric cancer cells in 14 cases (63.6%). Normal gastric mucosa showed negative staining for p53; thus, the immunoreactivity was likely to represent mutant forms. The correlation of band mobility shift and the immunoreactivity to anti-p53 was not significant (P = .90). There was no correlation of gene alterations with the disease severity. CONCLUSIONS: The inactivation of Rb and p53 genes is involved in gastric carcinogenesis in our environment. Loss of the Rb gene observed only in the intestinal-type gastric cancer should be further evaluated in association with Helicobacter pylori infection. The p53 gene was affected in both intestinal and diffuse histological types of gastric cancer.

Renal and urinary findings in 20 patients with Williams-Beuren syndrome diagnosed by fluorescence in situ hybridization (FISH)

PURPOSE: Williams-Beuren syndrome is a rare multiple anomalies/mental retardation syndrome caused by deletion of contiguous genes at chromosome region 7q11.23. The aim of this work was to determine the frequency and the types of renal and urinary tract anomalies in 20 patients with Williams-Beuren syndrome. METHODS: The fluorescence in situ hybridization test using a LSI Williams syndrome region DNA probe was performed for all 20 patients to confirm the diagnosis of Williams-Beuren syndrome. A prospective study was performed in order to investigate renal and urinary aspects using laboratory assays to check renal function, ultrasonography of the kidneys and urinary tract, voiding cystourethrogram and urodynamics. RESULTS: Deletion of the elastin gene (positive fluorescence in situ hybridization test) was found in 17 out of 20 patients. Renal alterations were diagnosed in 5 of 17 (29%) the patients with the deletion and in 1 of 3 patients without the deletion. Fourteen patients with the deletion presented dysfunctional voiding. Arterial hypertension was diagnosed in 3 patients with deletions and 1 of these presented bilateral stenosis of the renal arteries. CONCLUSIONS: Due to the high incidence of renal and urinary abnormalities in Williams-Beuren syndrome, performing a systematic laboratory and sonographic evaluation of the patients is recommended.

Regulação epigenética de um gene de resposta a stress de frio em arroz (OsDREB1B)

O arroz (Oryza sativa L.) constitui a principal fonte de alimento para mais de metade da população mundial. O frio tem um grande impacto no desenvolvimento da planta de arroz com efeitos negativos ao nível da produtividade e economia mundial. Os mecanismos epigenéticos associados a alterações estruturais da cromatina estão envolvidos nos mecanismos de resposta das plantas a stresses abióticos. O foco do presente trabalho consistiu na compreensão da influência de fatores epigenéticos, nomeadamente de modificações de histonas, na regulação transcricional de um gene específico (OsDREB1B) envolvido na resposta de arroz ao stress de frio. O estudo da regulação epigenética deste gene envolveu a utilização de plantas de arroz com mutações em enzimas epigenéticas e drogas remodeladoras da cromatina. Neste trabalho mostramos que o gene OsDREB1B é induzido pelo stress de frio (4 °C) em todas os genótipos de arroz analisados, no entanto, a indução do gene em resposta ao stress de frio é maior em Nipponbare que em Dongjin. Nos genótipos que apresentam epimutações, nomeadamente o knockout de osdrm2 (DMT706) e de oshac704 (HAC704), o gene OsDREB1B é mais expresso que no respetivo genótipo selvagem (Dongjin), sendo que a indução da expressão em resposta ao frio foi maior em DMT706 do que em HAC704. A análise do padrão de marcas histónicas ao longo do promotor de OsDREB1B, através da imunoprecipitação da cromatina (ChIP) com anticorpos específicos, revelou que, em resposta ao frio (4ºC), Dongjin apresenta uma menor predominância da marca H3K9ac na região de -519 a -355 pares de bases (antes do ATG), enquanto que Nipponbare possui maior presença de H4K5ac na região de -280 a -121 pares de bases, sugerindo que a regulação epigenética de OsDREB1B seja influenciada pelo genótipo. No caso dos epi-mutantes (HAC704 e DMT706) registou-se um empobrecimento das marcas histónicas testadas sugerindo que a indução da expressão do OsDREB1B em reposta ao stress provocado pelo frio não depende necessariamente da presença de OsDRM2 nem de OsHAC704. Uma visão integrada da paisagem epigenética e do transcriptoma, perspetiva uma melhor compreensão dos mecanismos envolvidos na resposta da planta a condições ambientais adversas, abrindo novas linhas de trabalho no sentido de obter plantas mais tolerantes a fatores de stress.

Hair coloration by gene regulation: fact or fiction?

The unravelling of hair pigmentation genetics and robust delivery systems to the hair follicle (HF) will allow the development of a new class of colouring products. The challenge will be changing hair colour from inside out by safely regulating the activity of target genes through the specific delivery of synthetic/natural compounds, proteins, genes, or small RNAs.

Association study of the Ile349val polymorphism of the gene ADH1C and alcohol dependence

OBJECTIVE: The aim of this study was to investigate the polymorphism Ile349Val of the enzyme alcohol dehydrogenase ADH1C gene among individuals with alcohol dependence syndrome (ADS) attending Alcoholics Anonymous (AA) meetings. METHODS: A total of 120 subjects residing in Rio de Janeiro city participated in this study. Subjects were divided into two groups: a group consisting of 54 individuals from the ADS group and 66 individuals that declared not having any alcohol dependence (control group). DNA was extracted from mouth epithelial cells by phenol-chloroform method and further submitted to amplification by polymerase chain reaction (PCR). RESULTS: Our results did not show differences between the genotypes of control individuals and ADS subjects. Nevertheless, we found increased rates of alcoholism in families of ADS subjects as compared to controls. CONCLUSIONS: Our results did not show any genotype difference on the ADH1C gene when control and AA genotypes are compared.

Evolution of the gene regulatory network controlling flower dorsoventral asymmetry

Tese de Doutoramento em Biologia de Plantas.

KIAA1549: BRAF gene fusion and FGFR1 hotspot mutations are prognostic factors in pilocytic astrocytomas

Up to 20% of patients with pilocytic astrocytoma (PA) experience a poor outcome. BRAF alterations and Fibroblast growth factor receptor 1 (FGFR1) point mutations are key molecular alterations in Pas, but their clinical implications are not established. We aimed to determine the frequency and prognostic role of these alterations in a cohort of 69 patients with PAs. We assessed KIAA1549:BRAF fusion by fluorescence in situ hybridization and BRAF (exon 15) mutations by capillary sequencing. In addition, FGFR1 expression was analyzed using immunohistochemistry, and this was compared with gene amplification and hotspot mutations (exons 12 and 14) assessed by fluorescence in situ hybridization and capillary sequencing. KIAA1549:BRAF fusion was identified in almost 60% of cases. Two tumors harbored mutated BRAF. Despite high FGFR1 expression overall, no cases had FGFR1 amplifications. Three cases harbored a FGFR1 p.K656E point mutation. No correlation was observed between BRAF and FGFR1 alterations. The cases were predominantly pediatric (87%), and no statistical differences were observed in molecular alterations-related patient ages. In summary, we confirmed the high frequency of KIAA1549:BRAF fusion in PAs and its association with a better outcome. Oncogenic mutations of FGFR1, although rare, occurred in a subset of patients with worse outcome. These molecular alterations may constitute alternative targets for novel clinical approaches, when radical surgical resection is unachievable.

Optimization of peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) for the detection of bacteria: the effect of pH, dextran sulfate and probe concentration

Fluorescence in situ hybridization (FISH) is a molecular technique widely used for the detection and characterization of microbial populations. FISH is affected by a wide variety of abiotic and biotic variables and the way they interact with each other. This is translated into a wide variability of FISH procedures found in the literature. The aim of this work is to systematically study the effects of pH, dextran sulfate and probe concentration in the FISH protocol, using a general peptide nucleic acid (PNA) probe for the Eubacteria domain. For this, response surface methodology was used to optimize these 3 PNA-FISH parameters for Gram-negative (Escherichia coli and Pseudomonas fluorescens) and Gram-positive species (Listeria innocua, Staphylococcus epidermidis and Bacillus cereus). The obtained results show that a probe concentration higher than 300 nM is favorable for both groups. Interestingly, a clear distinction between the two groups regarding the optimal pH and dextran sulfate concentration was found: a high pH (approx. 10), combined with lower dextran sulfate concentration (approx. 2% [w/v]) for Gram-negative species and near-neutral pH (approx. 8), together with higher dextran sulfate concentrations (approx. 10% [w/v]) for Gram-positive species. This behavior seems to result from an interplay between pH and dextran sulfate and their ability to influence probe concentration and diffusion towards the rRNA target. This study shows that, for an optimum hybridization protocol, dextran sulfate and pH should be adjusted according to the target bacteria.

Reconstruction of the regulatory network for Bacillus subtilis and reconciliation with gene expression data

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2016.00275

Experimental Model of Gene Transfection in Healthy Canine Myocardium: Perspectives of Gene Therapy for Ischemic Heart Disease

OBJECTIVE: To assess the transfection of the gene that encodes green fluorescent protein (GFP) through direct intramyocardial injection. METHODS: The pREGFP plasmid vector was used. The EGFP gene was inserted downstream from the constitutive promoter of the Rous sarcoma virus. Five male dogs were used (mean weight 13.5 kg), in which 0.5 mL of saline solution (n=1) or 0.5 mL of plasmid solution containing 0.5 µg of pREGFP/dog (n=4) were injected into the myocardium of the left ventricular lateral wall. The dogs were euthanized 1 week later, and cardiac biopsies were obtained. RESULTS: Fluorescence microscopy showed differences between the cells transfected and not transfected with pREGFP plasmid. Mild fluorescence was observed in the cardiac fibers that received saline solution; however, the myocardial cells transfected with pREGFP had overt EGFP expression. CONCLUSION: Transfection with the EGFP gene in healthy canine myocardium was effective. The reproduction of this efficacy using vascular endothelial growth factor (VEGF) instead of EGFP aims at developing gene therapy for ischemic heart disease.

Estrategias estadístico-computacionales avanzadas para análisis de expresión de proteínas/genes en cáncer. Advanced statistical and computational strategies for protein/gene expression analysis in cancer.

El objetivo de este proyecto, enmarcado en el área de metodología de análisis en bioingeniería-biotecnología aplicadas al estudio del cancer, es el análisis y caracterización a través modelos estadísticos con efectos mixtos y técnicas de aprendizaje automático, de perfiles de expresión de proteínas y genes de las vías metabolicas asociadas a progresión tumoral. Dicho estudio se llevará a cabo mediante la utilización de tecnologías de alto rendimiento. Las mismas permiten evaluar miles de genes/proteínas en forma simultánea, generando así una gran cantidad de datos de expresión. Se hipotetiza que para un análisis e interpretación de la información subyacente, caracterizada por su abundancia y complejidad, podría realizarse mediante técnicas estadístico-computacionales eficientes en el contexto de modelos mixtos y técnias de aprendizaje automático. Para que el análisis sea efectivo es necesario contemplar los efectos ocasionados por los diferentes factores experimentales ajenos al fenómeno biológico bajo estudio. Estos efectos pueden enmascarar la información subycente y así perder informacion relavante en el contexto de progresión tumoral. La identificación de estos efectos permitirá obtener, eficientemente, los perfiles de expresión molecular que podrían permitir el desarrollo de métodos de diagnóstico basados en ellos. Con este trabajo se espera poner a disposición de investigadores de nuestro medio, herramientas y procedimientos de análisis que maximicen la eficiencia en el uso de los recursos asignados a la masiva captura de datos genómicos/proteómicos que permitan extraer información biológica relevante pertinente al análisis, clasificación o predicción de cáncer, el diseño de tratamientos y terapias específicos y el mejoramiento de los métodos de detección como así tambien aportar al entendimieto de la progresión tumoral mediante análisis computacional intensivo.

O polimorfismo VNTR no gene codificador do antagonista do receptor da interleucina-1 está associado com a doença arterial coronariana

FUNDAMENTO: A Doença Arterial Coronariana (DAC) é a aterosclerose das artérias coronárias que transportam o sangue para o coração. A aterosclerose é uma doença inflamatória. As variações gênicas das citocinas - como as associadas à família IL1 - fazem parte da patogênese da aterosclerose. OBJETIVO: O objetivo deste estudo foi determinar a relação entre os polimorfismos da família IL1 (VNTR do IL1RN, posições -511 e +3953 do IL1B) e a DAC na população turca. MÉTODOS: Um total de 427 indivíduos foram submetidos à angiografia coronariana e em seguida divididos da seguinte forma: 170 no grupo controle e 257 no grupo de pacientes com DAC. Os sujeitos com DAC foram divididos em dois subgrupos: 91 no grupo de Doença Coronariana em um único vaso (Single Vessel Disease - SVD) e 166 no grupo Doença Coronariana em múltiplos vasos (Multiple Vessel Disease - MVD). Os genótipos de IL1RN e IL1B (-511, +3953) foram determinados por reação em cadeia da polimerase (RCP), seguida de análise da digestão por enzima de restrição. RESULTADOS: Não foram observadas diferenças significantes nas distribuições de genótipos de IL1RN e IL1B (-511 e +3953) entre os sujeitos com DAC e os controles, ou entre sujeitos com MVD e controles. No entanto, observou-se uma relação significante no genótipo IL1RN 2/2 entre sujeitos portadores de SVD e controles (P= 0,016, x2: 10,289, OR: 2,94IC 95% 1,183 - 7,229). Tampouco foi observada diferença estatisticamente significante nas freqüências dos alelos de IL1RN e IL1B (-511 e +3953) entre os sujeitos com DAC e controles, os sujeitos com MVD e controles, ou ainda os sujeitos SVD e controles. CONCLUSÃO: Não foi observada nenhuma relação na freqüência alélica e nem na distribuição genotípica dos polimorfismos de IL1RN e IL1B entre sujeitos com DAC e grupos controle. No entanto, o genótipo IL1RN 2/2 pode representar um fator de risco para sujeitos com SVD na população turca.