Genetic polymorphisms modulate the folate metabolism of Brazilian individuals with Down syndrome

Individuals with Down syndrome (DS) carry three copies of the Cystathionine beta-synthase (C beta S) gene. The increase in the dosage of this gene results in an altered profile of metabolites involved in the folate pathway, including reduced homocysteine (Hcy), methionine, S-adenosylhomocysteine (SAH) and S-adenosylmethionine (SAM). Furthermore, previous studies in individuals with DS have shown that genetic variants in genes involved in the folate pathway influence the concentrations of this metabolism's products. The purpose of this study is to investigate whether polymorphisms in genes involved in folate metabolism affect the plasma concentrations of Hcy and methylmalonic acid (MMA) along with the concentration of serum folate in individuals with DS. Twelve genetic polymorphisms were investigated in 90 individuals with DS (median age 1.29 years, range 0.07-30.35 years; 49 male and 41 female). Genotyping for the polymorphisms was performed either by polymerase chain reaction (PCR) based techniques or by direct sequencing. Plasma concentrations of Hcy and MMA were measured by liquid chromatography-tandem mass spectrometry as previously described, and serum folate was quantified using a competitive immunoassay. Our results indicate that the MTHFR C677T, MTR A2756G, TC2 C776G and BHMT G742A polymorphisms along with MMA concentration are predictors of Hcy concentration. They also show that age and Hcy concentration are predictors of MMA concentration. These findings could help to understand how genetic variation impacts folate metabolism and what metabolic consequences these variants have in individuals with trisomy 21.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [04/15944-5, 03/09931-5]Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) [302157/2008-5]Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES) [CGPP 046/2006

Mutational analysis of the GAP-related domain of the neurofibromatosis type 1 gene in Brazilian NF1 patients

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder caused by mutations in the NF1 gene. In the present study, a total of 55 unrelated NF1 patients were screened for mutations in the GAP-related domain/GRD (exons 20-27a) by single-strand conformation polymorphism (SSCP). Four different mutations were identified and, taken together, they comprise one nonsense substitution (Q1189X), one deletion (3525-3526delAA), one missense substitution (E1356G) and one mutation in the splice acceptor site (c.4111-1G>A). One novel polymorphism (c.4514+11C>G) and other three putative polymorphisms were also found (c.3315-27G>A, V1146I and V1317A). Genotype-phenotype correlations were investigated, but no particular association was detected.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

From Tissue Physoxia to Cancer Hypoxia, Cost-Effective Methods to Study Tissue-Specific O2 Levels in Cellular Biology

The human body is endowed with an extraordinary ability to maintain different oxygen levels in various tissues and organs. The maintenance of physiological levels of oxygen is known as physoxia. The development of hypoxic conditions plays an important role in the biology of several pathologies, including cancer. In vitro studies using normal and neoplastic cells require that culture conditions be carried out under appropriate oxygen levels, either physoxic or hypoxic conditions. Such requirements are difficult to widely implement in laboratory practice, mainly due to the high costs of specialized equipment. In this work, we present and characterize a cost-effective method to culture cells under a range of oxygen levels using deoxidizing pouches. Our results show that physoxic and hypoxic levels using deoxidizing absorbers can be achieved either by implementing a gradual change in oxygen levels or by a regimen of acute depletion of oxygen. This approach triggers the activation of an epithelial-mesenchymal transition in cancer cells while stimulating the expression of HIF-1α. Culturing cancer cells with deoxidizing agent pouches revealed PI3K oncogenic pathway exacerbations compared to tumor cells growing under atmospheric levels of oxygen. Similar to the PI3K signaling disturbance, we also observed augmented oxidative stress and superoxide levels and increased cell cycle arrest. Most interestingly, the culture of cancer cells under hypoxia resulted in the accumulation of cancer stem cells in a time-dependent manner. Overall, we present an attractive, cost-effective method of culturing cells under appropriate physoxic or hypoxic conditions that is easily implementable in any wet laboratory equipped with cell culture tools

Hypoxic niches are endowed with a protumorigenic mechanism that supersedes the protective function of PTEN

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide and is characterized by a fast-paced growth. Like other solid tumors, the HNSCC growth rate results in the development of hypoxic regions identified by the expression of hypoxia-inducible factor 1 alpha (HIF-1 alpha). Interestingly, clinical data have shown that pharmacological induction of intratumoral hypoxia caused an unexpected rise in tumor metastasis and the accumulation of cancer stem cells (CSCs). However, little is known on the molecular circuitries involved in the presence of intratumoral hypoxia and the augmented population of CSCs. Here we explore the impact of hypoxia on the behavior of HNSCC and define that the controlling function of phosphatase and tensin homolog (PTEN) over HIF-1 alpha expression and CSC accumulation are de-regulated during hypoxic events. Our findings indicate that hypoxic niches are poised to accumulate CSCs in a molecular process driven by the loss of PTEN activity. Furthermore, our data suggest that targeted therapies aiming at the PTEN/PI3K signaling may constitute an effective strategy to counteract the development of intratumoral hypoxia and the accumulation of CSCs33121343513449COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESBEX/88881.135014/2016-01 PDSEThis work was conducted during a visiting scholar period at the University of Michigan, sponsored by the Capes Foundation within the Brazilian Ministry of Education (Grant BEX/88881.135014/2016-01 PDSE). This work was partially supported by University of Michigan Cancer Center Support Grant P30 CA046592, and by the U.S. National Institutes of Health (NIH), National Institute of General Medical Sciences Research Grant 5R01GM120056. This grant was funded by the University of Michigan School of Dentistry faculty, and by The Robert Wood Johnson Foundation (AMFDP-72425). The monoclonal antibody AMF-17b, developed by A. B. Fulton, was obtained from the Developmental Studies Hybridoma Bank, created by the NIH Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) and maintained at The University of Iowa (Iowa City, IA, USA). The authors declare no conflict of interes