Buccal cells DNA extraction to obtain high quality human genomic DNA suitable for polymorphism genotyping by PCR-RFLP and Real-Time PCR

OBJECTIVE: The aim of this study was to evaluate, by PCR-RFLP and real-time PCR, the yield and quality of genomic DNA collected from buccal cells by mouthwash after different storage times at room temperature. MATERIAL AND METHODS: A group of volunteers was recruited to collect buccal cells using a mouthwash solution. The collected solution was divided into 3 tubes, one tube were used for immediate extraction and the remaining received ethanol and were kept at room temperature for 4 and 8 days followed by dna extraction. The concentration, purity and integrity of the dna were determined using spectrophotometry and electrophoresis. DNA quality differences among the three incubation times were also evaluated for genotyping EGF +61 a/g (rs 4444903) polymorphism by PCR-RFLP and for IRF6 polymorphism (rs 17015215) using real-time PCR. RESULTS: There was no significant difference of dna yield (p=0.75) and purity (p=0.86) among the three different incubation times. DNA obtained from different incubation times presented high-molecular weight. The PCR-RFLP and real time pcr reactions were successfully performed for all DNA samples, even those extracted after 8 days of incubation. All samples genotyped by real-time pcr presented c allele for irf6 gene polymorphism (homozygous: cc; heterozygous: Ct) and the C allele was used as a reference for Ct values. The samples presented the same genotype for the different times in both techniques. CONCLUSION: We demonstrated that the method described herein is simple and low cost, and that DNA can be extracted and pcr amplified after storage in mouthwash solution at room temperature

The functional EGF+61 polymorphism and nonsyndromic oral clefts susceptibility in a Brazilian population

AbstractNonsyndromic oral clefts are considered a problem of public health in Brazil, presenting a multifactorial etiology that involves genetic and environmental components, such as maternal alcohol consumption. Several candidate genes have been investigated to identify some association with nonsyndromic clefts risk. The epidermal growth factor (EGF) gene is implicated in the normal craniofacial development and its functional +61 A>;G polymorphism has been related to cancer susceptibility. It has been suggested that cancer and oral clefts may share the same molecular pathways.Objective Our goal was to evaluate the association between the EGF+61 A>;G polymorphism and nonsyndromic oral clefts susceptibility.Material and Methods The case-control study included 218 cleft cases and 253 controls from Brazil. The control group was comprised of individuals without congenital malformations, dental anomalies and family history of clefts. The cleft phenotypes and subphenotypes were determined based on clinical examination. Genomic DNA was extracted from oral mucosa cells obtained by mouthwash. The EGF+61 A>;G polymorphism genotype was determined by polymerase chain reaction-restriction fragment length polymorphism.Results We noticed the association between maternal alcohol consumption during pregnancy and cleft occurrence. The A allele and AA genotype were over-represented in cleft cases compared with control group when we considered the bilateral cleft lip with or without cleft palate (CL±P) cases, cleft cases with tooth agenesis and cleft cases presenting family history of cleft, but the differences were not statistically significant. Contradictorily, the G allele was higher in cleft palate only (CP) cases than in control group, showing a borderline p value. Comparing the different cleft phenotypes, we observed statistical differences between CP and CL±P cases. Our data suggest the EGF+61 A>;G polymorphism was not related with nonsyndromic oral clefts susceptibility in a Brazilian population, but supported the different genetic background between CL±P and CP. Moreover, we confirmed the potential effect of maternal alcohol intake on cleft risk in our population

XRCC1 gene polymorphisms in a population sample and in women with a family history of breast cancer from Rio de Janeiro (Brazil)

The X-ray repair cross-complementing Group1 (XRCC1) gene has been defined as essential in the base excision repair (BER) and single-strand break repair processes. This gene is highly polymorphic, and the most extensively studied genetic changes are in exon 6 (Arg194Trp) and in exon 10 (Arg399Gln). These changes, in conserved protein sites, may alter the base excision repair capacity, increasing the susceptibility to adverse health conditions, including cancer. In the present study, we estimated the frequencies of the XRCC1 gene polymorphisms Arg194Trp and Arg399Gln in healthy individuals and also in women at risk of breast cancer due to family history from Rio de Janeiro. The common genotypes in both positions (194 and 399) were the most frequent in this Brazilian sample. Although the 194Trp variant was overrepresented in women reporting familial cases of breast cancer, no statistically significant differences concerning genotype distribution or intragenic interactions were found between this group and the controls. Thus, in the population analyzed by us, variants Arg194Trp and Arg399Gln did not appear to have any impact on breast cancer susceptibility

XRCC1 gene polymorphisms in a population sample and in women with a family history of breast cancer from Rio de Janeiro (Brazil)

The X-ray repair cross-complementing Group1 (XRCC1) gene has been defined as essential in the base excision repair (BER) and single-strand break repair processes. This gene is highly polymorphic, and the most extensively studied genetic changes are in exon 6 (Arg194Trp) and in exon 10 (Arg399Gln). These changes, in conserved protein sites, may alter the base excision repair capacity, increasing the susceptibility to adverse health conditions, including cancer. In the present study, we estimated the frequencies of the XRCC1 gene polymorphisms Arg194Trp and Arg399Gln in healthy individuals and also in women at risk of breast cancer due to family history from Rio de Janeiro. The common genotypes in both positions (194 and 399) were the most frequent in this Brazilian sample. Although the 194Trp variant was overrepresented in women reporting familial cases of breast cancer, no statistically significant differences concerning genotype distribution or intragenic interactions were found between this group and the controls. Thus, in the population analyzed by us, variants Arg194Trp and Arg399Gln did not appear to have any impact on breast cancer susceptibility

Buccal cells DNA extraction to obtain high quality human genomic DNA suitable for polymorphism genotyping by PCR-RFLP and Real-Time PCR

OBJECTIVE: The aim of this study was to evaluate, by PCR-RFLP and Real-time PCR, the yield and quality of genomic DNA collected from buccal cells by mouthwash after different storage times at room temperature. MATERIAL AND METHODS: A group of volunteers was recruited to collect buccal cells using a mouthwash solution. The collected solution was divided into 3 tubes, one tube were used for immediate extraction and the remaining received ethanol and were kept at room temperature for 4 and 8 days followed by DNA extraction. The concentration, purity and integrity of the DNA were determined using spectrophotometry and electrophoresis. DNA quality differences among the three incubation times were also evaluated for genotyping EGF +61 A/G (rs 4444903) polymorphism by PCR-RFLP and for IRF6 polymorphism (rs 17015215) using Real-Time PCR. RESULTS: There was no significant difference of DNA yield (p=0.75) and purity (p=0.86) among the three different incubation times. DNA obtained from different incubation times presented high-molecular weight. The PCR-RFLP and Real time PCR reactions were successfully performed for all DNA samples, even those extracted after 8 days of incubation. All samples genotyped by Real-Time PCR presented C allele for IRF6 gene polymorphism (homozygous: CC; heterozygous: CT) and the C allele was used as a reference for Ct values. The samples presented the same genotype for the different times in both techniques. CONCLUSION: We demonstrated that the method described herein is simple and low cost, and that DNA can be extracted and PCR amplified after storage in mouthwash solution at room temperature

Poly-lactic acid nanoparticles (PLA-NP) promote physiological modifications in lung epithelial cells and are internalized by clathrin-coated pits and lipid rafts

BackgroundPoly-lactic acid nanoparticles (PLA-NP) are a type of polymeric NP, frequently used as nanomedicines, which have advantages over metallic NP such as the ability to maintain therapeutic drug levels for sustained periods of time. Despite PLA-NP being considered biocompatible, data concerning alterations in cellular physiology are scarce.MethodsWe conducted an extensive evaluation of PLA-NP biocompatibility in human lung epithelial A549 cells using high throughput screening and more complex methodologies. These included measurements of cytotoxicity, cell viability, immunomodulatory potential, and effects upon the cells’ proteome. We used non- and green-fluorescent PLA-NP with 63 and 66 nm diameters, respectively. Cells were exposed with concentrations of 2, 20, 100 and 200 µg/mL, for 24, 48 and 72 h, in most experiments. Moreover, possible endocytic mechanisms of internalization of PLA-NP were investigated, such as those involving caveolae, lipid rafts, macropinocytosis and clathrin-coated pits.ResultsCell viability and proliferation were not altered in response to PLA-NP. Multiplex analysis of secreted mediators revealed a low-level reduction of IL-12p70 and vascular epidermal growth factor (VEGF) in response to PLA-NP, while all other mediators assessed were unaffected. However, changes to the cells’ proteome were observed in response to PLA-NP, and, additionally, the cellular stress marker miR155 was found to reduce. In dual exposures of staurosporine (STS) with PLA-NP, PLA-NP enhanced susceptibility to STS-induced cell death. Finally, PLA-NP were rapidly internalized in association with clathrin-coated pits, and, to a lesser extent, with lipid rafts.ConclusionsThese data demonstrate that PLA-NP are internalized and, in general, tolerated by A549 cells, with no cytotoxicity and no secretion of pro-inflammatory mediators. However, PLA-NP exposure may induce modification of biological functions of A549 cells, which should be considered when designing drug delivery systems. Moreover, the pathways of PLA-NP internalization we detected could contribute to the improvement of selective uptake strategies

Mechanistic Insights into the Biological Effects of Engineered Nanomaterials: A Focus on Gold Nanoparticles

Nanotechnology has great potential to significantly advance the biomedical field for the benefit of human health. However, the limited understanding of nano–bio interactions leading to unknowns about the potential adverse health effects of engineered nanomaterials and to the poor efficacy of nanomedicines has hindered their use and commercialization. This is well evidenced considering gold nanoparticles, one of the most promising nanomaterials for biomedical applications. Thus, a fundamental understanding of nano–bio interactions is of interest to nanotoxicology and nanomedicine, enabling the development of safe-by-design nanomaterials and improving the efficacy of nanomedicines. In this review, we introduce the advanced approaches currently applied in nano–bio interaction studies—omics and systems toxicology—to provide insights into the biological effects of nanomaterials at the molecular level. We highlight the use of omics and systems toxicology studies focusing on the assessment of the mechanisms underlying the in vitro biological responses to gold nanoparticles. First, the great potential of gold-based nanoplatforms to improve healthcare along with the main challenges for their clinical translation are presented. We then discuss the current limitations in the translation of omics data to support risk assessment of engineered nanomaterials

Anticancer properties of carotenoids in prostate cancer. A review

Prostate cancer is the most common noncutaneous cancer of men in the world. Several epidemiological studies have linked increased carotenoids consumption with decreased prostate cancer risk. These findings are supported by in vitro and in vivo experiments showing that carotenoids not only enhance the antioxidant response of prostate cells, but that they are able to inhibit proliferation, induce apoptosis and decrease the metastatic capacity of prostate cancer cells. However, clear clinical evidence supporting the use of carotenoids in prevention or treatment of prostate cancer is not available, due to the limited number of published randomized clinical trials, and the varying protocols used in the existing studies. The scope of the present review is to discuss the potential impact of carotenoids on prostate cancer by giving an overview of the molecular mechanisms and in vitro / in vivo effects