Cytotoxicity of mini gold nanorods: intersection with extracellular vesicles.

It is well-known that there are size- and shape-dependencies to nanoparticle uptake and processing by living cells. Small gold nanorods have shown to exhibit low toxicity and high clearance rates when compared to larger ones, making smaller particles more desirable for biomedical applications. In this study gold mini-rods (approximately 9.5 × 23, 8 × 26, and 6 × 26 nm, corresponding to aspect ratios 2.5, 3.2 and 4.1) and gold nanospheres (15.6 nm average diameter) were synthesized, and wrapped with cationic and anionic polyelectrolytes. This library of colloidally stable nanomaterials was exposed to human dermal fibroblasts at the relatively low concentration of 1 nM for each nanoparticle type. The cytotoxic profile of these nanoparticles and their influence on the small extracellular vesicles released by the cells was assessed. It was observed that although the nanoparticles were found in vesicles inside the cells, the cell viability, the mitochondrial membrane potential and levels of reactive oxygen species were not markedly affected by the mini gold nanorods. The production of extracellular vesicles by the cells was unaffected by gold nanoparticle exposure; moreover, no gold nanoparticles were observed in extracellular vesicles in the exosomal size range. Taken together, these results suggest that these mini gold nanorods are suitable for a wide range of cellular applications for relatively short-term studies.

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.

Nanoparticles Interfere with Chemotaxis: An Example of Nanoparticles as Molecular "Knockouts" at the Cellular Level.

Engineered colloidal nanoparticles show great promise in biomedical applications. While much of the work of assessing nanoparticle impact on living systems has been focused on the direct interactions of nanoparticles with cells/organisms, indirect effects via the extracellular matrix have been observed and may provide deeper insight into nanoparticle fate and effects in living systems. In particular, the large surface area of colloidal nanoparticles may sequester molecules from the biological milieu, make these molecules less bioavailable, and therefore function indirectly as "molecular knockouts" to exert effects at the cellular level and beyond. In this paper, the hypothesis that molecules that control cellular behavior (in this case, chemoattract molecules that promote migration of a human monocytic cell line, THP-1) will be less bioavailable in the presence of appropriately functionalized nanoparticles, and therefore the cellular behavior will be altered, was investigated. Three-dimensional chemotaxis assays for the characterization and comparison of THP-1 cell migration upon exposure to a gradient of monocyte chemoattractant protein-1 (MCP-1), with and without gold nanoparticles with four different surface chemistries, were performed. By time-lapse microscopy, characteristic parameters for chemotaxis, along with velocity and directionality of the cells, were quantified. Anionic poly(sodium 4-styrenesulfonate)-coated gold nanoparticles were found to significantly reduce THP-1 chemotaxis. Enzyme-linked immunosorbent assay results show adsorption of MCP-1 on the poly(sodium 4-styrenesulfonate)-coated gold nanoparticle surface, supporting the hypothesis that adsorption of chemoattractants to nanoparticle surfaces interferes with chemotaxis. Free anionic sulfonated polyelectrolytes also interfered with cell migrational behavior, showing that nanoparticles can also act as carriers of chemotactic-interfering molecules.

Impact of Zero-Valent Iron on Freshwater Bacterioplankton Metabolism as Predicted from 16S rRNA Gene Sequence Libraries.

The application of zero-valent iron particles (ZVI) for the treatment of heavily polluted environment and its biological effects have been studied for at least two decades. Still, information on the impact on bacterial metabolic pathways is lacking. This study describes the effect of microscale and nanoscale ZVI (mZVI and nZVI) on the abundance of different metabolic pathways in freshwater bacterial communities. The metabolic pathways were inferred from metabolism modelling based on 16S rRNA gene sequence data using paprica pipeline. The nZVI changed the abundance of numerous metabolic pathways compared to a less influencing mZVI. We identified the 50 most affected pathways, where 31 were related to degradation, 17 to biosynthesis, and 2 to detoxification. The linkage between pathways was two times higher in nZVI samples compared to mZVI, and was specifically higher considering the arsenate detoxification II pathway. Limnohabitans and Roseiflexus were linked to the same pathways in both nZVI and mZVI. The prediction of metabolic pathways increases our knowledge of the impacts of nZVI and mZVI on freshwater bacterioplankton.

Network-based analysis implies critical roles of microRNAs in the long-term cellular responses to gold nanoparticles.

Since gold nanoparticles (AuNPs) have great potential to bring improvements to the biomedical field, their impact on biological systems should be better understood, particularly over the long term, using realistic doses of exposure. MicroRNAs (miRNAs) are small noncoding RNAs that play key roles in the regulation of biological pathways, from development to cellular stress responses. In this study, we performed genome-wide miRNA expression profiling in primary human dermal fibroblasts 20 weeks after chronic and acute (non-chronic) treatments to four AuNPs with different shapes and surface chemistries at a low dose. The exposure condition and AuNP surface chemistry had a significant impact on the modulation of miRNA levels. In addition, a network-based analysis was employed to provide a more complex, systems-level perspective of the miRNA expression changes. In response to the stress caused by AuNPs, miRNA co-expression networks perturbed in cells under non-chronic exposure to AuNPs were enriched for target genes implicated in the suppression of proliferative pathways, possibly in attempt to restore cell homeostasis, while changes in miRNA co-expression networks enriched for target genes related to activation of proliferative and suppression of apoptotic pathways were observed in cells chronically exposed to one specific type of AuNPs. In this case, miRNA dysregulation might be contributing to enforce a new cell phenotype during stress. Our findings suggest that miRNAs exert critical roles in the cellular responses to the stress provoked by a low dose of NPs in the long term and provide a fertile ground for further targeted experimental studies.

Virus-Sized Gold Nanorods: Plasmonic Particles for Biology.

Plasmons, collective oscillations of conduction-band electrons in nanoscale metals, are well-known phenomena in colloidal gold and silver nanocrystals that produce brilliant visible colors in these materials that depend on the nanocrystal size and shape. Under illumination at or near the plasmon bands, gold and silver nanocrystals exhibit properties that enable fascinating biological applications: (i) the nanocrystals elastically scatter light, providing a straightforward way to image them in complex aqueous environments; (ii) the nanocrystals produce local electric fields that enable various surface-enhanced spectroscopies for sensing, molecular diagnostics, and boosting of bound fluorophore performance; (iii) the nanocrystals produce heat, which can lead to chemical transformations at or near the nanocrystal surface and can photothermally destroy nearby cells. While all the above-mentioned applications have already been well-demonstrated in the literature, this Account focuses on several other aspects of these nanomaterials, in particular gold nanorods that are approximately the size of viruses (diameters of ∼10 nm, lengths up to 100 nm). Absolute extinction, scattering, and absorption properties are compared for gold nanorods of various absolute dimensions, and references for how to synthesize gold nanorods with four different absolute dimensions are provided. Surface chemistry strategies for coating nanocrystals with smooth or rough shells are detailed; specific examples include mesoporous silica and metal-organic framework shells for porous (rough) coatings and polyelectrolyte layer-by-layer wrapping for "smooth" shells. For self-assembled-monolayer molecular coating ligands, the smoothest shells of all, a wide range of ligand densities have been reported from many experiments, yielding values from less than 1 to nearly 10 molecules/nm2 depending on the nanocrystal size and the nature of the ligand. Systematic studies of ligand density for one particular ligand with a bulky headgroup are highlighted, showing that the highest ligand density occurs for the smallest nanocrystals, even though these ligand headgroups are the most mobile as judged by NMR relaxation studies. Biomolecular coronas form around spherical and rod-shaped nanocrystals upon immersion into biological fluids; these proteins and lipids can be quantified, and their degree of adsorption depends on the nanocrystal surface chemistry as well as the biophysical characteristics of the adsorbing biomolecule. Photothermal adsorption and desorption of proteins on nanocrystals depend on the enthalpy of protein-nanocrystal surface interactions, leading to light-triggered alteration in protein concentrations near the nanocrystals. At the cellular scale, gold nanocrystals exert genetic changes at the mRNA level, with a variety of likely mechanisms that include alteration of local biomolecular concentration gradients, changes in mechanical properties of the extracellular matrix, and physical interruption of key cellular processes-even without plasmonic effects. Microbiomes, both organismal and environmental, are the likely first point of contact of nanomaterials with natural living systems; we see a major scientific frontier in understanding, predicting, and controlling microbe-nanocrystal interactions, which may be augmented by plasmonic effects.

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

BACKGROUND: Poly-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. METHODS: We 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. RESULTS: Cell 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. CONCLUSIONS: These 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.

Are your results valid? Cellular authentication a need from the past, an emergency on the present.

The cultures of immortalized cells have been established in the 50s and become popular as a biological model for in vitro assays. The success and popularization brought side effects. Still, in the 60 years emerge the first cases of misidentification/contamination of cell line. Because of that, the scientific community has been oriented to authenticate their lines before performing assays. The use of cells with incorrect identification or contamination has been identified as responsible for an increasing number of unmatched results and a waste of resources. For this reason, we implemented the Cell Line Authentication Service at Brazilian Metrology Institute (Inmetro), open to Brazilian scientific community and society in general. From 2012 to 2014 were conducted 111 cell line authentication test, of which 13.8% had some problem. Here are the description and discussion of these data and simple guidelines to minimize the risk of contamination and misidentification, and invite the scientific community to maintain an alert system to avoid spending unnecessary resources and produce unreliable data.

New Advances in Nanotechnology-Based Diagnosis and Therapeutics for Breast Cancer: An Assessment of Active-Targeting Inorganic Nanoplatforms.

Breast cancer is a major cause of suffering and mortality among women. Limitations in the current diagnostic methods and treatment approaches have led to new strategies to positively impact the survival rates and quality of life of breast cancer patients. Nanotechnology offers a real possibility of mitigating breast cancer mortality by early-stage cancer detection and more precise diagnosis as well as more effective treatments with minimal side effects. The current nanoplatforms approved for breast cancer therapeutics are based on passive tumor targeting using organic nanoparticles and have not provided the expected significant improvements in the clinic. In this review, we present the emerging approaches in breast cancer nanomedicine based on active targeting using versatile inorganic nanoplatforms with biomedical relevance, such as gold, silica, and iron oxide nanoparticles, as well as their efficacy in breast cancer imaging, drug and gene delivery, thermal therapy, combinational therapy, and theranostics in preclinical studies. The main challenges for clinical translation and perspectives are discussed.

Successful Low-Cost Scaffold-Free Cartilage Tissue Engineering Using Human Cartilage Progenitor Cell Spheroids Formed by Micromolded Nonadhesive Hydrogel.

The scaffold-free tissue engineering using spheroids is pointed out as an approach for optimizing the delivery system of cartilage construct. In this study, we aimed to evaluate the micromolded nonadhesive hydrogel (MicroTissues®) for spheroid compaction (2-day culture) and spontaneous chondrogenesis (21-day culture) using cartilage progenitors cells (CPCs) from human nasal septum without chondrogenic stimulus. CPC spheroids showed diameter stability (486 µm ± 65), high percentage of viable cells (88.1 ± 2.1), and low percentage of apoptotic cells (2.3%). After spheroid compaction, the synthesis of TGF-ß1, TGF-ß2, and TGF-ß3 was significantly higher compared to monolayer (p < 0.005). Biomechanical assay revealed that the maximum forces applied to spheroids after chondrogenesis were 2.6 times higher than for those cultured for 2 days. After spontaneous chondrogenesis, CPC spheroids were entirely positive for N-cadherin, collagen type II and type VI, and aggrecan and chondroitin sulfate. Comparing to monolayer, the expression of SOX5 and SOX6 genes analyzed by qPCR was significantly upregulated (p < 0.01). Finally, we observed the capacity of CPC spheroids starting to fuse. To the best of our knowledge, this is the first time in the scientific literature that human CPC spheroids were formed by micromolded nonadhesive hydrogel, achieving a successful scaffold-free cartilage engineering without chondrogenic stimulus (low cost).

One low-dose exposure of gold nanoparticles induces long-term changes in human cells.

We report the in vitro long-term (20 wk) changes in cells exposed to well-characterized gold nanoparticles (Au NPs) with varying shapes and surface coatings under both chronic (exposure to Au NPs continuously over 20 wk) and nonchronic (initial acute cell exposure to Au NPs, followed by 20 wk in NP-free cell media) conditions. Both chronic and nonchronic Au NPs exposures at low dose induce modifications at the gene level after long periods. In attempt to overcome from the injuries caused by nanoparticle exposure, genes related to oxidative stress, cell cycle regulation, and inflammation are among those presenting differential expression levels. Surprisingly, the nonchronic exposure induced more gene expression changes than its chronic counterpart and the stress effects caused by this type of exposure were sustained even after 20 wk without any additional NP exposure. NP surface chemistry played an important role in the alteration of gene regulation. Overall, our data suggest that (i) cells can adaptively respond to chronic, low-level NP insults; (ii) the cell stress response is not reversible over time upon removal of NPs upon acute, nonchronic exposure; and (iii) polyethylene glycol is not as benign a surface chemistry as is generally supposed.

An international comparability study on quantification of mRNA gene expression ratios: CCQM-P103.1.

Measurement of RNA can be used to study and monitor a range of infectious and non-communicable diseases, with profiling of multiple gene expression mRNA transcripts being increasingly applied to cancer stratification and prognosis. An international comparison study (Consultative Committee for Amount of Substance (CCQM)-P103.1) was performed in order to evaluate the comparability of measurements of RNA copy number ratio for multiple gene targets between two samples. Six exogenous synthetic targets comprising of External RNA Control Consortium (ERCC) standards were measured alongside transcripts for three endogenous gene targets present in the background of human cell line RNA. The study was carried out under the auspices of the Nucleic Acids (formerly Bioanalysis) Working Group of the CCQM. It was coordinated by LGC (United Kingdom) with the support of National Institute of Standards and Technology (USA) and results were submitted from thirteen National Metrology Institutes and Designated Institutes. The majority of laboratories performed RNA measurements using RT-qPCR, with datasets also being submitted by two laboratories based on reverse transcription digital polymerase chain reaction and one laboratory using a next-generation sequencing method. In RT-qPCR analysis, the RNA copy number ratios between the two samples were quantified using either a standard curve or a relative quantification approach. In general, good agreement was observed between the reported results of ERCC RNA copy number ratio measurements. Measurements of the RNA copy number ratios for endogenous genes between the two samples were also consistent between the majority of laboratories. Some differences in the reported values and confidence intervals ('measurement uncertainties') were noted which may be attributable to choice of measurement method or quantification approach. This highlights the need for standardised practices for the calculation of fold change ratios and uncertainties in the area of gene expression profiling.

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

Nonsyndromic 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.

Performance of PCR-based and Bioluminescent assays for mycoplasma detection.

Contaminated eukaryotic cell cultures are frequently responsible for unreliable results. Regulatory entities request that cell cultures must be mycoplasma-free. Mycoplasma contamination remains a significant problem for cell cultures and may have an impact on biological analysis since they affect many cell parameters. The gold standard microbiological assay for mycoplasma detection involves laborious and time-consuming protocols. PCR-based and Bioluminescent assays have been considered for routine cell culture screening in research laboratories since they are fast, easy and sensitive. Thus, the aim of this work is to compare the performance of two popular commercial assays, PCR-based and Bioluminescent assays, by assessing the level of mycoplasma contamination in cell cultures from Rio de Janeiro Cell Bank (RJCB) and also from customers' laboratories. The results obtained by both performed assays were confirmed by scanning electron microscopy. In addition, we evaluated the limit of detection of the PCR kit under our laboratory conditions and the storage effects on mycoplasma detection in frozen cell culture supernatants. The performance of both assays for mycoplasma detection was not significantly different and they showed very good agreement. The Bioluminescent assay for mycoplasma detection was slightly more dependable than PCR-based due to the lack of inconclusive results produced by the first technique, especially considering the ability to detect mycoplasma contamination in frozen cell culture supernatants. However, cell lines should be precultured for four days or more without antibiotics to obtain safe results. On the other hand, a false negative result was obtained by using this biochemical approach. The implementation of fast and reliable mycoplasma testing methods is an important technical and regulatory issue and PCR-based and Bioluminescent assays may be good candidates. However, validation studies are needed.

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.

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.

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.

Prognostic and predictive value of TP53 mutations in node-positive breast cancer patients treated with anthracycline- or anthracycline/taxane-based adjuvant therapy: results from the BIG 02-98 phase III trial.

INTRODUCTION: Pre-clinical data suggest p53-dependent anthracycline-induced apoptosis and p53-independent taxane activity. However, dedicated clinical research has not defined a predictive role for TP53 gene mutations. The aim of the current study was to retrospectively explore the prognosis and predictive values of TP53 somatic mutations in the BIG 02-98 randomized phase III trial in which women with node-positive breast cancer were treated with adjuvant doxorubicin-based chemotherapy with or without docetaxel. METHODS: The prognostic and predictive values of TP53 were analyzed in tumor samples by gene sequencing within exons 5 to 8. Patients were classified according to p53 protein status predicted from TP53 gene sequence, as wild-type (no TP53 variation or TP53 variations which are predicted not to modify p53 protein sequence) or mutant (p53 nonsynonymous mutations). Mutations were subcategorized according to missense or truncating mutations. Survival analyses were performed using the Kaplan-Meier method and log-rank test. Cox-regression analysis was used to identify independent predictors of outcome. RESULTS: TP53 gene status was determined for 18% (520 of 2887) of the women enrolled in BIG 02-98. TP53 gene variations were found in 17% (90 of 520). Nonsynonymous p53 mutations, found in 16.3% (85 of 520), were associated with older age, ductal morphology, higher grade and hormone-receptor negativity. Of the nonsynonymous mutations, 12.3% (64 of 520) were missense and 3.6% were truncating (19 of 520). Only truncating mutations showed significant independent prognostic value, with an increased recurrence risk compared to patients with non-modified p53 protein (hazard ratio = 3.21, 95% confidence interval = 1.740 to 5.935, P = 0.0002). p53 status had no significant predictive value for response to docetaxel. CONCLUSIONS: p53 truncating mutations were uncommon but associated with poor prognosis. No significant predictive role for p53 status was detected. TRIAL REGISTRATION: ClinicalTrials.gov NCT00174655.

Polimorfismos nos genes MMP2, MMP13, CYP1A1, GSTM1 e EMX2 e endometriose / Polymorphisms in MMP2, MMP13, CYP1A1, GSTM1 and EMX2 and endometriosis

A endometriose é uma doença que afeta de 10 a 15% das mulheres em idade fértil. Trata-se de uma doença crônica dependente de estrogênio, caracterizada pela presença de tecido endometrial fora da cavidade uterina. É uma doença multifatorial e poligênica que ainda apresenta etiologia desconhecida. Estudos moleculares relacionam a suscetibilidade à endometriose com polimorfismos genéticos específicos. Os principais genes estudados são responsáveis pelos mecanismos biológicos da doença, como por exemplo, metabolismo do estrogênio, receptores hormonais e detoxificação celular. O objetivo do presente artigo é revisar as pesquisas relacionadas aos genes CYP1A1, MMP2, MMP13, GSTM1 e EMX2, para avaliar as possíveis implicações na patogênese da endometriose

Endometriosis is a disease that affects 10 to 15% of women on reproductive age. It is an estrogen-dependent chronic disease, which is characterized by the presence of endometrial tissue outside the uterine cavity. It is a polygenic and multifactorial disease that still has unknown etiology. Molecular studies relate susceptibility to specific genetic polymorphisms with endometriosis. The main studied genes are responsible for the biological mechanisms of disease, such as estrogen metabolism, hormone receptors, and cellular detoxification. The aim of this paper is to review the research related to CYP1A1, MMP2, MMP13, GSTM1, and EMX2 genes, in order to assess the possible implications in the pathogenesis of endometriosis

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.