Site‐Directed Mutagenesis by Polymerase Chain Reaction

Since genomic data are widely available, many strategies have been implemented to reveal the function of specific nucleotides or amino acids in promoter regions or proteins, respectively. One of the methods most commonly used to determine the impact of mutations is the site‐directed mutagenesis using the polymerase chain reaction (PCR). There are different published protocols to develop single or multiple site‐directed mutagenesis. In this chapter, we reviewed the enzymes commonly used in site‐directed mutagenesis, the methods for simple and multiple site‐directed mutagenesis in large constructs, mediated by insertion of restriction sites. Other methods reviewed include high‐throughput site‐directed mutagenesis using oligonucleotides synthesized on DNA chips, and those based on multi‐site‐directed mutagenesis, based on recombination. Software tools to design site‐directed mutagenesis primers are also presented

Drug Resistance in Mycobacterium tuberculosis

Tuberculosis (TB) remains to be a serious health problem worldwide. There is an increased transmission of Mycobacterium tuberculosis strains with drug resistance, hence complicating TB control. The deciphering of the M. tuberculosis genome, together with the implementation of new molecular biology tools, has allowed the identification of changes in nucleic acid sequences with a functional impact. These mutations have become important in the design of early‐diagnostic kits to identify the resistance profile of M. tuberculosis. Since the conventional methods to determine the identity of M. tuberculosis strains based in cultures are laborious, time‐consuming and performed by specialized technicians, the result is generated until 4 months after receiving the samples. During this time, patients with TB are not adequately treated, and resistant strains may be transmitted to the rest of the population. In this chapter, we describe the most relevant mutations in genes associated with drug resistance in M. tuberculosis, the analysis of gene expression to identify new markers of drug resistance strains, and the development of new antituberculosis drugs against drug‐resistant strains

La relación de la gestión de las cadenas de suministro con los procesos de producción para la competitividad de la pyme de Aguascalientes. Aguilera

Con el presente trabajo de investigación, aplicado a la pequeña y mediana empresa (pyme) del sector de manufactura se pretende explicar si existe una relación positiva entre la gestión de la cadena de suministro y los procesos de producción, con la finalidad de que sean más competitivas; para ello se aplicó un instrumento de evaluación dirigido a los gerentes, donde los resultados obtenidos se han analizado con apoyo del paquete estadístico eqs, donde a través de ecuaciones estructurales se ha dado respuesta al objetivo planteado. En este sentido se puede concluir que la gestión de las cadenas de suministro tiene una relación positiva con los procesos de producción, y a su vez esta relación muestra un impacto positivo en la competitividad de la pyme de Aguascalientes. Para esta investigación se ha utilizando una muestra de 120 empresas, las cuales contienen de cinco a 250 trabajadores

Selenite downregulates STAT3 expression and provokes lymphocytosis in the liver of chronically exposed Syrian golden hamsters

Arsenic is considered a worldwide pollutant that can be present in drinking water. Arsenic exposure is associated with various diseases, including cancer. Antioxidants as selenite and α-tocopherol-succinate have been shown to modulate arsenic toxic effects. Since changes in STAT3 and PSMD10 gene expression have been associated with carcinogenesis, the aim of this study was to evaluate the effect of arsenic exposure and co-treatments with selenite or α-tocopherol-succinate on the expression of these genes, in the livers of chronically exposed Syrian golden hamsters. Animals were divided into six groups: (i) control, (ii) chronically treated with 100 ppm arsenic, (iii) treated with 6 ppm α-tocopherol-succinate (α-TOS), (iv) treated with 8.5 ppm selenite, (v) treated with arsenic + α-TOS, and (vi) treated with arsenic + selenite. Urine samples and livers were collected after 20 weeks of continuous exposure. The urine samples were analyzed for arsenic species by atomic absorption spectrophotometry, and real-time RT-qPCR analysis was performed for gene expression evaluation. A reduction in STAT3 expression was observed in the selenite-treated group. No differences in PSMD10 expression were found among groups. Histopathological analysis revealed hepatic lymphocytosis in selenite-treated animals. As a conclusion, long-term exposure to arsenic does not significantly alter the expression of STAT3 and PSMD10 oncogenes in the livers of hamsters; however, selenite down-regulates STAT3 expression and provokes lymphocytosis

Occupational Toluene Exposure Induces Cytochrome P450 2E1 mRNA Expression in Peripheral Lymphocytes

Print workers are exposed to organic solvents, of which the systemic toxicant toluene is a main component. Toluene induces expression of cytochrome P450 2E1 (CYP2E1), an enzyme involved in its own metabolism and that of other protoxicants, including some procarcinogens. Therefore, we investigated the association between toluene exposure and the CYP2E1 response, as assessed by mRNA content in peripheral lymphocytes or the 6-hydroxychlorzoxazone (6OH-CHZ)/chlorzoxazone (CHZ) quotient (known as CHZ metabolic ratio) in plasma, and the role of genotype (5′-flanking region RsaI/PstI polymorphic sites) in 97 male print workers. The geometric mean (GM) of toluene concentration in the air was 52.80 ppm (10–760 ppm); 54% of the study participants were exposed to toluene concentrations that exceeded the maximum permissible exposure level (MPEL). The GM of urinary hippuric acid at the end of a work shift (0.041 g/g creatinine) was elevated relative to that before the shift (0.027 g/g creatinine; p < 0.05). The GM of the CHZ metabolic ratio was 0.33 (0–9.3), with 40% of the subjects having ratios below the GM. However, the average CYP2E1 mRNA level in peripheral lymphocytes was 1.07 (0.30–3.08), and CYP2E1 mRNA levels within subjects correlated with the toluene exposure ratio (environmental toluene concentration:urinary hippuric acid concentration) (p = 0.014). Genotype did not alter the association between the toluene exposure ratio and mRNA content. In summary, with further validation, CYP2E1 mRNA content in peripheral lymphocytes could be a sensitive and noninvasive biomarker for the continuous monitoring of toluene effects in exposed persons

The Role of Molecular Biology in the Biomonitoring of Human Exposure to Chemicals

Exposure to different substances in an occupational environment is of utmost concern to global agencies such as the World Health Organization and the International Labour Organization. Interest in improving work health conditions, particularly of those employees exposed to noxious chemicals, has increased considerably and has stimulated the search for new, more specific and selective tests. Recently, the field of molecular biology has been indicated as an alternative technique for monitoring personnel while evaluating work-related pathologies. Originally, occupational exposure to environmental toxicants was assessed using biochemical techniques to determine the presence of higher concentrations of toxic compounds in blood, urine, or other fluids or tissues; results were used to evaluate potential health risk. However, this approach only estimates the presence of a noxious chemical and its effects, but does not prevent or diminish the risk. Molecular biology methods have become very useful in occupational medicine to provide more accurate and opportune diagnostics. In this review, we discuss the role of the following common techniques: (1) Use of cell cultures; (2) evaluation of gene expression; (3) the “omic” sciences (genomics, transcriptomics, proteomics and metabolomics) and (4) bioinformatics. We suggest that molecular biology has many applications in occupational health where the data can be applied to general environmental conditions

High seroprevalence of Toxoplasma gondii infection in a subset of Mexican patients with work accidents and low socioeconomic status

<p>Abstract</p> <p>Background</p> <p><it>Toxoplasma gondii </it>has been associated with reflex impairment and traffic accidents. It is unknown whether <it>Toxoplasma </it>infection might be associated with work accidents. Therefore, using a case-control seroprevalence study design, 133 patients with a recent work accident and 266 control subjects of the general population from the same region were examined with enzyme-linked immunoassays for the presence and levels of anti-<it>Toxoplasma </it>IgG antibodies and anti-<it>Toxoplasma </it>IgM antibodies. Socio-demographic, work, clinical and behavioral characteristics from each worker were obtained.</p> <p>Results</p> <p>Eleven (8.3%) of 133 patients, and 14 (5.3%) of 266 controls had anti-<it>T. gondii </it>IgG antibodies. Anti-<it>T. gondii </it>IgG levels were higher than 150 IU/ml in 8 (6%) patients and 10 (3.8%) controls. Anti-<it>T. gondii </it>IgM antibodies were found in one (0.8%) of the workers, and in 6 (2.3%) of the controls. No statistically significant differences in the IgG seroprevalences, frequencies of high IgG levels, and IgM seroprevalences among patients and controls were found. In contrast, a low socio-economic level in patients with work accidents was associated with <it>Toxoplasma </it>seropositivity (<it>P </it>= 0.01). Patients with work accidents and low socioeconomic status showed a significantly (OR = 3.38; 95% CI: 0.84-16.06; <it>P </it>= 0.04) higher seroprevalence of <it>T. gondii </it>infection than controls of the same socioeconomic status (15.1% vs. 5%, respectively). Multivariate analysis showed a positive association of <it>T. gondii </it>infection with boar meat consumption (OR = 3.04; 95% CI: 1.03-8.94; <it>P </it>= 0.04). In contrast, a negative association between <it>T. gondii </it>infection and national trips (OR = 0.40; 95% CI: 0.17-0.96; <it>P </it>= 0.04), sausage consumption (OR = 0.20; 95% CI: 0.05-0.68; <it>P </it>= 0.01), and ham consumption (OR = 0.16; 95% CI: 0.05-0.51; <it>P </it>= 0.002) was found.</p> <p>Conclusions</p> <p>In the study described here seropositivity to <it>T. gondii </it>was associated to work accidents in a subset of patients with low socioeconomic status. This is the first report of an association of <it>T. gondii </it>infection and work accidents. Further studies to confirm our results are needed. Results may help in designing optimal prevention strategies to avoid <it>T. gondii </it>infection.</p

Establishment of triple-negative breast cancer cells based on BMI: A novel model in the correlation between obesity and breast cancer

IntroductionObesity has been associated with an increased risk of biologically aggressive variants in breast cancer. Women with obesity often have tumors diagnosed at later stages of the disease, associated with a poorer prognosis and a different response to treatment. Human cell lines have been derived from specific subtypes of breast cancer and have served to define the cell physiology of corresponding breast cancer subtypes. However, there are no current cell lines for breast cancer specifically derived from patients with different BMIs. The availability of those breast cancer cell lines should allow to describe and unravel functional alterations linked to these comorbidities. MethodsCell cultures were established from tumor explants. Once generated, the triple negative subtype in a patient with obesity and a patient with a normal BMI were chosen for comparison. For cellular characterization, the following assays were conducted: proliferation assays, chemo – sensitivity assays for doxorubicin and paclitaxel, wound healing motility assays, matrix invasion assays, breast cancer cell growth to estradiol by chronic exposure to leptin, induction of endothelial permeability and tumorigenic potential in athymic mice with normo - versus hypercaloric diets with an evaluation of the epithelium – mesenchymal transformation proteins.ResultsTwo different cell lines, were established from patients with breast cancer: DSG-BC1, with a BMI of 21.9 kg/m2 and DSG-BC2, with a BMI of 31.5 kg/m2. In vitro, these two cell lines show differential growth rates, motility, chemosensitivity, vascular permeability, response to leptin with an activation of the JAK2/STAT3/AKT signaling pathway. In vivo, they displayed distinct tumorigenic potential. In particular, DSG-BC2, presented higher tumorigenicity when implanted in mice fed with a hypercaloric diet.DiscussionTo our knowledge, these primary cultures are the first in vitro representation of both breast cancer and obesity. DSG – BC2 presented a more aggressive in vivo and in vitro phenotype. These results support the hypothesis that breast cancer generated in an obese metabolic state may represent a contrasting variant within the same disease. This new model will allow both further comprehension, functional studies and the analysis of altered molecular mechanisms under the comorbidity of obesity and breast cancer