Differential Expression of FXR and Genes Involved in Inflammation and lipid Metabolism Indicate Adipose Tissue Dysfunction in Gestational Diabetes.

BACKGROUND: Gestational diabetes mellitus (GDM) is the most frequent metabolic alteration in pregnancy. Several abnormalities in visceral adipose tissue (VAT) have been described as part of its pathophysiology including hypertrophy, inflammation and altered lipid metabolism. Farnesoid X receptor (FXR) is involved in adipocyte physiology and inflammation, so its expression may correlate with the expression of tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10), lipoprotein lipase (LPL), and two fatty acid transporters (SLC27A2, and SLC27A4). AIM: To compare the FXR, LPL, SLC27A2, SLC27A4, TNF-α, and IL-10 mRNA expression in VAT between women with GDM and healthy pregnant (HP) women. Secondarily, to evaluate the potential correlation between these expression levels. MATERIALS AND METHODS: Cross-sectional study of 50 GDM and 50 HP women. Conventional biochemical tests were performed and relative mRNA expression in VAT was measured by RT-qPCR. RESULTS: Gene expression levels of FXR and IL-10 were lower, whereas those of LPL, as well as the TNF-α/IL-10 ratio, were higher in women with GDM compared to HP. Pre-pregnancy BMI was the main significant independent variable for FXR levels in VAT from women with GDM. In all women, LPL expression levels correlated positively with those of SLC27A2. Only in women with GDM, IL-10 expression levels correlated negatively with those of SLC27A2, and SLC27A4. CONCLUSIONS: GDM is associated with decreased expression of FXR and IL-10 and increased expression of LPL, as well as a higher TNF/IL-10 ratio in VAT. These results suggest increased lipid storage and pro-inflammatory state indicating VAT dysfunction in this metabolic disorder.

The big challenge of SARS-CoV-2 latency: testes as reservoir.

In the efforts to explain COVID-19 pathophysiology, studies are being carried out on the correspondence between the expression of SARS-CoV-2 cell receptors and viral sequences. ACE2, CD147 and TMPRSS2 receptors expression could indicate poorly explored potential infection targets. For the genomic analysis of SARS-CoV-2 receptors, using BioGPS information was decided, which is a portal that centralizes genetic annotation resources, in combination with that of The Human Protein Atlas, the largest portal of human transcriptome and proteome data. We also reviewed the most recent articles on the subject. RNA and viral receptor proteins expression was observed in numerous anatomical sites, which partially coincides with the information reported in the literature. High expression in testicular cells markedly stood out, and it would be therefore important ruling out whether this anatomical site is a SARS-CoV-2 reservoir; otherwise, germ cell damage, as it is observed in infections with other RNA viruses, should be determined.

En el afán por explicar la fisiopatogenia de COVID-19 se están realizando estudios en torno a la correspondencia entre la expresión de receptores celulares de SARS-CoV-2 y las secuencias virales. La expresión de los receptores ACE2, CD147 y TMPRSS2 podría indicar blancos de infección poco explorados. Para el análisis genómico de los receptores de SARS-CoV-2 se optó por utilizar la información del BioGPS, un portal que centraliza los recursos de anotación genética, en combinación con la de The Human Protein Atlas, el portal más grande de datos del transcriptoma y proteoma humanos. También se revisaron los artículos más recientemente respecto al tema. En numerosos sitios anatómicos se observó la expresión de ARN y proteínas de los receptores del virus, que coinciden parcialmente con la información reportada en la literatura. Resaltó la alta expresión en las células de los testículos, por lo que sería importante descartar si este sitio anatómico es un reservorio de SARS-CoV-2; de no ser así, determinar el daño en las células germinales, tal como sucede en infecciones por otros virus ARN.

The big challenge of SARS-CoV-2 latency: testes as reservoir / El gran desafío de la latencia de SARS-CoV-2: el testículo como reservorio

Abstract In the efforts to explain COVID-19 pathophysiology, studies are being carried out on the correspondence between the expression of SARS-CoV-2 cell receptors and viral sequences. ACE2, CD147 and TMPRSS2 receptors expression could indicate poorly explored potential infection targets. For the genomic analysis of SARS-CoV-2 receptors, using BioGPS information was decided, which is a portal that centralizes genetic annotation resources, in combination with that of The Human Protein Atlas, the largest portal of human transcriptome and proteome data. We also reviewed the most recent articles on the subject. RNA and viral receptor proteins expression was observed in numerous anatomical sites, which partially coincides with the information reported in the literature. High expression in testicular cells markedly stood out, and it would be therefore important ruling out whether this anatomical site is a SARS-CoV-2 reservoir; otherwise, germ cell damage, as it is observed in infections with other RNA viruses, should be determined.

Resumen En el afán por explicar la fisiopatogenia de COVID-19 se están realizando estudios en torno a la correspondencia entre la expresión de receptores celulares de SARS-CoV-2 y las secuencias virales. La expresión de los receptores ACE2, CD147 y TMPRSS2 podría indicar blancos de infección poco explorados. Para el análisis genómico de los receptores de SARS-CoV-2 se optó por utilizar la información del BioGPS, un portal que centraliza los recursos de anotación genética, en combinación con la de The Human Protein Atlas, el portal más grande de datos del transcriptoma y proteoma humanos. También se revisaron los artículos más recientemente respecto al tema. En numerosos sitios anatómicos se observó la expresión de ARN y proteínas de los receptores del virus, que coinciden parcialmente con la información reportada en la literatura. Resaltó la alta expresión en las células de los testículos, por lo que sería importante descartar si este sitio anatómico es un reservorio de SARS-CoV-2; de no ser así, determinar el daño en las células germinales, tal como sucede en infecciones por otros virus ARN.

[Comparative genomic analysis: from SARS to SARS-CoV-2 virus-types. Similarities and differences]. / Análisis de genómica comparativa: del virus SARS al SARS-CoV-2. Similitudes y diferencias.

BACKGROUND: We are currently witnessing a worldwide event caused by the pandemic outbreak derived from the new SARS-CoV-2 virus, which requires the generation of knowledge. Due to its novelty, many hypotheses and theories are discussed daily regarding the origin of this new virus. Several studies are focused on demonstrating how similar it is to other viruses. OBJECTIVE: To highlight the differences of SARS-CoV-2 with other SARS viruses, from a comparative genomics analysis, and determine if these can be attributed to manipulation events. MATERIAL AND METHODS: Complete genomes of two SARS viruses were downloaded, along with other six of human coronaviruses, and 16 of SARS-type coronaviruses. These were analyzed using the BLAST Ring Image Generator tool; afterwards, the evident differences were examined by MAFFT and BLAST programs. RESULTS: High identity was observed in fragments of the mammalian SARS-like genomes with the SARS-CoV-1 and SARS-CoV-2 genomes, identifying three main nucleotide differences, in the ORF1ab nsp3 region gene, in the receptor recognition S gene, and in the ORF8 gene, with which the SARS-type strains of mammals can be separated into the SARS-CoV-1 and SARS-CoV-2 type. CONCLUSION: The complete SARS-CoV-2 genome has high identity with mammalian SARS-type strains, which is why its most probable appearance could be the result of natural evolution.

INTRODUCCIÓN: en este momento somos testigos de un evento de magnitud mundial provocado por el brote pandémico derivado del nuevo virus SARS-CoV-2, lo cual requiere la generación de conocimiento. Por lo novedoso que resulta, muchas hipótesis y teorías son discutidas a diario respecto al origen de este nuevo virus. Varios estudios están enfocados en demostrar la similitud que el SARS-CoV-2 tiene con otros virus. OBJETIVO: resaltar las diferencias del SARS-CoV-2 con otros virus SARS, a partir de un análisis de genómica comparativa, y determinar si se pueden atribuir a eventos de manipulación. MATERIAL Y MÉTODOS: se descargaron dos genomas completos de virus SARS, seis genomas completos de coronavirus humanos y 16 de coronavirus tipo SARS; fueron analizados en un estudio de genómica comparativa mediante la herramienta BLAST Ring Image Generator, y a continuación se examinaron las diferencias evidentes mediante el uso de los programas MAFFT y BLAST. RESULTADOS: se observó una alta identidad en fragmentos de los genomas tipo SARS de mamíferos con los genomas SARS-CoV-1 y SARS-CoV-2, y se identificaron tres diferencias nucleotídicas principales: en el gen ORF1ab región nsp3, en el gen S de reconocimiento al receptor y en el gen ORF8, con el cual se pueden separar las cepas tipo SARS de mamíferos en tipo SARS-CoV-1 y SARS-CoV-2. CONCLUSIÓN: el genoma completo de SARS-CoV-2 posee una alta identidad con cepas tipo SARS de mamíferos, por lo que su aparición más probable podría ser el resultado de la evolución natural.

Morphometric study of adipocytes on breast cancer by means of photonic microscopy and image analysis.

Worldwide, breast cancer (BrCa) is currently the leading cause of deaths associated to malignant lesions in adult women. Given that some studies have mentioned that peritumoral adipocytes may contribute to breast carcinogenesis, present work sought to quantitative evaluate the morphometry of these cells in a group of adult women. Three thousand six hundred sixty four breast adipocytes, that came from biopsies of a group of adult females with different types of breast carcinomas (ductal, lobular, and mixed) and one with normal tissues, were evaluated through an image analysis (IA) process regarding six morphometric descriptors: area (A), perimeter (P), Feret diameter (FD ), aspect ratio (AR), roundness factor (RF), and fractal dimension of cellular contour (FDC ). Data showed that the adipocytes of the normal tissues group were bigger (A: 3398 ± 2331 µm2 , P: 239 ± 83 µm, and FD : 79.9 ± 24.5 µm) than those from BrCa samples (A: 2860 ± 1933 µm2 , P: 214 ± 66 µm, and FD : 73.2 ± 22.5 µm), and presented a more irregular contour (FDC of 1.370 ± 0.037 for normal group and of 1.335 ± 0.049 for the oncologic one). Moreover, it could be accounted that adipocytes of mixed carcinomas were largest (FD : 75.1 ± 22.4 µm) than those of lobular lesions (FD : 61.6 ± 22.6 µm), while the adipocytes of ductal carcinomas were the most oval (AR: 1.421 ± 0.524) and roughest (FDC : 1.324 ± 0.050) cells. IA results suggest that BrCa lesions can be categorized through a quantitative morphometric evaluation of peritumoral adipocytes. These findings could let the development of an analytical tool to help the Pathologist to enhance the accuracy of the oncologic diagnose.

DNA Methylation of Cellular Retinoic Acid-Binding Proteins in Cervical Cancer.

This study determined the methylation status of cellular retinoic acid-binding protein (CRABP) gene promoters and associated them with demographic characteristics, habits, and the presence of human papilloma virus (HPV) in patients with cervical cancer (CC), low and high squamous intraepithelial lesions, and no intraepithelial lesion. Women (n = 158) were selected from the Colposcopy Clinic of Sanitary Jurisdiction II in Ciudad Juarez, Chihuahua, Mexico. Demographic characteristics and habit information were collected. Cervical biopsy and endocervical scraping were used to determine methylation in promoter regions by methylation-specific polymerase chain reaction technique. We found hemi-methylation patterns in the promoter regions of CRABP1 and CRABP2; there was 28.5% hemi-methylation in CRABP1 and 7.0% in that of CRABP2. Methylation in CRABP1 was associated with age (≥35 years, P = 0.002), family history of cancer (P = 0.032), the presence of HPV-16 (P = 0.013), and no alcohol intake (P = 0.035). These epigenetic changes could be involved in the CC process, and CRABP1 has the potential to be a predictive molecular marker of retinoid therapy response.

Deficiencia de micronutrientes en la dieta del paciente con lesiones precancerosas del cérvix de una clínica de colposcopía en Ciudad Juárez, México / Micronutrient deficiency in the diet of the patient with precancerous cervix lesions on a colposcopy clinic at Ciudad Juárez, Mexico

Introducción: las lesiones intraepiteliales escamosas (LIE) son un estado de transición hacia el cáncer cervicouterino (CaCu) y un déficit de cronutrientes puede acelerar este proceso. Por ello, determinar la existencia de este déficit y conocer qué factores se asocian permitiría una posible prevención en esta población de riesgo. Objetivo: determinar la presencia de alguna deficiencia de micronutrientes involucrados en el proceso anticancerígeno y asociar este déficit con hábitos y factores demográficos en pacientes con LIE de Ciudad Juárez, Chihuahua, México. Métodos: en un estudio transversal analítico fueron seleccionadas 102 pacientes con LIE. Se realizó una encuesta dietaría (recordatorio de 24 horas) para estimar la ingesta de micronutrientes. La deficiencia fue determinada con un consumo < 75% de la ingesta diaria recomendada o sugerida (IDR o IDS) en México. Algunos hábitos y factores demográficos fueron obtenidos mediante la entrevista con la paciente. Se realizó un modelo de regresión logística para asociar la presencia de deficiencia con factores que afectan a la ingesta o incrementan el requerimiento de micronutrientes. Resultados: el retinol, ácido fólico, zinc, vitaminas C y E, considerados como micronutrientes en el proceso anticancerígeno del CaCu, se encontraron por debajo del 75% de la IDR. Aquellas mujeres con sobrepeso, obesidad y amas de casa se asociaron significativamente con la deficiencia de micronutrientes. Conclusión: el sobrepeso, la obesidad y la ocupación han sido asociados para presentar deficiencias de micronutrientes en este estudio. Estas variables convergen en una posible inseguridad alimentaria, la cual podría asociarse al incremento de incidencia de CaCu en México (AU)

Introduction: Squamous Intraepithelial Lesions (SIL) is a state of transition to cervical cancer (CC), and micronutrient deficiencies can speed up this process. Therefore, determining the existence of this deficit and know what factors are associated would allow for possible prevention in this population at risk. Objective: To determine the presence of some micronutrient deficiencies involved in the anti-carcinogenic process, also associate this deficit with habits and demographic factors in patients with SIL in Ciudad Juarez, Chihuahua, Mexico. Methods: An analytic cross-sectional study, 102 patients were selected with SIL. A dietary survey (24-hour recall) was performed to estimate the intake of micronutrients. The deficiency was determined when the consumption was less than 75% of the Recommended Dietary Allowances (RDA) or suggested in Mexico. Some habits and demographic factors were obtained by interview with the patient. A logistic regression was performed to associate the presence of deficiencies with factors that affecting the intake or increase the requirement of micronutrients. Results: Retinol, folic acid, zinc, vitamins C and E, considered micronutrients in the anti-carcinogenic process CC, were less than 75% of the RDA. Women with overweight, obesity and housewives, were significantly associated with micronutrient deficiencies. Conclusion: Overweight, obesity and occupation have been associated to present micronutrient deficiencies in this study. These variables converge on a possible food insecurity, which could be associated with increased incidence of CC in Mexico (AU)

Genetic polymorphism of tumor necrosis factor promoter region and susceptibility to develop Hodgkin lymphoma in a Mexican population.

Hodgkin lymphoma (HL) is a rare neoplasm of the lymphatic system, in which inflammation and allelic variants in cytokines have been proposed as etiological factors. Epstein-Barr virus infection is often associated as a risk factor in HL and since cytokines are involved in the humoral response to viral infection. Our aim was to study the association between single nucleotide polymorphisms (SNPs) located in the tumor necrosis factor (TNF) gene (- 376G> A, - 238G> A and 581G> A) in a sample of Mexican patients (56 cases) and their susceptibility to develop HL, comparing these SNPs among healthy individuals (127 controls). Frequencies for TNF - 238G> A and TNF 581G> A showed no significant differences between cases and controls. However, the proportion of cases with the GA genotype of - 376 SNP showed a significant difference as compared to controls, odds ratio = 4.41 (95% confidence interval: 1.21-16.6), p = 0.02. We found that in this group of patients from Mexico the SNP - 376G> A in TNF shows an association with higher risk for HL.

Suplementos dietéticos como tratamiento en el cáncer cervicouterino; revisión sistemática / Dietary supplements as a treatment for cervical cancer; a systemic review

Introducción: En el cáncer cervicouterino (CaCu) y neoplasias intraepiteliales cervicales (NIC) se requiere de una cuidadosa selección de terapias convencionales y complementarias, la inclusión de suplementos dietéticos dentro de ellas marcaría la pauta dentro de los tratamientos. Sin embargo, no se tiene algún reporte de qué tipo de suplementos pudieran ser utilizados para brindar una mejor respuesta ante la enfermedad por estas pacientes. Objetivo: En la presente revisión se analizan los ensayos clínicos de los últimos 20 años que evaluaron suplementos dietéticos en esta población, con el objetivo de dar a conocer cuáles de ellos son viables para ser administrados. Metodología: Se realizó una búsqueda de ensayos clínicos que utilizaron algún suplemento dietario en mujeres con CaCu y algún grado de NIC analizando en ellos el modo de utilización del suplemento; el tratamiento convencional; si existe una deficiencia del nutriente ó compuesto a evaluar; las características de la población, del ensayo clínico y del suplemento; así como la dosis y los efectos esperados. Resultados: Veinte fueron en total el número de estudios analizados. Los artículos fueron clasificados según la naturaleza del suplemento: Retinoides, vitamina E, probióticos, indoles, multivitamínico, ácido fólico y selenio. Conclusión: Para el tratamiento de CaCu y NIC resultaron efectivos algunos de los suplementos encontrados en esta revisión, sin embargo su efecto dependerá de diferentes factores propios de la enfermedad. Debido a esto, es necesario el manejo correcto de los suplementos dietéticos para poder ser utilizados eficientemente como tratamiento complementario en esta población (AU)

Introduction: Cervical cancer and cervical intraepithelial neoplasia (CIN) require a careful selection of conventional and complementary therapies. The inclusion of dietary supplements within the aforementioned treatments set the tone within treatments. However, there are no reports of what kind of supplements could be used to provide a better response to the disease in these patients. Objective: In this review, we analyze clinical trials of the past 20 years that evaluated dietary supplements on this population, with the objective to raise awareness on which of them are viable to be administered. Methods: We performed a search for clinical trials that used a dietary supplement in women with cervical cancer and some degree of NIC analyzing them how to use the supplement, the conventional treatment, deficiency of the nutrient or compound to be evaluated, the characteristics of the population, the clinical trial and the supplement, as well as dosage and their effects. Results: Twenty were in total the number of studies reviewed. The articles were classified according to the nature of the supplement: Retinoids, vitamin E, probiotics, indoles, multivitamin, folic acid and selenium. Conclusion: Some supplements were found to be effective in the treatment of cervical cancer and CIN. However, their effect depends on specific factors of the disease. The correct management of dietary supplements is an effective aid to help the patient with cancer and therefore, it is important to define what complementary therapies can be used for this population (AU)

[Dietary supplements as a treatment for cervical cancer: a systematic review]. / Suplementos dietéticos como tratamiento en el cáncer cervicouterino: revisión sistemática.

INTRODUCTION: Cervical cancer and cervical intraepithelial neoplasia (CIN) require a careful selection of conventional and complementary therapies. The inclusion of dietary supplements within the aforementioned treatments set the tone within treatments. However, there are no reports of what kind of supplements could be used to provide a better response to the disease in these patients. OBJECTIVE: In this review, we analyze clinical trials of the past 20 years that evaluated dietary supplements on this population, with the objective to raise awareness on which of them are viable to be administered. METHODS: We performed a search for clinical trials that used a dietary supplement in women with cervical cancer and some degree of NIC analyzing them how to use the supplement, the conventional treatment, deficiency of the nutrient or compound to be evaluated, the characteristics of the population, the clinical trial and the supplement, as well as dosage and their effects. RESULTS: Twenty were in total the number of studies reviewed. The articles were classified according to the nature of the supplement: Retinoids, vitamin E, probiotics, indoles, multivitamin, folic acid and selenium. CONCLUSION: Some supplements were found to be effective in the treatment of cervical cancer and CIN. However, their effect depends on specific factors of the disease. The correct management of dietary supplements is an effective aid to help the patient with cancer and therefore, it is important to define what complementary therapies can be used for this population.

Introducción: En el cáncer cervicouterino (CaCu) y neoplasias intraepiteliales cervicales (NIC) se requiere de una cuidadosa selección de terapias convencionales y complementarias, la inclusión de suplementos dietéticos dentro de ellas marcaría la pauta dentro de los tratamientos. Sin embargo, no se tiene algún reporte de qué tipo de suplementos pudieran ser utilizados para brindar una mejor respuesta ante la enfermedad por estas pacientes. Objetivo: En la presente revisión se analizan los ensayos clínicos de los últimos 20 años que evaluaron suplementos dietéticos en esta población, con el objetivo de dar a conocer cuáles de ellos son viables para ser administrados. Metodología: Se realizó una búsqueda de ensayos clínicos que utilizaron algún suplemento dietario en mujeres con CaCu y algún grado de NIC analizando en ellos el modo de utilización del suplemento; el tratamiento convencional; si existe una deficiencia del nutriente ó compuesto a evaluar; las características de la población, del ensayo clínico y del suplemento; así como la dosis y los efectos esperados. Resultados: Veinte fueron en total el número de estudios analizados. Los artículos fueron clasificados según la naturaleza del suplemento: Retinoides, vitamina E, probióticos, indoles, multivitamínico, ácido fólico y selenio. Conclusión: Para el tratamiento de CaCu y NIC resultaron efectivos algunos de los suplementos encontrados en esta revisión, sin embargo su efecto dependerá de diferentes factores propios de la enfermedad. Debido a esto, es necesario el manejo correcto de los suplementos dietéticos para poder ser utilizados eficientemente como tratamiento complementario en esta población.

[The nanotechnology as a support for diagnosis and prognosis in cancer research]. / La nanotecnología en apoyo a la investigación del cáncer.

Recently, technological advances have greatly increased, generating the development of nanotechnology, which is responsible for the design of structures and materials in the nanometer scale. This creates one of the most important cutting-edge sciences, integrating physics, chemistry, engineering and biology sciences. Specifically the integration with biology results in a new science called nanobiotechnology, specifically nanomedicine, which has the goal of mainly looking for more precise molecular diagnostic and prognostic processes, as well as the new design of drugs in the personalized medicine field. On the other hand, at molecular level in medical research, the nanoparticles are most commonly used as tools. Molecular diagnostics uses gold nanoparticles, paramagnetic nanoparticles and quantum dots, which can be used for the diagnosis and treatment of several diseases, including cancer. Quantum dots are the most promising tools for diagnosis and therapy in cancer research.

Human leukocyte antigens I and II haplotypes associated with human papillomavirus 16-positive invasive cervical cancer in Mexican women.

Infection with human papillomavirus (HPV), mainly HPV type 16, is the major etiologic factor associated with cervical cancer (CC), but HPV infection alone is not sufficient for progression of precursor lesions. Host genetic susceptibility may lead to abnormal immune response resulting from virus persistence. Several studies have suggested a possible association with specific human leukocyte antigen (HLA) class I and II alleles and CC, but results are not consistent. The association of genetic HLA class I (A and B) and HLA class II (DR*B1 and DQ*B1) haplotypes with HPV16-positive CC (n = 104) and base population controls (n = 104) was evaluated in this Mexican population study. Sequence-specific primer HLA genes were determined by polymerase chain reaction (PCR)-based methods in peripheral blood cell counts (PCR sequence-specific oligonucleotides). The cervical swabs of 208 women were tested for HPV16 by Hybrid Capture II. Allele and haplotype HLA frequencies, Hardy-Weinberg tests, and a haplotype homogeneity test were estimated using the Arlequin software v. 3.01. Odds ratio (OR) was calculated to compare cases and control women. Consistent associations across other studies in women with CC and infected by HPV16 were observed for HLA-DRB1*15 (OR, 3.9; 95% CI, 1.6-10.2) and the haplotype DRB1*15 DQB1*0602 (OR, 4.1; 95% CI, 1.4-12.7) compared with control women. The HLA-A2-B44-DR4-DQ*0302, HLA-A24-B35-DR16-DQ*0301, and HLA-A2-B40-DR4-DQ*0302 haplotypes showed a positive association with CC (OR, >1), whereas HLA-A2-B39-DR4-DQ*0302, HLA-A24-B35-DR4-DQ*0302, and HLA-A68-B40-DR4-DQ*0302 showed a negative association (OR, <1). These results support the hypothesis that some HLA class I and II haplotypes could be involved with susceptibility for developing CC.

Low density DNA microarray for detection of most frequent TP53 missense point mutations.

BACKGROUND: We have developed an oligonucleotide microarray (genosensor) utilizing a double tandem hybridization technique to search for 9 point mutations located in the most frequently altered codons of the TP53 gene. Isolated and multiplexed PCR products, 108 and 92 bp long, from exons 7 and 8, respectively, were obtained from 24 different samples. Single-stranded target DNA was then prepared from isolated or multiplexed PCR products, through cyclic DNA synthesis. Independent ssDNA's were annealed with the corresponding pairs of labeled stacking oligonucleotides to create partially duplex DNA having a 7-nt gap, which contains the sequence that will be interrogated by the capture probes forming double tandem hybridization. In the case of multiplexed ssPCR products, only two stacking oligonucleotides were added per target, therefore the gap for the PCR products having two consecutive codons to be interrogated in exon 7 was 12 nt long, so only single tandem hybridization was produced with these respective probes. RESULTS: 18 codon substitutions were found by DNA sequencing. In 13 of them a perfect correlation with the pattern of hybridization was seen (In 5 no signal was seen with the wt probe while a new signal was seen with the appropriate mutant probe, and in 8 more, as expected, no signal was seen with any probe due to the absence of the corresponding probe in the array). In 3 other cases a mutation was falsely suggested by the combination of the absence of the wild type signal along with a false signal in the other probe. In the other 2 cases the presence of the mutation was not detected due to the production of a false hybridization signal with the wild type probe. In both cases (false mutation or no mutation detected) relatively stable mismatched target/probe duplexes should be formed. These problems could be avoided by the addition of probes to improve the performance of the array. CONCLUSION: Our results demonstrate that a simple TP53 microarray employing short (7-mer) probes, used in combination with single or double tandem hybridization approach and a simple or multiplex target preparation method, can identify common TP53 missense mutations from a variety of DNA sources with good specificity.

Detection of mutations in RET proto-oncogene codon 634 through double tandem hybridization.

We developed a procedure to detect the 7 point mutations at Cys634 of the proto-oncogene RET, which is responsible for medullary thyroid carcinoma (MTC). Genomic DNA was prepared from blood samples obtained from normal and MTC-affected individuals belonging to a family with a history of the disease. The RET genotype for each individual was first established by performing restriction and sequencing analyses. Single-stranded target DNA was prepared by asymmetric polymerase chain reaction (PCR) amplification of a 93-bp fragment containing Cys634. The target was annealed with pairs of prelabeled stacking oligonucleotides designed to create appropriate 7-nucleotide gaps, which served as the sites of subsequent hybridization with glass-immobilized 7-mer probes. The target-stacking oligonucleotide duplexes were hybridized with DNA chips containing a set of eight 7-mer probes designed to detect the wild-type sequence and the seven point mutations described. We tested two sets of immobilized probes containing internal or 5'-terminal codon-634 single-base variations. Both groups of probes were able to discriminatively identify the mutations. The hybridization patterns indicated that the disease in this family was due to the C634Y mutation, in accord with the original sequence analysis. The hybridization-based mutation assignment was additionally supported by determination of the control homozygous and heterozygous hybridization patterns produced with synthetic targets having the normal or codon 634 mutant sequences. The effects of mismatch type and nearest-neighbor sequences on the occurrence of false-positive (mismatched) hybridizations are discussed.

Association between high-risk human papillomavirus DNA load and precursor lesions of cervical cancer in Mexican women.

OBJECTIVE: Our objective was to determine the association between viral load of high risk human papilloma virus (HPV) using the Hybrid Capture II (HC II) system and cervical intraepithelial neoplasia (CIN) lesion stage. METHODS: A total of 182 consecutive women with confirmed diagnoses of CIN 1-3 and 182 healthy women with negative Pap were included. All subjects underwent structured interviews focused on socioeconomic and reproductive factors. HC II testing was used to detect human papilloma virus (HPV) DNA. Viral load was measured by light measurements expressed as relative lights unit (RLU) ratio (specimens/control). Log(10)RLU ratios were categorized for analysis into four groups: negative (

Diagnóstico molecular de la neoplasia endocrina múltiple tipo 2B en un caso de novo con la mutación 918 en el oncogen ret / Molecular diagnosis of de novo 918 mutation of ret oncogne in a case of multiple endocrine neoplasia type 2B

Introducción. La neoplasia endocrina múltiple tipo 2B (NEM2B) es un síndrome con carácter dominante hereditario que se caracteriza por el desarrollo de diversas neoplasias de origen neuroendocrino en distintos órganos, tales como carcinoma medular de tiroides (CMT), feocromocitomas, neuromas mucosales, ganglioneuromas del aparato gastrointestinal, también se observan anormalidades esqueléticas y oftálmicas. En más de 95 por ciento de los casos, este padecimiento se asocia con una mutación puntual específica en el dominio tirosina cinasa del proto-oncogen ret, en el codón 918 (METÕTHR), la cual surge de novo en 50 por ciento de los pacientes. Material y métodos. El probando fue un paciente masculino de 19 años de edad sin antecedentes de importancia para la enfermedad y que inició su padecimiento a los 5 años con neuromas submucosos en lengua y labios, así como habitus marfanoide que se acentuó a los 19 años. Determinándose la presencia de la mutación mencionada anteriormente en el DNA de leucocitos de sangre periférica y de carcinoma medular de tiroides de este paciente afectado por NEM2B y se realizó la búsqueda de la misma en leucocitos de sus familiares. Resultados. Los elevados niveles séricos de calcitonina basal (600 pg/mL) sugirieron, además del aspecto clínico y evolución, que el paciente era portador de NEM2B. El estudio histopatológico de tiroides reveló la presencia de CTM clásico. Al estudio del DNA de células de sangre periférica se observó una banda extra sugiriendo que contenía una mutación. Se confirmó la presencia de la mutación ATGÕACG en el codón 918. Conclusión. Al no encontrarse la mutación en los familiares del paciente sugiere que ésta surgió de novo en etapas tempranas del desarrollo embrionario.