Gaining Insights into Inherited Bleeding Disorders of Complex Etiology in Pediatric Patients: Whole-Exome Sequencing as First-Line Investigation Tool.

INTRODUCTION: Investigation of the molecular basis of inherited bleeding disorders (IBD) is mostly performed with gene panel sequencing. However, the continuous discovery of new related genes underlies the limitation of this approach. This study aimed to identify genetic variants responsible for IBD in pediatric patients using whole-exome sequencing (WES), and to provide a detailed description and reclassification of candidate variants. MATERIAL AND METHODS: WES was performed for 18 pediatric patients, and variants were filtered using a first-line list of 290 genes. Variant prioritization was discussed in a multidisciplinary team based on genotype-phenotype correlation, and segregation studies were performed with available family members. RESULTS: The study identified 22 candidate variants in 17 out of 18 patients (94%). Eleven patients had complete genotype-phenotype correlation, resulting in a diagnostic yield of 61%, 5 (28%) were classified as partially solved, and 2 (11%) remained unsolved. Variants were identified in platelet (ACTN1, ANKRD26, CYCS, GATA1, GFI1B, ITGA2, NBEAL2, RUNX1, SRC, TUBB1), bleeding (APOLD1), and coagulation (F7, F8, F11, VWF) genes. Notably, 9 out of 22 (41%) variants were previously unreported. Variant pathogenicity was assessed according to the American College of Medical Genetics and Genomics guidelines and reclassification of three variants based on family segregation evidence, resulting in the identification of 10 pathogenic or likely pathogenic variants, 6 variants of uncertain significance, and 6 benign or likely benign variants. CONCLUSION: This study demonstrated the high potential of WES in identifying rare molecular defects causing IBD in pediatric patients, improving their management, prognosis, and treatment, particularly for patients at risk of malignancy and/or bleeding due to invasive procedures.

DPPA3-HIF1α axis controls colorectal cancer chemoresistance by imposing a slow cell-cycle phenotype.

Tumor relapse is linked to rapid chemoresistance and represents a bottleneck for cancer therapy success. Engagement of a reduced proliferation state is a non-mutational mechanism exploited by cancer cells to bypass therapy-induced cell death. Through combining functional pulse-chase experiments in engineered cells and transcriptomic analyses, we identify DPPA3 as a master regulator of slow-cycling and chemoresistant phenotype in colorectal cancer (CRC). We find a vicious DPPA3-HIF1α feedback loop that downregulates FOXM1 expression via DNA methylation, thereby delaying cell-cycle progression. Moreover, downregulation of HIF1α partially restores a chemosensitive proliferative phenotype in DPPA3-overexpressing cancer cells. In cohorts of CRC patient samples, DPPA3 overexpression acts as a predictive biomarker of chemotherapeutic resistance that subsequently requires reduction in its expression to allow metastatic outgrowth. Our work demonstrates that slow-cycling cancer cells exploit a DPPA3/HIF1α axis to support tumor persistence under therapeutic stress and provides insights on the molecular regulation of disease progression.

Presence of human papillomavirus in the buccal mucosa of blood donors.

OBJECTIVE: To determine the presence of human papillomavirus (HPV) in the oral mucosa of blood donors (BD) and risk factors associated with HPV and oral cancer. MATERIALS AND METHODS: Prospective cross-sectional study, population matched to BD from the National Cancer Institute, Mexico for HPV identification in oral cytological samples using the CLART® Human Papillomavirus 2 Kit (35 genotypes) and risk factors. RESULTS: Of 352 BD with signed informed consent, 285 were selected by simple randomization. The prevalence of oral HPV was 17.5% (95% CI 13-21.9%), the genotype was identified in 13 cases, with a total of 16 genotypes (10 high-risk), the most common being 16 and 84. Five cases had multiple infections, three with at least one high-risk type. Associations were found for marital status (OR 3.3) and educational level (OR-1.9). CONCLUSIONS: The percentage of HPV-positive cases in blood donors with no risk practices was similar to that found in Spanish-speaking population studies in which at least one risk practice was described. The presence of other genotypes with high oncogenic risk and multitype infection, described as a marker of persistence of HPV infection, is highlighted.

Functional patient-derived organoid screenings identify MCLA-158 as a therapeutic EGFR × LGR5 bispecific antibody with efficacy in epithelial tumors.

Patient-derived organoids (PDOs) recapitulate tumor architecture, contain cancer stem cells and have predictive value supporting personalized medicine. Here we describe a large-scale functional screen of dual-targeting bispecific antibodies (bAbs) on a heterogeneous colorectal cancer PDO biobank and paired healthy colonic mucosa samples. More than 500 therapeutic bAbs generated against Wingless-related integration site (WNT) and receptor tyrosine kinase (RTK) targets were functionally evaluated by high-content imaging to capture the complexity of PDO responses. Our drug discovery strategy resulted in the generation of MCLA-158, a bAb that specifically triggers epidermal growth factor receptor degradation in leucine-rich repeat-containing G-protein-coupled receptor 5-positive (LGR5+) cancer stem cells but shows minimal toxicity toward healthy LGR5+ colon stem cells. MCLA-158 exhibits therapeutic properties such as growth inhibition of KRAS-mutant colorectal cancers, blockade of metastasis initiation and suppression of tumor outgrowth in preclinical models for several epithelial cancer types.

Validación de un instrumento para evaluar consejería adolescente diferenciada en un centro de salud / Validation of an instrument for assessing adolescent counseling differentiated in a health center

RESUMEN La OMS/OPS admite que los patrones de conducta sexuales de los adolescentes y jóvenes han cambiado. Se afirma la consejería adolescente ha mostrado buenos resultados. Por ello el propósito de este estudio es evaluar un cuestionario que evaluará este tipo de intervención. Objetivos Establecer la validez de constructo y la confiabilidad de un instrumento para evaluar consejería en adolescentes asistentes al centro de salud Rucahueche. Métodos Estudio exploratorio con una muestra de 151 adolescentes entre 15 y 19 años, a los que se les aplicó un cuestionario autoadministrado. La validez de constructo y de consistencia interna fue evaluada por análisis factorial y el Alfa de Cronbach, respectivamente. Resultados El análisis factorial identificó en 3 dimensiones 10 constructos, los cuales explicaron el 62% - 67% de su variabilidad. A su vez, el análisis de consistencia interna obtuvo una puntuación de alfa de 0,934. Conclusiones El instrumento mostró evidencias de validez de constructo y de confiabilidad. Dichos análisis indican la factibilidad de aplicación del instrumento. Sin embargo, la validación del instrumento aún es un reto, debido a su relevancia para una mirada más profunda sobre la atención y la gestión en la atención de adolescentes. Por tanto, se considerará seguir haciendo estudios para explorar su dimensionalidad y validez de contenido.

ABSTRACT The WHO/PAHO admits that the sexual behavior patterns of adolescents and young people have changed. It is claimed adolescent counseling has shown good results. Therefore, the purpose of this study is to evaluate a questionnaire that will evaluate this type of intervention. Objectives To establish the construct validity and reliability of an instrument for assessing adolescent counseling attending the health center Rucahueche. Methods An exploratory study with a sample of 151 adolescents aged 15 to 19 years, who were applied a self-administered questionnaire. Construct validity and internal consistency was assessed by factor analysis and Cronbach's alpha, respectively. Results: Factor analysis identified three dimensions 10 constructs, which accounted for 62% -67% of its variability. In turn, analysis of internal consistency score was alpha of 0.934. Conclusions The instrument showed evidence of construct validity and reliability. These analyzes indícate the feasibility of the instrument. However, validation of the instrument is still a challenge, because of its relevance to a deeper look on the care and management in adolescent care. Therefore, it is deemed to continue doing studies to explore the dimensionality and content validity.

TET2 controls chemoresistant slow-cycling cancer cell survival and tumor recurrence.

Dormant or slow-cycling tumor cells can form a residual chemoresistant reservoir responsible for relapse in patients, years after curative surgery and adjuvant therapy. We have adapted the pulse-chase expression of H2BeGFP for labeling and isolating slow-cycling cancer cells (SCCCs). SCCCs showed cancer initiation potential and enhanced chemoresistance. Cells at this slow-cycling status presented a distinctive nongenetic and cell-autonomous gene expression profile shared across different tumor types. We identified TET2 epigenetic enzyme as a key factor controlling SCCC numbers, survival, and tumor recurrence. 5-Hydroxymethylcytosine (5hmC), generated by TET2 enzymatic activity, labeled the SCCC genome in carcinomas and was a predictive biomarker of relapse and survival in cancer patients. We have shown the enhanced chemoresistance of SCCCs and revealed 5hmC as a biomarker for their clinical identification and TET2 as a potential drug target for SCCC elimination that could extend patients' survival.

Colorectal Cancer Consensus Molecular Subtypes Translated to Preclinical Models Uncover Potentially Targetable Cancer Cell Dependencies.

Purpose: Response to standard oncologic treatment is limited in colorectal cancer. The gene expression-based consensus molecular subtypes (CMS) provide a new paradigm for stratified treatment and drug repurposing; however, drug discovery is currently limited by the lack of translation of CMS to preclinical models.Experimental Design: We analyzed CMS in primary colorectal cancers, cell lines, and patient-derived xenografts (PDX). For classification of preclinical models, we developed an optimized classifier enriched for cancer cell-intrinsic gene expression signals, and performed high-throughput in vitro drug screening (n = 459 drugs) to analyze subtype-specific drug sensitivities.Results: The distinct molecular and clinicopathologic characteristics of each CMS group were validated in a single-hospital series of 409 primary colorectal cancers. The new, cancer cell-adapted classifier was found to perform well in primary tumors, and applied to a panel of 148 cell lines and 32 PDXs, these colorectal cancer models were shown to recapitulate the biology of the CMS groups. Drug screening of 33 cell lines demonstrated subtype-dependent response profiles, confirming strong response to EGFR and HER2 inhibitors in the CMS2 epithelial/canonical group, and revealing strong sensitivity to HSP90 inhibitors in cells with the CMS1 microsatellite instability/immune and CMS4 mesenchymal phenotypes. This association was validated in vitro in additional CMS-predicted cell lines. Combination treatment with 5-fluorouracil and luminespib showed potential to alleviate chemoresistance in a CMS4 PDX model, an effect not seen in a chemosensitive CMS2 PDX model.Conclusions: We provide translation of CMS classification to preclinical models and uncover a potential for targeted treatment repurposing in the chemoresistant CMS4 group. Clin Cancer Res; 24(4); 794-806. ©2017 AACR.

p38α regulates actin cytoskeleton and cytokinesis in hepatocytes during development and aging.

BACKGROUND: Hepatocyte poliploidization is an age-dependent process, being cytokinesis failure the main mechanism of polyploid hepatocyte formation. Our aim was to study the role of p38α MAPK in the regulation of actin cytoskeleton and cytokinesis in hepatocytes during development and aging. METHODS: Wild type and p38α liver-specific knock out mice at different ages (after weaning, adults and old) were used. RESULTS: We show that p38α MAPK deficiency induces actin disassembly upon aging and also cytokinesis failure leading to enhanced binucleation. Although the steady state levels of cyclin D1 in wild type and p38α knock out old livers remained unaffected, cyclin B1- a marker for G2/M transition- was significantly overexpressed in p38α knock out mice. Our findings suggest that hepatocytes do enter into S phase but they do not complete cell division upon p38α deficiency leading to cytokinesis failure and binucleation. Moreover, old liver-specific p38α MAPK knock out mice exhibited reduced F-actin polymerization and a dramatic loss of actin cytoskeleton. This was associated with abnormal hyperactivation of RhoA and Cdc42 GTPases. Long-term p38α deficiency drives to inactivation of HSP27, which seems to account for the impairment in actin cytoskeleton as Hsp27-silencing decreased the number and length of actin filaments in isolated hepatocytes. CONCLUSIONS: p38α MAPK is essential for actin dynamics with age in hepatocytes.

Efectividad de intervención preventiva y de limitación del daño por caries dental en la primera molar permanente en escolares de 7 a 8 años / Effectiveness of preventive intervention and limitation of damage due to dental caries in the first permanent molar in schoolchildren aged 7 to 8 years

Objetivo: Establecer la efectividad de intervención preventiva y limitación del daño por caries dental en la primera molar permanente, en escolares de 7 a 8 años; posterior a 3 y 6 meses de aplicada. Metodología: Esta investigación fue de tipo intervención cuasi experimental, dirigida a la prevención y limitación del daño por caries en las Primeras Molares Permanentes (PMP) de 150 escolares de centros educativos públicos. Fueron atendidas 568 PMP en total, en las que se aplicó 314 sellantes de fosas y fisuras preventivos, 124 sellantes curativos, 36 remineralizaciones con flúor barniz y 130 obturaciones. Posterior a 3 y 6 meses se efectuó la evaluación de caries y de la condición de los tratamientos (sellantes y obturaciones) para medir la efectividad. La prueba estadística utilizada para el análisis de los datos fue la de McNemar. Resultados: Posterior a 3 y 6 meses de aplicada la intervención, se encontraron respectivamente el 96.8% y 94.2% de molares sanas; en tanto que, en cuanto a supervivencia de tratamientos se encontró de 88% y 72.9% respectivamente. Conclusión: La intervención mostró a 3 y 6 meses, ser efectiva para prevenir y limitar el daño por caries dental en la primera molar permanente.

Objective: To establish the effectiveness of preventive intervention and limitation of damage by caries, in the first permanent molar (FPM), in scholar children from 7 to 8 years; after 3 and 6 months of being applied. Methodology: This research was a quasi-experimental dental intervention type, directed to the prevention and limitation of damage by caries in the first permanent molars of 150 scholar children, attending public schools. In total, 568 FPM were intervened; in which 314 preventive sealing of tooth cavities and fissures, were applied, as well as 124 healing sealants, 36 remineralization with varnish fluoride and 130 obturations (fillings). Afterwards to 3 and 6 months, the evaluation of caries and the condition of the treatments (sealants and obturations) was developed, to measure their effectiveness. The statistical test used for the analysis of the data was that of McNemar. Results: Afterwards to 3 and 6 months the dental intervention was applied; 96.8% and 94.2% of healthy molars was obtained, respectively; and, 88%; 72.9% permanence (survival) of treatments respectively. Conclusion: The intervention showed effectiveness to prevent and limit the damage by dental caries in first permanent molar; after 3 and 6 months of being done.

Evidence-based practice: beliefs, attitudes, knowledge, and skills among Colombian physical therapists / Práctica basada en la evidencia: creencias, actitudes, conocimientos y habilidades entre fisioterapeutas colombianos

Objective: The main purpose of this study was to describe a group of Colombian physical therapists' beliefs and attitudes towards Evidence-Based Practice (EBP), their education, knowledge and skills for implementing EBP, the use of relevant literature in clinical practice, access to and availability of scientific information and perceived barriers to including EBP in practice. Methods: This was a cross-sectional study which involved 1,064 Colombian physical therapists. The study used a 50-item screening questionnaire EBP developed to estimate attitudes, beliefs, knowledge and skills regarding. This instrument has been adapted and was validated previously in Colombia by Flórez-López et al. Results: The population mostly consisted of young females (77.2%) aged 22 to 29 years old (79.4%). Most respondents had an undergraduate degree (87.7%). The physical therapists stated that they had positive attitudes and beliefs regarding EBP, most of them answering that they agreed or strongly agreed that EBP is necessary (71.6%), the relevant literature is useful for practice (61.3%), EBP improves the quality of patient care (64.1%) and evidence helps in decision-making (44.5%). Forty-one percent of the respondents indicated that a lack of research skills was the most important barrier to the use of evidence in practice. Conclusion: The physical therapists reported that they had a positive attitude to EBP and were interested in learning about or improving the skills necessary to adopt EBP in their clinical practice.

Objetivo: Describir en un grupo colombiano de fisioterapeutas las i) creencias y actitudes hacia la practica basada en la evidencia (PBE), ii) la educación, el conocimiento y las habilidades para implementar la PBE; iii) el uso de la literatura relevante en la práctica clínica; iv) el acceso y la disponibilidad de información científica; y v) la percepción de las barreras para la inclusión de la PBE. Métodos: Se realizó un estudio transversal en 1,064 fisioterapeutas colombianos. El estudio usó el cuestionario que consta de 50-ítems para estimar las actitudes, creencias, conocimientos y habilidades hacia la PBE. Dicho instrumento fue adaptado y validado en Colombia por Flórez-López et al. Resultados: La mayoría de la población participante fueron mujeres (77.2%) en edades comprendidas entre 22 y 29 años (79.4%). El 87.7% de los encuestados eran titulados en fisioterapia. Los fisioterapeutas manifestaron tener actitudes y creencias positivas hacia la PBE. Una mayoría respondió que estaba de acuerdo o muy de acuerdo en que es necesaria la PBE (71.6%), en que la literatura es útil para la práctica clínica (61.3%), que la PBE mejora la calidad de la atención a los pacientes (64.1%), y en que la evidencia ayuda en la toma de decisiones clínicas (44.5%). El 41.0% de los encuestados indicaron que la falta de habilidades de investigación era la barrera más importante para el uso de la evidencia científica en la práctica clínica. Conclusiones: Los fisioterapeutas manifestaron una actitud positiva acerca la PBE y estaban interesados en aprender o mejorar las habilidades necesarias para adoptar la PBE en la práctica clínica.

Fas and FasL expression in leukocytes from chronic granulomatous disease patients.

Abstract. Chronic Granulomatous Disease (CGD) is a primary immunodeficiency characterized by defects in superoxide (O2-) production, which result from mutations in one of the four NADPH oxidase components, predisposing to bacterial and fungal infections. Besides the O2-defect, it has been described that neutrophils from CGD patients are resistant to cell death, a phenomenon that has been connected to chronic inflammation and predisposition to autoimmune diseases. A diminished expression of Fas and its counterpart FasL, molecules known to play a major role in cell death, has been described in lymphocytes depleted of O2-reactive oxygen species (ROS), suggesting an involvement of ROS in Fas/FasL expression. In this work, Fas and FasL expressions were analyzed in T cells and neutrophils from two CGD families, previously known to harbor two different molecular defects: absence of either p47-phox or p67-phox. We found that T lymphocytes from CGD patients express low levels of Fas and FasL, while a diminished FasL expression was observed on neutrophils from a CGD A470 patient. These defects may contribute to understand altered cell death in CGD patients.

Fas and FasL expression in leukocytes from Chronic Granulomatous Disease patients / Expresión de Fas y FasL en leucocitos de pacientes con Enfermedad Granulomatosa Crónica

Chronic Granulomatous Disease (CGD) is a primary immunodeficiency characterized by defects in superoxide (O2-) production, which result from mutations in one of the four NADPH oxidase components, predisposing to bacterial and fungal infections. Besides the O2-defect, it has been described that neutrophils from CGD patients are resistant to cell death, a phenomenon that has been connected to chronic inflammation and predisposition to autoimmune diseases. A diminished expression of Fas and its counterpart FasL, molecules known to play a major role in cell death, has been described in lymphocytes depleted of O2-reactive oxygen species (ROS), suggesting an involvement of ROS in Fas/FasL expression. In this work, Fas and FasL expressions were analyzed in T cells and neutrophils from two CGD families, previously known to harbor two different molecular defects: absence of either p47-phox or p67-phox. We found that T lymphocytes from CGD patients express low levels of Fas and FasL, while a diminished FasL expression was observed on neutrophils from a CGD A470 patient. These defects may contribute to understand altered cell death in CGD patients.

La Enfermedad Granulomatosa Crónica (EGC) es una inmunodeficiencia primaria caracterizada por un defecto en la producción de superóxido (O2-), que se genera como consecuencia de mutaciones en uno de los cuatro componentes del complejo NADPH oxidasa y predispone a infecciones por bacterias y hongos. Además de los defectos en la producción de O2-, se ha descrito que los neutrófilos de los pacientes con EGC exhiben una resistencia a la muerte celular, evento que se asocia con la inflamación crónica y predisposición a enfermedades autoinmunes. Se ha descrito que linfocitos en medios desprovistos de O2-especies reactivas del oxigeno (ROS), muestran reducida expresión de Fas y FasL, moléculas que juegan un papel relevante en el control de la muerte celular, sugiriendo la participación de los ROS su regulación. En este trabajo analizamos la expresión de Fas y FasL en linfocitos T y neutrófilos en dos familias portadores de dos defectos genéticos diferentes asociados con EGC: ausencia de p47-phox o de p67-phox. Evidenciamos una baja expresión de Fas y FasL en los linfocitos T de los pacientes con EGC, pero solo los neutrófilos de los pacientes con defecto de p47-phox, fueron incapaces de expresar FasL. Estos defectos pudieran contribuir a entender la alteración de la muerte celular observada en los pacientes con EGC.