An aspartate residue in the external vestibule of glycine transporter 2 (GLYT2) controls cation access and transport coupling.

Synaptic glycine levels are controlled by GLYTs (glycine transporters). GLYT1 is the main regulator of synaptic glycine concentrations and catalyses Na+-Cl--glycine co-transport with a 2:1:1 stoichiometry. In contrast, neuronal GLYT2 supplies glycine to the presynaptic terminal with a 3:1:1 stoichiometry. We subjected homology models of GLYT1 and GLYT2 to molecular dynamics simulations in the presence of Na+. Using molecular interaction potential maps and in silico mutagenesis, we identified a conserved region in the GLYT2 external vestibule likely to be involved in Na+ interactions. Replacement of Asp471 in this region reduced Na+ affinity and Na+ co-operativity of transport, an effect not produced in the homologous position (Asp295) in GLYT1. Unlike the GLYT1-Asp295 mutation, this Asp471 mutant increased sodium leakage and non-stoichiometric uncoupled ion movements through GLYT2, as determined by simultaneously measuring current and [3H]glycine accumulation. The homologous Asp471 and Asp295 positions exhibited distinct cation-sensitive external accessibility, and they were involved in Na+ and Li+-induced conformational changes. Although these two cations had opposite effects on GLYT1, they had comparable effects on accessibility in GLYT2, explaining the inhibitory and stimulatory responses to lithium exhibited by the two transporters. On the basis of these findings, we propose a role for Asp471 in controlling cation access to GLYT2 Na+ sites, ion coupling during transport and the subsequent conformational changes

Diabetes mellitus tipo 2 y toxicidad por quimioterapia en adultos mayores con cáncer prostático

Introduction: Prostate cancer is considered a predominant type of neoplasia and aging is a factor for chemotherapeutic toxicity, which can increase due to chronic diseases, particularly diabetes. Despite all this knowledge, there are no studies to evaluate the association between diabetes and the risk of chemotherapeutic toxicity in these patients.Objective: To determine the association between Type 2 Diabetes Mellitus and the risk of chemotherapy toxicity in in older adults with prostate cancer in the Geriatric Service of the Peruvian Naval Medical Center.Material and methods: Analytical retrospective cohort study and secondary database analysis.  The adverse effects of chemotherapy and the associated factors of 161 retired sailors with prostate cancer were evaluated between 2013 and 2015. Cox Regression Model for Adjusted Toxicity was constructed for antecedents of diabetes, age, pathological antecedents, smoking antecedents, calf circumference, physical activity, dependence on ABVD, falls, polypharmacy, fragility, and vulnerability.Results: The 23.6% of patients had diabetes. The prevalence of fragility was 39.7% and the one of vulnerability was 24.2% (G8) and 26.71% (VES-13). The frequent adverse effects were: gastrointestinal (13.04%) and hematological (8.07%). The most significant associations by adjusted regression model were the antecedent of Type 2 Diabetes Mellitus, 3 or more pathological antecedents, smoking antecedents, calf circumference, physical activity, dependence on ABVD, falls, polypharmacy, fragility, and vulnerability.Conclusions: The antecedent of Type 2 Diabetes Mellitus is a predictive factor for the risk of chemotherapy toxicity in older adults with prostate cancer.Keywords: Diabetes Mellitus, toxicity, chemotherapy, older adults, cancer, prostate, cohort.Introducción: Siendo el cáncer prostático una neoplasia prevalente, el envejecimiento es un factor para la toxicidad quimioterapéutica, adicionalmente puede incrementarse por enfermedades crónicas, destacando la diabetes. A pesar de estos conocimientos, no hay estudios que evalúen la asociación entre la diabetes y el riesgo de toxicidad quimioterapéutica en estos pacientes. Objetivo: Determinar la asociación entre diabetes mellitus tipo 2 y riesgo de toxicidad por quimioterapia en adultos mayores con cáncer prostático del Servicio de Geriatría del Centro Médico Naval del Perú.Material y métodos: Estudio analítico de cohorte retrospectiva, análisis secundario de una base de datos. Se evaluaron los efectos adversos de quimioterapia y factores asociados de 161 marinos retirados con cáncer prostático entre 2013 y 2015. Se construyó un modelo de regresión de Cox sobre la toxicidad ajustado por el antecedente de diabetes, edad, antecedentes patológicos, antecedentes de consumo de tabaco, circunferencia de pantorrilla, actividad física, dependencia para ABVD, caídas, polifarmacia, fragilidad y vulnerabilidad.Resultados: El 23.6% presentó diabetes. La prevalencia de fragilidad fue del 39.7% y de vulnerabilidad, del 24.2% (G8) y 26.71% (VES-13). Los efectos adversos frecuentes fueron gastrointestinales (13.04%) y hematológicos (8.07%). Mediante el modelo de regresión ajustado, el antecedente de diabetes mellitus tipo 2, 3 o más antecedentes patológicos, antecedente de consumo de tabaco, circunferencia de pantorrilla, actividad física, dependencia de ABVD, caídas, polifarmacia, vulnerabilidad y fragilidad presentaron asociación significativa.Conclusiones: El antecedente de diabetes mellitus tipo 2 es un factor predictivo para el riesgo de toxicidad por quimioterapia en adultos mayores con cáncer prostático

Inter-laboratory variation in DNA damage using a standard comet assay protocol.

There are substantial inter-laboratory variations in the levels of DNA damage measured by the comet assay. The aim of this study was to investigate whether adherence to a standard comet assay protocol would reduce inter-laboratory variation in reported values of DNA damage. Fourteen laboratories determined the baseline level of DNA strand breaks (SBs)/alkaline labile sites and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites in coded samples of mononuclear blood cells (MNBCs) from healthy volunteers. There were technical problems in seven laboratories in adopting the standard protocol, which were not related to the level of experience. Therefore, the inter-laboratory variation in DNA damage was only analysed using the results from laboratories that had obtained complete data with the standard comet assay protocol. This analysis showed that the differences between reported levels of DNA SBs/alkaline labile sites in MNBCs were not reduced by applying the standard assay protocol as compared with the laboratory's own protocol. There was large inter-laboratory variation in FPG-sensitive sites by the laboratory-specific protocol and the variation was reduced when the samples were analysed by the standard protocol. The SBs and FPG-sensitive sites were measured in the same experiment, indicating that the large spread in the latter lesions was the main reason for the reduced inter-laboratory variation. However, it remains worrying that half of the participating laboratories obtained poor results using the standard procedure. This study indicates that future comet assay validation trials should take steps to evaluate the implementation of standard procedures in participating laboratories

A novel dominant hyperekplexia mutation Y705C alters trafficking and biochemical properties of the presynaptic glycine transporter GlyT2

Hyperekplexia or startle disease is characterized by an exaggerated startle response, evoked by tactile or auditory stimuli, producing hypertonia and apnea episodes. Although rare, this orphan disorder can have serious consequences, including sudden infant death. Dominant and recessive mutations in the human glycine receptor (GlyR) α1 gene (GLRA1) are the major cause of this disorder. However, recessive mutations in the presynaptic Na+/Cl−-dependent glycine transporter GlyT2 gene (SLC6A5) are rapidly emerging as a second major cause of startle disease. In this study, systematic DNA sequencing of SLC6A5 revealed a new dominant GlyT2 mutation: pY705C (c.2114A→G) in transmembrane domain 11, in eight individuals from Spain and the United Kingdom. Curiously, individuals harboring this mutation show significant variation in clinical presentation. In addition to classical hyperekplexia symptoms, some individuals had abnormal respiration, facial dysmorphism, delayed motor development, or intellectual disability. We functionally characterized this mutation using molecular modeling, electrophysiology, [3H]glycine transport, cell surface expression, and cysteine labeling assays. We found that the introduced cysteine interacts with the cysteine pair Cys-311–Cys-320 in the second external loop of GlyT2. This interaction impairs transporter maturation through the secretory pathway, reduces surface expression, and inhibits transport function. Additionally, Y705C presents altered H+ and Zn2+ dependence of glycine transport that may affect the function of glycinergic neurotransmission in vivo

An ECVAG inter-laboratory validation study of the comet assay: inter-laboratory and intra-laboratory variations of DNA strand breaks and FPG-sensitive sites in human mononuclear cells

The alkaline comet assay is an established, sensitive method extensively used in biomonitoring studies. This method can be modified to measure a range of different types of DNA damage. However, considerable differences in the protocols used by different research groups affect the inter-laboratory comparisons of results. The aim of this study was to assess the inter-laboratory, intra-laboratory, sample and residual (unexplained) variations in DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites measured by the comet assay by using a balanced Latin square design. Fourteen participating laboratories used their own comet assay protocols to measure the level of DNA strand breaks and FPG-sensitive sites in coded samples containing peripheral blood mononuclear cells (PBMC) and the level of DNA strand breaks in coded calibration curve samples (cells exposed to different doses of ionising radiation) on three different days of analysis. Eleven laboratories found doseresponse relationships in the coded calibration curve samples on two or three days of analysis, whereas three laboratories had technical problems in their assay. In the coded calibration curve samples, the dose of ionising radiation, inter-laboratory variation, intra-laboratory variation and residual variation contributed to 60.9, 19.4, 0.1 and 19.5%, respectively, of the total variation. In the coded PBMC samples, the inter-laboratory variation explained the largest fraction of the overall variation of DNA strand breaks (79.2%) and the residual variation (19.9%) was much larger than the intra-laboratory (0.3%) and inter-subject (0.5%) variation. The same partitioning of the overall variation of FPG-sensitive sites in the PBMC samples indicated that the inter-laboratory variation was the strongest contributor (56.7%), whereas the residual (42.9%), intra-laboratory (0.2%) and inter-subject (0.3%) variations again contributed less to the overall variation. The results suggest that the variation in DNA damage, measured by comet assay, in PBMC from healthy subjects is assay variation rather than variation between subjects.</p