Mitochondrial DNA (mtDNA) haplogroups and serum levels of anti-oxidant enzymes in patients with osteoarthritis

BACKGROUND: Oxidative stress play a main role in the initiation and progression of the OA disease and leads to the degeneration of mitochondria. To prevent this, the chondrocytes possess a well-coordinated enzymatic antioxidant system. Besides, the mitochondrial DNA (mtDNA) haplogroups are associated with the OA disease. Thus, the main goal of this work is to assess the incidence of the mtDNA haplogroups on serum levels of two of the main antioxidant enzymes, Manganese Superoxide Dismutase (Mn-SOD or SOD2) and catalase, and to test the suitability of these two proteins for potential OA-related biomarkers. METHODS: We analyzed the serum levels of SOD2 and catalase in 73 OA patients and 77 healthy controls carrying the haplogroups J, U and H, by ELISA assay. Knee and hip radiographs were classified according to Kellgren and Lawrence (K/L) scoring from Grade 0 to Grade IV. Appropriate statistical analyses were performed to test the effects of clinical variables, including gender, body mass index (BMI), age, smoking status, diagnosis, haplogroups and radiologic K/L grade on serum levels of these enzymes. RESULTS: Serum levels of SOD2 appeared statistically increased in OA patients when compared with healthy controls (p < 0.001). Even in those OA patients with higher OA severity (K/L grade IV), the serum levels of this antioxidant enzyme appeared more significantly increased than in OA patients with lower K/L grade (p < 0.001). The mtDNA haplogroups showed an influence on serum levels of catalase (p = 0.054), being carriers of the mtDNA haplogroup J those who showed higher serum levels than non-J carriers (p = 0.057). CONCLUSIONS: The increased levels of SOD2 in OA patients indicate an increased oxidative stress OA-related, therefore this antioxidant enzyme could be a suitable candidate biomarker for diagnosis of OA. Mitochondrial haplogroups significantly correlates with serum levels of catalase

Hsp90 beta inhibition modulates nitric oxide production and nitric oxide-induced apoptosis in human chondrocytes

Background: Hsp90 beta is a member of the Hsp90 family of protein chaperones. This family plays essential roles in the folding, maturation and activity of many proteins that are involved in signal transduction and transcriptional regulation. The role of this protein in chondrocytes is not well understood, although its increase in osteoarthritic cells has been reported. The present study aimed to explore the role of Hsp90 beta in key aspects of OA pathogenesis. Methods: Human OA chondrocytes were isolated from cartilage obtained from patients undergoing joint replacement surgery, and primary cultured. Cells were stimulated with proinflammatory cytokines (IL-1 beta or TNF-alpha) and nitric oxide donors (NOC-12 or SNP). For Hsp90 beta inhibition, two different chemical inhibitors (Geldanamycin and Novobiocin) were employed, or siRNA transfection procedures were carried out. Gene expression was determined by real-time PCR, apoptosis was quantified by flow cytometry and ELISA, and nitric oxide (NO) production was evaluated by the Griess method. Indirect immunofluorescence assays were performed to evaluate the presence of Hsp90 beta in stimulated cells. Results: Hsp90 beta was found to be increased by proinflammatory cytokines. Inhibition of Hsp90 beta by the chemicals Geldanamycin (GA) and Novobiocin (NB) caused a dose-dependent decrease of the NO production induced by IL-1 beta in chondrocytes, up to basal levels. Immunofluorescence analyses demonstrate that the NO donors NOC-12 and SNP also increased Hsp90 beta. Chemical inhibition or specific gene silencing of this chaperone reduced the DNA condensation and fragmentation, typical of death by apoptosis, that is induced by NO donors in chondrocytes. Conclusions: The present results show how Hsp90 beta modulates NO production and NO-mediated cellular death in human OA chondrocytes

Sequential depletion of human serum for the search of osteoarthritis biomarkers

Background: The field of biomarker discovery, development and application has been the subject of intense interest and activity, especially with the recent emergence of new technologies, such as proteomics-based approaches. In proteomics, search for biomarkers in biological fluids such as human serum is a challenging issue, mainly due to the high dynamic range of proteins present in these types of samples. Methods for reducing the content of most highly abundant proteins have been developed, including immunodepletion or protein equalization. In this work, we report for the first time the combination of a chemical sequential depletion method based in two protein precipitations with acetonitrile and DTT, with a subsequent two-dimensional difference in-gel electrophoresis (2D-DIGE) analysis for the search of osteoarthritis (OA) biomarkers in human serum. The depletion method proposed is non-expensive, of easy implementation and allows fast sample throughput. Results: Following this workflow, we have compared sample pools of human serum obtained from 20 OA patients and 20 healthy controls. The DIGE study led to the identification of 16 protein forms (corresponding to 14 different proteins) that were significantly (p < 0.05) altered in OA when compared to controls (8 increased and 7 decreased). Immunoblot analyses confirmed for the first time the increase of an isoform of Haptoglobin beta chain (HPT) in sera from OA patients. Conclusions: Altogether, these data demonstrate the utility of the proposed chemical sequential depletion for the analysis of sera in protein biomarker discovery approaches, exhibit the usefulness of quantitative 2D gel-based strategies for the characterization of disease-specific patterns of protein modifications, and also provide a list of OA biomarker candidates for validation

Effects of severe hypoxia on bone marrow mesenchymal stem cells differentiation potential

Background. The interests in mesenchymal stem cells (MSCs) and their application in cell therapy have resulted in a better understanding of the basic biology of these cells. Recently hypoxia has been indicated as crucial for complete chondrogenesis. We aimed at analyzing bone marrow MSCs (BM-MSCs) differentiation capacity under normoxic and severe hypoxic culture conditions. Methods. MSCs were characterized by flow cytometry and differentiated towards adipocytes, osteoblasts, and chondrocytes under normoxic or severe hypoxic conditions. The differentiations were confirmed comparing each treated point with a control point made of cells grown in DMEM and fetal bovine serum (FBS). Results. BM-MSCs from the donors displayed only few phenotypical differences in surface antigens expressions. Analyzing marker genes expression levels of the treated cells compared to their control point for each lineage showed a good differentiation in normoxic conditions and the absence of this differentiation capacity in severe hypoxic cultures. Conclusions. In our experimental conditions, severe hypoxia affects the in vitro differentiation potential of BM-MSCs. Adipogenic, osteogenic, and chondrogenic differentiations are absent in severe hypoxic conditions. Our work underlines that severe hypoxia slows cell differentiation by means of molecular mechanisms since a decrease in the expression of adipocyte-, osteoblast-, and chondrocyte-specific genes was observed

Differences in performance indicator between winning and losing team in formative stages according to sex and final score differences

En este estudio se analizó la competición infantil de balonmano en función del género y la diferencia final de goles, identificando los indicadores de rendimiento que diferencian a los equipos ganadores de los perdedores en el Campeonato de España de Selecciones Autonómicas 2011. La muestra fue de 47 partidos en la competición masculina y de 45 en la femenina. El análisis se llevó a cabo mediante el SPSS 17.0 para Windows. Se realizó un clúster para agrupar los partidos en función del resultado final, y se aplicó la prueba t para muestras independientes o la U de Mann-Whitney según los resultados de las pruebas de normalidad, aleatoriedad y homocedasticidad. Los resultados reflejan que, en ambas competiciones, los equipos ganadores obtienen un mayor número de goles desde 6 m., un menor número de errores pase-recepción, al igual que poseen mejores valores en CEO (Coeficiente de Eficacia Ofensiva) y CRO (Coeficiente de Resolución ofensiva). Los lanzamientos fallados y parados desde 9 m. diferencian a los equipos ganadores en competición masculina. Las diferencias en femenino son las posesiones, los goles de 7 m y los pasos Los resultados sugieren la importancia de la condición fisca y la técnica individual en edades de formación.The aim of this study was to analyze an u´14 handball competition in function of gender and final score differences, trying to identify performance indicators that differentiate winning teams from losing team in the 2011 Spanish Championship An amount of 47 masculine and 45 feminine matches from the 2011 Spanish u´14 Championship were analyzed. The analysis was developed with specific software SPSS 17.0 for Windows. A cluster analysis was carried out in function of the final score differences, as well as a t test for independent samples or a Mann- Whitney U in relation of normality, random and homoscedasticity tests. Results show that, in both competitions, winning teams reach more goals from 6 m line, fewer errors in pass-reception, as well as better values of Offensive Efficacy Coefficient (OEC) and Offensive Realization Coefficcient (ORC) versus looser teams. Failed and stopped shoots from free throw line differentiate winner teams is male competitions. The amount of possession, penalty goals and steps differentiate winner teams from looser ones in female competitions. Results highlight how important are the physical condition and the individual technic for this ages.peerReviewe

Differences in performance indicator between winning and losing team in formative stages according to sex and final score differences

En este estudio se analizó la competición infantil de balonmano en función del género y la diferencia final de goles, identificando los indicadores de rendimiento que diferencian a los equipos ganadores de los perdedores en el Campeonato de España de Selecciones Autonómicas 2011. La muestra fue de 47 partidos en la competición masculina y de 45 en la femenina. El análisis se llevó a cabo mediante el SPSS 17.0 para Windows. Se realizó un clúster para agrupar los partidos en función del resultado final, y se aplicó la prueba t para muestras independientes o la U de Mann-Whitney según los resultados de las pruebas de normalidad, aleatoriedad y homocedasticidad. Los resultados reflejan que, en ambas competiciones, los equipos ganadores obtienen un mayor número de goles desde 6 m., un menor número de errores pase-recepción, al igual que poseen mejores valores en CEO (Coeficiente de Eficacia Ofensiva) y CRO (Coeficiente de Resolución ofensiva). Los lanzamientos fallados y parados desde 9 m. diferencian a los equipos ganadores en competición masculina. Las diferencias en femenino son las posesiones, los goles de 7 m y los pasos Los resultados sugieren la importancia de la condición fisca y la técnica individual en edades de formación.The aim of this study was to analyze an u´14 handball competition in function of gender and final score differences, trying to identify performance indicators that differentiate winning teams from losing team in the 2011 Spanish Championship An amount of 47 masculine and 45 feminine matches from the 2011 Spanish u´14 Championship were analyzed. The analysis was developed with specific software SPSS 17.0 for Windows. A cluster analysis was carried out in function of the final score differences, as well as a t test for independent samples or a Mann- Whitney U in relation of normality, random and homoscedasticity tests. Results show that, in both competitions, winning teams reach more goals from 6 m line, fewer errors in pass-reception, as well as better values of Offensive Efficacy Coefficient (OEC) and Offensive Realization Coefficcient (ORC) versus looser teams. Failed and stopped shoots from free throw line differentiate winner teams is male competitions. The amount of possession, penalty goals and steps differentiate winner teams from looser ones in female competitions. Results highlight how important are the physical condition and the individual technic for this ages.peerReviewe

A Study of the Prevalence of Asthma in the General Population in Spain

Asma; Epidemiología; PrevalenciaAsthma; Epidemilogy; PrevalenceAsma; Epidemiologia; PrevalençaIntroducción El asma es una enfermedad con elevada prevalencia, que afecta a todos los grupos de edad y genera elevados costes sociosanitarios. Estudios realizados en diversas poblaciones muestran gran variabilidad en su prevalencia, incluso en poblaciones cercanas geográficamente, con datos que sugieren una influencia relevante de factores socioeconómicos. Actualmente en población adulta de España no disponemos de datos poblacionales fiables sobre la prevalencia de esta enfermedad. Los objetivos de este estudio son estimar la prevalencia de asma en población española de 18-79 años, describir la variabilidad entre comunidades autónomas, estimar la prevalencia de infra y sobrediagnóstico, prevalencia de asma no controlada, de asma córticodependiente, conocer el consumo de recursos sanitarios, identificar los fenotipos más frecuentes y establecer un punto de partida para evaluar la tendencia temporal con estudios posteriores. Material y métodos Se realizará un estudio transversal, bietápico, incluyendo pacientes de 50 áreas sanitarias. El estudio se desarrollará en tres fases: 1) cribado y confirmación en historia clínica, en la cual se identificarán los pacientes con diagnóstico previo correctamente establecido de asma; 2) diagnóstico de asma, evaluando a los pacientes en los cuales no está claro el diagnóstico de asma con los datos disponibles en la historia clínica; 3) caracterización del asma, analizando las características de estos pacientes e identificando los fenotipos más frecuentes. Discusión Parece necesario y factible realizar un estudio epidemiológico del asma en España que permita identificar la prevalencia de asma, optimizar la planificación sanitaria, caracterizar los fenotipos más frecuentes de la enfermedad y evaluar los diagnósticos erróneos.Introduction Asthma is a disease with high prevalence, which affects all age groups and generates high health and social care costs. Studies carried out in a number of populations show great variability in its prevalence, even in geographically close populations, with data suggesting a relevant influence of socio-economic factors. At present, we do not have reliable data on the prevalence of this disease in the adult population of Spain. The objectives of this study are to estimate the prevalence of asthma in the Spanish population for those aged 18-79, to describe the variability between autonomous communities, to estimate the prevalence of under and overdiagnosis, to analyse the prevalence of uncontrolled asthma and steroid-dependent asthma, to evaluate the health care cost, to identify the most frequent phenotypes and to establish a starting point to evaluate the temporal trend with subsequent studies. Methods A cross-sectional, two-stage study will be carried out, including patients from 50 catchment areas. The study will be carried out in 3 phases: 1) screening and confirmation in the clinical history, in which patients with a previously correctly established diagnosis of asthma will be identified; 2) diagnosis of asthma to evaluate patients without a confirmed or excluded diagnosis; 3) characterization of asthma, where the characteristics of the asthmatic patients will be analysed, identifying the most frequent phenotypes. Discussion It seems necessary and feasible to carry out an epidemiological study of asthma in Spain to identify the prevalence of asthma, to optimize healthcare planning, to characterize the most frequent phenotypes of the disease, and to evaluate inaccurate diagnoses.Este trabajo ha sido financiado por el Foro Autonómico de Asma-SEPAR

A novel procedure for protein extraction from formalin-fixed paraffin-embedded tissues

Most of the archived pathological specimens in hospitals are kept as formalin-fixed paraffin-embedded tissues (FFPE) for long-term preservation. Up to now, these samples are only used for immunohistochemistry in a clinical routine as it is difficult to recover intact protein from these FFPE tissues. Here, we report a novel, short time-consuming and cost-effective method to extract full-length, non-degraded proteins from FFPE tissues. This procedure is combined with an effective and non-toxic deparaffinisation process and an extraction method based on antigen-retrieval, high concentration of SDS and high temperature. We have obtained enough intact protein to be detected by Western blotting analysis. This technique will allow utilising these stored FFPE tissues in several applications for protein analysis helping to advance the translational studies in cancer and other diseases

Impact of human-associated Escherichia coli clonal groups in Antarctic pinnipeds: presence of ST73, ST95, ST141 and ST131

There is growing concern about the spreading of human microorganisms in relatively untouched ecosystems such as the Antarctic region. For this reason, three pinniped species (Leptonychotes weddellii, Mirounga leonina and Arctocephalus gazella) from the west coast of the Antartic Peninsula were analysed for the presence of Escherichia spp. with the recovery of 158 E. coli and three E. albertii isolates. From those, 23 harboured different eae variants (α1, β1, β2, ε1, θ1, κ, ο), including a bfpA-positive isolate (O49:H10-A-ST206, eae-k) classified as typical enteropathogenic E. coli. Noteworthy, 62 of the 158 E. coli isolates (39.2%) exhibited the ExPEC status and 27 (17.1%) belonged to sequence types (ST) frequently occurring among urinary/bacteremia ExPEC clones: ST12, ST73, ST95, ST131 and ST141. We found similarities >85% within the PFGE-macrorrestriction profiles of pinniped and human clinic O2:H6-B2-ST141 and O16:H5/O25b:H4-B2-ST131 isolates. The in silico analysis of ST131 Cplx genomes from the three pinnipeds (five O25:H4-ST131/PST43-fimH22-virotype D; one O16:H5-ST131/PST506-fimH41; one O25:H4-ST6252/PST9-fimH22-virotype D1) identified IncF and IncI1 plasmids and revealed high core-genome similarities between pinniped and human isolates (H22 and H41 subclones). This is the first study to demonstrate the worrisome presence of human-associated E. coli clonal groups, including ST131, in Antarctic pinnipeds.The sampling was funded by the Spanish Ministry of Science and Innovation (CGL-2005-25073-E/ANT and CTM2008-00570) and the Sea World & Bush Gardens Conservation Fund. Work at USC-LREC was supported by projects AGL2013-47852-R from the Ministerio de Economía y Competitividad (MINECO, Spain) and Fondo Europeo de Desarrollo Regional (FEDER); AGL2016-79343-R from the Agencia Estatal de Investigación (AEI, Spain) and FEDER; PI16/01477 from Plan Estatal de I + D + I 2013-2016, Instituto de Salud Carlos III (ISCIII), Subdirección General de Evaluación y Fomento de la Investigación, and FEDER; CN2012/303 and ED431C 2017/57 from the Consellería de Cultura, Educación e Ordenación Universitaria, (Xunta de Galicia) and FEDER. We would like to thank the military personnel at the Spanish Antarctic Base “Gabriel de Castilla” for their help and assistance and the Marine Technology Unit (CSIC), the Spanish Navy’s Oceanographic Research Ship “Las Palmas” and the Unidad de Tecnología Marítima (UTM, CSIC) for logistics and transport. We also express our gratitude to J. Castro-Urda, F.T. García-Moreno, C. Rengifo-Herrera, S. Rojo-Montejo, I. Ferre, V. Navarro, M. Gómez-Bautista, T. Alvaro-Alvarez and J. Coello-Pérez for their invaluable help in the sample collection. A. Mora acknowledges the Ministerio de Educación, Cultura y Deporte (Spain) for the mobility grant for teachers and researchers from the Programa Estatal de Promoción del Talento y su Empleabilidad, Plan Estatal de Investigación Científica y Técnica y de Innovación 2013–2016.S

The Second Oncogenic Hit Determines the Cell Fate of ETV6-RUNX1 Positive Leukemia

ETV6-RUNX1 is almost exclusively associated with childhood B-cell acute lymphoblastic leukemia (B-ALL), but the consequences of ETV6-RUNX1 expression on cell lineage decisions during B-cell leukemogenesis are completely unknown. Clinically silent ETV6-RUNX1 preleukemic clones are frequently found in neonatal cord blood, but few carriers develop B-ALL as a result of secondary genetic alterations. The understanding of the mechanisms underlying the first transforming steps could greatly advance the development of non-toxic prophylactic interventions. Using genetic lineage tracing, we examined the capacity of ETV6-RUNX1 to instruct a malignant phenotype in the hematopoietic lineage by cell-specific Cre-mediated activation of ETV6-RUNX1 from the endogenous Etv6 gene locus. Here we show that, while ETV6-RUNX1 has the propensity to trigger both T- and B-lymphoid malignancies, it is the second hit that determines tumor cell identity. To instigate leukemia, both oncogenic hits must place early in the development of hematopoietic/precursor cells, not in already committed B-cells. Depending on the nature of the second hit, the resulting B-ALLs presented distinct entities that were clearly separable based on their gene expression profiles. Our findings give a novel mechanistic insight into the early steps of ETV6-RUNX1+ B-ALL development and might have major implications for the potential development of ETV6-RUNX1+ B-ALL prevention strategies