Análisis funcional de proteínas implicadas en el mantenimiento de la estabilidad cromosómica en mamíferos

[ES]Análisis funcional de las proteínas implicadas en el mantenimiento de la estabilidad cromosómica en mamíferos. La liberación secuencial de los complejos de cohesina tanto durante la mitosis y la meiosis es esencial para la segregación cromosómica precisa. Esta protección centromérica Shugoshin dependiente de la vía durante la profase mitótica y la primera división meiótica es, pues, crucial. Por otra parte, la cleaveage proteolítica por la separación en subunidades de los kleisin, que cierran estos complejos, es esencial para su disociación del ADN durante la mitosis y la meiosis. En el presente trabajo, hemos abordado la función mitótica de los mamíferos SGOL2 Shugoshin mediante la generación de un modelo de ratón deficiente en SGOL2. El análisis funcional del fenotipo del ratón y el estudio detallado de la mitosis de las células primarias, nos permitió determinar que SGOL2 es prescindible para la protección de la cohesión centromérica durante la profase de la vía y para la segregación correcta de los cromosomas durante la mitosis. Un análisis de transcriptoma de una pérdida prematura del modelo meiótica centroméricas cohesión debido a la ausencia de SGOL2, reveló que HDAC11 es uno de los genes que son regulados a la baja en los testículos. Hemos generado ratones knock-out para este histona desacetilasa, y se muestra que HDAC11 no interfiere con la progresión de la meiosis. Además, hemos determinado que HDAC11 modula la expresión de genes implicados en la transducción de los estímulos olfativos en testículos. Posteriormente, hemos abordado la búsqueda de nuevos paralogs de cohesin, que ha conducido a la identificación y caracterización bioquímica de un nuevo miembro de la familia kleisin. Esta nueva kleisin constituye un complejo específico de meiosis cohesin que colocaliza con elementos axiales del complejo sinaptonémico de meiocytes. Finalmente, se ha determinado que la separasa desempeña un papel protector contra la progresión de los tumores de piel inducidos químicamente.[EN]Functional Analysis of the Proteins Involved in Maintaining Chromosome Stability in Mammals. The sequential release of the cohesin complexes during both vertebrate mitosis and meiosis is essential for accurate chromosome segregation. This shugoshin-dependent centromeric protection during the mitotic prophase pathway and the first meiotic division is thus crucial. Moreover, the proteolytic cleaveage by Separase of the kleisin-subunits, which close these complexes, is essential for their dissociation of the DNA during mitosis and meiosis. In the present work, we have addressed the mitotic function of the mammalian shugoshin SGOL2 by generating a SGOL2-deficient mouse model. The functional analysis of the mouse phenotype and the detailed study of the mitosis of its primary cells, enabled us to determine that SGOL2 is dispensable for the protection of centromeric cohesion during the prophase pathway and for the correct segregation of chromosomes during mitosis. A transcriptomic analysis of a premature loss of meiotic centromeric cohesion model due to the absence of SGOL2, revealed that HDAC11 is one of the genes that are downregulated in testis. We have generated a constitutive knock-out mice for this histone deacetylase, and shown that HDAC11 does not interfere with meiosis progression. Moreover, we have determined that HDAC11 modulates the expression of genes involved in the transduction of olfactory stimuli on testes. Subsequently, we have addressed the search of new paralogs of cohesin, which has led to the identification and biochemical characterization of a new member of the kleisin family. This new kleisin constitutes a meiosis-specific cohesin complex that colocalizes with axial elements of the synaptonemal complex of meiocytes. Finally, we have determined that Separase plays a protective role against progression of chemically induced skin tumors

Análisis funcional de proteínas implicadas en el mantenimiento de la estabilidad cromosómica en mamíferos

Tesis Doctoral presentada por Cristina Gutiérrez Caballero, licenciada en Biología para optar al grado de Doctor por la Universidad de Salamanca.[ES]: La liberación secuencial de los complejos de cohesina tanto durante la mitosis y la meiosis es esencial para la segregación cromosómica precisa. Esta protección centromérica Shugoshin dependiente de la vía durante la profase mitótica y la primera división meiótica es, pues, crucial. Por otra parte, la cleaveage proteolítica por la separación en subunidades de los kleisin, que cierran estos complejos, es esencial para su disociación del ADN durante la mitosis y la meiosis. En el presente trabajo, hemos abordado la función mitótica de los mamíferos SGOL2 Shugoshin mediante la generación de un modelo de ratón deficiente en SGOL2. El análisis funcional del fenotipo del ratón y el estudio detallado de la mitosis de las células primarias, nos permitió determinar que SGOL2 es prescindible para la protección de la cohesión centromérica durante la profase de la vía y para la segregación correcta de los cromosomas durante la mitosis. Un análisis de transcriptoma de una pérdida prematura del modelo meiótica centroméricas cohesión debido a la ausencia de SGOL2, reveló que HDAC11 es uno de los genes que son regulados a la baja en los testículos. Hemos generado ratones knock-out para este histona desacetilasa, y se muestra que HDAC11 no interfiere con la progresión de la meiosis. Además, hemos determinado que HDAC11 modula la expresión de genes implicados en la transducción de los estímulos olfativos en testículos. Posteriormente, hemos abordado la búsqueda de nuevos paralogs de cohesin, que ha conducido a la identificación y caracterización bioquímica de un nuevo miembro de la familia kleisin. Esta nueva kleisin constituye un complejo específico de meiosis cohesin que colocaliza con elementos axiales del complejo sinaptonémico de meiocytes. Finalmente, se ha determinado que la separasa desempeña un papel protector contra la progresión de los tumores de piel inducidos químicamente[EN]: The sequential release of the cohesin complexes during both vertebrate mitosis and meiosis is essential for accurate chromosome segregation. This shugoshin-dependent centromeric protection during the mitotic prophase pathway and the first meiotic division is thus crucial. Moreover, the proteolytic cleaveage by Separase of the kleisin-subunits, which close these complexes, is essential for their dissociation of the DNA during mitosis and meiosis. In the present work, we have addressed the mitotic function of the mammalian shugoshin SGOL2 by generating a SGOL2-deficient mouse model. The functional analysis of the mouse phenotype and the detailed study of the mitosis of its primary cells, enabled us to determine that SGOL2 is dispensable for the protection of centromeric cohesion during the prophase pathway and for the correct segregation of chromosomes during mitosis. A transcriptomic analysis of a premature loss of meiotic centromeric cohesion model due to the absence of SGOL2, revealed that HDAC11 is one of the genes that are downregulated in testis. We have generated a constitutive knock-out mice for this histone deacetylase, and shown that HDAC11 does not interfere with meiosis progression. Moreover, we have determined that HDAC11 modulates the expression of genes involved in the transduction of olfactory stimuli on testes. Subsequently, we have addressed the search of new paralogs of cohesin, which has led to the identification and biochemical characterization of a new member of the kleisin family. This new kleisin constitutes a meiosis-specific cohesin complex that colocalizes with axial elements of the synaptonemal complex of meiocytes. Finally, we have determined that Separase plays a protective role against progression of chemically induced skin tumors.Peer Reviewe

Real-time human action recognition using raw depth video-based recurrent neural networks

This work proposes and compare two different approaches for real-time human action recognition (HAR) from raw depth video sequences. Both proposals are based on the convolutional long short-term memory unit, namely ConvLSTM, with differences in the architecture and the long-term learning. The former uses a video-length adaptive input data generator (stateless) whereas the latter explores the stateful ability of general recurrent neural networks but is applied in the particular case of HAR. This stateful property allows the model to accumulate discriminative patterns from previous frames without compromising computer memory. Furthermore, since the proposal uses only depth information, HAR is carried out preserving the privacy of people in the scene, since their identities can not be recognized. Both neural networks have been trained and tested using the large-scale NTU RGB+D dataset. Experimental results show that the proposed models achieve competitive recognition accuracies with lower computational cost compared with state-of-the-art methods and prove that, in the particular case of videos, the rarely-used stateful mode of recurrent neural networks significantly improves the accuracy obtained with the standard mode. The recognition accuracies obtained are 75.26% (CS) and 75.45% (CV) for the stateless model, with an average time consumption per video of 0.21 s, and 80.43% (CS) and 79.91%(CV) with 0.89 s for the stateful one.Agencia Estatal de InvestigaciónUniversidad de Alcal

3DFCNN: real-time action recognition using 3D deep neural networks with raw depth information

This work describes an end-to-end approach for real-time human action recognition from raw depth image-sequences. The proposal is based on a 3D fully convolutional neural network, named 3DFCNN, which automatically encodes spatio-temporal patterns from raw depth sequences. The described 3D-CNN allows actions classification from the spatial and temporal encoded information of depth sequences. The use of depth data ensures that action recognition is carried out protecting people"s privacy, since their identities can not be recognized from these data. The proposed 3DFCNN has been optimized to reach a good performance in terms of accuracy while working in real-time. Then, it has been evaluated and compared with other state-of-the-art systems in three widely used public datasets with different characteristics, demonstrating that 3DFCNN outperforms all the non-DNNbased state-of-the-art methods with a maximum accuracy of 83.6% and obtains results that are comparable to the DNN-based approaches, while maintaining a much lower computational cost of 1.09 seconds, what significantly increases its applicability in real-world environments.Agencia Estatal de InvestigaciónUniversidad de Alcal

Lipopolysacharide-induced neuroinflammation leads to the accumulation of ubiquitinated proteins and increases susceptibility to neurodegeneration induced by proteasome inhibition in rat hippocampus

BACKGROUND: Neuroinflammation and protein accumulation are characteristic hallmarks of both normal aging and age-related neurodegenerative diseases. However, the relationship between these factors in neurodegenerative processes is poorly understood. We have previously shown that proteasome inhibition produced higher neurodegeneration in aged than in young rats, suggesting that other additional age-related events could be involved in neurodegeneration. We evaluated the role of lipopolysaccharide (LPS)-induced neuroinflammation as a potential synergic risk factor for hippocampal neurodegeneration induced by proteasome inhibition. METHODS: Young male Wistar rats were injected with 1 μL of saline or LPS (5 mg/mL) into the hippocampus to evaluate the effect of LPS-induced neuroinflammation on protein homeostasis. The synergic effect of LPS and proteasome inhibition was analyzed in young rats that first received 1 μL of LPS and 24 h later 1 μL (5 mg/mL) of the proteasome inhibitor lactacystin. Animals were sacrificed at different times post-injection and hippocampi isolated and processed for gene expression analysis by real-time polymerase chain reaction; protein expression analysis by western blots; proteasome activity by fluorescence spectroscopy; immunofluorescence analysis by confocal microscopy; and degeneration assay by Fluoro-Jade B staining. RESULTS: LPS injection produced the accumulation of ubiquitinated proteins in hippocampal neurons, increased expression of the E2 ubiquitin-conjugating enzyme UB2L6, decreased proteasome activity and increased immunoproteasome content. However, LPS injection was not sufficient to produce neurodegeneration. The combination of neuroinflammation and proteasome inhibition leads to higher neuronal accumulation of ubiquitinated proteins, predominant expression of pro-apoptotic markers and increased neurodegeneration, when compared with LPS or lactacystin (LT) injection alone. CONCLUSIONS: Our results identify neuroinflammation as a risk factor that increases susceptibility to neurodegeneration induced by proteasome inhibition. These results highlight the modulation of neuroinflammation as a mechanism for neuronal protection that could be relevant in situations where both factors are present, such as aging and neurodegenerative diseases

A Semiautomated Classification System for Producing Service Directories in Social and Health Care (DESDE-AND): Maturity Assessment Study

Background: DESDE-LTC (Description and Evaluation of Services and DirectoriEs for Long-Term Care) is an international classification system that allows standardized coding and comparisons between different territories and care sectors, such as health and social care, in defined geographic areas. We adapted DESDE-LTC into a computer tool (DESDE-AND) for compiling a directory of care services in Andalucia, Spain. Objective: The aim of this study was to evaluate the maturity of DESDE-AND. A secondary objective of this study is to show the practicality of a new combined set of standard evaluation tools for measuring the maturity of health technology products. Methods: A system for semiautomated coding of service provision has been co-designed. A panel of 23 domain experts and a group of 68 end users participated in its maturity assessment that included its technology readiness level (TRL), usability, validity, adoption (Adoption Impact Ladder [AIL]), and overall degree of maturity [implementation maturity model [IMM]). We piloted the prototype in an urban environment (Seville, Spain). Results: The prototype was demonstrated in an operational environment (TRL 7). Sixty-eight different care services were coded, generating fact sheets for each service and its geolocation map. The observed agreement was 90%, with moderate reliability. The tool was partially adopted by the regional government of Andalucia (Spain), reaching a level 5 in adoption (AIL) and a level 4 in maturity (IMM) and is ready for full implementation. Conclusions: DESDE-AND is a usable and manageable system for coding and compiling service directories and it can be used as a core module of decision support systems to guide planning in complex cross-sectoral areas such as combined social and health care

Low-count monoclonal B-cell lymphocytosis persists after seven years of follow up and is associated with a poorer outcome

Low-count monoclonal B-cell lymphocytosis is defined by the presence of very low numbers of circulating clonal B cells, usually phenotypically similar to chronic lymphocytic leukemia cells, whose biological and clinical significance remains elusive. Herein, we re-evaluated 65/91 low-count monoclonal B-cell lymphocytosis cases (54 chronic lymphocytic leukemia-like and 11 non-chronic lymphocytic leukemia-like) followed-up for a median of seven years, using high-sensitivity flow cytometry and interphase fluorescence in situ hybridization. Overall, the clone size significantly increased in 69% of low-count monoclonal B-cell lymphocytosis cases, but only one subject progressed to high-count monoclonal B-cell lymphocytosis. In parallel, the frequency of cytogenetic alterations increased over time (32% vs. 61% of cases, respectively). The absolute number of the major T-cell and natural killer cell populations also increased, but only among chronic lymphocytic leukemia-like cases with increased clone size vs. age- and sex-matched controls. Although progression to chronic lymphocytic leukemia was not observed, the overall survival of low-count monoclonal B-cell lymphocytosis individuals was significantly reduced vs. non-monoclonal B-cell lymphocytosis controls (P=0.03) plus the general population from the same region (P≤0.001), particularly among females (P=0.01); infection and cancer were the main causes of death in low-count monoclonal B-cell lymphocytosis. In summary, despite the fact that mid-term progression from low-count monoclonal B-cell lymphocytosis to high-count monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia appears to be unlikely, these clones persist at increased numbers, usually carrying more genetic alterations, and might thus be a marker of an impaired immune system indirectly associated with a poorer outcome, particularly among females.This work was supported by the RD06/0020/0035 and RD12/0036/0048 grants from Red Temática de Investigación Cooperativa en Cáncer (RTICC), Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, (Madrid, Spain and FONDOS FEDER); CB16/12/00400 grant (CIBERONC, Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, (Madrid, Spain and FONDOS FEDER); the FIS PI06/0824-FEDER, PS09/02430-FEDER, PI12/00905- FEDER, DTS15/00119-FEDER, PI16/00787-FEDER and PI17/00399-FEDER grants, from the Fondo de Investigación Sanitaria of Instituto de Salud Carlos III; the GRS206/A/08 grant, (Ayuda al Grupo GR37 de Excelencia, SAN/1778/2009) from the Gerencia Regional de Salud (Consejería de Educación and Consejería de Sanidad of Castilla y León, Valladolid, Spain) and the SA079U14 grant (Consejería de Educación and Consejería de Sanidad of Castilla y León, Valladolid, Spain). ML Gutiérrez is supported by grant PTA2014-09963-I from the Instituto de Salud Carlos III

High-resolution copy number analysis of paired normal-tumor samples from diffuse large B cell lymphoma

Copy number analysis can be useful for assessing prognosis in diffuse large B cell lymphoma (DLBCL). We analyzed copy number data from tumor samples of 60 patients diagnosed with DLBCL de novo and their matched normal samples. We detected 63 recurrent copy number alterations (CNAs), including 33 gains, 30 losses, and nine recurrent acquired copy number neutral loss of heterozygosity (CNN-LOH). Interestingly, 20 % of cases acquired CNN-LOH of 6p21 locus, which involves the HLA region. In normal cells, there were no CNAs but we observed CNN-LOH involving some key lymphoma regions such as 6p21 and 9p24.1 (5 %) and 17p13.1 (2.5 %) in DLBCL patients. Furthermore, a model with some specific CNA was able to predict the subtype of DLBCL, 1p36.32 and 10q23.31 losses being restricted to germinal center B cell-like (GCB) DLBCL. In contrast, 8p23.3 losses and 11q24.3 gains were strongly associated with the non-GCB subtype. A poor prognosis was associated with biallelic inactivation of TP53 or 18p11.32 losses, while prognosis was better in cases carrying 11q24.3 gains. In summary, CNA abnormalities identify specific DLBCL groups, and we describe CNN-LOH in germline cells from DLBCL patients that are associated with genes that probably play a key role in DLBCL development.This work was supported by research funding from the Health Council of Castilla y León (GRS265/A/08), the Health Research Program (PS09/01382), and the Red Temática de Investigación Cooperativa en Cáncer (RTICC) grant RD12/0036 (groups 0069, 0029, 0036, 0058, and 0060) included in the National Plan I+D+I supported by the Instituto Carlos III and the Fondo Europeo de Desarrollo Regional (FEDER), the Spanish Ministry of Economy and Competitiveness, and the European Regional Development Fund (ERDF) 'Una manera de hacer Europa' (Innocampus; CEI-2010-1-0010). ES was supported by CM10/00078-Río Hortega, an ISCIII contract, FEHH grant 2013–2014 and JR14/00025-Juan Rodés, an ISCIII contract. IS was supported by the Subprograma Juan de la Cierva (JCI-2011-10232) and a Miguel Servet contract (CP13/00159).Peer Reviewe

Podocyte and tubular involvement in AngioJet-induced kidney injury

Altres ajuts: Sociedad Española de Nefrología, Fundacion Renal Iñigo Álvarez de Toledo and Comunidad de Madrid CIFRA2 B2017/BMD-3686.The AngioJet technique combines localized thrombolysis and percutaneous mechanical thrombectomy (PMT). However, PMT may cause acute kidney injury (AKI), which has been ascribed to severe mechanical haemolysis, although no renal biopsies have been reported. We now report the first renal biopsy in a patient with AKI following PMT. There is histological evidence of haemoglobin (Hb)-induced tubular injury and podocyte stress characterized by intracellular Hb and staining for ferritin and hemo-oxygenase-1, suggestive of an adaptive response to oxidative stress. This confirms that Hb is involved in kidney cell injury and supports the existence of several different kidney cellular targets

Mitotic spindle association of TACC3 requires Aurora-A-dependent stabilization of a cryptic α-helix.

Aurora-A regulates the recruitment of TACC3 to the mitotic spindle through a phospho-dependent interaction with clathrin heavy chain (CHC). Here, we describe the structural basis of these interactions, mediated by three motifs in a disordered region of TACC3. A hydrophobic docking motif binds to a previously uncharacterized pocket on Aurora-A that is blocked in most kinases. Abrogation of the docking motif causes a delay in late mitosis, consistent with the cellular distribution of Aurora-A complexes. Phosphorylation of Ser558 engages a conformational switch in a second motif from a disordered state, needed to bind the kinase active site, into a helical conformation. The helix extends into a third, adjacent motif that is recognized by a helical-repeat region of CHC, not a recognized phospho-reader domain. This potentially widespread mechanism of phospho-recognition provides greater flexibility to tune the molecular details of the interaction than canonical recognition motifs that are dominated by phosphate binding