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espanolObjetivo: describir el proceso de validacion psicometrica del instrumento COPE-Index en poblacion espanola. Metodo: estudio de validacion del cuestionario COPE-Index, que cuenta con 15 items divididos en tres subescalas: valoracion del impacto negativo, valoracion del impacto positivo y calidad del apoyo. Los sujetos de estudio fueron 165 figuras cuidadoras de personas mayores, pertenecientes a la Asociacion de Familiares de Alzheimer de la Comunidad de Madrid. Mediciones principales: factibilidad del instrumento COPE, consistencia interna, efecto techo y suelo, analisis factorial exploratorio, validez convergente y divergente (para ello se usaron los cuestionarios PACS, Duke-Unc y SF-12). Resultados: la muestra de estudio estuvo formaba por 150 sujetos (tasa de respuesta del 90,9%). El 65,33% (n= 98) era mujer; la edad media (DE) fue de 64 (12,23). Los resultados se mostraron fiables en cuanto a su homogeneidad interna en relacion con la subescala negativa y la subescala de calidad (alfas de Cronbach > 0,7) y menor en la subescala positiva (alfa de Cronbach: 0,61). No se aprecio efecto techo ni suelo. La validez de constructo confirmo tres dimensiones del cuestionario COPE, que explicaban el 52% de la varianza total. En las pruebas de validez convergente/divergente se correlacionaron las puntuaciones de la subescala positiva del cuestionario COPE con los items del cuestionario PACS, la subescala negativa del cuestionario COPE se relaciono con las preguntas del cuestionario Duke-Unc y, finalmente, la subescala de calidad del cuestionario COPE se correlaciono con los items del instrumento SF-12 version dos. Conclusion: la version espanola del cuestionario COPE-Index fue valida y confiable para identificar los aspectos positivos, negativos y de calidad del cuidado en cuidadoras de personas mayores. EnglishObjective: to describe the psychometric validation process of the COPE-Index instrument for the Spanish population. Method: a validation study of the COPE-Index questionnaire, which consists of 15 items classified into three sub-scales: assessment of negative impact, assessment of positive impact, and quality of support. The study subjects were 165 carers for older people, from the Madrid Association of Relatives of Alzheimer’s Patients. The main measurements were: the feasibility of the COPE instrument, internal consistency, ceiling and floor effect, exploratory factor analysis, convergent and divergent validity (the PACS, Duke-Unc and SF-12 questionnaires were used for this). Results: the study sample was formed by 150 subjects (90.9% response rate). Of these, 65.33% (n= 98) were female, and their mean age (SD) was 64 (12.23). Results appeared reliable in terms of internal homogeneity regarding the negative sub-scale and the quality sub-scale (Cronbach’s alphas > 0.7), and lower in the positive sub-scale (Cronbach’s alpha: 0,61). Neither ceiling nor floor effect were observed. The construct validity confirmed three dimensions of the COPE questionnaire, which explained 52% of the total variance. In the convergent / divergent validity tests, the scores from the positive sub-scale of the COPE questionnaire were correlated with the PACS questionnaire items, the negative sub-scale of the COPE questionnaire was associated with the questions from the Duke-Unc questionnaire, and finally, the quality sub-scale of the COPE questionnaire was correlated with the items from the second version of the SF-12 instrument. Conclusion: the Spanish version of the COPE-Index questionnaire was valid and reliable to identify the positive, negative and quality aspects of care in caregivers for elderly persons.

Latin American Congress on Automation and Robotics LACAR 2019, 30/10/2019-01/11/2019, Cali, Colombia., Detection at an early stage of an infection is a major clinical challenge. An infection that is not diagnosed in time can not only seriously affect the health of the infected patient, but also spread and initiate a contagious approach towards other people. This paper deals with mHealth system for medical care and pre-diagnosis. The developed mHealth system use an Android App that collects physiological signals from the patients with a portable and easy-to-use sensors kit. The focus of the work is put on being able to build a low-cost system that using a very small amounts of data (one set record per patient and day). The processed data are uploaded to an online database to train a clinical decision support system to automatically diagnose infections. The mHealth system may be operated by the same personnel on site not requiring to be medical or computational skilled at all. The implementation takes five kinds of measures simultaneously (Electrodermal Activity, Body Temperature, Blood Pressure, Heart Beat Rate and Oxygen Saturation (SPO2)). A real implementation has been tested and results confirm that the sampling process can be done very fast and steadily Finally, the App usability was tested, showing a fast learning curve and no significant differences are observable in learning time by people with different skills or age. These usability factors are key for the mHealth system success.

Background: In the last decades, there have been important developments in the scientific and technological areas of healthcare. On certain occasions this provokes conflict between the patients' rights and the values of healthcare professionals which brings about, within this clinical relationship, the problem of conscientious objection. Aims: To learn the opinions that the Nurses of the Madrid Autonomous Community have regarding conscientious objection. Research design: Cross-cutting descriptive study. Participants and research context: The nurses of 9 hospitals and 12 Health Centers in the Madrid Autonomous Community. The study was done by means of an auto completed anonymous questionnaire. The variables studied were social-demographical and their opinions about conscientious objections. Ethical considerations: The study was approved by the Ethical Community of Clinical Research of the University Hospital Príncipe de Asturias. Participants were assured of maximum confidentiality and anonymity. Findings: A total of 421 nurses answered the questionnaire. In total, 55.6% of the nurses confirmed they were religious believers, and 64.3% declared having poor knowledge regarding conscientious objection. The matters that caused the greatest objections were voluntary abortions, genetic embryo selection, refusal of blood transfusions, and therapy refusal. Discussion: Different authors state that the most significant cases of conscientious objections for health professionals are those regarding carrying out or assisting in abortions, euthanasia, the practice of assisted reproduction and, finally, the prescription and dispensing of the morning-after pill. In our study, the most significant cases in which the nurses would declare conscientious objections would be the refusal to accept treatment, the selection of embryos after genetic diagnosis preimplantation, the patient’s refusal to receive blood transfusions due to religious reasons and pregnant women’s request for voluntary abortions within the first 14 weeks. Conclusion: Nurses’ religious beliefs influence their opinions regarding conscientious objection. The nurses who declare themselves as religious believers object in a higher percentage than those without religious beliefs.

Studying the evolution of catalytically promiscuous enzymes like those from the N-succinylamino acid racemase/o-succinylbenzoate synthase (NSAR/OSBS) subfamily can reveal mechanisms by which new functions evolve. Some enzymes in this subfamily only have OSBS activity, while others catalyze OSBS and NSAR reactions. We characterized several NSAR/OSBS subfamily enzymes as a step toward determining the structural basis for evolving NSAR activity. Three enzymes were promiscuous, like most other characterized NSAR/OSBS subfamily enzymes. However, Alicyclobacillus acidocaldarius OSBS (AaOSBS) efficiently catalyzes OSBS activity but lacks detectable NSAR activity. Competitive inhibition and molecular modeling show that AaOSBS binds N-succinylphenylglycine with moderate affinity in a site that overlaps its normal substrate. Based on possible steric conflicts identified by molecular modeling and sequence conservation within the NSAR/OSBS subfamily, we identified one mutation, Y299I, which increased NSAR activity from undetectable to 1.2 x 10(2) M(−1)s(−1) without affecting OSBS activity. This mutation does not appear to affect binding affinity, but instead affects k(cat), by reorienting the substrate or modifying conformation changes to allow both catalytic lysines to access the proton that is moved during the reaction. This is the first site known to affect reaction specificity in the NSAR/OSBS subfamily. However, this gain of activity was obliterated by a second mutation, M18F. Epistatic interference by M18F was unexpected because a phenylalanine at this position is important in another NSAR/OSBS enzyme. Together, modest NSAR activity of Y299I AaOSBS and epistasis between sites 18 and 299 indicate that additional sites influenced the evolution of NSAR reaction specificity in the NSAR/OSBS subfamily.

Gene regulatory analysis of highly diverse human tissues in vivo is essentially constrained by the challenge of performing genome-wide, integrated epigenetic and transcriptomic analysis in small selected groups of specific cell types. Here we performed genome-wide bisulfite sequencing and RNA-seq from the same small groups of bronchial and alveolar cells isolated by laser capture microdissection from flash-frozen lung tissue of 12 donors and their peripheral blood T cells. Methylation and transcriptome patterns differed between alveolar and bronchial cells, while each of these epithelia showed more differences from mesodermally-derived T cells. Differentially methylated regions (DMRs) between alveolar and bronchial cells tended to locate at regulatory regions affecting promoters of 4,350 genes. A large number of pathways enriched for these DMRs including GTPase signal transduction, cell death, and skeletal muscle. Similar patterns of transcriptome differences were observed: 4,108 differentially expressed genes (DEGs) enriched in GTPase signal transduction, inflammation, cilium assembly, and others. Prioritizing using DMR-DEG regulatory network, we highlighted genes, e.g., ETS1, PPARG, and RXRG, at prominent alveolar vs. bronchial cell discriminant nodes. Our results show that multi-omic analysis of small, highly specific cells is feasible and yields unique physiologic loci distinguishing human lung cell types in situ.

Pmi1525, an enzyme of unknown function from Proteus mirabilis HI4320 and the amidohydrolase superfamily, was cloned, purified to homogeneity, and functionally characterized. The three-dimensional structure of Pmi1525 was determined with zinc and cacodylate bound in the active site (PDB id: 3RHG ). The structure was also determined with manganese and butyrate in the active site (PDB id: 4QSF ). Pmi1525 folds as a distorted (β/α)8-barrel that is typical for members of the amidohydrolase superfamily and cog1735. The substrate profile for Pmi1525 was determined via a strategy that marshaled the utilization of bioinformatics, structural characterization, and focused library screening. The protein was found to efficiently catalyze the hydrolysis of organophosphonate and carboxylate esters. The best substrates identified for Pmi1525 are ethyl 4-nitrophenylmethyl phosphonate (kcat and kcat/Km values of 580 s(-1) and 1.2 × 10(5) M(-1) s(-1), respectively) and 4-nitrophenyl butyrate (kcat and kcat/Km values of 140 s(-1) and 1.4 × 10(5) M(-1) s(-1), respectively). Pmi1525 is stereoselective for the hydrolysis of chiral methylphosphonate esters. The enzyme hydrolyzes the (SP)-enantiomer of isobutyl 4-nitrophenyl methylphosphonate 14 times faster than the corresponding (RP)-enantiomer. The catalytic properties of this enzyme make it an attractive template for the evolution of novel enzymes for the detection, destruction, and detoxification of organophosphonate nerve agents.

Introduction A prolonged third stage of labor is considered to be a risk factor for postpartum hemorrhage. The objective of this study was to determine the ability of acupuncture to reduce the length of the third stage of labor. Methods Seventy-six puerperal women who had a normal spontaneous birth at the Hospital Universitario Principe de Asturias, Alcala de Henares, Spain, were included in a single-blind randomized trial and evaluated by a third party. Women were randomly assigned to receive true acupuncture or placebo acupuncture (also known as sham acupuncture). In the first group, a sterilized steel needle was inserted at the Ren Mai 6 point, which is located on the anterior midline between the umbilicus and the upper part of the pubic symphysis. In the second group, the insertion site was located at the same horizontal level as the Ren Mai 6 point but shifted slightly to the left of the anterior midline. The management of the third stage of labor was the same in both groups. Results Statistically significant differences were found, with an average time to placental expulsion of 15.2 minutes in the placebo group and 5.2 minutes in the acupuncture group. No major complications occurred in either group. Discussion These results confirm that acupuncture at the Ren Mai 6 point can decrease the time to placental expulsion. This treatment represents a simple, safe, and inexpensive way of decreasing the duration of the third stage of labor.

Part 1: Algorithms; International audience; In this article we address the problem of contaminated data in pattern recognition tasks, where apart from native patterns we may have foreign ones that do not belong to any native class. We present a novel approach to image classification with foreign pattern rejection based on cellular automata. The method is based only on native patterns, so no knowledge about characteristics of foreign patterns is required at the stage of model construction. The proposed approach is evaluated in a study of handwritten digits recognition. As foreign patterns we use distorted digits. Experiments show that the proposed model classifies native patterns with a high success rate and rejects foreign patterns as well.

The rate of protein evolution is determined by a combination of selective pressure on protein function and biophysical constraints on protein folding and structure. Determining the relative contributions of these properties is an unsolved problem in molecular evolution with broad implications for protein engineering and function prediction. As a case study, we examined the structural divergence of the rapidly evolving o-succinylbenzoate synthase (OSBS) family, which catalyzes a step in menaquinone synthesis in diverse microorganisms and plants. On average, the OSBS family is much more divergent than other protein families from the same set of species, with the most divergent family members sharing

Background Papillary thyroid carcinoma is the most common thyroid malignancy. Most papillary thyroid carcinomas contain BRAF mutations or RET/PTC rearrangements, thus providing targets for biologic therapy. Our previous studies had suggested papillary thyroid carcinomas (PTCs) with a BRAF mutation and the RET/PTC1 rearrangement have different sensitivities to MEK1/2 inhibitors, suggesting different signaling transduction pathways were involved. Methods Src signaling transduction pathway in PTC cells was examined using Src inhibitors (PP2, SU6656, or dasatinib) and si-Src RNA in vitro by Western blot analysis and proliferation analysis. An orthotopic xenograft mouse model was used for the in vivo studies using dasatinib. Results In PTC cells, Src inhibitors suppressed p-Src and p-FAK and inhibited cell growth. In addition, significant suppression and extension of the p-ERK1/2 dephosphorylation were detected in RET/PTC1-rearranged cells in combination with an MEK inhibitor (CI-1040). The Src family kinase/ABL inhibitor, dasatinib, significantly decreased tumor volume in mice inoculated with PTC cells carrying the RET/PTC1 rearrangement. In BRAF-mutated PTC cells, Src inhibitors effectively suppressed p-Src expression and dasatinib significantly decreased tumor volume with twice daily treatment. Conclusion Src inhibitors effectively inhibited the Src signaling transduction pathway in PTC cells in vitro and dasatinib suppressed tumor growth in vivo. These results suggested that Src signaling transduction pathway plays an important role in regulating growth in PTC cells. Combination of Src and MEK1/2 inhibitors extended the dephosphorylation of extracellular signal-regulated kinase (ERK)1/2 in PTCs carrying the RET/PTC1 rearrangement suggesting that combination therapy with complementary inhibitors of other signaling transduction pathways may be needed to effectively suppress growth and induce apoptosis in these cells. © 2013 Wiley Periodicals, Inc. Head Neck 36: 375–384, 2014

Import-Karyopherin or Importin proteins bind nuclear localization signals (NLSs) to mediate the import of proteins into the cell nucleus. Karyopherin β2 or Kapβ2, also known as Transportin, is a member of this transporter family responsible for the import of numerous RNA binding proteins. Kapβ2 recognizes a targeting signal termed the PY-NLS that lies within its cargos to target them through the nuclear pore complex. The recognition of PY-NLS by Kapβ2 is conserved throughout eukaryotes. Kap104, the Kapβ2 homolog in Saccharomyces cerevisiae, recognizes PY-NLSs in cargos Nab2, Hrp1, and Tfg2. We have determined the crystal structure of Kapβ2 bound to the PY-NLS of the mRNA processing protein Nab2 at 3.05-Å resolution. A seven-residue segment of the PY-NLS of Nab2 is observed to bind Kapβ2 in an extended conformation and occupies the same PY-NLS binding site observed in other Kapβ2·PY-NLS structures.

Software agents are a recognized way of introducing computer simulations to board games. Here, we consider one of the classic board games, called “Scotland Yard”, which belongs to the category of hider-seeker games, and involves six policemen that try to capture Mr. X. The aim of this paper is to discuss how software agents and AI-based strategies can be used to develop simulation in a hider-seeker game, named Warsaw's Backyard. Specifically, it is shown how A∗ and IDDFS algorithms influence the seekers and hider performance. Furthermore, a Move Filtering is introduced. It allows to better approximate the position of the hider. Performance of the proposed approach is experimentally evaluated.

The apoptotic effect of FasL:Fas signaling is disrupted by DcR3, a unique secreted member of the tumor necrosis factor receptor superfamily, which also binds and neutralizes TL1A and LIGHT. DcR3 is highly elevated in patients with various tumors and contributes to mechanisms by which tumor cells to evade host immune surveillance. Here we report the crystal structure of FasL in complex with DcR3. Comparison of FasL:DcR3 structure with our earlier TL1A:DcR3 and LIGHT:DcR3 structures supports a paradigm involving the recognition of invariant main-chain and conserved side-chain functionalities, which is responsible for the recognition of multiple TNF ligands exhibited by DcR3. The FasL:DcR3 structure also provides insight into the FasL:Fas recognition surface. We demonstrate that the ability of recombinant FasL to induce Jurkat cell apoptosis is significantly enhanced by native glycosylation or by structure-inspired mutations, both of which result in reduced tendency to aggregate. All of these activities are efficiently inhibited by recombinant DcR3.

Marburg virus (MARV) and Ebola virus (EBOV) are members of the family Filoviridae ("filoviruses") and cause severe hemorrhagic fever with human case fatality rates of up to 90%. Filovirus infection requires fusion of the host cell and virus membranes, a process that is mediated by the envelope glycoprotein (GP). GP contains two subunits, the surface subunit (GP1), which is responsible for cell attachment, and the transmembrane subunit (GP2), which catalyzes membrane fusion. The GP2 ectodomain contains two heptad repeat regions, N-terminal and C-terminal (NHR and CHR, respectively), that adopt a six-helix bundle during the fusion process. The refolding of this six-helix bundle provides the thermodynamic driving force to overcome barriers associated with membrane fusion. Here we report the crystal structure of the MARV GP2 core domain in its postfusion (six-helix bundle) conformation at 1.9 Å resolution. The MARV GP2 core domain backbone conformation is virtually identical to that of EBOV GP2 (reported previously), and consists of a central NHR core trimeric coiled coil packed against peripheral CHR α-helices and an intervening loop and helix-turn-helix segments. We previously reported that the stability of the MARV GP2 postfusion structure is highly pH-dependent, with increasing stability at lower pH [Harrison, J. S., Koellhoffer, J. K., Chandran, K., and Lai, J. R. (2012) Biochemistry51, 2515-2525]. We hypothesized that this pH-dependent stability provides a mechanism for conformational control such that the postfusion six-helix bundle is promoted in the environments of appropriately mature endosomes. In this report, a structural rationale for this pH-dependent stability is described and involves a high-density array of core and surface acidic side chains at the midsection of the structure, termed the "anion stripe". In addition, many surface-exposed salt bridges likely contribute to the stabilization of the postfusion structure at low pH. These results provide structural insights into the mechanism of MARV GP2-mediated membrane fusion.

5-Carboxyvanillate decarboxylase (LigW) catalyzes the conversion of 5-carboxyvanillate to vanillate in the biochemical pathway for the degradation of lignin. This enzyme was shown to require Mn(2+) for catalytic activity and the kinetic constants for the decarboxylation of 5-carboxyvanillate by the enzymes from Sphingomonas paucimobilis SYK-6 (kcat = 2.2 s(-1) and kcat/Km = 4.0 × 10(4) M(-1) s(-1)) and Novosphingobium aromaticivorans (kcat = 27 s(-1) and kcat/Km = 1.1 × 10(5) M(-1) s(-1)) were determined. The three-dimensional structures of both enzymes were determined in the presence and absence of ligands bound in the active site. The structure of LigW from N. aromaticivorans, bound with the substrate analogue, 5-nitrovanillate (Kd = 5.0 nM), was determined to a resolution of 1.07 Å. The structure of this complex shows a remarkable enzyme-induced distortion of the nitro-substituent out of the plane of the phenyl ring by approximately 23°. A chemical reaction mechanism for the decarboxylation of 5-carboxyvanillate by LigW was proposed on the basis of the high resolution X-ray structures determined in the presence ligands bound in the active site, mutation of active site residues, and the magnitude of the product isotope effect determined in a mixture of H2O and D2O. In the proposed reaction mechanism the enzyme facilitates the transfer of a proton to C5 of the substrate prior to the decarboxylation step.

In the last two years there has been a major legislative developments on the individual rights at the end of life process. The aim of this paper is to analyze the content of these new laws in order to know how are regulated the individual rights during the process of death, the duties of health workers who treat these patients and guarantees that public authorities and health institutions are required to provide to ensure the proper exercise of these rights.

BACKGROUND: Mutations in the PITX2 homeobox gene are known to contribute to Axenfeld-Rieger syndrome (ARS), an autosomal-dominant developmental disorder. Although most mutations are in the homeodomain and result in a loss of function, there is a growing subset in the C-terminal domain that has not yet been characterized. These mutations are of particular interest because the C-terminus has both inhibitory and stimulatory activities. METHODS: In this study we used a combination of in vitro DNA binding and transfection reporter assays to investigate the fundamental issue of whether C-terminal mutations result in gain or loss of function at a cellular level. RESULTS: We report a new frameshift mutation in the PITX2 allele that predicts a truncated protein lacking most of the C-terminal domain (D122FS). This newly reported mutant and another ARS C-terminal mutant (W133Stop) both have greater binding than wild-type to the bicoid element. Of interest, the mutants yielded ! 5-fold greater activation of the prolactin promoter in CHO cells, even though the truncated proteins were expressed at lower levels than the wild-type protein. The truncated proteins also had greater than wild-type activity in 2 other cell lines, including the LS8 oral epithelial line that expresses the endogenous Pitx2 gene. CONCLUSIONS: The results indicate that the PITX2 C-terminal domain has inhibitory activity and support the notion that ARS may also be caused by gain-of-function mutations. Birth Defects Research (Part A) 76:175‐181, 2006. © 2006 Wiley-Liss, Inc.

Hace 10 anos tuve la oportunidad de participar en un estudio sobre perspectivas de la educacion superior en ingenieria y ciencias en el pais. Recuerdo el enfasis que haciamos en el hecho de que el proceso educativo de una persona, hasta llegar al nivel profesional o postgraduado, es una larga cadena caracterizada por profundos "desencuentros" entre las diversas etapas. Las -a veces inexplicables- diferencias pedagogicas entre la educacion primaria y secundaria; las concepciones encontradas en...

Global networks of the cytosolic glutathione S-transferases illuminate sequence-structure-function relationships across more than 13,000 members of this superfamily, including experimental confirmation of enzymatic activity for 82 members and new crystal structures for 27., The cytosolic glutathione transferase (cytGST) superfamily comprises more than 13,000 nonredundant sequences found throughout the biosphere. Their key roles in metabolism and defense against oxidative damage have led to thousands of studies over several decades. Despite this attention, little is known about the physiological reactions they catalyze and most of the substrates used to assay cytGSTs are synthetic compounds. A deeper understanding of relationships across the superfamily could provide new clues about their functions. To establish a foundation for expanded classification of cytGSTs, we generated similarity-based subgroupings for the entire superfamily. Using the resulting sequence similarity networks, we chose targets that broadly covered unknown functions and report here experimental results confirming GST-like activity for 82 of them, along with 37 new 3D structures determined for 27 targets. These new data, along with experimentally known GST reactions and structures reported in the literature, were painted onto the networks to generate a global view of their sequence-structure-function relationships. The results show how proteins of both known and unknown function relate to each other across the entire superfamily and reveal that the great majority of cytGSTs have not been experimentally characterized or annotated by canonical class. A mapping of taxonomic classes across the superfamily indicates that many taxa are represented in each subgroup and highlights challenges for classification of superfamily sequences into functionally relevant classes. Experimental determination of disulfide bond reductase activity in many diverse subgroups illustrate a theme common for many reaction types. Finally, sequence comparison between an enzyme that catalyzes a reductive dechlorination reaction relevant to bioremediation efforts with some of its closest homologs reveals differences among them likely to be associated with evolution of this unusual reaction. Interactive versions of the networks, associated with functional and other types of information, can be downloaded from the Structure-Function Linkage Database (SFLD; http://sfld.rbvi.ucsf.edu)., Author Summary Cytosolic glutathione transferases (cytGSTs) are a large and diverse superfamily of enzymes that have important roles in metabolism and defense against oxidative damage. They have been studied for several decades but because of the synthetic nature of the chemicals used to test these proteins to determine if they have cytGST activity, little is known about the physiological reactions and roles of cytGSTs. In this large, collaborative study, we constructed networks where more than 13,000 cytGST sequences were grouped by sequence similarity and then used these networks to prioritize new targets for experimental characterization in relatively unexplored regions of the superfamily. We report here experimental results confirming GST-like activity for 82 of them, along with 37 new three-dimensional molecular structures determined for 27 targets. These new data, along with experimental data previously reported in the literature, were painted onto the networks to generate a global view of their sequence-structure-function relationships. The results show how proteins of both known and unknown function relate to each other across the entire superfamily and illuminate the complex ways in which their variations in sequence and structure affect our ability to predict unknown functional properties.

Fusion of the viral and host cell membranes is a necessary first step for infection by enveloped viruses, and is mediated by the envelope glycoprotein. The transmembrane subunits from the structurally defined “class I” glycoproteins adopt an α-helical “trimer- of-hairpins” conformation during the fusion pathway. Here we present our studies on the envelope glycoprotein transmembrane subunit, GP2, of the CAS virus (CASV). CASV was recently identified from annulated tree boas (Corallus annulatus) with inclusion body disease and is implicated in the disease etiology. We have generated and characterized two protein constructs consisting of the predicted CASV GP2 core domain. The crystal structure of the CASV GP2 post-fusion conformation indicates a trimeric α-helical bundle that is highly similar to those of Ebola Virus (EBOV) and Marburg Virus (MARV) GP2, despite CASV genome homology to arenaviruses. Denaturation studies demonstrate that the stability of CASV GP2 is pH-dependent with higher stability at lower pH; we propose that this behavior is due to a network of interactions among acidic residues that would destabilize the α-helical bundle under conditions where the side chains are deprotonated. The pH-dependent stability of the post-fusion structure has been observed in EBOV and MARV GP2, as well as other viruses that enter via the endosome. Infection experiments with CASV and the related Golden Gate Virus (GGV) support a mechanism of entry that requires endosomal acidification. Our results suggest that despite being primarily arenavirus-like, the transmembrane subunit of CASV is extremely similar to the filoviruses.

Putative 'protein nitratases,' which catalyze denitration of peroxynitrite (PN)-treated proteins, were detected in the homogenate/crude extract of rat brains and hearts. Nitratase activity was monitored by the decreased intensity of nitrotyrosine immunoreactive-bands in Western blot and increased nitrate level in dialysate of incubation mixture, which contained homogenate/crude extract, protease inhibitors and a PN-treated substrate, such as treated histone (III-S), BSA or invertase. Enhanced activity of nitratases was noted by preincubating crude extract with Ca2+. In addition, at least two types of nitratases may occur: type I, reductant-dependent, and type II, reductant- independent. Furthermore, upon denitration, the activity of PN-treated invertase increased to the same activity level of the untreated invertase. The overall reaction catalyzed by nitratases for denitration of nitrotyrosine residues in protein could be as follows: Protein-Tyr-NO2 + H2O --> Protein-Tyr-H + H+ + NO3-. The nitration/denitration of protein-tyrosine may be crucial in regulating signal transduction.

Proteins of unknown function belonging to cog1816 and cog0402 were characterized. Sav2595 from Steptomyces avermitilis MA-4680, Acel0264 from Acidothermus cellulolyticus 11B, Nis0429 from Nitratiruptor sp. SB155-2 and Dr0824 from Deinococcus radiodurans R1 were cloned, purified, and their substrate profiles determined. These enzymes were previously incorrectly annotated as adenosine deaminases or chlorohydrolases. It was shown here that these enzymes actually deaminate 6-aminodeoxyfutalosine. The deamination of 6-aminodeoxyfutalosine is part of an alternative menaquinone biosynthetic pathway that involves the formation of futalosine. 6-Aminodeoxyfutalosine is deaminated by these enzymes with catalytic efficiencies greater than 10(5) M(-1) s(-1), Km values of 0.9-6.0 μM, and kcat values of 1.2-8.6 s(-1). Adenosine, 2'-deoxyadenosine, thiomethyladenosine, and S-adenosylhomocysteine are deaminated at least an order of magnitude slower than 6-aminodeoxyfutalosine. The crystal structure of Nis0429 was determined and the substrate, 6-aminodeoxyfutalosine, was positioned in the active site on the basis of the presence of adventitiously bound benzoic acid. In this model, Ser-145 interacts with the carboxylate moiety of the substrate. The structure of Dr0824 was also determined, but a collapsed active site pocket prevented docking of substrates. A computational model of Sav2595 was built on the basis of the crystal structure of adenosine deaminase and substrates were docked. The model predicted a conserved arginine after β-strand 1 to be partially responsible for the substrate specificity of Sav2595.

Resumen Objetivos Describir y comparar los conocimientos y actitudes de médicos y enfermeras sobre las instrucciones previas. Diseño Estudio piloto descriptivo transversal. Emplazamiento Área Asistencial Este de la Comunidad de Madrid. Participantes Médicos y enfermeras de atención primaria y especializada. Mediciones principales Cuestionario autocumplimentado acerca de conocimientos y actitudes sobre las instrucciones previas, compuesto por variables dicotómicas y de escala tipo Likert (0-10). Resultados Respondieron al cuestionario un total de 192 médicos y enfermeras (tasa de respuesta = 83,4%); 72,4% eran mujeres y 27,6% hombres. La media de edad fue de 39,6 años (DE = 10,9). Para el conocimiento general sobre las instrucciones previas la mediana fue de 5 (rango intercuartílico: 3-7). El 60,1% conocía la regulación por ley, pero solo el 22,8% había leído el documento. Conclusiones El conocimiento de médicos y enfermeras sobre las instrucciones previas es bajo, por lo que es necesario mejorar este conocimiento. Los médicos y enfermeras de ambos niveles muestran actitudes favorables hacia el uso, la utilidad y el respeto del contenido del documento de instrucciones previas. El diseño metodológico propuesto es eficaz para aplicar en un estudio más amplio, aunque se debe mejorar el sistema de distribución y recogida de cuestionarios.

The rapid advance in genome sequencing presents substantial challenges for protein functional assignment, with half or more of new protein sequences inferred from these genomes having uncertain assignments. The assignment of enzyme function in functionally diverse superfamilies represents a particular challenge, which we address through a combination of computational predictions, enzymology, and structural biology. Here we describe the results of a focused investigation of a group of enzymes in the enolase superfamily that are involved in epimerizing dipeptides. The first members of this group to be functionally characterized were Ala-Glu epimerases in Eschericiha coli and Bacillus subtilis , based on the operon context and enzymological studies; these enzymes are presumed to be involved in peptidoglycan recycling. We have subsequently studied more than 65 related enzymes by computational methods, including homology modeling and metabolite docking, which suggested that many would have divergent specificities;, i.e., they are likely to have different (unknown) biological roles. In addition to the Ala-Phe epimerase specificity reported previously, we describe the prediction and experimental verification of: ( i ) a new group of presumed Ala-Glu epimerases; ( ii ) several enzymes with specificity for hydrophobic dipeptides, including one from Cytophaga hutchinsonii that epimerizes D-Ala-D-Ala; and ( iii ) a small group of enzymes that epimerize cationic dipeptides. Crystal structures for certain of these enzymes further elucidate the structural basis of the specificities. The results highlight the potential of computational methods to guide experimental characterization of enzymes in an automated, large-scale fashion.

The flow in cavities studies the dynamics of motion of a viscous fluid confined within a cavity in which the lower wall has a horizontal motion at constant speed. There exist two important reasons whichmotivate the study of cavity flows. First is the use of this particular geometry as a benchmark to verify the formulation and implementation of numerical methods and second the study of the dynamics of the flow inside the cavity which become very particular as the Reynolds (Re) number is increased, i.e. decreasing the fluid viscosity. Most of the studies, concerning flow dynamics inside the cavity, focus their efforts on the steady state, but very few study the mechanisms of evolution or transients until the steady state is achieved (Gustafson, 1991). Own to the latter aproach it was considered interesting to understand the mechanisms associated with the flow evolution until the steady state is reached and the steady state per se, since for different Re numbers (1,000 and 10,000) steady states are ”similar” but the transients to reach them are completely different. In order to study the flow dynamics and the evolution mechanisms to steady state the Lattice Boltzmann Method (LBM) was chosen to solve the dynamic system. The LBM was created in the late 90’s as a derivation of the Lattice Gas Automata (LGA). The idea that governs the method is to build simple mesoscale kinetic models that replicate macroscopic physics and after recovering the macro-level (continuum) it obeys the equations that governs it i.e. the Navier Stokes (NS) equations. The motivation for using LBM lies in a computational reason: Is easier to simulate fluid dynamics through a microscopic approach, more general than the continuum approach (Texeira, 1998) and the computational cost is lower than other NS equations solvers. Also is worth to mention that the prime characteristic of the present study and themethod itself was that the primitive variables were the vorticity-stream function not as the usual pressure-velocity variables. It was intended, by chosing this approach, to understand in a better way the fluid dynamics because what characterizes the cavity flow is the lower wall movement which creates itself an impulse of vorticiy which is transported within the cavity by diffusion and advection. This transport and the vorticity itself create the different vortex within the cavity and are responsible for its interaction. In the next sections steady states, periodic flows and feeding mechanisms for different Re numbers are going to be studied within square and deep cavities. 17

The explosion of protein sequence information requires that current strategies for function assignment evolve to complement experimental approaches with computationally based function prediction. This necessitates the development of strategies based on the identification of sequence markers in the form of specificity determinants and a more informed definition of orthologues. Herein, we have undertaken the function assignment of the unknown haloalkanoate dehalogenase superfamily member BT2127 (Uniprot accession code Q8A5 V9) from Bacteroides thetaiotaomicron using an integrated bioinformatics-structure-mechanism approach. The substrate specificity profile and steady-state rate constants of BT2127 (with a k(cat)/K(m) value for pyrophosphate of ~1 × 10(5) M(-1) s(-1)), together with the gene context, support the assigned in vivo function as an inorganic pyrophosphatase. The X-ray structural analysis of wild-type BT2127 and several variants generated by site-directed mutagenesis shows that substrate discrimination is based, in part, on active site space restrictions imposed by the cap domain (specifically by residues Tyr76 and Glu47). Structure-guided site-directed mutagenesis coupled with kinetic analysis of the mutant enzymes identified the residues required for catalysis, substrate binding, and domain-domain association. On the basis of this structure-function analysis, the catalytic residues Asp11, Asp13, Thr113, and Lys147 as well the metal binding residues Asp171, Asn172, and Glu47 were used as markers to confirm BT2127 orthologues identified via sequence searches. This bioinformatic analysis demonstrated that the biological range of BT2127 orthologue is restricted to the phylum Bacteroidetes/Chlorobi. The key structural determinants in the divergence of BT2127 and its closest homologue, β-phosphoglucomutase, control the leaving group size (phosphate vs glucose phosphate) and the position of the Asp acid/base in the open versus closed conformations. HADSF pyrophosphatases represent a third mechanistic and fold type for bacterial pyrophosphatases.

Human milk has been and it is an important means of survival for the human being. The history of the breastfeeding has been linked to the woman's social and cultural situation and it has gone by different vicissitudes. During a long period the breastfeeding was considered as an unsightly unworthy practice and characteristic of low classes, women that had resources nurses used. In the XIX century artificial nursing appears that will be developed significantly starting from half-filled of the following century relegating, again, to a second plane to the natural nursing. With the beginning of the XX century the first rights of the workers appear and among them it is regulated the right for the first time to the permission for nursing. With the advance of the century diverse norms enlarge their content. Arriving to the current moment in which the right enjoys a wide legal recognition inside the norms that regulate the family reconciliation.

Flow Evolution Mechanisms of Lid-Driven Cavities

Members of the enolase superfamily catalyze mechanistically diverse reactions, each initiated by abstraction of the α-proton of a carboxylate substrate by an active site base to form an enolate intermediate (1, 2). The active sites are located at the interface between an N-terminal α+β capping domain and a C-terminal (β/α)7β-barrel domain (modified (β/α)8- or TIM-barrel2). Residues responsible for binding the essential Mg2+ and the acid/base catalysts are located at the C-terminal ends of the β-strands of the barrel domain or in the loops that connect the β-strands with the following α-helices; loops in the capping domain contain residues that provide shape and polarity determinants for the active site, thereby determining the identity of the substrate. A conserved strategy is used to stabilize the enolate intermediate: one or both carboxylate oxygens of the substrate is coordinated to a Mg2+ so that the increased negative charge in the enolate can be stabilized. The superfamily is divided into subgroups on the basis of the identities of the ligands for the Mg2+ (located at the ends of the third, fourth, and fifth β-strands of the barrel domain) and the acid/base catalysts (located at the ends of the second, third, sixth, and/or seventh β-strands of the barrel domain). Six subgroups currently are recognized (designated by the name of the paradigm enzyme/structure): enolase, mandelate racemase (MR), cis,cis-muconate lactonizing enzyme (MLE), D-glucarate dehydratase (GlucD), D-mannonate dehydratase (ManD), and β-methylaspartate ammonia lyase (3). To date, nineteen distinct chemical reactions are catalyzed by members of the enolase superfamily, with these involving elimination of water or ammonia, intramolecular β-elimination (cycloisomerization), and 1,1-proton transfer (epimerization or racemization). As illustrated in the sequence similarity network (4) shown in Figure 1, the enolase superfamily can be partitioned into sequence “clusters” that aid identification of isofunctional families. In Figure 1 the members of each of the separate clusters share a BLASTP e-value of 35% sequence identity). The sequences in this network are color-coded by function, with grey sequences having unknown or uncertain function. The results of these and other bioinformatic analyses indicate that at least 50% of the currently known members of the enolase superfamily have unknown, uncertain, or incorrect functional assigments. If the structural bases for divergent evolution of function in the superfamily are to be understood and, perhaps, utilized for rational (re)design of enzymatic activities, the functions of all members of the superfamily must be assigned. Figure 1 Sequence similarity network for the enolase superfamily. Sequences are shown schematically as nodes (dots); BLASTP connections with E-values ≤ 10−80 are shown as edges (lines). At this E-value (sequence identities > ~35%), most ... Our goal is to use the sequences and/or structures of functionally uncharacterized members to enable computation-directed prediction of their substrate specificities, thereby focusing experimental efforts to accomplish their in vitro functional assignments. In the enolase superfamily, we have used comparative protein structure modeling and docking to guide discovery of a novel N-succinylamino acid racemase (5) and dipeptide epimerases with novel substrate specificities (6) in the absence of X-ray structures. In the mechanistically diverse amidohydrolase superfamily, Shoichet, Raushel, and coworkers have used in silico docking of “high-energy intermediates” to X-ray structures of an uncharacterized member to direct discovery of the 5-methylthioadenosine/S-adenosyl-homocysteine deaminase function for an uncharacterized protein from Thermotoga maritima (7). These examples establish the utility of an integrated sequence/structure-based computational strategy to direct experimental assignment of function. Herein we describe computation-facilitated assignment of the galactarate dehydratase function to a divergent member of the enolase superfamily from Oceanobacillus iheyensis (GI:23100298; IMG locus tag Ob2843). In the sequence similarity network in Figure 1, Ob2843 is “clustered” (highlighted with red circle) with one additional presumed orthologue from Bacillus clausii KSM-K16 (GI:56964777). The structure of Ob2843 (PDB Code 2OQY) was determined in the presence of Mg2+, but the absence of an organic ligand, by the New York SGX Research Center for Structural Genomics (NYSGXRC; PSI-2) as one of their community-nominated targets. In silico screening of a virtual library of acid sugars to the active site of Ob2843 predicted that a diacid sugar was a plausible substrate; activity screening with a physical library of acid sugars identified galactarate as the substrate. Dehydration of galactarate (as well as of L-talarate) was previously assigned as the enzymatic function of a divergent family by physical library screening (GalrD/TalrD; highlighted with blue circle in Figure 1) (8). However, the “old” (GalrD/TalrD) and “new” (GalrD-II) galactarate dehydratases differ in regioselectivity so the products obtained from (meso-)galactarate are enantiomers. The structure of the catalytically impaired Y90F mutant of GalrD-II was determined in the presence of galactarate and Mg2+, thereby allowing identification of novel active site residues: 1) an Arg-x-Tyr dyad at the end of the second β-strand of the barrel domain is the base that initiates the reaction, 2) a Tyr in the capping domain is the acid that facilitates departure of the 3-OH leaving group, and 3) a His (not Asp, Glu, or Asn as in other characterized members of the superfamily) located at the end of the fifth β-strand as a ligand for the essential Mg2+. This active site motif defines the seventh subgroup in the enolase superfamily (GalrD-II). The assignment of the GalrD-II function to Ob2843 provides additional evidence that an integrated sequence/structure-based strategy to direct functional assignment (using computational prediction to focus experimental activity measurements) is a viable approach for enabling nontrivial functional assignments.

TNF-like 1A (TL1A) is a newly described member of the TNF superfamily that is directly implicated in the pathogenesis of autoimmune diseases, including inflammatory bowel disease, atherosclerosis, and rheumatoid arthritis. We report the crystal structure of the human TL1A extracellular domain at a resolution of 2.5 A, which reveals a jelly-roll fold typical of the TNF superfamily. This structural information, in combination with complementary mutagenesis and biochemical characterization, provides insights into the binding interface and the specificity of the interactions between TL1A and the DcR3 and DR3 receptors. These studies suggest that the mode of interaction between TL1A and DcR3 differs from other characterized TNF ligand/receptor complexes. In addition, we have generated functional TL1A mutants with altered disulfide bonding capability that exhibit enhanced solution properties, which will facilitate the production of materials for future cell-based and whole animal studies. In summary, these studies provide insights into the structure and function of TL1A and provide the basis for the rational manipulation of its interactions with cognate receptors.

Death is one of the few certainties in human beings. This reality generates uneasiness regarding how death will occur especially in circumstances of loneliness or suffering which can become agony. In this report, the author first analyzes the existence of the right to die with dignity as a human right which takes on the nature of being a subjective right. In continuation, the author describes the existing problematic in the application of this right to die in relationship to the so-called double effect mechanism and euthanasia. The author concludes this article by proposing previous instructions or anticipated desires as ideal measures to make the right to die with dignity valid.

Heterochromatin protein 1 Hsalpha (HP1(Hsalpha)) is one of three human proteins that share sequence similarity with Drosophila HP1. HP1 proteins are enriched at centric heterochromatin and play a role in chromatin packaging and gene regulation. In humans, HP1(Hsalpha) is down-regulated in highly invasive/metastatic breast cancer cells, compared to poorly invasive/non-metastatic breast cancer cells. To gain insight into this differential regulation, we have cloned the HP1(Hsalpha) gene and characterized its genomic region. HP1(Hsalpha) is located on human chromosome 12q13.13, 589 bp upstream of the divergently transcribed hnRNPA1 gene. Analysis of the promoter region revealed that differential regulation of HP1(Hsalpha) between the two types of breast cancer cells is lost upon mutation of an USF/c-myc transcription factor binding site located 172 bp upstream of the predicted HP1(Hsalpha) transcription start site. These findings provide insights into the down-regulation of HP1(Hsalpha) in highly invasive/metastatic breast cancer cells. To examine the functional properties of HP1(Hsalpha), experiments were performed using Drosophila melanogaster as a genetic system. When human HP1(Hsalpha) was expressed in transgenic Drosophila, silencing of reporter genes inserted at centric and telomeric locations was enhanced. Furthermore, expression of HP1(Hsalpha) rescued the lethality of homozygous Su(var)2-5 mutants lacking HP1. Taken together, these results demonstrate the participation of HP1(Hsalpha) in silent chromatin formation and that HP1(Hsalpha) is a functional homologue of Drosophila HP1.

ResumenObjetivosDescribir y comparar los conocimientos y actitudes de médicos y enfermeras sobre las instrucciones previas.DiseñoEstudio piloto descriptivo transversal.EmplazamientoÁrea Asistencial Este de la Comunidad de Madrid.ParticipantesMédicos y enfermeras de atención primaria y especializada.Mediciones principalesCuestionario autocumplimentado acerca de conocimientos y actitudes sobre las instrucciones previas, compuesto por variables dicotómicas y de escala tipo Likert (0-10).ResultadosRespondieron al cuestionario un total de 192 médicos y enfermeras (tasa de respuesta=83,4%); 72,4% eran mujeres y 27,6% hombres. La media de edad fue de 39,6 años (DE=10,9). Para el conocimiento general sobre las instrucciones previas la mediana fue de 5 (rango intercuartílico: 3-7). El 60,1% conocía la regulación por ley, pero solo el 22,8% había leído el documento.ConclusionesEl conocimiento de médicos y enfermeras sobre las instrucciones previas es bajo, por lo que es necesario mejorar este conocimiento. Los médicos y enfermeras de ambos niveles muestran actitudes favorables hacia el uso, la utilidad y el respeto del contenido del documento de instrucciones previas. El diseño metodológico propuesto es eficaz para aplicar en un estudio más amplio, aunque se debe mejorar el sistema de distribución y recogida de cuestionarios.AbstractObjectivesTo describe and compare the knowledge and attitudes of the physicians and nurses towards the advance directives.DesignA descriptive, cross-sectional pilot study.SettingEast healthcare area of the Community of Madrid (Spain)ParticipantsPrimary care and specialized care physicians and nurses.Main measurementsQuestionnaire about knowledge, use and attitudes from the healthcare professionals over the advance directives given, with dichotomous and Likert scale (0-10) variables.ResultsReplies were received from a total of 192 physicians and nurses (response rate=83,4%), 72,4% were women and 27,6% were men. The mean age was 39,6 years (SD=10,86). For general knowledge on advance directives the median was 5 (RI=3-7). 60,1% were aware of the regulation by law, but only 22,8% had read the document.ConclusionsThe knowledge of physicians and nurses on advance directives is low, so it is necessary to improve this knowledge. Physicians and nurses from both levels show positive attitude towards the use and usefulness and respect the contents of advance directive. The methodology proposed is efficient to implement in a larger study, but should improve the distribution and collection of questionnaires.