Energy-dispersive EXAFS to study chemical reactions: the case of electron transfer reaction of hydroquinone

3 páginas, 3 figurasMost of the chemical processes that occur in nature take place in liquid media. For this reason, chemical reactions in solution have been widely studied for many years. Such studies have focused mainly on the mechanism of the reactions, whilst the structures of the species involved have received considerably less attention. This is mainly due to the difficulties of determining experimentally the structures of chemical species in liquid media. To date, this important question has largely been addressed by the standard techniques of UV-Vis and infrared spectroscopies. Unfortunately these techniques are seldom structurally specific, so the determination of the detailed chemical form of the majority of reactants has awaited the arrival of a fast and more structurally-focused method. Energy dispersive X-ray absorption spectroscopy (EDXAS), is ideal for this new class of experiments.Peer reviewe

Dimerization model of the C-terminal RNA Recognition Motif of HuR

In PressHuman antigen R (HuR) is a ubiquitous 32kDa protein comprising three RNA Recognition Motifs (RRMs), whose main function is to bind Adenylate and uridylate Rich Elements (AREs) in 3′ UnTranslated Regions (UTRs) of mRNAs. In addition to binding RNA molecules, the third domain (RRM3) is involved in HuR oligomerization and apoptotic signaling. The RRM3 monomer is able to dimerize, with its self-binding affinity being dependent on ionic strength. Here we provide a deeper structural insight into the nature of the encounter complexes leading to the formation of RRM3 dimers by using Brownian Dynamics and Molecular Dynamics. Our computational data show that the initial unspecific encounter follows a downhill pathway until reaching an optimum conformation stabilized by hydrophobic interactions.I.D.-M. wishes to thank the Andalusian Government (P07-CVI-02896, P11-CVI-07216 and BIO198) for financial support.Peer reviewe

HuR biological function involves RRM3-mediated dimerization and RNA binding by all three RRMs

HuR/ELAVL1 is an RNA-binding protein involved in differentiation and stress response that acts primarily by stabilizing messenger RNA (mRNA) targets. HuR comprises three RNA recognition motifs (RRMs) where the structure and RNA binding of RRM3 and of full-length HuR remain poorly understood. Here, we report crystal structures of RRM3 free and bound to cognate RNAs. Our structural, NMR and biochemical data show that RRM3 mediates canonical RNA interactions and reveal molecular details of a dimerization interface localized on the α-helical face of RRM3. NMR and SAXS analyses indicate that the three RRMs in full-length HuR are flexibly connected in the absence of RNA, while they adopt a more compact arrangement when bound to RNA. Based on these data and crystal structures of tandem RRM1,2-RNA and our RRM3-RNA complexes, we present a structural model of RNA recognition involving all three RRM domains of full-length HuR. Mutational analysis demonstrates that RRM3 dimerization and RNA binding is required for functional activity of full-length HuR in vitro and to regulate target mRNAs levels in human cells, thus providing a fine-tuning for HuR activity in vivo.España, MINECO BFU2015-71017España, Junta de Andalucía CVI-BIO198; P11-CVI7216 to I.D.M

TIA-1 RRM23 binding and recognition of target oligonucleotides

TIA-1 (T-cell restricted intracellular antigen-1) is an RNA-binding protein involved in splicing and translational repression. It mainly interacts with RNA via its second and third RNA recognition motifs (RRMs), with specificity for U-rich sequences directed by RRM2. It has recently been shown that RRM3 also contributes to binding, with preferential binding for C-rich sequences. Here we designed UC-rich and CU-rich 10-nt sequences for engagement of both RRM2 and RRM3 and demonstrated that the TIA-1 RRM23 construct preferentially binds the UC-rich RNA ligand (5΄-UUUUUACUCC-3΄). Interestingly, this binding depends on the presence of Lys274 that is C-terminal to RRM3 and binding to equivalent DNA sequences occurs with similar affinity. Small-angle X-ray scattering was used to demonstrate that, upon complex formation with target RNA or DNA, TIA-1 RRM23 adopts a compact structure, showing that both RRMs engage with the target 10-nt sequences to form the complex. We also report the crystal structure of TIA-1 RRM2 in complex with DNA to 2.3 Å resolution providing the first atomic resolution structure of any TIA protein RRM in complex with oligonucleotide. Together our data support a specific mode of TIA-1 RRM23 interaction with target oligonucleotides consistent with the role of TIA-1 in binding RNA to regulate gene expressio

Structural basis of mitochondrial dysfunction in response to cytochrome c phosphorylation at tyrosine 48

Regulation of mitochondrial activity allows cells to adapt to changing conditions and to control oxidative stress, and its dysfunction can lead to hypoxia-dependent pathologies such as ischemia and cancer. Although cytochrome c phosphorylation—in particular, at tyrosine 48—is a key modulator of mitochondrial signaling, its action and molecular basis remain unknown. Here we mimic phosphorylation of cytochrome c by replacing tyrosine 48 with p-carboxy-methyl-L-phenylalanine (pCMF). The NMR structure of the resulting mutant reveals significant conformational shifts and enhanced dynamics around pCMF that could explain changes observed in its functionality: The phosphomimetic mutation impairs cytochrome c diffusion between respiratory complexes, enhances hemeprotein peroxidase and reactive oxygen species scavenging activities, and hinders caspase-dependent apoptosis. Our findings provide a framework to further investigate the modulation of mitochondrial activity by phosphorylated cytochrome c and to develop novel therapeutic approaches based on its prosurvival effects.Financial support was provided by the Spanish Ministry of Economy and Competitiveness (Grants BFU2015-71017-P/BMC and BFU2015-19451/BMC, cofounded by FEDER EU), European Union (Bio-MR-00130 and CALIPSO-312284), Ramon Areces Foundation, and Andalusian Government (BIO198). B.M.-B. was awarded a PhD fellowship from the Spanish Ministry of Education (AP2009-4092) and a short-term traveling fellowship from the European Bio-NMR Project. A.G.-C. was awarded a PhD fellowship from the CSIC (JaePre-2011-01248).Peer reviewe

Fatores que influenciam o cuidado de enfermagem omitido em pacientes de um hospital particular

Objetivo: determinar os fatores que influenciam o cuidado de enfermagem omitido em pacientes hospitalizados. Método: estudo descritivo correlacional, desenvolvido em um hospital particular do México. Para identificar o cuidado omitido e fatores relacionados, utilizou-se o instrumento MISSCARE, que mede o cuidado omitido e os fatores associados. O cuidado omitido e os fatores foram agrupados em índices globais e por dimensões. Para fins de análise, foi utilizada estatística descritiva, correlação de Spearman e regressão linear simples. O estudo recebeu aprovação de comité de ética. Resultados: participaram 71 enfermeiras dos serviços de urgências, terapia intensiva e hospitalização. O índice global de cuidado omitido mostrou um coeficiente M=7,45 (DE=10,74); o índice com maior cuidado omitido correspondeu à dimensão de intervenções de cuidado básico (M=13,02, DE=17,60). O principal fator que contribuiu ao cuidado omitido foi o de recursos humanos (M=56,13, DE=21,38). Os fatores relacionados ao cuidado omitido foram os recursos humanos (rs=0,408, pObjetivo: determinar los factores que influyen en el cuidado de enfermería perdido en pacientes hospitalizados. Método: estudio descriptivo correlacional, se realizó en un hospital privado de México. Para identificar el cuidado perdido y factores relacionados se utilizó el instrumento MISSCARE que mide el cuidado perdido y los factores asociados. El cuidado perdido y los factores se agruparon en índices globales y por dimensiones. Para el análisis se utilizó estadística descriptiva, correlación de Spearman y regresión lineal simple. El estudio fue aprobado por el comité de ética. Resultados: participaron 71 enfermeras de los servicios de urgencias, terapia intensiva y hospitalización. El índice global de cuidado perdido mostró una M=7,45 (DE=10,74); el índice con mayor cuidado perdido correspondió a la dimensión de intervenciones de cuidado básico (M=13,02, DE=17,60). El principal factor que contribuyó en el cuidado perdido, fue el de recursos humanos (M=56,13, DE=21,38). Los factores relacionados con el cuidado perdido fueron los de recursos humanos (rs=0,408, pObjective: to determine the factors that influence the missed nursing care in hospitalized patients. Methods: descriptive correlational study developed at a private hospital in Mexico. To identify the missed nursing care and related factors, the MISSCARE survey was used, which measures the care missed and associated factors. The care missed and the factors were grouped in global and dimension rates. For the analysis, descriptive statistics, Spearman’s correlation and simple linear regression were used. Approval for the study was obtained from the ethics committee. Results: the participants were 71 nurses from emergency, intensive care and inpatient services. The global missed care index corresponded to M=7.45 (SD=10.74); the highest missed care index was found in the dimension basic care interventions (M=13.02, SD=17.60). The main factor contributing to the care missed was human resources (M=56.13, SD=21.38). The factors related to the care missed were human resources (rs=0.408,

Factors influencing the missed nursing care in patients from a private hospital

Objective: to determine the factors that influence the missed nursing care in hospitalized patients. Methods: descriptive correlational study developed at a private hospital in Mexico. To identify the missed nursing care and related factors, the MISSCARE survey was used, which measures the care missed and associated factors. The care missed and the factors were grouped in global and dimension rates. For the analysis, descriptive statistics, Spearman’s correlation and simple linear regression were used. Approval for the study was obtained from the ethics committee. Results: the participants were 71 nurses from emergency, intensive care and inpatient services. The global missed care index corresponded to M=7.45 (SD=10.74); the highest missed care index was found in the dimension basic care interventions (M=13.02, SD=17.60). The main factor contributing to the care missed was human resources (M=56.13, SD=21.38). The factors related to the care missed were human resources (rs =0.408, p<0.001) and communication (rs =0.418, p<0.001). Conclusions: the nursing care missed is mainly due to the human resource factor; these study findings will permit the strengthening of nursing care continuity. Descriptors: Nursing Care; Patient Care; Human Resources; Communication; Hospitals, Private

Oxidative stress is tightly regulated by cytochrome c phosphorylation and respirasome factors in mitochondria

Respiratory cytochrome c has been found to be phosphorylated at tyrosine 97 in the postischemic brain upon neuroprotective insulin treatment, but how such posttranslational modification affects mitochondrial metabolism is unclear. Here, we report the structural features and functional behavior of a phosphomimetic cytochrome c mutant, which was generated by site-specific incorporation at position 97 of p-carboxymethyl-l-phenylalanine using the evolved tRNA synthetase method. We found that the point mutation does not alter the overall folding and heme environment of cytochrome c, but significantly affects the entire oxidative phosphorylation process. In fact, the electron donation rate of the mutant heme protein to cytochrome c oxidase, or complex IV, within respiratory supercomplexes was higher than that of the wild-type species, in agreement with the observed decrease in reactive oxygen species production. Direct contact of cytochrome c with the respiratory supercomplex factor HIGD1A (hypoxia-inducible domain family member 1A) is reported here, with the mutant heme protein exhibiting a lower affinity than the wild-type species. Interestingly, phosphomimetic cytochrome c also exhibited a lower caspase-3 activation activity. Altogether, these findings yield a better understanding of the molecular basis for mitochondrial metabolism in acute diseases, such as brain ischemia, and thus could allow the use of phosphomimetic cytochrome c as a neuroprotector with therapeutic applications.España, Junta de Andalucía BIO-198España MINECO BFU2015-71017/BM

Structural basis of mitochondrial dysfunction in response to cytochrome c phosphorylation at tyrosine 48

Regulation of mitochondrial activity allows cells to adapt to changing conditions and to control oxidative stress, and its dysfunction can lead to hypoxia-dependent pathologies such as ischemia and cancer. Although cytochrome c phosphorylation—in particular, at tyrosine 48—is a key modulator of mitochondrial signaling, its action and molecular basis remain unknown. Here we mimic phosphorylation of cytochrome c by replacing tyrosine 48 with p-carboxy-methylL-phenylalanine (pCMF). The NMR structure of the resulting mutant reveals significant conformational shifts and enhanced dynamics around pCMF that could explain changes observed in its functionality: The phosphomimetic mutation impairs cytochrome c diffusion between respiratory complexes, enhances hemeprotein peroxidase and reactive oxygen species scavenging activities, and hinders caspase-dependent apoptosis. Our findings provide a framework to further investigate the modulation of mitochondrial activity by phosphorylated cytochrome c and to develop novel therapeutic approaches based on its prosurvival effects.España, MINECO BFU2015-71017-P/BMC and BFU2015- 19451/BMCUnión Europea, Bio-NMR-00130 and CALIPSO-312284España, Ministerio de Educación AP2009-409

Propuesta de estrategia comercial para empresa dedicada a la alta repostería, ubicada en el Área Metropolitana de Guadalajara

El desarrollo del proyecto se divide en tres partes, en la primera fase consiste en el conocimiento y entendimiento de la empresa de alta repostería “Desserty” así como el panorama general en el que se encuentra la industria de la compañía. En la fase 2, se habla sobre el diagnóstico de la empresa a profundidad donde se analiza en lo particular las áreas con las que cuentan, así como en general todo lo que generan. Por último, la fase 3, la cual incluye las propuestas de mejora, así como la estrategia de propuesta comercial.ITESO, A.C