Structural insights into the human RyR2 N-terminal region involved in cardiac arrhythmias

Human ryanodine receptor 2 (hRyR2) mediates calcium release from the sarcoplasmic reticulum, enabling cardiomyocyte contraction. The N-terminal region of hRyR2 (amino acids 1–606) is the target of >30 arrhythmogenic mutations and contains a binding site for phosphoprotein phosphatase 1. Here, the solution and crystal structures determined under near-physiological conditions, as well as a homology model of the hRyR2 N-terminal region, are presented. The N-terminus is held together by a unique network of interactions among its three domains, A, B and C, in which the central helix (amino acids 410–437) plays a prominent stabilizing role. Importantly, the anion-binding site reported for the mouse RyR2 N-terminal region is notably absent from the human RyR2. The structure concurs with the differential stability of arrhythmogenic mutations in the central helix (R420W, I419F and I419F/R420W) which are owing to disparities in the propensity of mutated residues to form energetically favourable or unfavourable contacts. In solution, the N-terminus adopts a globular shape with a prominent tail that is likely to involve residues 545–606, which are unresolved in the crystal structure. Docking the N-terminal domains into cryo-electron microscopy maps of the closed and open RyR1 conformations reveals C atom movements of up to 8 A ° upon channel gating, and predicts the location of the leucine– isoleucine zipper segment and the interaction site for spinophilin and phosphoprotein phosphatase 1 on the RyR surface

A regulatory component of the human ryanodine receptor 2 N-terminus

Human cardiac ryanodine receptor (hRyR2) is a channel mediating Ca2+ release from the sarcoplasmic reticulum during excitation-contraction coupling. The N-terminal (1-655) and central (2100-2500) regions of hRyR2 are thought to be involved in regulating channel gating. Mutations linked to several heart diseases are clustered within these two, as well as in the channel pore-containing C-terminal regions. High resolution structures of key regions involved in the regulation of RyR2 activity could further the understanding of the gating mechanism of hRyR2 and of its malfunction in disease

Coordination of multiple enzyme activities by a single PCNA in archaeal Okazaki fragment maturation

In vitro reconstitution of Okazaki fragment processing shows that DNA polymerase, flap endonuclease and DNA ligase need to simultaneously bind to the same PCNA-sliding clamp molecule during DNA lagging strand replication

The architecture of an Okazaki fragment-processing holoenzyme from the archaeon Sulfolobus solfataricus

DNA replication on the lagging strand occurs via the synthesis and maturation of Okazaki fragments. In archaea and eukaryotes, the enzymatic activities required for this process are supplied by a replicative DNA polymerase, Flap endonuclease 1 (Fen1) and DNA ligase 1 (Lig1). These factors interact with the sliding clamp PCNA (proliferating cell nuclear antigen) providing a potential means of co-ordinating their sequential actions within a higher order assembly. In hyperthermophilic archaea of the Sulfolobus genus, PCNA is a defined heterotrimeric assembly and each subunit interacts preferentially with specific client proteins. We have exploited this inherent asymmetry to assemble a PCNA-polymerase-Fen1-ligase complex on DNA and have visualized it by electron microscopy. Our studies reveal the structural basis of co-occupancy of a single PCNA ring by the three distinct client proteins

Reduced Stability and Increased Dynamics in the Human Proliferating Cell Nuclear Antigen (PCNA) Relative to the Yeast Homolog

Proliferating Cell Nuclear Antigen (PCNA) is an essential factor for DNA replication and repair. PCNA forms a toroidal, ring shaped structure of 90 kDa by the symmetric association of three identical monomers. The ring encircles the DNA and acts as a platform where polymerases and other proteins dock to carry out different DNA metabolic processes. The amino acid sequence of human PCNA is 35% identical to the yeast homolog, and the two proteins have the same 3D crystal structure. In this report, we give evidence that the budding yeast (sc) and human (h) PCNAs have highly similar structures in solution but differ substantially in their stability and dynamics. hPCNA is less resistant to chemical and thermal denaturation and displays lower cooperativity of unfolding as compared to scPCNA. Solvent exchange rates measurements show that the slowest exchanging backbone amides are at the β-sheet, in the structure core, and not at the helices, which line the central channel. However, all the backbone amides of hPCNA exchange fast, becoming undetectable within hours, while the signals from the core amides of scPCNA persist for longer times. The high dynamics of the α-helices, which face the DNA in the PCNA-loaded form, is likely to have functional implications for the sliding of the PCNA ring on the DNA since a large hole with a flexible wall facilitates the establishment of protein-DNA interactions that are transient and easily broken. The increased dynamics of hPCNA relative to scPCNA may allow it to acquire multiple induced conformations upon binding to its substrates enlarging its binding diversity

Multinational prospective cohort study of rates and risk factors for ventilator-associated pneumonia over 24 years in 42 countries of Asia, Africa, Eastern Europe, Latin America, and the Middle East: Findings of the International Nosocomial Infection Control Consortium (INICC)

Objective: Rates of ventilator-associated pneumonia (VAP) in low- and middle-income countries (LMIC) are several times above those of high-income countries. The objective of this study was to identify risk factors (RFs) for VAP cases in ICUs of LMICs. Design: Prospective cohort study. Setting: This study was conducted across 743 ICUs of 282 hospitals in 144 cities in 42 Asian, African, European, Latin American, and Middle Eastern countries. Participants: The study included patients admitted to ICUs across 24 years. Results: In total, 289,643 patients were followed during 1,951,405 patient days and acquired 8,236 VAPs. We analyzed 10 independent variables. Multiple logistic regression identified the following independent VAP RFs: male sex (adjusted odds ratio [aOR], 1.22; 95% confidence interval [CI], 1.16-1.28; P <.0001); longer length of stay (LOS), which increased the risk 7% per day (aOR, 1.07; 95% CI, 1.07-1.08; P <.0001); mechanical ventilation (MV) utilization ratio (aOR, 1.27; 95% CI, 1.23-1.31; P <.0001); continuous positive airway pressure (CPAP), which was associated with the highest risk (aOR, 13.38; 95% CI, 11.57-15.48; P <.0001)Revisión por pare

The impact of diagnosis on the psychosocial and spiritual needs of the patients

Introduction: Holistic care focuses on the relationship between the body, mind, and spirit. Spiritual needs are among the basic needs of the individual. From the physical and spiritual dimension and the interaction of these two dimensions, the spiritual needs of the person are developed. Aim:The aim of our study was to analyze if the patient's knowledge regarding their diagnosis and possible proximity of death have significant impact on satisfaction of psychological and spiritual needs. Material and Methods:In our study we used standardized questionnaire The Spiritual Needs Assessment for Patient – SNAP constructed by Sharma Rashmi, applied for 113 respondents and processed by SPSS statistical program. Results:Reliability of questionnaire is given by Cronbach's Alpha 0.945 for the total SNAP. A statistically significant difference at the level p < 0.01 has been confirmed in all questions among respondents with fatal diagnosis and respondents with not life-threatening diagnosis. In domain of psychosocial needs, significant difference was not confirmed regarding to gender, age, education, income, religion and place of residency. In domain of spiritual needs, significant difference was partially confirmed regarding religion in 4 questions from 13. Conclusion:Faced with chronic or fatal diseases, many patients rely on dealing with spiritual and religious issues. In fact, spirituality/religiosity can be considered an important source of support and management of severe chronic diseases. It is possible to identify the four basic dimensions of spiritual need: interconnection, peace, meaning/purpose, and transcendence. Patients often have the problem addressing their needs for related psychosocial and physical problems such as physical disability, fatigue, sleep disorders, side effects of treatment, etc. Importance of satisfaction of psychosocial and spiritual needs is increased in time of threat, fatal diagnosis

Effect of fertilization and variety on the beta-glucan content in the grain of oats

The object of the study was to investigate the influence fertilization on the β-glucan content of the common oats varieties (Vendelin, Zvolen) and the naked oats varieties (Detvan, Avenuda). The trial was established in the years 2007 and 2008 in the potato growing area in the centre of Slovakia in VígľašPstruša. The field treatments were realized in natural conditions without irrigation with four variants of fertilization. Nitrogen fertilization was applied before sowing and foliar application of selenium together with nitrogen was done in the growth phase at the end of stooling (BBCH 29). The amount of β-glucan in the samples was determined by using the β-glucan assay kit (Megazyme, Ldt. Ireland). The total average of β-glucan content in the experiment was 4.08 %. The naked oats varieties reached higher content of β-glucan, when we compare it with the common oats. The variety Avenuda reached highest of β-glucan content (5.20 %). The nitrogen fertilization together with selenium fertilization statistically significantly increased the content of this polysaccharides. The influence of a particular year was statistically significant in β-glucan content

Quality of Life in Patients with Diabetic Foot Ulcer in the Visegrad Countries

AIM AND OBJECTIVES: The aim of the present study was to identify the quality of life of patients with diabetic foot ulcers in the Visegrad countries. BACKGROUND: The diabetics with foot ulcers are principally evaluated on the basis of physical parameters, but this does not always reveal much about the patient's experience of life with ulceration. DESIGN: The cross-sectional study. METHODS: The standardized generic questionnaire World Health Organisation Quality of Life Bref was used. The sample was made up of 525 participants and the calculations were performed using the IBM SPSS statistical program. RESULTS: The significant negative correlations between demographic data such as age, duration of diabetes mellitus, duration of diabetes ulceration treatment and a lower level of quality of life were found across the sample. The statistically significant differences according to clinical characteristics such as Wagner classification, frequency of foot ulcers, present peripheral vascular diseas and pain in terms of quality of life were also revealed. Significant differences of quality of life among Visegrad countries were revealed: Hungary's participants had a worse quality of life than others, while Slovak participants expressed lower satisfaction with their health than Czech. CONCLUSIONS: Socio-demographic factors and clinical characteristics influence the quality of life of patients with diabetic foot ulcer. Significant differences between patients of Visegrad countries were found in all domains of quality of life: physical, psychological, social and environmental. RELEVANCE TO CLINICAL PRACTICE: The quality of life of patients with diabetic foot ulcer reflects the conditions and health care system in each of the Visegrad countries. We have to respect socio-demographic factors and clinical characteristics in nursing care. This could have an impact on managing patient care not only with regard to their diabetic foot ulcer but also with regard to the patient as a personality with their own problems in relation to physical, psychosocial and environmental conditions. This article is protected by copyright. All rights reserved