Structural and functional analysis of the human Rad50 and Mre11 DNA repair complex : reaching out for flexible solutions

DNA, the genetic material, contains essential information for the proper function of cells. lt is therefore of great importance that the integrity of the genome is carefully maintained during metabolic processes. Special challenges for genome maintenance pathways are DNA ends that arise in cells due to different causes. DNA ends, in the form of telomeres, naturally occur at the termini of the linear eukaryotic chromosomes. Random DNA double-strand breaks (DSBs) can be induced by various exogenous and endogenous DNA darnaging agents. In contrast, the cell can intentionally induce sitespecific DSBs in specialized processes to create genetic diversity during immune system development and meiosis. Impraper processing of these DNA ends imposes a high risk for genetic rearrangements that can lead to cell death, cell maltunetion or carcinogenesis. Therefore, it is of great importance that the DNA ends are processed and/or repaired accurately. For this purpose several mechanisms exist that depend on specific specialized protein machineries. The DSB repair pathway used depends on the circumstances under which the break occurs. One protein complex that is involved in multiple genome maintenance pathways that deal with DNA ends consists of three proteins: Rad50, Mre11 and Nbs1. The aim of this thesis is to ga in insights in the role of this Rad50 protein complex in DNA end metabolis

Multiple Aspects of ATP-Dependent Nucleosome Translocation by RSC and Mi-2 Are Directed by the Underlying DNA Sequence

Contains fulltext : 129351.pdf (publisher's version ) (Open Access)Background Chromosome structure, DNA metabolic processes and cell type identity can all be affected by changing the positions of nucleosomes along chromosomal DNA, a reaction that is catalysed by SNF2-type ATP-driven chromatin remodelers. Recently it was suggested that in vivo, more than 50% of the nucleosome positions can be predicted simply by DNA sequence, especially within promoter regions. This seemingly contrasts with remodeler induced nucleosome mobility. The ability of remodeling enzymes to mobilise nucleosomes over short DNA distances is well documented. However, the nucleosome translocation processivity along DNA remains elusive. Furthermore, it is unknown what determines the initial direction of movement and how new nucleosome positions are adopted. Methodology/Principal Findings We have used AFM imaging and high resolution PAGE of mononucleosomes on 600 and 2500 bp DNA molecules to analyze ATP-dependent nucleosome repositioning by native and recombinant SNF2-type enzymes. We report that the underlying DNA sequence can control the initial direction of translocation, translocation distance, as well as the new positions adopted by nucleosomes upon enzymatic mobilization. Within a strong nucleosomal positioning sequence both recombinant Drosophila Mi-2 (CHD-type) and native RSC from yeast (SWI/SNF-type) repositioned the nucleosome at 10 bp intervals, which are intrinsic to the positioning sequence. Furthermore, RSC-catalyzed nucleosome translocation was noticeably more efficient when beyond the influence of this sequence. Interestingly, under limiting ATP conditions RSC preferred to position the nucleosome with 20 bp intervals within the positioning sequence, suggesting that native RSC preferentially translocates nucleosomes with 15 to 25 bp DNA steps. Conclusions/Significance Nucleosome repositioning thus appears to be influenced by both remodeler intrinsic and DNA sequence specific properties that interplay to define ATPase-catalyzed repositioning. Here we propose a successive three-step framework consisting of initiation, translocation and release steps to describe SNF2-type enzyme mediated nucleosome translocation along DNA. This conceptual framework helps resolve the apparent paradox between the high abundance of ATP-dependent remodelers per nucleus and the relative success of sequence-based predictions of nucleosome positioning in vivo

Perceptions, experiences, barriers and facilitators regarding nutritional intake of patients with chronic limb threatening ischemia:a qualitative study

Objective: Patients with chronic limb threatening ischemia (CLTI) are at high risk for amputation and other cardiovascular adverse events. Nutrition-related symptoms and malnutrition are common in the CLTI population, and lead to worse clinical outcomes. Understanding of the factors influencing nutritional intake is required to determine whether optimization of nutritional intake in this population requires interventions. Therefore, this study aimed to describe perceptions and experiences on nutrition of patients with CLTI, and to identify perceived barriers and facilitators influencing their nutritional intake.Methods: In this phenomenological qualitative study, individual semi-structured, face-to-face interviews were conducted with patients with CLTI who lived independently. Interviews were transcribed verbatim, and reflexive thematic analysis was performed.Results: Twelve participants were interviewed. Five themes were generated: (1) lack of nutritional risk perception, (2) role of nutrition for health, functioning and surviving, (3) multiple factors influencing nutritional intake, (4) limited nutritional advice, and (5) no intention to change current nutritional intake.Conclusion: Patients with CLTI perceive nutritional intake as a necessity to survive and function. Patients express limited risk perception regarding adequate nutritional intake and undernutrition. Nutritional intake is mainly based on non-health related factors, as habits and taste, and multiple barriers hinder nutritional intake. Patients received no or only limited nutritional advice. Together this leads to an expressed lack of intention to change nutritional intake. Findings of this study stress the urgency for patient-centered nutritional support, to increase nutrition-related knowledge and motivation, to prevent or treat undernutrition, and may improve clinical outcomes in patients with CLTI

Gastric Acid Suppressive Therapy and Community-Acquired Pneumonia, Etiology and Outcome

Background: Community acquired pneumonia (CAP) is an infection of the pulmonary parenchyma that can be caused by various microbial pathogens. Co-morbidity and medication are related to specific pathogens. Patients on gastric acid suppressive therapy have an increased risk to develop CAP. We aimed to assess whether there are specific pathogens independently associated with gastric acid suppressive therapy and its impact on infection severity. Methods: From December 2007 to January 2010, all subjects consulting the emergency care unit of a general hospital in the south of the Netherlands with a suspected CAP were prospectively registered. Each patient underwent chest radiography. Sputum, urine, nose swabs and blood samples were obtained for microbial culture, antigen detection and polymerase chain reaction techniques, respectively. To study the severity of CAP upon presentation, the validated CURB-65 score was calculated. Furthermore, we assessed hospital or intensive care admission, length of hospitalization and in-hospital mortality. We evaluated the association between use of acid suppressive therapy and microbial aetiology of CAP and severity of illness with logistic regression analysis. Results: The final cohort comprised 463 patients with CAP, defined as presence of infiltrate on chest radiography and/ or microbial aetiology. Overall 136 patients (29%) used acid suppressive therapy, mainly proton pump inhibitors (97%). Patients with acid suppressive therapy more frequently had an infection with Streptococcus pneumoniae (28% vs. 14%) and Haemophilus influenzae (10% vs. 6%), and less frequently with Coxiella burnetii (8% vs. 19%) or H1N1 influenza A virus (2% vs. 7%) in comparison to those without acid suppressive therapy. After adjustment for baseline differences, the risk of proton pump inhibitor users being infected with S. pneumonia was 2.18 times (95%Confidence Interval(CI): 1.2-3.6) higher compared to those not on acid suppressive therapy. Patients using more than one defined daily dose of a PPI had a 1.48-fold increased risk of a S. pneumoniae infection compared with patients using the defined daily dose (95%CI:1.1-2.0). No risk between PPI use and any other microbial pathogen was found. Patients with acid suppressive therapy had on average higher CURB-65 scores, longer hospital stay and subsequently a case fatality rate of 11% vs. 4% compared to those not using acid suppressive therapy. Conclusions. Proton pump inhibitor therapy predisposes with community acquired S. pneumoniae pneumonia, and was associated with higher morbidity

Omineca Herald, April, 10, 1925

Purpose: The purpose of the study is to identify demographic, clinical, lifestyle-related, and social-cognitive correlates of physical activity (PA) intention and behavior in head and neck cancer (HNC) survivors using the theory of planned behavior (TPB). Methods: Data from two cross-sectional studies on correlates of PA in HNC survivors were pooled. Both studies used self-reports to assess PA and social-cognitive correlates. Potential correlates were collected via self-report or medical records. Univariable and multivariable multilevel linear mixed-effects models were built to identify correlates of PA intention and PA behavior (Z scores). Structural equation model analyses were conducted to study the full TPB model in one analysis, taking into account relevant covariates. Results: In total, 416 HNC survivors were surveyed. Their mean (SD) age was 66.6 (9.4) years; 64% were men, and 78% were diagnosed with laryngeal cancer. The structural equation model showed that PA intention was significantly higher in HNC survivors with a history of exercising, who had a more positive attitude, subjective norm, and perceived behavioral control. Patients with higher PA intention, higher PBC, a lower age, and without unintentional weight loss or comorbidities had higher PA behavior. The model explained 22.9% of the variance in PA intention and 16.1% of the variance in PA behavior. Conclusions: Despite significant pathways of the TPB model, the large proportion variance in PA intention and behavior remaining unexplained suggests the need for better PA behavior (change) models to guide the development of PA promotion programs, particularly for the elderly. Such programs should be tailored to comorbidities and nutritional status

Two-year cost effectiveness between two gradual tapering strategies in rheumatoid arthritis: Cost-utility analysis of the TARA trial

Objective The aim of the current study was to evaluate the 2-year cost-utility ratio between tapering conventional synthetic disease-modifying antirheumatic drugs (csDMARD) first followed by the tumour necrosis factor (TNF)-inhibitor, or vice versa, in patients with rheumatoid arthritis (RA). Methods Two-year data of the Tapering strategies in Rheumatoid Arthritis trial were used. Patients with RA, who used both a csDMARD and a TNF-i

Age-related difference in health care use and costs of patients with chronic kidney disease and matched controls

_Background_ The financial burden of chronic kidney disease (CKD) is increasing due to the ageing population and increased prevalence of comorbid diseases. Our aim was to evaluate age-related differences in health care use and costs in Stage G4/G5 CKD without renal replacement therapy (RRT), dialysis and kidney transplant patients and compare them to the general population. _Methods_ Using Dutch health care claims, we identified CKD patients and divided them into three groups: CKD Stage G4/G5 without RRT, dialysis and kidney transplantation. We matched them with two controls per patient. Total health care costs and hospital costs unrelated to CKD treatment are presented in four age categories (19–44, 45–64, 65–74 and ≥75 years). _Results_ Overall, health care costs of CKD patients ≥75 years of age were lower than costs of patients 65–74 years of age. In dialysis patients, costs were highest in patients 45–64 years of age. Since costs of controls increased gradually with age, the cost ratio of patients versus controls was highest in young patients (19–44 years). CKD patients were in greater need of additional specialist care than the general population, which was already evident in young patients. _Conclusion_ Already at a young age and in the earlier stages of CKD, patients are in need of additional care with corresponding health care costs far exceeding those of the general population. In contrast to the general population, the oldest patients (≥75 years) of all CKD patient groups have lower costs than patients 65–74 years of age, which is largely explained by lower hospital and medication costs

Healthcare costs of patients on different renal replacement modalities – Analysis of Dutch health insurance claims data

Background The aim of this study is to present average annual healthcare costs for Dutch renal replacement therapy (RRT) patients for 7 treatment modalities. Methods Health insurance claims data from 2012–2014 were used. All patients with a 2014 claim for dialysis or kidney transplantation were selected. The RRT related and RRT unrelated average annual healthcare costs were analysed for 5 dialysis modalities (in-centre haemodialysis (CHD), home haemodialysis (HHD), continuous ambulatory peritoneal dialysis (CAPD), automated peritoneal dialysis (APD) and multiple dialysis modalities in a year (Mix group)) and 2 transplant modalities (kidney from living and deceased donor, respectively). Results The total average annual healthcare costs in 2014 ranged from €77,566 (SD = €27,237) for CAPD patients to €105,833 (SD = €30,239) for patients in the Mix group. For all dialysis modalities, the vast majority (72–84%) of costs was RRT related. Patients on haemodialysis 4x/week had significantly higher average annual costs compared to those dialyzing 3x/ week (Δ€19,122). Costs for kidney transplant recipients were €85,127 (SD = €39,679) in the year of transplantation and rapidly declined in the first and second year after successful transplantation (resp. €29,612 (SD = €34,099) and €15,018 (SD = €16,186)). Transplantation with a deceased donor kidney resulted in higher costs (€99,450, SD = €36,036)) in the year of transplantation compared to a living donor kidney transplantation (€73,376, SD = €38,666). Conclusions CAPD patients have the lowest costs compared to other dialysis modalities. Costs in the year of transplantation are 25% lower for patients with kidneys from living vs. deceased donor. After successful transplantation, annual costs decline substantially to a level that is approximately 14–19% of annual dialysis costs

Delivery of modified mRNA to damaged myocardium by systemic administration of lipid nanoparticles

Lipid Nanoparticles (LNPs) are a promising drug delivery vehicle for clinical siRNA delivery. Modified mRNA (modRNA) has recently gained great attention as a therapeutic molecule in cardiac regeneration. However, for mRNA to be functional, it must first reach the diseased myocardium, enter the target cell, escape from the endosomal compartment into the cytosol and be translated into a functional protein. However, it is unknown if LNPs can effectively deliver mRNA, which is much larger than siRNA, to the ischemic myocardium. Here, we evaluated the ability of LNPs to deliver mRNA to the myocardium upon ischemia-reperfusion injury functionally. By exploring the bio-distribution of fluorescently labeled LNPs, we observed that, upon reperfusion, LNPs accumulated in the infarct area of the heart. Subsequently, the functional delivery of modRNA was evaluated by the administration of firefly luciferase encoding modRNA. Concomitantly, a significant increase in firefly luciferase expression was observed in the heart upon myocardial reperfusion when compared to sham-operated animals. To characterize the targeted cells within the myocardium, we injected LNPs loaded with Cre modRNA into Cre-reporter mice. Upon LNP infusion, Tdtomato+ cells, derived from Cre mediated recombination, were observed in the infarct region as well as the epicardial layer upon LNP infusion. Within the infarct area, most targeted cells were cardiac fibroblasts but also some cardiomyocytes and macrophages were found. Although the expression levels were low compared to LNP-modRNA delivery into the liver, our data show the ability of LNPs to functionally deliver modRNA therapeutics to the damaged myocardium, which holds great promise for modRNA-based cardiac therapies