Illuminating the functional and structural repertoire of human TBC/RABGAPs

The Tre2-Bub2-Cdc16 (TBC) domain-containing RAB-specific GTPase-activating proteins (TBC/RABGAPs) are characterized by the presence of highly conserved TBC domains and act as negative regulators of RABs. The importance of TBC/RABGAPs in the regulation of specific intracellular trafficking routes is now emerging, as is their role in different diseases. Importantly, TBC/RABGAPs act as key regulatory nodes, integrating signalling between RABs and other small GTPases and ensuring the appropriate retrieval, transport and delivery of different intracellular vesicles.     

The Relay/Converter Interface Influences Hydrolysis of ATP by Skeletal Muscle Myosin II

The interface between relay and converter domain of muscle myosin is critical for optimal myosin performance. Using Drosophila melanogaster indirect flight muscle S1, we performed a kinetic analysis of the effect of mutations in the converter and relay domain. Introduction of a mutation (R759E) in the converter domain inhibits the steady-state ATPase of myosin S1, whereas an additional mutation in the relay domain (N509K) is able to restore the ATPase toward wild-type values. The R759E S1 construct showed little effect on most steps of the actomyosin ATPase cycle. The exception was a 25–30% reduction in the rate constant of the hydrolysis step, the step coupled to the cross-bridge recovery stroke that involves a change in conformation at the relay/converter domain interface. Significantly, the double mutant restored the hydrolysis step to values similar to the wild-type myosin. Modeling the relay/converter interface suggests a possible interaction between converter residue 759 and relay residue 509 in the actin-detached conformation, which is lost in R759E but is restored in N509K/R759E. This detailed kinetic analysis of Drosophila myosin carrying the R759E mutation shows that the interface between the relay loop and converter domain is important for fine-tuning myosin kinetics, in particular ATP binding and hydrolysis.     

Implementatie van een proactief zorgprogramma voor kwetsbare ouderen in de huisartsenpraktijk: van onderzoek naar praktijk

Background

Multimorbidity, functional impairment and frailty among community-dwelling older people are causing increasing complexity in primary care. A proactive integrated primary care approach is therefore essential. Between October 2014–2015, an evidence-based proactive care program for frail older people was implemented in the region Noord-West Veluwe en Zeewolde, the Netherlands. This study evaluated the feasibility of the implementation, having a strong focus on the collaboration between the medical and social domain.

Methods

Using a mixed-methods design we evaluated several process indicators. Data were obtained from electronic routine medical record data within primary care, questionnaires, and interviews with older adults. The questionnaires provided information regarding the expectations and experiences towards the program and were sent to health care professionals at baseline and six months follow-up. Stakeholders from various domains were asked to fill in the questionnaire at baseline and twelve months follow-up. Interviews were conducted to explore the experiences of older adults with the program. Regional work groups were set up in each municipality to enhance the interdisciplinary and domain transcending collaboration.

Results

The proactive primary care program was implemented in 42 general practices who provided care to 7904 older adults aged 75 years or older. A total of 101 health care professionals and 44 stakeholders filled in the questionnaires. The need for better structure and interdisciplinary cooperation seemed widespread among the participants. The implementation resulted in a positive significant change in the demand for a better regional healthcare-framework (34% p ≤ .001) among health care professionals, and the needs for transparency regarding the possibilities for referral improved (27% , p = .009). Half of the participants reported that the regional collaboration has been improved after the implementation. Health care professionals and stakeholders gained increased attention and awareness of frail elderly in their area compared to before the implementation. Older people and their caregivers were positive about the proactive approach. The nurses reported that the screenings questionnaire was too lengthy and therefore time consuming.

Conclusions

The implementation of the proactive primary care approach in daily practice was feasible. A strong interdisciplinary collaboration was realized. The program was easily adapted to the local context.

     

Effect and Process Evaluation of a Cluster Randomized Control Trial on Water Intake and Beverage Consumption in Preschoolers from Six European Countries: The ToyBox-Study

BackgroundWithin the ToyBox-study, a kindergarten-based, family-involved intervention was developed to prevent overweight and obesity in European preschoolers, targeting four key behaviours related to early childhood obesity, including water consumption. The present study aimed to examine the effect of the ToyBox-intervention (cluster randomized controlled trial) on water intake and beverage consumption in European preschoolers and to investigate if the intervention effects differed by implementation score of kindergartens and parents/caregivers.MethodA sample of 4964 preschoolers (4.7±0.4 years; 51.5% boys) from six European countries (Belgium, Bulgaria, Germany, Greece, Poland, Spain) was included in the data analyses. A standardized protocol was used and parents/caregivers filled in socio-demographic data and a food-frequency questionnaire. To assess intervention effects, multilevel repeated measures analyses were conducted for the total sample and for the six country-specific samples. Based on the process evaluation questionnaire of teachers and parents/caregivers, an implementation score was constructed. To assess differences in water intake and beverage consumption by implementation score in the total sample, multilevel repeated measures analyses were performed.ResultsLimited intervention effects on water intake from beverages and overall beverage consumption were found. However, important results were found on prepacked fruit juice consumption, with a larger decrease in the intervention group compared to the control group. However, also a decline in plain milk consumption was found. Implementation scores were rather low in both kindergartens and parents/caregivers. Nevertheless, more favorable effects on beverage choices were found in preschoolers whose parents/caregivers and kindergarten teachers had higher implementation scores compared to those with lower implementation scores.ConclusionThe ToyBox-intervention can provide the basis for the development of more tailor-made interventions. However, new strategies to improve implementation of interventions should be created.     

Health Related Behaviours in Normal Weight and Overweight Preschoolers of a Large Pan-European Sample: The ToyBox-Study

The aim of this study was to investigate the associations of health related behaviours (HRB) with Body Mass Index (BMI) in preschoolers, and to study the likelihood of being overweight/obese in relation to compliance with recommended HRB. The sample consisted of 3301 normal weight and overweight/obese preschoolers (mean age: 4.7 years; 52% boys, 85% normal weight) from six European countries (Belgium, Bulgaria, Germany, Greece, Poland, Spain). Height and weight were measured, total daily step counts were registered during six days, and HRB were assessed with validated parental surveys in 2012. Multiple linear and logistic regression analyses were performed. Only few HRB were significantly associated with BMI. In boys, higher water intake and higher soft drink and higher fruit consumption were significantly associated with higher BMI. Boys drinking less water than recommended were less likely to be overweight/obese (OR = 0.60), while boys who consume soft drinks were more likely to be overweight/obese (OR = 1.52). In girls, higher water intake, higher vegetable consumption, and more TV time on weekend days were significantly associated with higher BMI. Girls eating less vegetables than recommended were less likely to be overweight/obese (OR = 0.62), and girls who engaged in quiet play for more than 90 minutes on weekend days were more likely to be overweight/obese (OR = 1.64). In general, the associations between HRB and BMI or being overweight/obese were limited and mainly related to dietary intake. Awareness campaigns for caregivers should stress that HRB of young children are important and independent of children’s weight status.     

Homocysteine affects cardiomyocyte viability: concentration-dependent effects on reversible flip-flop, apoptosis and necrosis

Background Hyperhomocysteinaemia (HHC) is thought to be a risk factor for cardiovascular disease including heart failure. While numerous studies have analyzed the role of homocysteine (Hcy) in the vasculature, only a few studies investigated the role of Hcy in the heart. Therefore we have analyzed the effects of Hcy on isolated cardiomyocytes. Methods H9c2 cells (rat cardiomyoblast cells) and adult rat cardiomyocytes were incubated with Hcy and were analyzed for cell viability. Furthermore, we determined the effects of Hcy on intracellular mediators related to cell viability in cardiomyocytes, namely NOX2, reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨ _m) and ATP concentrations. Results We found that incubation of H9c2 cells with 0.1 mM D,L-Hcy (= 60 μM l-Hcy) resulted in an increase of ΔΨ _m as well as ATP concentrations. 1.1 mM d,l-Hcy (= 460 μM l-Hcy) induced reversible flip-flop of the plasma membrane phospholipids, but not apoptosis. Incubation with 2.73 mM d,l-Hcy (= 1.18 mM l-Hcy) induced apoptosis and necrosis. This loss of cell viability was accompanied by a thread-to-grain transition of the mitochondrial reticulum, ATP depletion and nuclear NOX2 expression coinciding with ROS production as evident from the presence of nitrotyrosin residues. Notably, only at this concentration we found a significant increase in S-adenosylhomocysteine which is considered the primary culprit in HHC. Conclusion We found concentration-dependent effects of Hcy in cardiomyocytes, varying from induction of reversible flip-flop of the plasma membrane phospholipids, to apoptosis and necrosis.     

Mrp8 and Mrp14 are endogenous activators of Toll-like receptor 4, promoting lethal, endotoxin-induced shock

To identify new components that regulate the inflammatory cascade during sepsis, we characterized the functions of myeloid-related protein-8 (Mrp8, S100A8) and myeloid-related protein-14 (Mrp14, S100A9), two abundant cytoplasmic proteins of phagocytes. We now demonstrate that mice lacking Mrp8-Mrp14 complexes are protected from endotoxin-induced lethal shock and Escherichia coli-induced abdominal sepsis. Both proteins are released during activation of phagocytes, and Mrp8-Mrp14 complexes amplify the endotoxin-triggered inflammatory responses of phagocytes. Mrp8 is the active component that induces intracellular translocation of myeloid differentiation primary response protein 88 and activation of interleukin-1 receptor-associated kinase-1 and nuclear factor-kappaB, resulting in elevated expression of tumor necrosis factor-alpha (TNF-alpha). Using phagocytes expressing a nonfunctional Toll-like receptor 4 (TLR4), HEK293 cells transfected with TLR4, CD14 and MD2, and by surface plasmon resonance studies in vitro, we demonstrate that Mrp8 specifically interacts with the TLR4-MD2 complex, thus representing an endogenous ligand of TLR4. Therefore Mrp8-Mrp14 complexes are new inflammatory components that amplify phagocyte activation during sepsis upstream of TNFalpha-dependent effects.