Yeast growth selection system for the identification of cell-active inhibitors of [beta]-secretase

The production and deposition of the cytotoxic Aβ peptide is a central event in the pathogenesis of Alzheimer’s disease (AD). Aβ is excised from the amyloid precursor protein (APP) through sequential actions of the β-secretase, which cleaves at the socalled β-site, and the γ-secretase, which cleaves at the so-called γ-site of APP. Inhibition the β-secretase BACE1 is a promising approach for AD therapy, but the search for small molecule inhibitors has proven to be challenging. In this thesis I describe a novel screening assay to identify cell-active BACE1 inhibitors by a positive yeast growth selection system, which combines the practicability of in vitro assays with the advantages of a cell-based assay. Analogue to the situation in mammalian cells, the β-site cleavage reaction was reconstituted in the secretory pathway of yeast cells using membrane-bound BACE1 and a membrane-bound APP-derived substrate. In contrast to conventional mammalian cell-based assays, false positive compounds can be rapidly excluded by the use of simple specificity controls that mimic the readout in the absence of BACE1. Furthermore, there is counter-selection for toxic compounds due to the positive growth readout upon inhibition of BACE1. The system was initially validated with two bona fide BACE1 inhibitors that stimulated the growth of BACE1-expressing cultures in a concentration-dependent manner, whereas the growth of control cultures remained unaffected in the presence of these inhibitors. In order to identify novel BACE1 inhibitors and to further validate the system, we screened a library of 15’000 small molecules. This screening revealed six compounds, which significantly reduced the secretion of Aβ from a human cell line overexpressing APP

Knowledge, attitudes and practices of medical staff towards obesity management in patients with spinal cord injuries: an International survey of four western European countries

Objective: To (1) examine the opinions of medical staff working in spinal cord injury (SCI) centres (SCICs); (2) evaluate their knowledge, attitudes and practices towards obesity prevention and management; (3) report the number of beds and dietitians available at each SCIC. Methods: A 37-item questionnaire was sent to 23 SCICs in the UK, the Netherlands, Belgium and the Republic of Ireland between September 2012 and January 2013. Results: Eighteen SCICs returned the questionnaires for analysis. All respondents stated that they had an interest in obesity treatment but only 2.3% of the respondents received training in obesity management. Sixty-one percent of staff did not consider body mass index (BMI) to be appropriate for use in SCI patients and subsequently less than half of the respondents use BMI routinely. The majority of respondents reported that they are confident in dealing with overweight (74.5%) and obese (66.1%) SCI adults, less than half (44.1%) are confident in treating overweight and obese SCI children. Respondents also indicated the need for nationally adopted guidelines and a lack of physical activity provision. There were 17.5 whole-time equivalent (WTE) dietitians recorded in 22 SCICs, equivalent to 47.8 beds per WTE dietitians (range 10–420). Non-UK SCIC dietitians are significantly better resourced than in UK SCICs (beds per WTE dietitian: 36 vs 124, P=0.035). Conclusion: Medical staff expressed the need to participate in obesity prevention and management. Appropriate training should be considered for all medical staff and the development of specific weight management guidelines and dietetic provision should be considered

Can verbal suggestions strengthen the effects of a relaxation intervention?

Short stress management interventions such as relaxation therapy have demonstrated preliminary effectiveness in reducing stress-related problems. A promising tool to strengthen the effectiveness of relaxation-based interventions is the use of verbal suggestions, as previous research provided evidence that verbal suggestions can induce positive outcome expectancies, facilitate adaptive responses to stress and improve health outcomes. The present experimental proof-of-concept study aimed to investigate the effects of a brief relaxation intervention and specifically the role of verbal suggestions on stress-related outcomes assessed by self-report questionnaires and psychophysiological data. 120 participants (mean age = 22.1 years) were randomized to one of four intervention conditions: a brief relaxation intervention plus verbal suggestions condition, a brief relaxation intervention only condition, a verbal suggestions only condition, and a control condition. Afterwards, participants were subjected to a psychosocial stress challenge to assess reactivity to a stressful event. Immediately after both relaxation interventions (with and without verbal suggestions), lower self-reported state anxiety was found compared to the control condition, but no differences were observed in response to the stressor. The verbal suggestions only condition did not impact state anxiety. No significant effects were found for verbal suggestion interventions on cortisol, alpha amylase, heart rate and skin conductance. This is the first study investigating the role of verbal suggestions in the effectiveness of a brief relaxation intervention. Although this experimental proof-of-concept study provides support for the effectiveness of a brief relaxation intervention in lowering state anxiety directly after the intervention, the effects did not impact the response to a subsequent stressor and we did not observe any evidence for the add-on effectiveness of verbal suggestions. The effectiveness of brief relaxation interventions on stress responses should be investigated further in future research by incorporating interventions that are tailored to the specific stress challenge and various types of verbal suggestions

The role of outcome expectancies for a training program consisting of meditation, breathing exercises, and cold exposure on the response to endotoxin administration: A proof-of-principle study.

Expectancies play a major role for the treatment outcome of a broad variety of immune-mediated conditions and may strengthen or mimic the effects of regular long-term therapies. This study adds to a recently published study of Kox et al. (PNAS 111:7379-7384, 2014) on the ability to voluntarily influence the physiological stress response in healthy men after a training program consisting of meditation, breathing techniques, and exposure to cold, which found highly promising results on the clinical, autonomic, and immune response to experimentally induced inflammation (using the experimental human endotoxemia model). Within this project, a number of variables were included to assess the role of generalized (optimism, neuroticism) and specific outcome expectancies (related to the effects of the training on health) on the response to endotoxin administration after training. Indications were found that especially the generalized outcome expectancy optimism is a potential determinant of the autonomic (epinephrine: rho = 0.76, p

Applying the Common Sense Model to predicting quality of life in alopecia areata: The role of illness perceptions and coping strategies

Item does not contain fulltextApplying the Common Sense Model, this cross-sectional study examines associations between illness perceptions and quality of life and the mediating role of coping in 243 adults with alopecia areata, a chronic dermatological condition. At least some QoL impairment was reported by 84 percent of participants, with 31 percent reporting very to extremely large impairment. Stronger perceptions of consequences, emotional representations, identity, and lower attribution to chance were related to more impairment, with avoidant coping acting as (partial) mediator. Illness perceptions and avoidant coping seem to play an important role in QoL and are relevant intervention targets in alopecia areata

The Edwin Smith papyrus: a clinical reappraisal of the oldest known document on spinal injuries

Dating from the seventeenth century b.c. the Edwin Smith papyrus is a unique treatise containing the oldest known descriptions of signs and symptoms of injuries of the spinal column and spinal cord. Based on a recent “medically based translation” of the Smith papyrus, its enclosed treasures in diagnostic, prognostic and therapeutic reasoning are revisited. Although patient demographics, diagnostic techniques and therapeutic options considerably changed over time, the documented rationale on spinal injuries can still be regarded as the state-of-the-art reasoning for modern clinical practice

Bruton's Tyrosine Kinase Cooperates with the B Cell Linker Protein SLP-65 as a Tumor Suppressor in Pre-B Cells

Expression of the pre-B cell receptor (pre-BCR) leads to activation of the adaptor molecule SLP-65 and the cytoplasmic kinase Btk. Mice deficient for one of these signaling proteins have an incomplete block in B cell development at the stage of large cycling pre-BCR+CD43+ pre-B cells. Our recent findings of defective SLP-65 expression in ∼50% of childhood pre-B acute lymphoblastic leukemias and spontaneous pre-B cell lymphoma development in SLP-65−/− mice demonstrate that SLP-65 acts as a tumor suppressor. To investigate cooperation between Btk and SLP-65, we characterized the pre-B cell compartment in single and double mutant mice, and found that the two proteins have a synergistic role in the developmental progression of large cycling into small resting pre-B cells. We show that Btk/SLP-65 double mutant mice have a dramatically increased pre-B cell tumor incidence (∼75% at 16 wk of age), as compared with SLP-65 single deficient mice (<10%). These findings demonstrate that Btk cooperates with SLP-65 as a tumor suppressor in pre-B cells. Furthermore, transgenic low-level expression of a constitutive active form of Btk, the E41K-Y223F mutant, prevented tumor formation in Btk/SLP-65 double mutant mice, indicating that constitutive active Btk can substitute for SLP-65 as a tumor suppressor