Nationwide comprehensive gastro-intestinal cancer cohorts: the 3P initiative.

BACKGROUND: The increasing sub-classification of cancer patients due to more detailed molecular classification of tumors, and limitations of current trial designs, require innovative research designs. We present the design, governance and current standing of three comprehensive nationwide cohorts including pancreatic, esophageal/gastric, and colorectal cancer patients (NCT02070146). Multidisciplinary collection of clinical data, tumor tissue, blood samples, and patient-reported outcome (PRO) measures with a nationwide coverage, provides the infrastructure for future and novel trial designs and facilitates research to improve outcomes of gastrointestinal cancer patients. MATERIAL AND METHODS: All patients aged ≥18 years with pancreatic, esophageal/gastric or colorectal cancer are eligible. Patients provide informed consent for: (1) reuse of clinical data; (2) biobanking of primary tumor tissue; (3) collection of blood samples; (4) to be informed about relevant newly identified genomic aberrations; (5) collection of longitudinal PROs; and (6) to receive information on new interventional studies and possible participation in cohort multiple randomized controlled trials (cmRCT) in the future. RESULTS: In 2015, clinical data of 21,758 newly diagnosed patients were collected in the Netherlands Cancer Registry. Additional clinical data on the surgical procedures were registered in surgical audits for 13,845 patients. Within the first two years, tumor tissue and blood samples were obtained from 1507 patients; during this period, 1180 patients were included in the PRO registry. Response rate for PROs was 90%. The consent rate to receive information on new interventional studies and possible participation in cmRCTs in the future was >85%. The number of hospitals participating in the cohorts is steadily increasing. CONCLUSION: A comprehensive nationwide multidisciplinary gastrointestinal cancer cohort is feasible and surpasses the limitations of classical study designs. With this initiative, novel and innovative studies can be performed in an efficient, safe, and comprehensive setting.

Monocyte gene expression in childhood obesity is associated with obesity and complexity of atherosclerosis in adults.

Childhood obesity coincides with increased numbers of circulating classical CD14++CD16- and intermediate CD14++CD16+ monocytes. Monocytes are key players in the development and exacerbation of atherosclerosis, which prompts the question as to whether the monocytosis in childhood obesity contributes to atherogenesis over the years. Here, we dissected the monocyte gene expression profile in childhood obesity using an Illumina microarray platform on sorted monocytes of 35 obese children and 16 lean controls. Obese children displayed a distinctive monocyte gene expression profile compared to lean controls. Upon validation with quantitative PCR, we studied the association of the top 5 differentially regulated monocyte genes in childhood obesity with obesity and complexity of coronary atherosclerosis (SYNTAX score) in a cohort of 351 adults at risk for ischemic cardiovascular disease. The downregulation of monocyte IMPDH2 and TMEM134 in childhood obesity was also observed in obese adults. Moreover, downregulation of monocyte TMEM134 was associated with a higher SYNTAX atherosclerosis score in adults. In conclusion, childhood obesity entails monocyte gene expression alterations associated with obesity and enhanced complexity of coronary atherosclerosis in adults.

Altered Levels of Salivary 8-oxo-7-hydrodeoxyguanosine in Breast Cancer.

Oxidative stress has been implicated in the pathogenesis of breast cancer (BC). To determine whether BC is associated with altered salivary redox homeostasis, we performed a case-control study assessing the relationship between BC and 8-oxo-7-hydrodeoxyguanosine (8-oxodG), a marker for oxidative damage to DNA. Enzyme-linked immunosorbent assay for 8-oxodG was used on whole, unstimulated saliva of 134 BC patients and 226 healthy controls. Associations of the redox data were assessed by analysis of variance and logistic regression analysis. Our results revealed that there were 1) significantly lower mean levels of salivary 8-oxodG in BC patients versus controls ( P = 0.0005), 2) significantly lower levels among participants who did not receive radiotherapy and/or chemotherapy as compared with controls ( P < 0.0001), 3) significantly lower levels among BC patients who did not receive these treatments than among those who did ( P < 0.02), 4) and no significant differences in mean 8-oxodG levels among BC patients positive or negative for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 ( P ≥ 0.08). Our results suggest that BC is associated with decreased levels of oxidatively modified DNA in saliva. Knowledge Transfer Statement: The results of our current case-control study indicate that the molecular biomarker of oxidative stress 8-oxo-7-hydrodeoxyguanosine, measured from saliva, is associated with breast cancer. Our findings may provide the basis for future studies on molecular biomarkers of oxidative stress and breast cancer using saliva as an accessible and noninvasive tissue.

What is a new drug worth? An innovative model for performance-based pricing.

This article focuses on a novel method to derive prices for new pharmaceuticals by making price a function of drug performance. We briefly review current models for determining price for a new product and discuss alternatives that have historically been favoured by various funding bodies. The progressive approach to drug pricing, proposed herein, may better address the views and concerns of multiple stakeholders in a developed healthcare system by acknowledging and incorporating input from disparate parties via comprehensive and successive negotiation stages. In proposing a valid construct for performance-based pricing, the following model seeks to achieve several crucial objectives: earlier and wider access to new treatments; improved transparency in drug pricing; multi-stakeholder involvement through phased pricing negotiations; recognition of innovative product performance and latent changes in value; an earlier and more predictable return for developers without sacrificing total return on investment (ROI); more involved and informed risk sharing by the end-user.

Neural HO-1/sterol interactions in vivo: implications for Alzheimer's disease.

BACKGROUND: Up-regulation of heme oxygenase-1 (HO-1) and altered cholesterol (CH) metabolism are characteristic of Alzheimer-diseased (AD) neural tissues. We previously provided evidence of significant HO-1/sterol interactions in vitro (cultured rat astroglia) and in post-mortem human AD brain (Religious Orders Study). METHODS: The current experiments were designed to further delineate these interactions in vivo by comparing the behavior of HO-1/sterol interactions in two mouse models; (1) a novel HO-1 transgenic mouse (GFAP.HMOX1) engineered to selectively express human HO-1 in the astrocytic compartment and (2) the previously described triple transgenic AD mouse (3xTg-AD). In samples of frontal cortex, total CH, CH precursors and relevant oxysterols were quantified by gas chromatography-mass spectrometry (GC-MS) and HO-1 protein expression was assessed by ELISA. The relationships of HO-1 expression to total CH, CH precursors and total oxysterols were determined for both mouse models using linear regression analysis. RESULTS: HO-1 expression is increased in GFAP.HMOX1 mice relative to wild type and in 11-12-month-old 3xTg-AD mice (with AD-like phenotype) relative to control mice and 5-6-month-old 3xTg-AD mice (no AD-like phenotype). Total oxysterols significantly decreased as HO-1 expression increased in GFAP.HMOX1 mice expressing high levels of HO-1, whereas total oxysterols increased as HO-1 expression increased in aged 3xTg-AD mice. Total CH and total CH precursors increased as HO-1 protein expression increased in 11-12-month-old 3xTg-AD mice relative to 5-6-month old 3xTg-AD mice. CONCLUSIONS: Our findings indicate a differential impact of HO-1 on patterns of brain sterol and redox homeostasis that is contingent on the presence or absence of AD-like neuropathology. These data provide fresh insight concerning the regulation of sterol homeostasis within the aging and degenerating CNS which may inform the development of novel therapeutic and preventive strategies for the management of AD and related conditions.

Vitamin D deficiency in childhood obesity is associated with high levels of circulating inflammatory mediators, and low insulin sensitivity.

HYPOTHESIS: Childhood obesity is accompanied by low-grade systemic inflammation, which contributes to the development of insulin resistance and cardiovascular complications later in life. As vitamin D exhibits profound immunomodulatory functions and vitamin D deficiency is highly prevalent in childhood obesity, we hypothesized that vitamin D deficiency in childhood obesity coincides with enhanced systemic inflammation and reduced insulin sensitivity. METHODS: In a cross-sectional study of 64 obese and 32 healthy children aged 6-16 years, comprehensive profiling of 32 circulating inflammatory mediators was performed, together with assessment of 25-hydroxyvitamin D (25(OH)D) levels and measures for insulin sensitivity. RESULTS: Severe vitamin D insufficiency, which is further referred to as vitamin D deficiency, was defined as a 25(OH)D level ≤37.5 nmol l(-1), and was highly prevalent in obese (56%) versus healthy control children (16%). Throughout the study, 25(OH)D-deficient children were compared with the other children, including 25(OH)D insufficient (37.5-50 nmol l(-1)) and 25(OH)D sufficient children (≥50 nmol l(-1)). First, 25(OH)D-deficient obese children showed a lower insulin sensitivity than other obese children, as measured by a lower quantitative insulin sensitivity check index. Second, the association between 25(OH)D deficiency and insulin resistance in childhood obesity was confirmed with multiple regression analysis. Third, 25(OH)D-deficient obese children showed higher levels of the inflammatory mediators cathepsin S, chemerin and soluble vascular adhesion molecule (sVCAM), compared with the other obese children. Finally, hierarchical cluster analysis revealed an over-representation of 25(OH)D deficiency in obese children expressing inflammatory mediator clusters with high levels of cathepsin S, sVCAM and chemerin. CONCLUSION: 25(OH)D deficiency in childhood obesity was associated with enhanced systemic inflammation and reduced insulin sensitivity. The high cathepsin S and sVCAM levels may reflect activation of a pro-inflammatory, pro-diabetic and atherogenic pathway, which could be inhibited by vitamin D supplementation.

The novel arsenical Darinaparsin circumvents BRG1-dependent, HO-1-mediated cytoprotection in leukemic cells.

Darinaparsin (Dar) is a more potent cytotoxic arsenical than arsenic trioxide (ATO). We hypothesized that the increased cytotoxicity of Dar may be because of a decreased cytoprotective response. We observed that, unlike ATO, Dar does not induce heme oxygenase-1 (HO-1), even though it induces expression of other nuclear factor (erythroid-derived 2)-like 2 (NRF2)-dependent detoxifying enzymes to a greater extent than ATO, in both cancer cell lines and patient-derived leukemic cells. This strengthens the emerging evidence, showing that response to reactive oxygen species (ROS) is stimuli specific. Dar treatment prevents recruitment of the transcriptional coregulator Brahma-related gene 1 (BRG1) to the HMOX1 promoter, which is required for HMOX1 expression. The inability of Dar to induce HO-1 correlates with arrest in G2/M cell cycle phase and BRG1 phosphorylation. Inhibition of HO-1 increases the toxicity of ATO, but has no effect on Dar-induced apoptosis. Accordingly, the lack of HO-1 induction is involved in Dar's enhanced antileukemic properties. Our data highlight cytoprotective responses mediated by HO-1 and BRG1 as a novel target for enhancing the therapeutic range of arsenicals.

Systemic inflammation in childhood obesity: circulating inflammatory mediators and activated CD14++ monocytes.

AIMS/HYPOTHESIS: In adults, circulating inflammatory mediators and activated CD14(++) monocytes link obesity to its metabolic and cardiovascular complications. However, it is largely unknown whether these inflammatory changes already occur in childhood obesity. To survey inflammatory changes during the early stages of obesity, we performed a comprehensive analysis of circulating inflammatory mediators, monocyte populations and their function in childhood obesity. METHODS: In lean and obese children aged 6 to 16 years (n = 96), 35 circulating inflammatory mediators including adipokines were measured. Hierarchical cluster analysis of the inflammatory mediator profiles was performed to investigate associations between inflammatory mediator clusters and clinical variables. Whole-blood monocyte phenotyping and functional testing with the toll-like receptor 4 ligand, lipopolysaccharide, were also executed. RESULTS: First, next to leptin, the circulating mediators chemerin, tissue inhibitor of metalloproteinase 1, EGF and TNF receptor 2 were identified as novel inflammatory mediators that are increased in childhood obesity. Second, cluster analysis of the circulating mediators distinguished two obesity clusters, two leanness clusters and one mixed cluster. All clusters showed distinct inflammatory mediator profiles, together with differences in insulin sensitivity and other clinical variables. Third, childhood obesity was associated with increased CD14(++) monocyte numbers and an activated phenotype of the CD14(++) monocyte subsets. CONCLUSIONS/INTERPRETATION: Inflammatory mediator clusters were associated with insulin resistance in obese and lean children. The activation of CD14(++) monocyte subsets, which is associated with increased development of atherosclerosis in obese adults, was also readily detected in obese children. Our results indicate that inflammatory mechanisms linking obesity to its metabolic and cardiovascular complications are already activated in childhood obesity.

Altered plasma adipokine levels and in vitro adipocyte differentiation in pediatric type 1 diabetes.

CONTEXT: Type 1 diabetes (T1D) is considered a proinflammatory condition. Adipose tissue involvement seems evident because adiponectin levels correlate with disease remission and administration of leptin suppresses the low-grade systemic inflammation in mice with T1D. Whether adipose tissue involvement in T1D already occurs at a young age is yet unknown. OBJECTIVE: The aim was to explore the extent of adipokine alterations in pediatric T1D and gain more insight into the mechanisms underlying the involvement of adipose tissue. DESIGN AND PARTICIPANTS: First, plasma adipokine profiling (24 adipokines) of 20 children with onset T1D, 20 children with long-standing T1D, and 17 healthy controls was performed using a recently developed and validated multiplex immunoassay. Second, the effects of diabetic plasma factors on preadipocyte proliferation and differentiation were studied in vitro. RESULTS: In children with onset and long-standing T1D, plasma adipokine profiling showed increased levels of various adipokines acting at the crossroads of adipose tissue function and inflammation, including CCL2/monocyte chemoattractant protein-1 and the novel adipokines cathepsin S, chemerin, and tissue inhibitor of metalloproteinase-1 (P < 0.05). Furthermore, onset and long-standing diabetic plasma significantly induced preadipocyte proliferation and adipocyte differentiation in vitro (P < 0.05). Two candidate plasma factors, glucose and the saturated fatty acid palmitic acid, did not affect proliferation or adipocyte differentiation in vitro but were found to increase CCL2 (monocyte chemoattractant protein-1) secretion by adipocytes. CONCLUSIONS: The adipogenic effects of diabetic plasma in vitro and the altered adipokine levels in vivo suggest adipose tissue involvement in the low-grade inflammation associated with T1D, already in pediatric patients.

Adipose tissue quantity and composition contribute to adipokine concentrations in the subclavian vein and the inferior mesenteric vein.

BACKGROUND: Adipose tissue dysfunction is associated with inflammation, type 2 diabetes mellitus and vascular diseases. Visceral adipose tissue (VAT)-derived adipokines, which are released in the portal circulation may influence liver metabolism. OBJECTIVES: (1) To estimate the contribution of VAT and subcutaneous adipose tissue (SAT) on adipokine levels by measuring differences in adipokine concentrations between the portal draining inferior mesenteric vein and the subclavian vein. (2) To determine the relation of both VAT and SAT quantity and composition to mesenteric and systemic concentrations of adipokines. DESIGN: Cross-sectional cohort study. SUBJECTS: A total of 32 patients undergoing abdominal aortic surgery. MEASUREMENTS: A panel of 18 adipokines was measured in perioperatively obtained blood samples from the subclavian vein and the inferior mesenteric vein. Adipocyte size, macrophage infiltration and capillary density were measured in subcutaneous and mesenteric adipose tissue biopsies; SAT and VAT areas were measured on computed tomography images. RESULTS: Serum interferon-γ-inducible protein 10 (IP-10) and hepatocyte growth factor (HGF) concentrations were significantly higher in the inferior mesenteric vein vs the subclavian vein. SAT area (ß -18; 95% confidence interval (CI) -35 to -2), subcutaneous adipocyte size (ß -488; 95% CI -938 to -38) and SAT macrophages quantity (ß -1439; 95% CI -2387 to -491) were negatively associated with adiponectin levels in the systemic circulation. SAT area was related to systemic concentrations of leptin. Mesenteric adiponectin concentrations were related to VAT area (ß -20; 95% CI -35 to -5) and visceral adipocyte size (ß -1076; 95% CI -1624 to -527). VAT area, adipocyte size and capillary density were related to systemic adiponectin concentrations. CONCLUSION: SAT and VAT quantities as well as morphologic characteristics of both adipose tissue depots are related to systemic and mesenteric adipokine concentrations. There were no differences in adipokine concentrations between the mesenteric and subclavian vein, except for higher IP-10 and HGF concentrations in the inferior mesenteric vein, indicating a possible contribution of VAT to IP-10 and HGF levels.

Effect of different time intervals between examinations on the reproducibility of ICDAS-II for occlusal caries.

AIM: To evaluate intra- and interexaminer reproducibility of ICDAS-II on occlusal caries diagnosis when different time intervals were allowed to elapse between examinations. A subsidiary aim was to determine whether collapsing the codes would influence this reproducibility. METHODS: The occlusal surfaces of 50 permanent posterior teeth were investigated by 3 trained examiners using ICDAS-II at baseline, 1 day, 1 week and 4 weeks after baseline. RESULTS: Weighted kappa values for intra- and interexaminer reproducibility were 0.76-0.93. CONCLUSION: The time span did not have a major impact on assessing intra- and interexaminer reproducibility. Collapsing ICDAS-II codes had no impact on examiner reproducibility.

Early life experiences affect the adaptive capacity of rearing hens during infectious challenges.

This study aimed to investigate whether pre- and early postnatal experiences of rearing hens contribute to the ability to cope with infectious challenges at an older age. In a 2 × 2 factorial arrangement, 352 Lohmann Brown chicks were exposed to either suboptimal or optimized incubation plus hatch conditions, and to cage or enriched rearing from week 0 to 7 of age. After week 7 all rearing conditions were similar until the end of the experiment. The development of adaptive capacity to infectious challenges was investigated by introducing an Eimeria and Infectious Bronchitis (IB) infection on day 53 and day 92, respectively. BW gain and feed intake during the infections, duodenal lesions and amount of positive stained CD4+ T cells, CD8+ T cells and macrophages at day 4 and day 7 after Eimeria infection, as well as the IB antibody titer throughout the experimental period were determined. The results showed a significant interaction between incubation plus hatch and rearing environment. Optimized incubation plus hatch conditions followed by an enriched rearing environment resulted in the least weight loss (P < 0.05) and the highest feed intake (P < 0.01) from day 3 to day 7 after the Eimeria infection (day 56 to 60 of age), compared with all other treatments. In addition, the optimized × enriched chicks had the highest BW gain from day 7 to day 14 after IB infection (day 99 to 106 of age), compared with chicks housed in a cage environment (P < 0.01). Besides the interaction, optimized incubation plus hatch alone resulted in reduced macrophage numbers in the duodenal tissue at day 4 after Eimeria infection, compared with suboptimal incubation plus hatch, whereas the enriched rearing environment stimulated the recovery of intestinal damage caused by Eimeria (P < 0.05) and reduced the production of specific antibodies after IB infection (P < 0.05), compared with the cage environment. In conclusion, this study shows that early life experiences can indeed affect the capacity of rearing hens to cope with an Eimeria and IB infection at an older age, in which performance of chicks is best maintained after optimized incubation plus hatch followed by enriched rearing. This suggests that the development of adaptive capacity to infectious challenges can be influenced with management during a short period in pre- or early postnatal life, but that effects last for a considerable time after cessation of the specific management.

Systemic and local interferon-gamma production following Mycobacterium ulcerans infection.

Buruli ulcer disease (BUD) is an emerging predominantly tropical disease caused by Mycobacterium ulcerans. The initial pre-ulcerative skin lesion often breaks down into an ulcer with undermined edges. Healing is common but may require considerable time, and scarring often results in functional limitations. Considerable evidence has now emerged that patients with early BUD cannot mount a sufficient protective T helper 1 (Th1) cell response to M. ulcerans, but uncertainty remains as to whether immune protection is restored over time. This study investigates the Th1 cell response of patients with various stages of BUD on mycobacterial antigens. We measured interferon (IFN)-gamma levels after ex vivo whole blood stimulation with tuberculin purified protein derivative (PPD), and compared the Th1 cell response of individuals with pre-ulcerative, ulcerative and healed BUD as well as healthy controls. Moreover, the systemic Th1 cell response was related to histopathological features in the various stages of surgically resected BUD lesions. We show that patients with ulcerative and healed BUD produce significantly higher IFN-gamma levels after mycobacterial ex vivo whole blood stimulation than healthy controls, and that patients with a granulomatous tissue response produce higher IFN-gamma levels than individuals without. We therefore suggest that the mounted Th1 cell response in ulcerative BUD patients might be related to their histopathological tissue response.

Trolox enhances the anti-lymphoma effects of arsenic trioxide, while protecting against liver toxicity.

Arsenic trioxide (As2O3) is an effective therapy in acute promyelocytic leukemia (APL), but its use in other malignancies is limited by the higher concentrations required to induce apoptosis. We have reported that trolox, an analogue of alpha-tocopherol, increases As2O3-mediated apoptosis in a variety of APL, myeloma and breast cancer cell lines, while non-malignant cells may be protected. In the present study, we extended previous results to show that trolox increases As2O3-mediated apoptosis in the P388 lymphoma cell line in vitro, as evidenced by decrease of mitochondrial membrane potential and release of cytochrome c. We then sought to determine whether this combination can enhance antitumor effects while protecting normal cells in vivo. In BDF1 mice, trolox treatment decreased As2O3-induced hepatomegaly, markers of oxidative stress and hepatocellular damage. In P388 tumor-bearing mice, As2O3 treatment prolonged survival, and the addition of trolox provided a further significant increase in lifespan. In addition, the combination of As2O3 and trolox inhibited metastatic spread, and protected the tumor-bearing mice from As2O3 liver toxicity. Our results suggest, for the first time, that trolox might prevent some of the clinical manifestations of As2O3-related toxicity while increasing its pro-apoptotic capacity and clinical efficacy in hematological malignancies.

Endurance exercise differentially stimulates heart and axial muscle development in zebrafish (Danio rerio).

Mechanical load is an important factor in the differentiation of cells and tissues. To investigate the effects of increased mechanical load on development of muscle and bone, zebrafish were subjected to endurance swim training for 6 h/day for 10 wk starting at 14 days after fertilization. During the first 3 wk of training, trained fish showed transiently increased growth compared with untrained (control) fish. Increased expression of proliferating cell nuclear antigen suggests that this growth is realized in part through increased cell proliferation. Red and white axial muscle fiber diameter was not affected. Total cross-sectional area of red fibers, however, was increased. An improvement in aerobic muscle performance was supported by an increase in myoglobin expression. At the end of 10 wk of training, heart and axial muscle showed increased expression of the muscle growth factor myogenin and proliferating cell nuclear antigen, but there were major differences between cardiac and axial muscle. In axial muscle, expression of the "slow" types of myosin and troponin C was increased, together with expression of erythropoietin and myoglobin, which enhance oxygen transport, indicating a shift toward a slow aerobic phenotype. In contrast, the heart muscle shifts to a faster phenotype but does not become more aerobic. This suggests that endurance training differentially affects heart and axial muscle.

Syringeal muscles fit the trill in ring doves (Streptopelia risoria L.).

In contrast to human phonation, the virtuoso vocalizations of most birds are modulated at the level of the sound generator, the syrinx. We address the hypothesis that syringeal muscles are physiologically capable of controlling the sound-generating syringeal membranes in the ring dove (Streptopelia risoria) syrinx. We establish the role of the tracheolateralis muscle and propose a new function for the sternotrachealis muscle. The tracheolateralis and sternotrachealis muscles have an antagonistic mechanical effect on the syringeal aperture. Here, we show that both syringeal muscles can dynamically control the full syringeal aperture. The tracheolateralis muscle is thought to directly alter position and tension of the vibrating syringeal membranes that determine the gating and the frequency of sound elements. Our measurements of the muscle's contractile properties, combined with existing electromyographic and endoscopic evidence, establish its modulating role during the dove's trill. The muscle delivers the highest power output at cycle frequencies that closely match the repetition rates of the fastest sound elements in the coo. We show that the two syringeal muscles share nearly identical contraction characteristics, and that sternotrachealis activity does not clearly modulate during the rapid trill. We propose that the sternotrachealis muscle acts as a damper that stabilizes longitudinal movements of the sound-generating system induced by tracheolateralis muscle contraction. The extreme performance of both syringeal muscles implies that they play an important role in fine-tuning membrane position and tension, which determines the quality of the sound for a conspecific mate.

Effects of decreased muscle activity on developing axial musculature in nicb107 mutant zebrafish (Danio rerio).

The present paper discusses the effects of decreased muscle activity (DMA) on embryonic development in the zebrafish. Wild-type zebrafish embryos become mobile around 18 h post-fertilisation, long before the axial musculature is fully differentiated. As a model for DMA, the nic(b107) mutant was used. In nic(b107) mutant embryos, muscle fibres are mechanically intact and able to contract, but neuronal signalling is defective and the fibres are not activated, rendering the embryos immobile. Despite the immobility, distinguished slow and fast muscle fibres developed at the correct location in the axial muscles, helical muscle fibre arrangements were detected and sarcomere architecture was generated. However, in nic(b107) mutant embryos the notochord is flatter and the cross-sectional body shape more rounded, also affecting muscle fibre orientation. The stacking of sarcomeres and myofibril arrangement show a less regular pattern. Finally, expression levels of several genes were changed. Together, these changes in expression indicate that muscle growth is not impeded and energy metabolism is not changed by the decrease in muscle activity but that the composition of muscle is altered. In addition, skin stiffness is affected. In conclusion, the lack of muscle fibre activity did not prevent the basal muscle components developing but influenced further organisation and differentiation of these components.

Identification and characterisation of two runx2 homologues in zebrafish with different expression patterns.

Genome and gene duplications are considered to be the impetus to generate new genes, as the presence of multiple copies of a gene allows for paralogues to adopt novel function. After at least two rounds of genome/gene duplication, the Runt gene family consists of three members in vertebrates, instead of one in invertebrates. One of the family members, Runx2, plays a key role in the development of bone, a tissue that first occurs in vertebrates. The family has thus gained new gene function in the course of evolution. Two Runx2 genes were cloned in the vertebrate model system the zebrafish (Danio rerio). The expression patterns of the two genes differ and their kinetics differ up to four fold. In addition, splice forms exist that are novel when compared with mammals. Together, these findings comprise opportunities for selection and retention of the paralogues towards divergent and possibly new function.

[Clinical reasoning and decision making in practice. A depressive foreign woman with symptoms of malaise]. / Klinisch denken en beslissen in de praktijk. Een depressieve buitenlandse vrouw met klachten van malaise.

A 27-year-old woman was admitted to the hospital with a depression, anaemia and fatigue. She had come from Angola to the Netherlands as a refugee 2 years before this evaluation. As an explanation for her symptoms tropical infectious diseases of parasitic origin were considered, but no clues were found in this direction. The test for trypanosomiasis was considered to be suggestive for an infection in the past (persistent titre 1:200). She was discharged but readmitted 6 months later because of a deterioration of her clinical condition. Magnetic resonance imaging showed bilateral signal abnormalities within the white matter of the brain. On examination no neurological signs or abnormalities were found. Again, no definite diagnosis could be made and the patient was discharged. Because of a further deterioration of her clinical condition she was readmitted a short time later for the third time. On the MRI the white matter lesions had increased. The serum protein electrophoresis was markedly abnormal with an elevated IgM Level. Finally, at a repeated lumbar puncture mobile trypanosomes were found. The diagnosis of 'West African sleeping sickness' was made and the patient was treated with eflornithine. She recovered completely during the next 18 months.

Effects of dietary restriction and metal supplementation on the accumulation of iron-laden glial inclusions in the aging rat hippocampus.

The mechanisms responsible for the pathological deposition of iron and other redox-active metals in the aging and degenerating mammalian CNS remain poorly understood. We previously demonstrated that normal aging and pharmacological (oxidative) stressors promote the transformation of astroglial mitochondria to iron-laden, diaminobenzidine (DAB)-positive cytoplasmic inclusions in sub-cortical regions of the rat brain. In the current study, we demonstrate that (1) numbers of DAB-positive glial granules in the rat dorsal hippocampus, an area implicated in learning and memory, progressively increase between 3, 12 and 22 months of age; (2) dietary restriction (40%), a manipulation that attenuates many mammalian aging processes, has no effect on the age-related accumulation of these gliosomes in the rat hippocampus; and (3) the latter can be accelerated by dietary supplementation of iron and copper. Our data support the view that dietary exposure to iron and/or copper in adult life can impact the sequestration of redox-active metals in aging hippocampal astroglia.