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1.  Impact of peer feedback on the performance of lecturers in emergency medicine: a prospective observational study 
Background
Although it is often criticised, the lecture remains a fundamental part of medical training because it is an economical and efficient method for teaching both factual and experimental knowledge. However, if administered incorrectly, it can be boring and useless.
Feedback from peers is increasingly recognized as an effective method of encouraging self-reflection and continuing professional development. The aim of this observational study is to analyse the impact of written peer feedback on the performance of lecturers in an emergency medicine lecture series for undergraduate students.
Methods
In this prospective study, 13 lecturers in 15 lectures on emergency medicine for undergraduate medical students were videotaped and analysed by trained peer reviewers using a 21-item assessment instrument. The lecturers received their written feedback prior to the beginning of the next years’ lecture series and were assessed in the same way.
Results
In this study, we demonstrated a significant improvement in the lecturers’ scores in the categories ‘content and organisation’ and ‘visualisation’ in response to written feedback. The highest and most significant improvements after written peer feedback were detected in the items ‘provides a brief outline’, ‘provides a conclusion for the talk’ and ‘clearly states goal of the talk’.
Conclusion
This study demonstrates the significant impact of a single standardized written peer feedback on a lecturer’s performance.
doi:10.1186/s13049-014-0071-1
PMCID: PMC4264246  PMID: 25472430
Peer feedback; Lecture; Faculty development; Emergency medicine; Undergraduate education
3.  Cross-Species Comparison of Genes Related to Nutrient Sensing Mechanisms Expressed along the Intestine 
PLoS ONE  2014;9(9):e107531.
Introduction
Intestinal chemosensory receptors and transporters are able to detect food-derived molecules and are involved in the modulation of gut hormone release. Gut hormones play an important role in the regulation of food intake and the control of gastrointestinal functioning. This mechanism is often referred to as “nutrient sensing”. Knowledge of the distribution of chemosensors along the intestinal tract is important to gain insight in nutrient detection and sensing, both pivotal processes for the regulation of food intake. However, most knowledge is derived from rodents, whereas studies in man and pig are limited, and cross-species comparisons are lacking.
Aim
To characterize and compare intestinal expression patterns of genes related to nutrient sensing in mice, pigs and humans.
Methods
Mucosal biopsy samples taken at six locations in human intestine (n = 40) were analyzed by qPCR. Intestinal scrapings from 14 locations in pigs (n = 6) and from 10 locations in mice (n = 4) were analyzed by qPCR and microarray, respectively. The gene expression of glucagon, cholecystokinin, peptide YY, glucagon-like peptide-1 receptor, taste receptor T1R3, sodium/glucose cotransporter, peptide transporter-1, GPR120, taste receptor T1R1, GPR119 and GPR93 was investigated. Partial least squares (PLS) modeling was used to compare the intestinal expression pattern between the three species.
Results and conclusion
The studied genes were found to display specific expression patterns along the intestinal tract. PLS analysis showed a high similarity between human, pig and mouse in the expression of genes related to nutrient sensing in the distal ileum, and between human and pig in the colon. The gene expression pattern was most deviating between the species in the proximal intestine. Our results give new insights in interspecies similarities and provide new leads for translational research and models aiming to modulate food intake processes in man.
doi:10.1371/journal.pone.0107531
PMCID: PMC4162619  PMID: 25216051
4.  Sexually dimorphic characteristics of the small intestine and colon of prepubescent C57BL/6 mice 
Background
There is increasing appreciation for sexually dimorphic effects, but the molecular mechanisms underlying these effects are only partially understood. In the present study, we explored transcriptomics and epigenetic differences in the small intestine and colon of prepubescent male and female mice. In addition, the microbiota composition of the colonic luminal content has been examined.
Methods
At postnatal day 14, male and female C57BL/6 mice were sacrificed and the small intestine, colon and content of luminal colon were isolated. Gene expression of both segments of the intestine was analysed by microarray analysis. DNA methylation of the promoter regions of selected sexually dimorphic genes was examined by pyrosequencing. Composition of the microbiota was explored by deep sequencing.
Results
Sexually dimorphic genes were observed in both segments of the intestine of 2-week-old mouse pups, with a stronger effect in the small intestine. Amongst the total of 349 genes displaying a sexually dimorphic effect in the small intestine and/or colon, several candidates exhibited a previously established function in the intestine (i.e. Nts, Nucb2, Alox5ap and Retnlγ). In addition, differential expression of genes linked to intestinal bowel disease (i.e. Ccr3, Ccl11 and Tnfr) and colorectal cancer development (i.e. Wt1 and Mmp25) was observed between males and females. Amongst the genes displaying significant sexually dimorphic expression, nine genes were histone-modifying enzymes, suggesting that epigenetic mechanisms might be a potential underlying regulatory mechanism. However, our results reveal no significant changes in DNA methylation of analysed CpGs within the selected differentially expressed genes. With respect to the bacterial community composition in the colon, a dominant effect of litter origin was found but no significant sex effect was detected. However, a sex effect on the dominance of specific taxa was observed.
Conclusions
This study reveals molecular dissimilarities between males and females in the small intestine and colon of prepubescent mice, which might underlie differences in physiological functioning and in disease predisposition in the two sexes.
doi:10.1186/s13293-014-0011-9
PMCID: PMC4169057  PMID: 25243059
Small intestine; Colon; Sexually dimorphism; Gene expression; Microbiota; Histone modification; DNA methylation; Epigenetics; Chromosomes; Prepubescent
5.  Hypothalamic food intake regulation in a cancer-cachectic mouse model 
Background
Appetite is frequently affected in cancer patients leading to anorexia and consequently insufficient food intake. In this study, we report on hypothalamic gene expression profile of a cancer-cachectic mouse model with increased food intake. In this model, mice bearing C26 tumour have an increased food intake subsequently to the loss of body weight. We hypothesise that in this model, appetite-regulating systems in the hypothalamus, which apparently fail in anorexia, are still able to adapt adequately to changes in energy balance. Therefore, studying changes that occur on appetite regulators in the hypothalamus might reveal targets for treatment of cancer-induced eating disorders. By applying transcriptomics, many appetite-regulating systems in the hypothalamus could be taken into account, providing an overview of changes that occur in the hypothalamus during tumour growth.
Methods
C26-colon adenocarcinoma cells were subcutaneously inoculated in 6 weeks old male CDF1 mice. Body weight and food intake were measured three times a week. On day 20, hypothalamus was dissected and used for transcriptomics using Affymetrix chips.
Results
Food intake increased significantly in cachectic tumour-bearing mice (TB), synchronously to the loss of body weight. Hypothalamic gene expression of orexigenic neuropeptides NPY and AgRP was higher, whereas expression of anorexigenic genes CCK and POMC were lower in TB compared to controls.
In addition, serotonin and dopamine signalling pathways were found to be significantly altered in TB mice. Serotonin levels in brain showed to be lower in TB mice compared to control mice, while dopamine levels did not change. Moreover, serotonin levels inversely correlated with food intake.
Conclusions
Transcriptomic analysis of the hypothalamus of cachectic TB mice with an increased food intake showed changes in NPY, AgRP and serotonin signalling. Serotonin levels in the brain showed to correlate with changes in food intake. Further research has to reveal whether targeting these systems will be a good strategy to avoid the development of cancer-induced eating disorders.
Electronic supplementary material
The online version of this article (doi:10.1007/s13539-013-0121-y) contains supplementary material.
doi:10.1007/s13539-013-0121-y
PMCID: PMC4053566  PMID: 24222472
Cancer; Hypothalamus; Appetite; Serotonin; Transcriptomics; Anorexia
6.  Genome-wide age-related changes in DNA methylation and gene expression in human PBMCs 
Age  2014;36(3):9648.
Aging is a progressive process that results in the accumulation of intra- and extracellular alterations that in turn contribute to a reduction in health. Age-related changes in DNA methylation have been reported before and may be responsible for aging-induced changes in gene expression, although a causal relationship has yet to be shown. Using genome-wide assays, we analyzed age-induced changes in DNA methylation and their effect on gene expression with and without transient induction with the synthetic transcription modulating agent WY14,643. To demonstrate feasibility of the approach, we isolated peripheral blood mononucleated cells (PBMCs) from five young and five old healthy male volunteers and cultured them with or without WY14,643. Infinium 450K BeadChip and Affymetrix Human Gene 1.1 ST expression array analysis revealed significant differential methylation of at least 5 % (ΔYO > 5 %) at 10,625 CpG sites between young and old subjects, but only a subset of the associated genes were also differentially expressed. Age-related differential methylation of previously reported epigenetic biomarkers of aging including ELOVL2, FHL2, PENK, and KLF14 was confirmed in our study, but these genes did not display an age-related change in gene expression in PBMCs. Bioinformatic analysis revealed that differentially methylated genes that lack an age-related expression change predominantly represent genes involved in carcinogenesis and developmental processes, and expression of most of these genes were silenced in PBMCs. No changes in DNA methylation were found in genes displaying transiently induced changes in gene expression. In conclusion, aging-induced differential methylation often targets developmental genes and occurs mostly without change in gene expression.
Electronic supplementary material
The online version of this article (doi:10.1007/s11357-014-9648-x) contains supplementary material, which is available to authorized users.
doi:10.1007/s11357-014-9648-x
PMCID: PMC4082572  PMID: 24789080
Molecular aging; Epigenetics; DNA methylation; Gene expression; PBMCs; Epigenetic biomarkers of aging
7.  Screw placement in percutaneous acetabular surgery: gender differences of anatomical landmarks in a cadaveric study 
International Orthopaedics  2012;37(4):673-679.
Purpose
Percutaneous reduction and periarticular screw implantation techniques have been successfully introduced in acetabular surgery. The advantages of this less invasive approach are attenuated by higher risks of screw misplacement. Anatomical landmarks are strongly needed to prevent malplacement. This cadaver study was designed to identify reliable anatomical osseous landmarks in the pelvic region for screw placement in acetabular surgery. Gender differences were specifically addressed.
Methods
Twenty-seven embalmed cadaveric hemipelvic specimens (13 male, 14 female) were used. After soft-tissue removal, anterior and posterior column acetabular screw placement was conducted by one orthopaedic trauma surgeon under direct vision. Each column was addressed by antegrade and retrograde screw insertion. Radiographic verification of ideal screw placement was followed by assessment of the distance from the different entry points to adjoining anatomical osseous structures.
Results
For anterior column screw positioning, the posterior superior iliac spine (PSIS), posterior inferior iliac spine (PIIS), iliopectineal eminence and centre of the symphysis were most reliable regarding gender differences. For posterior column screw positioning, the distance to the anterior superior iliac spine (ASIS) and the ischial tuberosity showed the lowest deviation between the different gender specimens. Highest gender differences were seen in relation to the cranial rim of the superior pubic ramus in retrograde anterior column screw positioning (p = 0.002). Most landmarks could be targeted within a 2.5-cm range in all specimens.
Conclusions
The findings emphasise the relevance of osseous landmarks in acetabular surgery. By adhering to easily identifiable structures, screw placement can be safely performed. Significant gender differences must be taken into consideration during preoperative planning.
doi:10.1007/s00264-012-1740-1
PMCID: PMC3609967  PMID: 23250351
8.  The free radical scavenger Trolox dampens neuronal hyperexcitability, reinstates synaptic plasticity, and improves hypoxia tolerance in a mouse model of Rett syndrome 
Rett syndrome (RS) causes severe cognitive impairment, loss of speech, epilepsy, and breathing disturbances with intermittent hypoxia. Also mitochondria are affected; a subunit of respiratory complex III is dysregulated, the inner mitochondrial membrane is leaking protons, and brain ATP levels seem reduced. Our recent assessment of mitochondrial function in MeCP2 (methyl-CpG-binding protein 2)-deficient mouse (Mecp2-/y) hippocampus confirmed early metabolic alterations, an increased oxidative burden, and a more vulnerable cellular redox balance. As these changes may contribute to the manifestation of symptoms and disease progression, we now evaluated whether free radical scavengers are capable of improving neuronal and mitochondrial function in RS. Acute hippocampal slices of adult mice were incubated with the vitamin E derivative Trolox for 3–5 h. In Mecp2-/y slices this treatment dampened neuronal hyperexcitability, improved synaptic short-term plasticity, and fully restored synaptic long-term potentiation (LTP). Furthermore, Trolox specifically attenuated the increased hypoxia susceptibility of Mecp2-/y slices. Also, the anticonvulsive effects of Trolox were assessed, but the severity of 4-aminopyridine provoked seizure-like discharges was not significantly affected. Adverse side effects of Trolox on mitochondria can be excluded, but clear indications for an improvement of mitochondrial function were not found. Since several ion-channels and neurotransmitter receptors are redox modulated, the mitochondrial alterations and the associated oxidative burden may contribute to the neuronal dysfunction in RS. We confirmed in Mecp2-/y hippocampus that Trolox dampens neuronal hyperexcitability, reinstates synaptic plasticity, and improves the hypoxia tolerance. Therefore, radical scavengers are promising compounds for the treatment of neuronal dysfunction in RS and deserve further detailed evaluation.
doi:10.3389/fncel.2014.00056
PMCID: PMC3932407  PMID: 24605086
oxidative stress; redox signaling; reactive oxygen species (ROS); mitochondrial metabolism; free radical scavenger; neurodevelopmental disorder; synaptic dysfunction; vitamin E
9.  Inhibitor of differentiation proteins do not influence prognosis of biliary tract cancer 
AIM: To investigate the expression and clinical relevance of inhibitor of differentiation (ID) proteins in biliary tract cancer.
METHODS: ID protein expression was analyzed in 129 samples from patients with advanced biliary tract cancer (BTC) (45 extrahepatic, 50 intrahepatic, and 34 gallbladder cancers), compared to normal controls and correlated with clinical an pathological parameters.
RESULTS: ID1-3 proteins are frequently overexpressed in all BTC subtypes analyzed. No correlation between increased ID protein expression and tumor grading, tumor subtype or treatment response was detected. Survival was influenced primary tumor localization (extrahepatic vs intrahepatic and gall bladder cancer, OS 1.5 years vs 0.9 years vs 0.7 years, P = 0.002), by stage at diagnosis (OS 2.7 years in stage I vs 0.6 years in stage IV, P < 0.001), resection status and response to systemic chemotherapy. In a multivariate model, ID protein expression did not correlate with clinical prognosis. Nevertheless, there was a trend of shorter OS in patients with loss of cytoplasmic ID4 protein expression (P = 0.076).
CONCLUSION: ID protein expression is frequently deregulated in BTC but does not influence clinical prognosis. Their usefulness as prognostic biomarkers in BTC is very limited.
doi:10.3748/wjg.v19.i48.9334
PMCID: PMC3882406  PMID: 24409060
Biliary tract cancer; Cholangiocarcinoma; Inhibitor of differentiation; Prognostic factors
10.  Maternal Western-Style High Fat Diet Induces Sex-Specific Physiological and Molecular Changes in Two-Week-Old Mouse Offspring 
PLoS ONE  2013;8(11):e78623.
Maternal diet is associated with the development of metabolism-related and other non-communicable diseases in offspring. Underlying mechanisms, functional profiles, and molecular markers are only starting to be revealed. Here, we explored the physiological and molecular impact of maternal Western-style diet on the liver of male and female offspring. C57BL/6 dams were exposed to either a low fat/low cholesterol diet (LFD) or a Western-style high fat/high cholesterol diet (WSD) for six weeks before mating, as well as during gestation and lactation. Dams and offspring were sacrificed at postnatal day 14, and body, liver, and blood parameters were assessed. The impact of maternal WSD on the pups’ liver gene expression was characterised by whole-transcriptome microarray analysis. Exclusively male offspring had significantly higher body weight upon maternal WSD. In offspring of both sexes of WSD dams, liver and blood parameters, as well as hepatic gene expression profiles were changed. In total, 686 and 604 genes were differentially expressed in liver (p≤0.01) of males and females, respectively. Only 10% of these significantly changed genes overlapped in both sexes. In males, in particular alterations of gene expression with respect to developmental functions and processes were observed, such as Wnt/beta-catenin signalling. In females, mainly genes important for lipid metabolism, including cholesterol synthesis, were changed. We conclude that maternal WSD affects physiological parameters and induces substantial changes in the molecular profile of the liver in two-week-old pups. Remarkably, the observed biological responses of the offspring reveal pronounced sex-specificity.
doi:10.1371/journal.pone.0078623
PMCID: PMC3818485  PMID: 24223833
11.  Short-Chain Fatty Acids Stimulate Angiopoietin-Like 4 Synthesis in Human Colon Adenocarcinoma Cells by Activating Peroxisome Proliferator-Activated Receptor γ 
Molecular and Cellular Biology  2013;33(7):1303-1316.
Angiopoietin-like protein 4 (ANGPTL4/FIAF) has been proposed as a circulating mediator between the gut microbiota and fat storage. Here, we show that transcription and secretion of ANGPTL4 in human T84 and HT29 colon adenocarcinoma cells is highly induced by physiological concentrations of short-chain fatty acids (SCFA). SCFA induce ANGPTL4 by activating the nuclear receptor peroxisome proliferator activated receptor γ (PPARγ), as demonstrated using PPARγ antagonist, PPARγ knockdown, and transactivation assays, which show activation of PPARγ but not PPARα and PPARδ by SCFA. At concentrations required for PPARγ activation and ANGPTL4 induction in colon adenocarcinoma cells, SCFA do not stimulate PPARγ in mouse 3T3-L1 and human SGBS adipocytes, suggesting that SCFA act as selective PPARγ modulators (SPPARM), which is supported by coactivator peptide recruitment assay and structural modeling. Consistent with the notion that fermentation leads to PPAR activation in vivo, feeding mice a diet rich in inulin induced PPAR target genes and pathways in the colon. We conclude that (i) SCFA potently stimulate ANGPTL4 synthesis in human colon adenocarcinoma cells and (ii) SCFA transactivate and bind to PPARγ. Our data point to activation of PPARs as a novel mechanism of gene regulation by SCFA in the colon, in addition to other mechanisms of action of SCFA.
doi:10.1128/MCB.00858-12
PMCID: PMC3624264  PMID: 23339868
12.  Cartilage labelling for mechanical testing in T-peel configuration 
International Orthopaedics  2012;36(7):1493-1499.
Purpose
The purpose of this study was to find a suitable method of labelling cartilage samples for the measurement of distraction distances in biomechanical testing.
Methods
Samples of bovine cartilage were labelled using five different methods: hydroquinone and silver nitrate (AgNO3), potassium permanganate (KMnO4) with sodium thiosulphate (Na2S2O3), India ink, heat, and laser energy. After the labelling, we analysed the cartilage samples with regard to cytotoxity by histochemical staining with ethidiumbromide homodimer (EthD-1) and calcein AM. Furthermore, we tested cartilages labelled with India ink and heat in a T-peel test configuration to analyse possible changes in the mechanical behaviour between marked and unlabelled samples.
Results
Only the labelling methods with Indian ink or a heated needle showed acceptable results in the cytotoxity test with regard to labelling persistence, accuracy, and the influence on consistency and viability of the chondrocytes. In the biomechanical T-peel configuration, heat-labelled samples collapsed significantly earlier than unlabelled samples.
Conclusion
Labelling bovine cartilage samples with Indian ink in biomechanical testing is a reliable, accurate, inexpensive, and easy-to-perform method. This labelling method influenced neither the biomechanical behaviour nor the viability of the tissue compared to untreated bovine cartilage.
doi:10.1007/s00264-011-1468-3
PMCID: PMC3385900  PMID: 22237920
13.  The Inflammasome-Mediated Caspase-1 Activation Controls Adipocyte Differentiation and Insulin Sensitivity 
Cell metabolism  2010;12(6):593-605.
SUMMARY
Obesity-induced inflammation originating from expanding adipose tissue interferes with insulin sensitivity. Important metabolic effects have been recently attributed to IL-1β and IL-18, two members of the IL-1 family of cytokines. Processing of IL-1β and IL-18 requires cleavage by caspase-1, a cysteine protease regulated by a protein complex called the inflammasome. We demonstrate that the inflamma-some/caspase-1 governs adipocyte differentiation and insulin sensitivity. Caspase-1 is upregulated during adipocyte differentiation and directs adipocytes toward a more insulin-resistant phenotype. Treatment of differentiating adipocytes with recombinant IL-1β and IL-18, or blocking their effects by inhibitors, reveals that the effects of caspase-1 on adipocyte differentiation are largely conveyed by IL-1β. Caspase-1 and IL-1β activity in adipose tissue is increased both in diet-induced and genetically induced obese animal models. Conversely, mice deficient in caspase-1 are more insulin sensitive as compared to wild-type animals. In addition, differentiation of preadipocytes isolated from caspase-1−/− or NLRP3−/− mice resulted in more metabolically active fat cells. In vivo, treatment of obese mice with a caspase-1 inhibitor significantly increases their insulin sensitivity. Indirect calorimetry analysis revealed higher fat oxidation rates in caspase-1−/− animals. In conclusion, the inflammasome is an important regulator of adipocyte function and insulin sensitivity, and caspase-1 inhibition may represent a novel therapeutic target in clinical conditions associated with obesity and insulin resistance.
doi:10.1016/j.cmet.2010.11.011
PMCID: PMC3683568  PMID: 21109192
14.  Simple Process-Based Simulators for Generating Spatial Patterns of Habitat Loss and Fragmentation: A Review and Introduction to the G-RaFFe Model 
PLoS ONE  2013;8(5):e64968.
Landscape simulators are widely applied in landscape ecology for generating landscape patterns. These models can be divided into two categories: pattern-based models that generate spatial patterns irrespective of the processes that shape them, and process-based models that attempt to generate patterns based on the processes that shape them. The latter often tend toward complexity in an attempt to obtain high predictive precision, but are rarely used for generic or theoretical purposes. Here we show that a simple process-based simulator can generate a variety of spatial patterns including realistic ones, typifying landscapes fragmented by anthropogenic activities. The model “G-RaFFe” generates roads and fields to reproduce the processes in which forests are converted into arable lands. For a selected level of habitat cover, three factors dominate its outcomes: the number of roads (accessibility), maximum field size (accounting for land ownership patterns), and maximum field disconnection (which enables field to be detached from roads). We compared the performance of G-RaFFe to three other models: Simmap (neutral model), Qrule (fractal-based) and Dinamica EGO (with 4 model versions differing in complexity). A PCA-based analysis indicated G-RaFFe and Dinamica version 4 (most complex) to perform best in matching realistic spatial patterns, but an alternative analysis which considers model variability identified G-RaFFe and Qrule as performing best. We also found model performance to be affected by habitat cover and the actual land-uses, the latter reflecting on land ownership patterns. We suggest that simple process-based generators such as G-RaFFe can be used to generate spatial patterns as templates for theoretical analyses, as well as for gaining better understanding of the relation between spatial processes and patterns. We suggest caution in applying neutral or fractal-based approaches, since spatial patterns that typify anthropogenic landscapes are often non-fractal in nature.
doi:10.1371/journal.pone.0064968
PMCID: PMC3665680  PMID: 23724108
15.  Increased Plasma Citrulline in Mice Marks Diet-Induced Obesity and May Predict the Development of the Metabolic Syndrome 
PLoS ONE  2013;8(5):e63950.
In humans, plasma amino acid concentrations of branched-chain amino acids (BCAA) and aromatic amino acids (AAA) increase in states of obesity, insulin resistance and diabetes. We here assessed whether these putative biomarkers can also be identified in two different obesity and diabetic mouse models. C57BL/6 mice with diet-induced obesity (DIO) mimic the metabolic impairments of obesity in humans characterized by hyperglycemia, hyperinsulinemia and hepatic triglyceride accumulation. Mice treated with streptozotocin (STZ) to induce insulin deficiency were used as a type 1 diabetes model. Plasma amino acid profiling of two high fat (HF) feeding trials revealed that citrulline and ornithine concentrations are elevated in obese mice, while systemic arginine bioavailability (ratio of plasma arginine to ornithine + citrulline) is reduced. In skeletal muscle, HF feeding induced a reduction of arginine levels while citrulline levels were elevated. However, arginine or citrulline remained unchanged in their key metabolic organs, intestine and kidney. Moreover, the intestinal conversion of labeled arginine to ornithine and citrulline in vitro remained unaffected by HF feeding excluding the intestine as prime site of these alterations. In liver, citrulline is mainly derived from ornithine in the urea cycle and DIO mice displayed reduced hepatic ornithine levels. Since both amino acids share an antiport mechanism for mitochondrial import and export, elevated plasma citrulline may indicate impaired hepatic amino acid handling in DIO mice. In the insulin deficient mice, plasma citrulline and ornithine levels also increased and additionally these animals displayed elevated BCAA and AAA levels like insulin resistant and diabetic patients. Therefore, type 1 diabetic mice but not DIO mice show the “diabetic fingerprint” of plasma amino acid changes observed in humans. Additionally, citrulline may serve as an early indicator of the obesity-dependent metabolic impairments.
doi:10.1371/journal.pone.0063950
PMCID: PMC3653803  PMID: 23691124
16.  User-friendly solutions for microarray quality control and pre-processing on ArrayAnalysis.org 
Nucleic Acids Research  2013;41(Web Server issue):W71-W76.
Quality control (QC) is crucial for any scientific method producing data. Applying adequate QC introduces new challenges in the genomics field where large amounts of data are produced with complex technologies. For DNA microarrays, specific algorithms for QC and pre-processing including normalization have been developed by the scientific community, especially for expression chips of the Affymetrix platform. Many of these have been implemented in the statistical scripting language R and are available from the Bioconductor repository. However, application is hampered by lack of integrative tools that can be used by users of any experience level. To fill this gap, we developed a freely available tool for QC and pre-processing of Affymetrix gene expression results, extending, integrating and harmonizing functionality of Bioconductor packages. The tool can be easily accessed through a wizard-like web portal at http://www.arrayanalysis.org or downloaded for local use in R. The portal provides extensive documentation, including user guides, interpretation help with real output illustrations and detailed technical documentation. It assists newcomers to the field in performing state-of-the-art QC and pre-processing while offering data analysts an integral open-source package. Providing the scientific community with this easily accessible tool will allow improving data quality and reuse and adoption of standards.
doi:10.1093/nar/gkt293
PMCID: PMC3692049  PMID: 23620278
17.  Vascular and Inflammatory High Fat Meal Responses in Young Healthy Men; A Discriminative Role of IL-8 Observed in a Randomized Trial 
PLoS ONE  2013;8(2):e53474.
Background
High fat meal challenges are known to induce postprandial low-grade inflammation and endothelial dysfunction. This assumption is largely based on studies performed in older populations or in populations with a progressed disease state and an appropriate control meal is often lacking. Young healthy individuals might be more resilient to such challenges. We therefore aimed to characterize the vascular and inflammatory response after a high fat meal in young healthy individuals.
Methods
In a double-blind randomized cross-over intervention study, we used a comprehensive phenotyping approach to determine the vascular and inflammatory response after consumption of a high fat shake and after an average breakfast shake in 20 young healthy subjects. Both interventions were performed three times.
Results
Many features of the vascular postprandial response, such as FMD, arterial stiffness and micro-vascular skin blood flow were not different between shakes. High fat/high energy shake consumption was associated with a more pronounced increase in blood pressure, heart rate, plasma concentrations of IL-8 and PBMCs gene expression of IL-8 and CD54 (ICAM-1), whereas plasma concentrations of sVCAM1 were decreased compared to an average breakfast.
Conclusion
Whereas no difference in postprandial response were observed on classical markers of endothelial function, we did observe differences between consumption of a HF/HE and an average breakfast meal on blood pressure and IL-8 in young healthy volunteers. IL-8 might play an important role in dealing with high fat challenges and might be an early marker for endothelial stress, a stage preceding endothelial dysfunction.
Trial Registration
ClinicalTrials.gov NCT00766623
doi:10.1371/journal.pone.0053474
PMCID: PMC3566159  PMID: 23405070
18.  Dietary Heme Alters Microbiota and Mucosa of Mouse Colon without Functional Changes in Host-Microbe Cross-Talk 
PLoS ONE  2012;7(12):e49868.
Colon cancer is a major cause of cancer deaths in Western countries and is associated with diets high in red meat. Heme, the iron-porphyrin pigment of red meat, induces cytotoxicity of gut contents which injures surface cells leading to compensatory hyperproliferation of crypt cells. This hyperproliferation results in epithelial hyperplasia which increases the risk of colon cancer. In humans, a high red-meat diet increases Bacteroides spp in feces. Therefore, we simultaneously investigated the effects of dietary heme on colonic microbiota and on the host mucosa of mice. Whole genome microarrays showed that heme injured the colonic surface epithelium and induced hyperproliferation by changing the surface to crypt signaling. Using 16S rRNA phylogenetic microarrays, we investigated whether bacteria play a role in this changed signaling. Heme increased Bacteroidetes and decreased Firmicutes in colonic contents. This shift was most likely caused by a selective susceptibility of Gram-positive bacteria to heme cytotoxic fecal water, which is not observed for Gram-negative bacteria, allowing expansion of the Gram-negative community. The increased amount of Gram-negative bacteria most probably increased LPS exposure to colonocytes, however, there is no appreciable immune response detected in the heme-fed mice. There was no functional change in the sensing of the bacteria by the mucosa, as changes in inflammation pathways and Toll- like receptor signaling were not detected. This unaltered host-microbe cross-talk indicates that the changes in microbiota did not play a causal role in the observed hyperproliferation and hyperplasia.
doi:10.1371/journal.pone.0049868
PMCID: PMC3519815  PMID: 23239972
19.  Pronounced Effects of Acute Endurance Exercise on Gene Expression in Resting and Exercising Human Skeletal Muscle 
PLoS ONE  2012;7(11):e51066.
Regular physical activity positively influences whole body energy metabolism and substrate handling in exercising muscle. While it is recognized that the effects of exercise extend beyond exercising muscle, it is unclear to what extent exercise impacts non-exercising muscles. Here we investigated the effects of an acute endurance exercise bouts on gene expression in exercising and non-exercising human muscle. To that end, 12 male subjects aged 44–56 performed one hour of one-legged cycling at 50% Wmax. Muscle biopsies were taken from the exercising and non-exercising leg before and immediately after exercise and analyzed by microarray. One-legged cycling raised plasma lactate, free fatty acids, cortisol, noradrenalin, and adrenalin levels. Surprisingly, acute endurance exercise not only caused pronounced gene expression changes in exercising muscle but also in non-exercising muscle. In the exercising leg the three most highly induced genes were all part of the NR4A family. Remarkably, many genes induced in non-exercising muscle were PPAR targets or related to PPAR signalling, including PDK4, ANGPTL4 and SLC22A5. Pathway analysis confirmed this finding. In conclusion, our data indicate that acute endurance exercise elicits pronounced changes in gene expression in non-exercising muscle, which are likely mediated by changes in circulating factors such as free fatty acids. The study points to a major influence of exercise beyond the contracting muscle.
doi:10.1371/journal.pone.0051066
PMCID: PMC3511348  PMID: 23226462
20.  Dietary Protein Affects Gene Expression and Prevents Lipid Accumulation in the Liver in Mice 
PLoS ONE  2012;7(10):e47303.
Background and Aims
High protein (HP) diets are suggested to positively modulate obesity and associated increased prevalence of non-alcoholic fatty liver (NAFLD) disease in humans and rodents. The aim of our study was to detect mechanisms by which a HP diet affects hepatic lipid accumulation.
Methods
To investigate the acute and long term effect of high protein ingestion on hepatic lipid accumulation under both low and high fat (HF) conditions, mice were fed combinations of high (35 energy%) or low (10 energy%) fat and high (50 energy%) or normal (15 energy%) protein diets for 1 or 12 weeks. Effects on body composition, liver fat, VLDL production rate and the hepatic transcriptome were investigated.
Results
Mice fed the HP diets displayed a lower body weight, developed less adiposity and decreased hepatic lipid accumulation, which could be attributed to a combination of several processes. Next to an increased hepatic VLDL production rate, increased energy utilisation due to enhanced protein catabolic processes, such as transamination, TCA cycle and oxidative phosphorylation was found upon high protein ingestion.
Conclusion
Feeding a HP diet prevented the development of NAFLD by enhancing lipid secretion into VLDL particles and a less efficient use of ingested calories.
doi:10.1371/journal.pone.0047303
PMCID: PMC3479095  PMID: 23110065
21.  5-HT7R/G12 Signaling Regulates Neuronal Morphology and Function in an Age-Dependent Manner 
The common neurotransmitter serotonin controls different aspects of early neuronal differentiation, although the underlying mechanisms are poorly understood. Here we report that activation of the serotonin 5-HT7 receptor promotes synaptogenesis and enhances synaptic activity in hippocampal neurons at early postnatal stages. An analysis of Gα12-deficient mice reveals a critical role of G12-protein for 5-HT7 receptor-mediated effects in neurons. In organotypic preparations from the hippocampus of juvenile mice, stimulation of 5-HT7R/G12 signaling potentiates formation of dendritic spines, increases neuronal excitability, and modulates synaptic plasticity. In contrast, in older neuronal preparations, morphogenetic and synaptogenic effects of 5-HT7/G12 signaling are abolished. Moreover, inhibition of 5-HT7 receptor had no effect on synaptic plasticity in hippocampus of adult animals. Expression analysis reveals that the production of 5-HT7 and Gα12-proteins in the hippocampus undergoes strong regulation with a pronounced transient increase during early postnatal stages. Thus, regulated expression of 5-HT7 receptor and Gα12-protein may represent a molecular mechanism by which serotonin specifically modulates formation of initial neuronal networks during early postnatal development.
doi:10.1523/JNEUROSCI.2765-11.2012
PMCID: PMC3369253  PMID: 22378867
22.  Temporo-Spectral Imaging of Intrinsic Optical Signals during Hypoxia-Induced Spreading Depression-Like Depolarization 
PLoS ONE  2012;7(8):e43981.
Spreading depression (SD) is characterized by a sustained near-complete depolarization of neurons, a massive depolarization of glia, and a negative deflection of the extracellular DC potential. These electrophysiological signs are accompanied by an intrinsic optical signal (IOS) which arises from changes in light scattering and absorption. Even though the underlying mechanisms are unclear, the IOS serves as non-invasive tool to define the spatiotemporal dynamics of SD in brain slices. Usually the tissue is illuminated by white light, and light reflectance or transmittance is monitored. Using a polychromatic, fast-switchable light source we now performed temporo-spectral recordings of the IOS associated with hypoxia-induced SD-like depolarization (HSD) in rat hippocampal slices kept in an interface recording chamber. Recording full illumination spectra (320–680 nm) yielded distinct reflectance profiles for the different phases of HSD. Early during hypoxia tissue reflectance decreased within almost the entire spectrum due to cell swelling. HSD was accompanied by a reversible reflectance increase being most pronounced at 400 nm and 460 nm. At 440 nm massive porphyrin absorption (Soret band) was detected. Hypotonic solutions, Ca2+-withdrawal and glial poisoning intensified the reflectance increase during HSD, whereas hypertonic solutions dampened it. Replacement of Cl- inverted the reflectance increase. Inducing HSD by cyanide distorted the IOS and reflectance at 340–400 nm increased irreversibly. The pronounced changes at short wavelengths (380 nm, 460 nm) and their cyanide sensitivity suggest that block of mitochondrial metabolism contributes to the IOS during HSD. For stable and reliable IOS recordings during HSD wavelengths of 460–560 nm are recommended.
doi:10.1371/journal.pone.0043981
PMCID: PMC3430631  PMID: 22952835
23.  Dietary Heme-Mediated PPARα Activation Does Not Affect the Heme-Induced Epithelial Hyperproliferation and Hyperplasia in Mouse Colon 
PLoS ONE  2012;7(8):e43260.
Red meat consumption is associated with an increased colon cancer risk. Heme, present in red meat, injures the colon surface epithelium by luminal cytotoxicity and reactive oxygen species. This surface injury is overcompensated by hyperproliferation and hyperplasia of crypt cells. Transcriptome analysis of mucosa of heme-fed mice showed, besides stress- and proliferation-related genes, many upregulated lipid metabolism-related PPARα target genes. The aim of this study was to investigate the role of PPARα in heme-induced hyperproliferation and hyperplasia. Male PPARα KO and WT mice received a purified diet with or without heme. As PPARα is proposed to protect against oxidative stress and lipid peroxidation, we hypothesized that the absence of PPARα leads to more surface injury and crypt hyperproliferation in the colon upon heme-feeding. Heme induced luminal cytotoxicity and lipid peroxidation and colonic hyperproliferation and hyperplasia to the same extent in WT and KO mice. Transcriptome analysis of colonic mucosa confirmed similar heme-induced hyperproliferation in WT and KO mice. Stainings for alkaline phosphatase activity and expression levels of Vanin-1 and Nrf2-targets indicated a compromised antioxidant defense in heme-fed KO mice. Our results suggest that the protective role of PPARα in antioxidant defense involves the Nrf2-inhibitor Fosl1, which is upregulated by heme in PPARα KO mice. We conclude that PPARα plays a protective role in colon against oxidative stress, but PPARα does not mediate heme-induced hyperproliferation. This implies that oxidative stress of surface cells is not the main determinant of heme-induced hyperproliferation and hyperplasia.
doi:10.1371/journal.pone.0043260
PMCID: PMC3419209  PMID: 22905243
24.  Markers of Endogenous Desaturase Activity and Risk of Coronary Heart Disease in the CAREMA Cohort Study 
PLoS ONE  2012;7(7):e41681.
Background
Intakes of n-3 polyunsaturated fatty acids (PUFAs), especially EPA (C20∶5n-3) and DHA (C22∶6n-3), are known to prevent fatal coronary heart disease (CHD). The effects of n-6 PUFAs including arachidonic acid (C20∶4n-6), however, remain unclear. δ-5 and δ-6 desaturases are rate-limiting enzymes for synthesizing long-chain n-3 and n-6 PUFAs. C20∶4n-6 to C20∶3n-6 and C18∶3n-6 to C18∶2n-6 ratios are markers of endogenous δ-5 and δ-6 desaturase activities, but have never been studied in relation to incident CHD. Therefore, the aim of this study was to investigate the relation between these ratios as well as genotypes of FADS1 rs174547 and CHD incidence.
Methods
We applied a case-cohort design within the CAREMA cohort, a large prospective study among the general Dutch population followed up for a median of 12.1 years. Fatty acid profile in plasma cholesteryl esters and FADS1 genotype at baseline were measured in a random subcohort (n = 1323) and incident CHD cases (n = 537). Main outcome measures were hazard ratios (HRs) of incident CHD adjusted for major CHD risk factors.
Results
The AA genotype of rs174547 was associated with increased plasma levels of C204n-6, C20∶5n-3 and C22∶6n-3 and increased δ-5 and δ-6 desaturase activities, but not with CHD risk. In multivariable adjusted models, high baseline δ-5 desaturase activity was associated with reduced CHD risk (P for trend = 0.02), especially among those carrying the high desaturase activity genotype (AA): HR (95% CI) = 0.35 (0.15–0.81) for comparing the extreme quintiles. High plasma DHA levels were also associated with reduced CHD risk.
Conclusion
In this prospective cohort study, we observed a reduced CHD risk with an increased C20∶4n-6 to C20∶3n-6 ratio, suggesting that δ-5 desaturase activity plays a role in CHD etiology. This should be investigated further in other independent studies.
doi:10.1371/journal.pone.0041681
PMCID: PMC3402436  PMID: 22911844
25.  The association between the sagittal femoral stem alignment and the resulting femoral head centre in total hip arthroplasty 
International Orthopaedics  2010;35(7):981-987.
Adequate stem alignment is essential for the success of Total Hip Arthroplasty (THA) to avoid dislocation and impingement. One factor that has not been sufficiently investigated so far is the stem tilting in the sagittal plane, which has an influence on the position of the centre of the femoral head and thus also on prosthesis torsion. We aimed to evaluate sagittal stem position using 3D-CTs in patients with THA and to develop a mathematical-geometrical model to simulate the functional correlation between sagittal stem tilting and the influence on functional anteversion. Thirty patients with THA underwent a CT-scan. By 3D-reconstruction of the CT-data, femoral-/prosthesis-axis, torsion and sagittal tilt were determined. In accordance with the position of the femoral and prosthesis axes, the rotatory (rAV) (surgically adjusted) and functional (depending on sagittal tilt) anteversion (fAV) was measured. A three dimentional-coordinate transformation was also performed using the Euler-angles to derive a mathematical-geometrical correlation between sagittal stem tilting and corresponding influence on anteversion. The mean rAV was 8° (-11.6 - 26°), the fAV 18° (6.2 - 37°), and the difference 10° (8.8 - 18°). The mean degree of stem tilting was 5.2° (0.7 - 9°) anterior towards the femoral axis. The individually measured parameters are reflected in the mathematical-geometrical model. Depending on the extent of the sagittal deviation, a clear influence on the torsion emerges. For example, a stem implanted at a 15° anteverted angle with a sagittal tilt by two degrees towards anterior results in a fAV of 20°. A clear association between the sagittal stem alignment and the impact on the fAV was demonstrated. Hence, the rotatory anteversion intended by the surgeon may be functionally significantly different. This might pose an increased risk of dislocation or impingement. The sagittal tilt of the prosthesis should therefore be considered in the context of impingement and dislocation diagnosis. In this respect, we recommend a 3D-analysis of stem alignment.
doi:10.1007/s00264-010-1047-z
PMCID: PMC3167395  PMID: 20549502

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