Intensity-Based Camera Setup for Refractometric and Biomolecular Sensing with a Photonic Crystal Microfluidic Chip.

Label-free sensing is a promising approach for point-of-care testing devices. Among optical transducers, photonic crystal slabs (PCSs) have positioned themselves as an inexpensive yet versatile platform for label-free biosensing. A spectral resonance shift is observed upon biomolecular binding to the functionalized surface. Commonly, a PCS is read out by a spectrometer. Alternatively, the spectral shift may be translated into an intensity change by tailoring the system response. Intensity-based camera setups (IBCS) are of interest as they mitigate the need for postprocessing, enable spatial sampling, and have moderate hardware requirements. However, they exhibit modest performance compared with spectrometric approaches. Here, we show an increase of the sensitivity and limit of detection (LOD) of an IBCS by employing a sharp-edged cut-off filter to optimize the system response. We report an increase of the LOD from (7.1 ± 1.3) × 10-4 RIU to (3.2 ± 0.7) × 10-5 RIU. We discuss the influence of the region of interest (ROI) size on the achievable LOD. We fabricated a biochip by combining a microfluidic and a PCS and demonstrated autonomous transport. We analyzed the performance via refractive index steps and the biosensing ability via diluted glutathione S-transferase (GST) antibodies (1:250). In addition, we illustrate the speed of detection and demonstrate the advantage of the additional spatial information by detecting streptavidin (2.9 µg/mL). Finally, we present the detection of immunoglobulin G (IgG) from whole blood as a possible basis for point-of-care devices.

Spatially Resolved Protein Binding Kinetics Analysis in Microfluidic Photonic Crystal Sensors.

Organ-on-a-Chip systems are emerging as an important in vitro analysis method for drug screening and medical research. For continuous biomolecular monitoring of the cell culture response, label-free detection within the microfluidic system or in the drainage tube is promising. We study photonic crystal slabs integrated with a microfluidic chip as an optical transducer for label-free biomarker detection with a non-contact readout of binding kinetics. This work analyzes the capability of same-channel reference for protein binding measurements by using a spectrometer and 1D spatially resolved data evaluation with a spatial resolution of 1.2 µm. A cross-correlation-based data-analysis procedure is implemented. First, an ethanol-water dilution series is used to obtain the limit of detection (LOD). The median of all row LODs is (2.3±0.4)×10-4 RIU with 10 s exposure time per image and (1.3±0.24)×10-4 RIU with 30 s exposure time. Next, we used a streptavidin-biotin binding process as a test system for binding kinetics. Time series of optical spectra were recorded while constantly injecting streptavidin in DPBS at concentrations of 1.6 nM, 3.3 nM, 16.6 nM and 33.3 nM into one channel half as well as the whole channel. The results show that localized binding within a microfluidic channel is achieved under laminar flow. Furthermore, binding kinetics are fading out at the microfluidic channel edge due to the velocity profile.

Four decades of experience of prosthetic valve endocarditis reflect a high variety of diverse pathogens.

Prosthetic valve endocarditis (PVE) remains a serious condition with a high mortality rate. Precise identification of the PVE-associated pathogen/s and their virulence is essential for successful therapy and patient survival. The commonly described PVE-associated pathogens are staphylococci, streptococci, and enterococci, with Staphylococcus aureus being the most frequently diagnosed species. Furthermore, multi-drug resistance pathogens are increasing in prevalence and continue to pose new challenges mandating a personalized approach. Blood cultures in combination with echocardiography are the most common methods to diagnose PVE, often being the only indication, it exists. In many cases, the diagnostic strategy recommended in the clinical guidelines does not identify the precise microbial agent, and frequently, false-negative blood cultures are reported. Despite the fact that blood culture findings are not always a good indicator of the actual PVE agent in the valve tissue, only a minority of re-operated prostheses are subjected to microbiological diagnostic evaluation. In this review, we focus on the diversity and the complete spectrum of PVE-associated bacterial, fungal, and viral pathogens in blood and prosthetic heart valve, their possible virulence potential, and their challenges in making a microbial diagnosis. We are curious to understand if the unacceptable high mortality of PVE is associated with the high number of negative microbial findings in connection with a possible PVE. Herein, we discuss the possibilities and limits of the diagnostic methods conventionally used and make recommendations for enhanced pathogen identification. We also show possible virulence factors of the most common PVE-associated pathogens and their clinical effects. Based on blood culture, molecular biological diagnostics, and specific valve examination, better derivations for the antibiotic therapy as well as possible preventive intervention can be established in the future.

Bacterial infiltration in structural heart valve disease.

OBJECTIVES: The pathology of structural valvular heart disease (sVHD) ranges from basic diseases of rheumatologic origin to chronic degenerative remodeling processes after acute bacterial infections. Molecular genetic methods allow detection of the complete microbial spectrum in heart valve tissues independent of microbiological cultivation. In particular, whole-metagenome analysis is a sensitive and highly specific analytical method that allows a deeper insight into the pathogenicity of the diseases. In the present study we assessed the pathogen spectrum in heart valve tissue from 25 sVHD patients using molecular and microbiological methods. METHODS: Twenty-five sVHD patients were selected randomly from an observational cohort study (March 2016 to January 2017). The explanted native heart valves were examined using microbiological methods and immunohistological structural analysis. In addition, the bacterial metagenome of the heart valve tissue was determined using next-generation sequencing. RESULTS: The use of sonication as a pretreatment of valve tissue from 4 sVHD patients permitted successful detection of Clostridium difficile, Enterococcus faecalis, Staphylococcus saccharolyticus, and Staphylococcus haemolyticus using microbial cultivation. Histological staining revealed intramural localization. Metagenome analysis identified a higher rate of bacterial infiltration in 52% of cases. The pathogen spectrum included both gram-positive and gram-negative bacteria. CONCLUSIONS: Microbiological and molecular biological studies are necessary to detect the spectrum of bacteria in a calcified heart valve. Metagenome analysis is a valid method to gain new insight into the polymicrobial pathophysiology of sVHD. Our results suggest that an undetected proportion of sVHD might be triggered by chronic inflammation or influenced by secondary bacterial infiltration.

Quantification of Multiple Bacteria in Calcified Structural Valvular Heart Disease.

Genome studies of heart valve tissue (HVT) in patients with structural valvular heart disease (sVHD) and acute infective endocarditis (aIE) showed polymicrobial infections. Subject of this study is the quantification of bacterial DNA in HVT of sVHD in comparison to aIE. It will be examined whether the bacterial DNA concentration can be used as surrogate marker to differentiate chronic and acute infections. DNA was isolated from HVT of 100 patients with sVHD and 23 microbiologically positively tested patients with aIE. Selected pathogens (Cutibacterium acnes, Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Streptococcus pyogenes, Streptococcus agalactiae, Clostridium difficile, and Klebsiella pneumoniae) were quantified using TaqMan-qPCR. Polymicrobial infiltration of HVT was investigated by immunohistologic methods. Of 100 sVHD patients, 94 tested positive for bacteria by 16S-rDNA and 72 by TaqMan-qPCR. In 29% of the sVHD cohort and in 70% of the aIE cohort, a coinfection with more than 2 bacteria was observed as indication of a polymicrobial infection. The most common pathogens in the sVHD patients were C. acnes (59%; 5-4074 pg/mL), E. faecalis (16%, 174-2781 pg/mL), and S. aureus (15%, 8-105 pg/mL). The DNA concentration of E. faecalis (P = 0.0285) and S. aureus (P = 0.0149) is significantly lower in the sVHD cohort than in the aIE cohort. sVHD is associated with bacterial infection and infiltration of the HVT in a majority of cases. TaqMan-qPCR is a valid instrument for the specific detection of bacteria in HVT and allows discrimination between sVHD and aIE for E. faecalis and S. aureus.

Gastric mucosal devitalization improves blood pressure, renin and cardiovascular lipid deposition in a rat model of obesity.

Background and study aims In lieu of the drawbacks of metabolic surgery, a method of mimicking resection of the gastric mucosa could be of value to those with obesity-related cardiovascular disease (CVD). Our study aims to investigate the effect of gastric mucosal devitalization (GMD) on blood pressure (BP) and cardiovascular lipid deposition in a rat model of obesity. Methods GMD of 70 % of the stomach was achieved by argon plasma coagulation. GMD was compared to sleeve gastrectomy (SG) and sham (SH) in a high-fat-diet-induced rat model of obesity (48 rats). At 8 weeks, we measured noninvasive BP, renin, vessel relaxation and ghrelin receptor regulation in the aorta. In addition, we quantified cardiac lipid deposition and lipid droplet deposition in cardiac muscle and aorta. Results GMD and SG were observed to have similar reductions in body weight, visceral adiposity, and serum lipid profile compared to SH rats. GMD resulted in a significant reduction in arterial BP compared to SH. Furthermore, there were significant reductions in plasma renin activity and percentage of phenylnephrine constriction to acetylcholine at the aortic ring in GMD rats compared to SH, providing insights into the mechanisms behind the reduced BP. Interestingly, the reduced BP occurred despite a reduction in endothelial ghrelin recteptor activation. Cardiac lipid content was significantly reduced in GMD rats. Lipid deposition, as illustrated by Nile Red stain, was reduced in cardiac muscle and the aorta. Conclusion GMD resulted in a significant improvement in BP, renin and cardiovascular lipid deposition. GMD deserves further attention as a method of treating obesity-related CVD.

Effects of Weight Loss on Glutathione Peroxidase 3 Serum Concentrations and Adipose Tissue Expression in Human Obesity.

BACKGROUND/AIMS: Altered expression and circulating levels of glutathione peroxidase 3 (GPX3) have been observed in obesity and type 2 diabetes (T2D) across species. Here, we investigate whether GPX3 serum concentrations and adipose tissue (AT) GPX3 mRNA expression are related to obesity and weight loss. METHODS: GPX3 serum concentration was measured in 630 individuals, including a subgroup (n = 293) for which omental and subcutaneous (SC) GPX3 mRNA expression has been analyzed. GPX3 analyses include three interventions: 6 months after bariatric surgery (n = 80) or combined exercise/hypocaloric diet (n = 20) or two-step bariatric surgery (n = 24) studies. RESULTS: Bariatric surgery-induced weight loss (-25.8 ± 8.4%), but not a moderate weight reduction of -8.8 ± 6.5% was associated with significantly reduced GPX3 serum concentrations. GPX3 mRNA is significantly higher expressed in AT from individuals with normal glucose metabolism compared to T2D patients. SC AT GPX3 expression is significantly higher in lean compared to obese as well as in insulin-sensitive compared insulin-resistant individuals with obesity. Weight loss after bariatric surgery causes a significant increase in SC AT GPX3 expression. AT GPX3 expression significantly correlates with age, BMI, fat distribution, insulin sensitivity (only SC AT), but not with circulating GPX3. CONCLUSION: Our data support the notion that SC AT GPX3 expression is associated with obesity, fat distribution and related to whole body insulin resistance.

MRI assessment of changes in adipose tissue parameters after bariatric surgery.

Bariatric surgery and other therapeutic options for obese patients are often evaluated by the loss of weight, reduction of comorbidities or improved quality of life. However, little is currently known about potential therapy-related changes in the adipose tissue of obese patients. The aim of this study was therefore to quantify fat fraction (FF) and T1 relaxation time by magnetic resonance imaging (MRI) after Roux-en-Y gastric bypass surgery and compare the resulting values with the preoperative ones. Corresponding MRI data were available from 23 patients (16 females and 7 males) that had undergone MRI before (M0) and one month after (M1) bariatric surgery. Patients were 22-59 years old (mean age 44.3 years) and their BMI ranged from 35.7-54.6 kg/m2 (mean BMI 44.6 kg/m2) at M0. Total visceral AT volumes (VVAT-T, in L) were measured by semi-automatic segmentation of axial MRI images acquired between diaphragm and femoral heads. MRI FF and T1 relaxation times were measured in well-defined regions of visceral (VAT) and subcutaneous (SAT) adipose tissue using two custom-made analysis tools. Average BMI values were 45.4 kg/m2 at time point M0 and 42.4 kg/m2 at M1. Corresponding VVAT-T values were 5.94 L and 5.33 L. Intraindividual differences in both BMI and VVAT-T were highly significant (p<0.001). Average relaxation times T1VAT were 303.7 ms at M0 and 316.9 ms at M1 (p<0.001). Corresponding T1SAT times were 283.2 ms and 280.7 ms (p = 0.137). Similarly, FFVAT differences (M0: 85.7%, M1: 83.4%) were significant (p <0.01) whereas FFSAT differences (M0: 86.1, M1: 85.9%) were not significant (p = 0.517). In conclusion, bariatric surgery is apparently not only related to a significant reduction in common parameters of adipose tissue distribution, here BMI and total visceral fat volume, but also significant changes in T1 relaxation time and fat fraction of visceral adipose tissue. Such quantitative MRI measures may potentially serve as independent biomarkers for longitudinal and cross-sectional measurements in obese patients.

Gastric mucosal devitalization is safe and effective in reducing body weight and visceral adiposity in a porcine model.

BACKGROUND AND AIMS: The early improvement in metabolic profile after sleeve gastrectomy (SG) indicates that the significant benefits of metabolic surgery are gastric in origin. We have previously demonstrated that devitalization of the gastric mucosa (without a reduction in gastric volume) in metabolically disturbed obese rats results in an improvement of obesity and its associated comorbidities. The aims of this study were to assess the technical feasibility, efficacy, and safety of gastric mucosal devitalization (GMD) in a large animal (porcine) model. METHODS: A 3-arm (GMD versus SG versus sham [SH]) prospective randomized controlled trial with an 8-week follow-up period was performed. The primary endpoint was relative weight loss. Secondary endpoints were absolute body weight, abdominal visceral adiposity, abdominal subcutaneous adiposity, organ lipid content, and serum ghrelin level. RESULTS: GMD resulted in a significant relative weight loss of 36% over SH at 8 weeks (P < .05). There was no significant difference in relative weight loss between GMD and SG at 4 weeks; however, SG resulted in a 29% superior relative weight loss at 8 weeks (P < .05). With regard to visceral adiposity, there was a significant benefit of GMD over SH at 8 weeks. Despite differences in relative weight loss, there was no significant difference in visceral adiposity between SG and GMD at 8 weeks. Significant improvements in GMD over SH were noted with regard to skeletal and heart muscle lipid content. GMD pigs at 8 weeks demonstrated regeneration of the gastric mucosa without ulceration or significant scarring. Despite mucosal regeneration, the abundance of serum ghrelin was significantly lower in the GMD cohort compared with the SG and SH cohorts. CONCLUSIONS: GMD was technically feasible and resulted in relative weight loss and an improvement in visceral adiposity. The benefits noted were out of proportion to what would be expected with weight loss alone.

Gastric mucosal devitalization reduces adiposity and improves lipid and glucose metabolism in obese rats.

BACKGROUND AND AIMS: The gastric mucosa is an endocrine organ that regulates satiation pathways by expression of orexigenic and anorexigenic hormones. Vertical sleeve gastrectomy (VSG) excludes gastric mucosa and reduces gastric volume. Our study aimed to investigate the independent effects of altering gastric mucosa on obesity and its related comorbidities. METHODS: Gastric mucosa devitalization (GMD) of 70% of the stomach was achieved by argon plasma coagulation in a high-fat diet rat model and was compared with VSG and sham surgery. In an 8-week follow-up study, we quantified body weight, visceral adiposity, insulin resistance index, cholesterol profiles, and free fatty acid profiles by enzyme-linked immunosorbent assay (ELISA). Following a 2-hour oral glucose tolerance test, the kinetics of ghrelin, glucagon-like peptide-1, peptide YY, and serum and liver bile acid levels were measured. Liver lipid content was quantified by ELISA. RESULTS: GMD resulted in significant reductions in body weight, visceral and subcutaneous adipose tissue, and hepatic steatosis as well as an improvement in lipid metabolism. GMD resulted in significant reductions in food intake and intestinal malabsorption of free fatty acids, both contributing to improved body composition and metabolic profile. Mechanistically, GMD resulted in a significant reduction in serum palmitate levels as well as an increase in serum and liver bile acid levels, known to alter glucose and lipid metabolism. Similar changes were noted when VSG rats were compared with sham surgery rats. CONCLUSIONS: Devitalization of gastric mucosa, independent of altering gastric volume, was able to reduce obesity-related comorbidities. The gastric mucosa may be a potential target for treating obesity and its associated comorbidities.

New insights into valve-related intramural and intracellular bacterial diversity in infective endocarditis.

AIMS: In infective endocarditis (IE), a severe inflammatory disease of the endocardium with an unchanged incidence and mortality rate over the past decades, only 1% of the cases have been described as polymicrobial infections based on microbiological approaches. The aim of this study was to identify potential biodiversity of bacterial species from infected native and prosthetic valves. Furthermore, we compared the ultrastructural micro-environments to detect the localization and distribution patterns of pathogens in IE. MATERIAL AND METHODS: Using next-generation sequencing (NGS) of 16S rDNA, which allows analysis of the entire bacterial community within a single sample, we investigated the biodiversity of infectious bacterial species from resected native and prosthetic valves in a clinical cohort of 8 IE patients. Furthermore, we investigated the ultrastructural infected valve micro-environment by focused ion beam scanning electron microscopy (FIB-SEM). RESULTS: Biodiversity was detected in 7 of 8 resected heart valves. This comprised 13 bacterial genera and 16 species. In addition to 11 pathogens already described as being IE related, 5 bacterial species were identified as having a novel association. In contrast, valve and blood culture-based diagnosis revealed only 4 species from 3 bacterial genera and did not show any relevant antibiotic resistance. The antibiotics chosen on this basis for treatment, however, did not cover the bacterial spectra identified by our amplicon sequencing analysis in 4 of 8 cases. In addition to intramural distribution patterns of infective bacteria, intracellular localization with evidence of bacterial immune escape mechanisms was identified. CONCLUSION: The high frequency of polymicrobial infections, pathogen diversity, and intracellular persistence of common IE-causing bacteria may provide clues to help explain the persistent and devastating mortality rate observed for IE. Improved bacterial diagnosis by 16S rDNA NGS that increases the ability to tailor antibiotic therapy may result in improved outcomes.

Metabolic in Vivo Labeling Highlights Differences of Metabolically Active Microbes from the Mucosal Gastrointestinal Microbiome between High-Fat and Normal Chow Diet.

The gastrointestinal microbiota in the gut interacts metabolically and immunologically with the host tissue in the contact zone of the mucus layer. For understanding the details of these interactions and especially their dynamics it is crucial to identify the metabolically active subset of the microbiome. This became possible by the development of stable isotope probing techniques, which have only sparsely been applied to microbiome research. We applied the in vivo stable isotope approach using 15N-labeled diet with subsequent identification of metabolically active bacterial species. Four-week old male Sprague-Dawley rats were randomly assigned to chow diet (CD, n =15) and high-fat diet (HFD, n =15). After 11 weeks, three animals from each group were sacrificed for baseline characterization of anthropometric and metabolic obesity. The remaining animals were exposed to either a 15N-labeled (n =9) or a 14N-unlabeled experimental diet (n =3). Three rats from each cohort (HFD and CD) were sacrificed at 12, 24, and 72 h. The remaining three animals from each cohort, which received the 14N-unlabeled diet, were sacrificed after 72 h. The colon was harvested and divided into three equal sections (proximal, medial, and distal), and the mucus layer of each specimen was sampled by scraping. We identified the active subset in an HFD model of obesity in comparison with lean controls rats using metaproteomics. In addition, all samples were investigated by 16S rRNA amplicon gene sequencing. The active microbiome of the HFD group showed an increase in bacterial taxa for Verrucomicrobia and Desulfovibrionaceae. In contrast with no significant changes in alpha diversity, time- and localization-dependent effects in beta-diversity were clearly observed. In terms of enzymatic functions the HFD group showed strong affected metabolic pathways such as energy production and carbohydrate metabolism. In vivo isotope labeling combined with metaproteomics provides a valuable method to distinguish the active from the non-active bacterial phylogenetic groups that are relevant for microbiota-host interaction. For morbid obesity such analysis may provide potentially new strategies for targeted pre- or probiotic treatments.

Age and gender specific estimation of visceral adipose tissue amounts from radiological images in morbidly obese patients.

Image-based quantifications of visceral adipose tissue (VAT) volumes from segmented VAT areas are increasingly considered for risk assessment in obese patients. The goal of this study was to determine the power of partial VAT areas to predict total VAT volume in morbidly obese patients (BMI > 40 kg/m(2)) as a function of gender, age and anatomical landmarks. 130 morbidly obese patients (mean BMI 46.5 kg/m(2); 94 females) underwent IRB-approved MRI. Total VAT volumes were predicted from segmented VAT areas (of single or five adjacent slices) at common axial landmark levels and compared with the measured ones (VVAT-T, about 40 slices between diaphragm and pelvic floor). Standard deviations σ1 and σ5 of the respective VAT volume differences served as measures of agreement. Mean VVAT-T was 4.9 L for females and 8.1 L for males. Best predictions were found at intervertebral spaces L3-L4 for females (σ5 = 688 ml, σ1 = 832 ml) and L1-L2 for males (σ5 = 846 ml, σ1 = 992 ml), irrespective of age. In conclusion, VAT volumes in morbidly obese patients can be reliably predicted by multiplying the segmented VAT area at a gender-specific lumbar reference level with a fixed scaling factor and effective slice thickness.

Proteome profiles of HDL particles of patients with chronic heart failure are associated with immune response and also include bacteria proteins.

Besides modulation of reverse cholesterol transport, high density lipoprotein (HDL) is able to modulate vascular function by stimulating endothelial nitric oxide synthase. Recently, it could be documented that this function of HDL was significantly impaired in patients with chronic heart failure (CHF). We investigated alterations in the HDL proteome in CHF patients. Therefore, HDL was isolated from 5 controls (HDLhealthy) and 5 CHF patients of NYHA-class IIIb (HDLCHF). Proteome analysis of HDL particles was performed by two-dimensional liquid chromatography-mass spectrometry (SCX/RP LC-MS/MS). In total, we identified 494 distinct proteins, of which 107 proteins were commonly found in both groups (HDLCHF and HDLhealthy) indicating a high inter-subject variability across HDL particles. Several important proteins (e.g. ITGA2, APBA1 or A2M) varied in level. Functional analysis revealed regulated pathways. A minor proportion of bacteria-derived proteins were also identified in the HDL-particles. The extension of the list of HDL-associated proteins allows besides their mere description new insights into alterations in HDL function in diseases. In addition, the detection of bacterial proteins bound to HDL will broaden our view of HDL not only as a cholesterol carrier but also as a carrier of proteins.

Establishing a reliable multiple reaction monitoring-based method for the quantification of obesity-associated comorbidities in serum and adipose tissue requires intensive clinical validation.

Multiple reaction monitoring (MRM)-based mass spectrometric quantification of peptides and their corresponding proteins has been successfully applied for biomarker validation in serum. The option of multiplexing offers the chance to analyze various proteins in parallel, which is especially important in obesity research. Here, biomarkers that reflect multiple comorbidities and allow monitoring of therapy outcomes are required. Besides the suitability of established MRM assays for serum protein quantification, it is also feasible for analysis of tissues secreting the markers of interest. Surprisingly, studies comparing MRM data sets with established methods are rare, and therefore the biological and clinical value of most analytes remains questionable. A MRM method using nano-UPLC-MS/MS for the quantification of obesity related surrogate markers for several comorbidities in serum, plasma, visceral and subcutaneous adipose tissue was established. Proteotypic peptides for complement C3, adiponectin, angiotensinogen, and plasma retinol binding protein (RBP4) were quantified using isotopic dilution analysis and compared to the standard ELISA method. MRM method variabilities were mainly below 10%. The comparison with other MS-based approaches showed a good correlation. However, large differences in absolute quantification for complement C3 and adiponectin were obtained compared to ELISA, while less marked differences were observed for angiotensinogen and RBP4. The verification of MRM in obesity was performed to discriminate first lean and obese phenotype and second to monitor excessive weight loss after gastric bypass surgery in a seven-month follow-up. The presented MRM assay was able to discriminate obese phenotype from lean and monitor weight loss related changes of surrogate markers. However, inclusion of additional biomarkers was necessary to interpret the MRM data on obesity phenotype properly. In summary, the development of disease-related MRMs should include a step of matching the MRM data with clinically approved standard methods and defining reference values in well-sized representative age, gender, and disease-matched cohorts.

Molecular fingerprint of high fat diet induced urinary bladder metabolic dysfunction in a rat model.

AIMS/HYPOTHESIS: Diabetic voiding dysfunction has been reported in epidemiological dimension of individuals with diabetes mellitus. Animal models might provide new insights into the molecular mechanisms of this dysfunction to facilitate early diagnosis and to identify new drug targets for therapeutic interventions. METHODS: Thirty male Sprague-Dawley rats received either chow or high-fat diet for eleven weeks. Proteomic alterations were comparatively monitored in both groups to discover a molecular fingerprinting of the urinary bladder remodelling/dysfunction. Results were validated by ELISA, Western blotting and immunohistology. RESULTS: In the proteome analysis 383 proteins were identified and canonical pathway analysis revealed a significant up-regulation of acute phase reaction, hypoxia, glycolysis, ß-oxidation, and proteins related to mitochondrial dysfunction in high-fat diet rats. In contrast, calcium signalling, cytoskeletal proteins, calpain, 14-3-3η and eNOS signalling were down-regulated in this group. Interestingly, we found increased ubiquitin proteasome activity in the high-fat diet group that might explain the significant down-regulation of eNOS, 14-3-3η and calpain. CONCLUSIONS/INTERPRETATION: Thus, high-fat diet is sufficient to induce significant remodelling of the urinary bladder and alterations of the molecular fingerprint. Our findings give new insights into obesity related bladder dysfunction and identified proteins that may indicate novel pathophysiological mechanisms and therefore constitute new drug targets.

Combined serum proteomic and metabonomic profiling after laparoscopic sleeve gastrectomy in children and adolescents.

AIM: The consequences of bariatric surgery on important metabolic and signaling pathways are still poorly understood. The aim of our study was to unravel the network of metabolic changes and obesity-related protein biomarkers in morbidly obese children and adolescents undergoing laparoscopic sleeve gastrectomy (LSG). METHODS: In a prospective study, 6 children with morbid obesity who had failed a well-established conservative weight loss therapy underwent LSG. Pre- and 6 months postoperatively, a metabonomic profiling of 163 metabolites by mass spectrometry and protein profiling by ELISA (clusterin [CLU], pigment epithelium-derived factor [PEDF], retinol binding protein 4 [RBP4], paraoxonase 1 [PON1]) was performed to identify biomarkers of important pathways. RESULTS: At referral for surgery, the mean age was 14.5 years (range 8-17), mean body mass index (BMI in kg/m(2)) was 48.13 (range 41.1-56.3). All patients showed various metabolic comorbidities. LSG was uneventful in all of them. At 6 months postsurgery, the mean BMI has dropped to 37.1 (range 28.4-40.6). Targeted serum metabonomics resulted in 7 metabolites, which were significantly affected by LSG. Among those, the amino acid phenylalanine was increased, and methionine decreased. The glycero-phospatidylcholine PCaaC38:5 were upregulated, and PCaaC40:2 and PCaaC42:1 were down regulated after 6 months compared with baseline. Further, sphingomyoline (SM) C26:0 and SM C26:1 were decreased after 6 months. The obesity-related biomarkers CLU, PEDF, and RBP4 were decreased, whereas PON1 levels were increased. CONCLUSION: LSG leads to changes in amino acids and in lipid metabolism indicated by glycerol-phosphatidylcholines and SM. The pattern of protein biomarkers and metabolites might provide measures for the induced physiological changes and for therapy monitoring.

Serum vaspin concentrations are decreased after exercise-induced oxidative stress.

Elevated visceral adipose tissue-derived serpin (vaspin) serum concentrations are associated with impaired insulin sensitivity, but increase unexpectedly after long-term physical training. We therefore investigated the effect of an acute exercise bout and the effects of vitamin supplementation on chronic exercise effect and on serum vaspin concentrations. We measured serum vaspin and thiobarbituric acid-reactive substances (TBARS) concentrations in 80 individuals before and after a 1-hour acute exercise bout and independently in 40 healthy young men who were randomly assigned to either antioxidant (vitamin C (1,000 mg/day) and vitamin E (400 IU/day)) or to no supplementation after a standardized 4-week physical training program as a post hoc analysis. Serum vaspin concentrations significantly decreased after acute physical exercise as well as after 4 weeks of training in individuals without antioxidants. Changes in vaspin serum concentration correlate with increased TBARS serum concentrations both in response to a 1-hour exercise bout (r = -0.42, p < 0.01) and to the 4-week training (r = -0.31, p < 0.05). Interestingly, supplementation with antioxidants rather increased circulating vaspin levels in response to 4 weeks of exercise. In conclusion, vaspin serum concentrations are decreased by exercise-induced oxidative stress, but not by exercise-associated improvement in insulin sensitivity.

An induction gene trap screen in neural stem cells reveals an instructive function of the niche and identifies the splicing regulator sam68 as a tenascin-C-regulated target gene.

Neural stem cells (NSCs) reside in a niche that abounds in extracellular matrix (ECM) molecules. The ECM glycoprotein tenascin-C (Tnc) that occurs in more than 25 isoforms represents a major constituent of the privileged NSC milieu. To understand its role for NSCs, the induction gene trap technology was successfully applied to mouse embryonic NSCs, and a library of more than 500 NSC lines with independent gene trap vector integrations was established. Our pilot screen identified Sam68 as a target of Tnc signaling in NSCs. The Tnc-mediated downregulation of Sam68, which we found expressed at low levels in the niche along with Tnc, was independently confirmed on the protein level. Sam68 is a multifunctional RNA-binding protein, and its potential significance for cultured NSCs was studied by overexpression. Increased Sam68 levels caused a marked reduction in NSC cell proliferation. In addition, Sam68 is a signal-dependent regulator of alternative splicing, and its overexpression selectively increased the larger Tnc isoforms, whereas a mutated phosphorylation-deficient Sam68 variant did not. This emphasizes the importance of Sam68 for NSC biology and implicates an instructive rather than a purely permissive role for Tnc in the neural stem cell niche.

Long-term exercise training decreases interleukin-6 (IL-6) serum levels in subjects with impaired glucose tolerance: effect of the -174G/C variant in IL-6 gene.

OBJECTIVE: Exercise training has been shown to have anti-inflammatory effects in patients with type 2 diabetes. Changes in interleukin-6 (IL-6) serum concentrations in response to training could contribute to these beneficial effects. However, there are heterogeneous data on whether circulating IL-6 is altered by exercise training. We therefore hypothesize that genetic factors modify the individual changes in IL-6 levels after long-term training. RESEARCH DESIGN AND METHODS: The -174G/C variant in the IL-6 gene was genotyped in 60 subjects with impaired glucose tolerance. For a 12-month interventional study, patients were randomized into three groups: a control group (n=16) was compared with one group, which underwent a standardized training program (n=24) and another group, which was treated with 4 mg rosiglitazone once daily (n=20). At baseline, after 1, 6, and 12 months, we measured anthropometric parameters and serum concentration of IL-6 and, at baseline and after 12 months, we determined glucose tolerance and fitness level. RESULTS: Only in subjects carrying the SNP -174C allele did long-term exercise training result in significantly reduced IL-6 serum concentrations. Multivariate linear regression analysis identified the IL-6 genotype as a significant predictor of changes in IL-6 serum concentrations independent of age, gender and improvement in body mass index, hemoglobin (Hb)A(1c), and fitness level in response to training. CONCLUSIONS: Genetic variants in the IL-6 gene significantly modify changes in IL-6 serum concentrations in response to long-term exercise training programs. Our data suggest that genetic factors are important determinants for the individual response to anti-inflammatory effects of exercise training.