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.

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.

Exercise Training Prevents Diaphragm Contractile Dysfunction in Heart Failure.

PURPOSE: Patient studies have demonstrated the efficacy of exercise training in attenuating respiratory muscle weakness in chronic heart failure (HF), yet direct assessment of muscle fiber contractile function together with data on the underlying intracellular mechanisms remains elusive. The present study, therefore, used a mouse model of HF to assess whether exercise training could prevent diaphragm contractile fiber dysfunction by potentially mediating the complex interplay between intracellular oxidative stress and proteolysis. METHODS: Mice underwent sham operation (n = 10) or a ligation of the left coronary artery and were randomized to sedentary HF (n = 10) or HF with aerobic exercise training (HF + AET; n = 10). Ten weeks later, echocardiography and histological analyses confirmed HF. RESULTS: In vitro diaphragm fiber bundles demonstrated contractile dysfunction in sedentary HF compared with sham mice that was prevented by AET, with maximal force 21.0 ± 0.7 versus 26.7 ± 1.4 and 25.4 ± 1.4 N·cm, respectively (P < 0.05). Xanthine oxidase enzyme activity and MuRF1 protein expression, markers of oxidative stress and protein degradation, were ~20% and ~70% higher in sedentary HF compared with sham mice (P < 0.05) but were not different when compared with the HF + AET group. Oxidative modifications to numerous contractile proteins (i.e., actin and creatine kinase) and markers of proteolysis (i.e., proteasome and calpain activity) were elevated in sedentary HF compared with HF + AET mice (P < 0.05); however, these indices were not significantly different between sedentary HF and sham mice. Antioxidative enzyme activities were also not different between groups. CONCLUSION: Our findings demonstrate that AET can protect against diaphragm contractile fiber dysfunction induced by HF, but it remains unclear whether alterations in oxidative stress and/or protein degradation are primarily responsible.

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.

Diaphragm muscle weakness in mice is early-onset post-myocardial infarction and associated with elevated protein oxidation.

Heart failure induced by myocardial infarction (MI) causes diaphragm muscle weakness, with elevated oxidants implicated. We aimed to determine whether diaphragm muscle weakness is 1) early-onset post-MI (i.e., within the early left ventricular remodeling phase of 72 h); and 2) associated with elevated protein oxidation. Ligation of the left coronary artery to induce MI (n = 10) or sham operation (n = 10) was performed on C57BL6 mice. In vitro contractile function of diaphragm muscle fiber bundles was assessed 72 h later. Diaphragm mRNA and protein expression, enzyme activity, and individual carbonylated proteins (by two-dimensional differential in-gel electrophoresis and mass spectrometry) were subsequently assessed. Infarct size averaged 57 ± 1%. Maximal diaphragm function was reduced (P < 0.01) by 20% post-MI, with the force-frequency relationship depressed (P < 0.01) between 80 and 300 Hz. The mRNA expression of inflammation, atrophy, and regulatory Ca(2+) proteins remained unchanged post-MI, as did the protein expression of key contractile proteins. However, enzyme activity of the oxidative sources NADPH oxidase and xanthine oxidase was increased (P < 0.01) by 45 and 33%, respectively. Compared with sham, a 57 and 45% increase (P < 0.05) was observed in the carbonylation of sarcomeric actin and creatine kinase post-MI, respectively. In conclusion, diaphragm muscle weakness was rapidly induced in mice during the early left ventricular remodeling phase of 72 h post-MI, which was associated with increased oxidation of contractile and energetic proteins. Collectively, these findings suggest diaphragm muscle weakness may be early onset in heart failure, which is likely mediated in part by posttranslational oxidative modifications at the myofibrillar level.

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.

A global proteome approach in uric acid stimulated human aortic endothelial cells revealed regulation of multiple major cellular pathways.

BACKGROUND: Uric acid (UA) has been identified as one major risk factor for cardiovascular diseases. Lowering of serum UA levels improves endothelial function. The present study investigates for the first time concentration-dependent effects of UA on human aortic endothelial cells (HAEC) and the cellular pathways involved in global proteomic analysis. METHODS: The concentration dependent effects of UA on HAEC were investigated by nanoLC-MS/MS and ingenuity pathway analysis to reveal putative cellular pathways. For verification of the identified pathways the abundance or activity of key proteins was measured using ELISA or Western blotting. NO production was quantified by confocal laser microscopy. RESULTS: We identified ubiquitin-proteasome system (UPS) and eIF4 signaling as the major pathways regulated by UA. K-means clustering analysis revealed 11 additional pathways, of which NO, superoxide signaling and hypoxia were further analyzed. A complex regulatory network was detected demonstrating that 500µmol/L UA, which is well above the concentration regarded as pathological in clinical settings, led to diminishing of NO bioavailability. In addition a UA-dependent downregulation of eIF4, an upregulation of UPS and an increase in HIF-1α were detected. CONCLUSIONS: Here we show for the first time, that increasing UA levels activate different sets of proteins representing specific cellular pathways important for endothelial function. This indicates that UA may alter far more pathways in HAEC than previously assumed. This regulation occurs in a complex manner depending on UA concentration. Further studies in knockout and overexpression models of the identified proteins are necessary to prove the correlation with endothelial dysfunction.

Bariatric surgery in severely obese adolescents improves major comorbidities including hyperuricemia.

OBJECTIVE: Serum uric acid (sUA) is believed to contribute to the pathogenesis of metabolic comorbidities like hypertension, insulin-resistance (IR) and endothelial dysfunction (EDF) in obese children. The present pilot study investigated the association between sUA concentrations and loss of body weight following laparoscopic sleeve gastrectomy (LSG) or laparoscopic Roux-en-Y-gastric bypass (RYGB) in severely obese adolescents. MATERIALS/METHODS: 10 severely obese adolescents underwent either LSG (n=5) or RYGB (n=5). 17 normal weight, healthy, age- and gender-matched adolescents served as a normal weight peer group (NWPG). Pre- and 12 months postoperatively, sUA and relevant metabolic parameters (glucose homeostasis, transaminases, lipids) were compared. RESULTS: Preoperatively, sUA was significantly elevated in patients with severe obesity compared to NWPG. Twelve months after LSG and RYGB, a significant decrease in sUA, BMI, CVD risk factors, hepatic transaminases, and HOMA-IR was observed. Reduction in SDS-BMI significantly correlated with changes in sUA. CONCLUSIONS: sUA levels and metabolic comorbidities improved following bariatric surgery in severely obese adolescents. The impact of changes in sUA on long-term clinical complications of childhood obesity deserves further study.

Exercise training prevents TNF-α induced loss of force in the diaphragm of mice.

RATIONALE: Inflammatory cytokines like tumor necrosis factor alpha (TNF-α) are elevated in congestive heart failure and are known to induce the production of reactive oxygen species as well as to deteriorate respiratory muscle function. OBJECTIVES: Given the antioxidative effects of exercise training, the aim of the present study was to investigate if exercise training is capable of preventing a TNF-α induced loss of diaphragmatic force in mice and, if so, to elucidate the potential underlying mechanisms. METHODS: Prior to intraperitoneal injection of TNF-α or saline, C57Bl6 mice were assigned to four weeks of exercise training or sedentary behavior. Diaphragmatic force and power generation were determined in vitro. Expression/activity of radical scavenger enzymes, enzymes producing reactive oxygen species and marker of oxidative stress were measured in the diaphragm. MAIN RESULTS: In sedentary animals, TNF-α reduced specific force development by 42% concomitant with a 2.6-fold increase in the amount of carbonylated α-actin and creatine kinase. Furthermore, TNF-α led to an increased NAD(P)H oxidase activity in both sedentary and exercised mice whereas xanthine oxidase activity and intramitochondrial ROS production was only enhanced in sedentary animals by TNF-α. Exercise training prevented the TNF-α induced force reduction and led to an enhanced mRNA expression and activity of glutathione peroxidase. Carbonylation of proteins, in particular of α-actin and creatine kinase, was diminished by exercise training. CONCLUSION: TNF-α reduces the force development in the diaphragm of mice. This effect is almost abolished by exercise training. This may be a result of reduced carbonylation of proteins due to the antioxidative properties of exercise training.

Combined proteomic and metabolomic profiling of serum reveals association of the complement system with obesity and identifies novel markers of body fat mass changes.

Obesity is associated with multiple adverse health effects and a high risk of developing metabolic and cardiovascular diseases. Therefore, there is a great need to identify circulating parameters that link changes in body fat mass with obesity. This study combines proteomic and metabolomic approaches to identify circulating molecules that discriminate healthy lean from healthy obese individuals in an exploratory study design. To correct for variations in physical activity, study participants performed a one hour exercise bout to exhaustion. Subsequently, circulating factors differing between lean and obese individuals, independent of physical activity, were identified. The DIGE approach yielded 126 differentially abundant spots representing 39 unique proteins. Differential abundance of proteins was confirmed by ELISA for antithrombin-III, clusterin, complement C3 and complement C3b, pigment epithelium-derived factor (PEDF), retinol binding protein 4 (RBP4), serum amyloid P (SAP), and vitamin-D binding protein (VDBP). Targeted serum metabolomics of 163 metabolites identified 12 metabolites significantly related to obesity. Among those, glycine (GLY), glutamine (GLN), and glycero-phosphatidylcholine 42:0 (PCaa 42:0) serum concentrations were higher, whereas PCaa 32:0, PCaa 32:1, and PCaa 40:5 were decreased in obese compared to lean individuals. The integrated bioinformatic evaluation of proteome and metabolome data yielded an improved group separation score of 2.65 in contrast to 2.02 and 2.16 for the single-type use of proteomic or metabolomics data, respectively. The identified circulating parameters were further investigated in an extended set of 30 volunteers and in the context of two intervention studies. Those included 14 obese patients who had undergone sleeve gastrectomy and 12 patients on a hypocaloric diet. For determining the long-term adaptation process the samples were taken six months after the treatment. In multivariate regression analyses, SAP, CLU, RBP4, PEDF, GLN, and C18:2 showed the strongest correlation to changes in body fat mass. The combined serum proteomic and metabolomic profiling reveals a link between the complement system and obesity and identifies both novel (C3b, CLU, VDBP, and all metabolites) and confirms previously discovered markers (PEDF, RBP4, C3, ATIII, and SAP) of body fat mass changes.

Conserved lysin and arginin residues in the extracellular loop of P2X(3) receptors are involved in agonist binding.

Wild-type human (h) P2X(3) receptors expressed in HEK293 cells responded to the prototypic agonist alpha,beta-methylene ATP (alpha,beta-meATP) with rapidly desensitizing inward currents and an increase in the intracellular Ca(2+) concentration. In contrast to electrophysiological recordings, Ca(2+) microfluorimetry showed a lower maximum of the concentration-response curve of alpha,beta-meATP in the transiently than in the permanently transfected HEK293 cells. However, the concentrations causing 50% of the maximum possible effect (EC(50) values) were identical, when measured with either method. In order to determine the role of certain conserved, positively charged amino acids in the nucleotide binding domains (NBD-1-4) of hP2X(3) receptors for agonist binding, the lysine-63, -65, -176 and -299 as well as the arginine-281 and -295 residues were substituted by the neutral amino acid alanine. We observed no effect of alpha,beta-meATP at the K63A, K176A, R295A, and K299A mutants, and a marked decrease of agonist potency at the K65A and R281A mutants. The P2X(3) receptor antagonist 2',3'-O-trinitrophenyl-ATP (TNP-ATP) blocked the effect of alpha,beta-meATP at the wild-type hP2X(3) receptor with lower affinity than at the mutant K65A, indicating an interference of this mutation with the docking of the antagonist with its binding sites. The use of confocal fluorescence microscopy in conjunction with an antibody raised against the extracellular loop of the hP2X(3) receptor documented the expression of all mutants in the plasma membrane of HEK293 cells. Eventually, we modelled the possible agonist and antagonist binding sites NBD-1-4 of the hP2X(3) subunit by using structural bioinformatics. This model is in complete agreement with the available data and integrates results from mutagenesis studies with geometry optimization of the tertiary structure predictions of the receptor.