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- ItemMulti-capillary column-ion mobility spectrometry (MCC-IMS) as a new method for the quantification of occupational exposure to sevoflurane in anaesthesia workplaces: an observational feasibility study(London : BioMed Central, 2015) Kunze, Nils; Weigel, Cathrin; Vautz, Wolfgang; Schwerdtfeger, Katrin; JĂ¼nger, Melanie; Quintel, Michael; Perl, ThorstenBackground: Occupational exposure to sevoflurane has the potential to cause health damage in hospital personnel. Workplace contamination with the substance mostly is assessed by using photoacoustic infrared spectrometry with detection limits of 10 ppbv. Multi-capillary column-ion mobility spectrometry (MCC-IMS) could be an alternative technology for the quantification of sevoflurane in the room air and could be even more accurate because of potentially lower detection limits. The aim of this study was to test the hypothesis that MCC-IMS is able to detect and monitor very low concentrations of sevoflurane (<10 ppbv) and to evaluate the exposure of hospital personnel to sevoflurane during paediatric anaesthesia and in the post anaesthesia care unit (PACU). Methods: A MCC-IMS device was calibrated to several concentrations of sevoflurane and limits of detection (LOD) and quantification (LOQ) were calculated. Sevoflurane exposure of hospital personnel was measured at two anaesthesia workplaces and time-weighted average (TWA) values were calculated. Results: The LOD was 0.0068 ppbv and the LOQ was 0.0189 ppbv. During paediatric anaesthesia the mean sevoflurane concentration was 46.9 ppbv (8.0 - 314.7 ppbv) with TWA values between 5.8 and 45.7 ppbv. In the PACU the mean sevoflurane concentration was 27.9 ppbv (8.0 – 170.2 ppbv) and TWA values reached from 8.3 to 45.1 ppbv. Conclusions: MCC-IMS shows a significantly lower LOD and LOQ than comparable methods. It is a reliable technology for monitoring sevoflurane concentrations at anaesthesia workplaces and has a particular strength in quantifying low-level contaminations of sevoflurane. The exposure of the personnel working in these areas did not exceed recommended limits and therefore adverse health effects are unlikely.
- ItemUsing SRM-MS to quantify nuclear protein abundance differences between adipose tissue depots of insulin-resistant mice(Rockville : American Society for Biochemistry and Molecular Biology, 2015) Ota, Asuka; Kovary, Kyle M.; Wu, Olivia H.; Ahrends, Robert; Shen, Wen-Jun; Costa, Maria J.; Feldman, Brian J.; Kraemer, Fredric B.; Teruel, Mary N.Insulin resistance (IR) underlies metabolic disease. Visceral, but not subcutaneous, white adipose tissue (WAT) has been linked to the development of IR, potentially due to differences in regulatory protein abundance. Here we investigate how protein levels are changed in IR in different WAT depots by developing a targeted proteomics approach to quantitatively compare the abundance of 42 nuclear proteins in subcutaneous and visceral WAT from a commonly used insulin-resistant mouse model, Lepr(db/db), and from C57BL/6J control mice. The most differentially expressed proteins were important in adipogenesis, as confirmed by siRNA-mediated depletion experiments, suggesting a defect in adipogenesis in visceral, but not subcutaneous, insulin-resistant WAT. Furthermore, differentiation of visceral, but not subcutaneous, insulin-resistant stromal vascular cells (SVCs) was impaired. In an in vitro approach to understand the cause of this impaired differentiation, we compared insulin-resistant visceral SVCs to preadipocyte cell culture models made insulin resistant by different stimuli. The insulin-resistant visceral SVC protein abundance profile correlated most with preadipocyte cell culture cells treated with both palmitate and TNFα. Together, our study introduces a method to simultaneously measure and quantitatively compare nuclear protein expression patterns in primary adipose tissue and adipocyte cell cultures, which we show can reveal relationships between differentiation and disease states of different adipocyte tissue types.
- ItemProteome analyses of hepatocellular carcinoma(Sugar Land, TX : Xia & He Publishing, 2014) Megger, Dominik A.; Naboulsi, Naboulsi; Meyer, Helmut E.; Sitek, BarbaraProteomics has evolved into a powerful and widely used bioanalytical technique in the study of cancer, especially hepatocellular carcinoma (HCC). In this review, we provide an up to date overview of feasible proteome-analytical techniques for clinical questions. In addition, we present a broad summary of proteomic studies of HCC utilizing various technical approaches for the analysis of samples derived from diverse sources like HCC cell lines, animal models, human tissue and body fluids.
- ItemIdentification of Eps15 as antigen recognized by the monoclonal antibodies aa2 and ab52 of the wuerzburg hybridoma library against Drosophila brain(San Francisco, CA : Public Library of Science, 2011) Halder, Partho; Chen, Yi-chun; Brauckhoff, Janine; Hofbauer, Alois; Dabauvalle, Marie-Christine; Lewandrowski, Urs; Winkler, Christiane; Sickmann, Albert; Buchner, ErichThe Wuerzburg Hybridoma Library against the Drosophila brain represents a collection of around 200 monoclonal antibodies that bind to specific structures in the Drosophila brain. Here we describe the immunohistochemical staining patterns, the Western blot signals of one- and two-dimensional electrophoretic separation, and the mass spectrometric characterization of the target protein candidates recognized by the monoclonal antibodies aa2 and ab52 from the library. Analysis of a mutant of a candidate gene identified the Drosophila homolog of the Epidermal growth factor receptor Pathway Substrate clone 15 (Eps15) as the antigen for these two antibodies.
- ItemBiological pathways modulated by antipsychotics in the blood plasma of schizophrenia patients and their association to a clinical response(London : Nature Publishing Group, 2015) Martins-de-Souza, Daniel; Solari, Fiorella A.; Guest, Paul C.; Zahedi, René P.; Steiner, JohannProteomics is a valuable tool to unravel molecular mechanisms involved in human disorders. Considering the mediocre effectiveness of antipsychotics, which are the main class of drug used to treat schizophrenia, we analyzed a cohort of 58 schizophrenia patients who had blood collected before and after 6 weeks of antipsychotic treatment using a shotgun mass spectrometry proteomic profiling approach. Our aim was to unravel molecular pathways involved with an effective drug response. The results showed that all patients had essentially the same biochemical pathways triggered Independent of the antipsychotic response outcome. However, we observed that these pathways were regulated in different directions in blood samples from those who responded well to antipsychotics, compared with those who had a poorer outcome. These data are novel, timely and may help to guide new research efforts in the design of new treatments or medications for schizophrenia based on biologically relevant pathways.
- ItemThe proteome of human liver peroxisomes: Identification of five new peroxisomal constituents by a label-free quantitative proteomics survey(San Francisco, CA : Public Library of Science, 2013) Gronemeyer, Thomas; Wiese, Sebastian; Ofman, Rob; Bunse, Christian; Pawlas, Magdalena; Hayen, Heiko; Eisenacher, Martin; Stephan, Christian; Meyer, Helmut E.; Waterham, Hans R.; Erdmann, Ralf; Wanders, Ronald J.; Warscheid, BettinaThe peroxisome is a key organelle of low abundance that fulfils various functions essential for human cell metabolism. Severe genetic diseases in humans are caused by defects in peroxisome biogenesis or deficiencies in the function of single peroxisomal proteins. To improve our knowledge of this important cellular structure, we studied for the first time human liver peroxisomes by quantitative proteomics. Peroxisomes were isolated by differential and Nycodenz density gradient centrifugation. A label-free quantitative study of 314 proteins across the density gradient was accomplished using high resolution mass spectrometry. By pairing statistical data evaluation, cDNA cloning and in vivo colocalization studies, we report the association of five new proteins with human liver peroxisomes. Among these, isochorismatase domain containing 1 protein points to the existence of a new metabolic pathway and hydroxysteroid dehydrogenase like 2 protein is likely involved in the transport or b-oxidation of fatty acids in human peroxisomes. The detection of alcohol dehydrogenase 1A suggests the presence of an alternative alcohol-oxidizing system in hepatic peroxisomes. In addition, lactate dehydrogenase A and malate dehydrogenase 1 partially associate with human liver peroxisomes and enzyme activity profiles support the idea that NAD+ becomes regenerated during fatty acid b-oxidation by alternative shuttling processes in human peroxisomes involving lactate dehydrogenase and/or malate dehydrogenase. Taken together, our data represent a valuable resource for future studies of peroxisome biochemistry that will advance research of human peroxisomes in health and disease.
- ItemDipA, a pore-forming protein in the outer membrane of lyme disease spirochetes exhibits specificity for the permeation of dicarboxylate(San Francisco, CA : Public Library of Science, 2012) Thein, Marcus; Bonde, Mari; Bunikis, Ignas; Denker, Katrin; Sickmann, Albert; Bergström, Sven; Benz, RolandLyme disease Borreliae are highly dependent on the uptake of nutrients provided by their hosts. Our study describes the identification of a 36 kDa protein that functions as putative dicarboxylate-specific porin in the outer membrane of Lyme disease Borrelia. The protein was purified by hydroxyapatite chromatography from Borrelia burgdorferi B31 and designated as DipA, for dicarboxylate-specific porin A. DipA was partially sequenced, and corresponding genes were identified in the genomes of B. burgdorferi B31, Borrelia garinii PBi and Borrelia afzelii PKo. DipA exhibits high homology to the Oms38 porins of relapsing fever Borreliae. B. burgdorferi DipA was characterized using the black lipid bilayer assay. The protein has a singlechannel conductance of 50 pS in 1 M KCl, is slightly selective for anions with a permeability ratio for cations over anions of 0.57 in KCl and is not voltage-dependent. The channel could be partly blocked by different di- and tricarboxylic anions. Particular high stability constants up to about 28,000 l/mol (in 0.1 M KCl) were obtained among the 11 tested anions for oxaloacetate, 2-oxoglutarate and citrate. The results imply that DipA forms a porin specific for dicarboxylates which may play an important role for the uptake of specific nutrients in different Borrelia species.
- ItemHighly immunoreactive IgG antibodies directed against a set of twenty human proteins in the sera of patients with amyotrophic lateral sclerosis identified by protein array(San Francisco, CA : Public Library of Science, 2014) May, Caroline; Nordhoff, Eckhard; Casjens, Swaantje; Turewicz, Michael; Eisenacher, Martin; Gold, Ralf; BrĂ¼ning, Thomas; Pesch, Beate; Stephan, Christian; Woitalla, Dirk; Penke, Botond; JanĂ¡ky, TamĂ¡s; VirĂ³k, DezsÅ‘; SiklĂ³s, LĂ¡szlĂ³; Engelhardt, Jozsef I.; Meyer, Helmut E.Amyotrophic lateral sclerosis (ALS), the most common adult-onset motor neuron disorder, is characterized by the progressive and selective loss of upper and lower motor neurons. Diagnosis of this disorder is based on clinical assessment, and the average survival time is less than 3 years. Injections of IgG from ALS patients into mice are known to specifically mark motor neurons. Moreover, IgG has been found in upper and lower motor neurons in ALS patients. These results led us to perform a case-control study using human protein microarrays to identify the antibody profiles of serum samples from 20 ALS patients and 20 healthy controls. We demonstrated high levels of 20 IgG antibodies that distinguished the patients from the controls. These findings suggest that a panel of antibodies may serve as a potential diagnostic biomarker for ALS.
- ItemVolatile organic compounds in uremia(San Francisco, CA : Public Library of Science, 2012) Pagonas, Nikolaos; Vautz, Wolfgang; Seifert, Luzia; Slodzinski, Rafael; Jankowski, Joachim; Zidek, Walter; Westhoff, Timm H.Background: Although ‘‘uremic fetor’’ has long been felt to be diagnostic of renal failure, the compounds exhaled in uremia remain largely unknown so far. The present work investigates whether breath analysis by ion mobility spectrometry can be used for the identification of volatile organic compounds retained in uremia. Methods: Breath analysis was performed in 28 adults with an eGFR $60 ml/min per 1.73 m2, 26 adults with chronic renal failure corresponding to an eGFR of 10–59 ml/min per 1.73 m2, and 28 adults with end-stage renal disease (ESRD) before and after a hemodialysis session. Breath analysis was performed by ion mobility spectrometryafter gas-chromatographic preseparation. Identification of the compounds of interest was performed by thermal desorption gas chromatography/mass spectrometry. Results: Breath analyses revealed significant differences in the spectra of patients with and without renal failure. Thirteen compounds were chosen for further evaluation. Some compounds including hydroxyacetone, 3-hydroxy-2-butanone and ammonia accumulated with decreasing renal function and were eliminated by dialysis. The concentrations of these compounds allowed a significant differentiation between healthy, chronic renal failure with an eGFR of 10–59 ml/min, and ESRD (p,0.05 each). Other compounds including 4-heptanal, 4-heptanone, and 2-heptanone preferentially or exclusively occurred in patients undergoing hemodialysis. Conclusion: Impairment of renal function induces a characteristic fingerprint of volatile compounds in the breath. The technique of ion mobility spectrometry can be used for the identification of lipophilic uremic retention molecules.
- ItemCytomegalovirus downregulates IRE1 to repress the unfolded protein response(San Francisco, CA : Public Library of Science, 2013) Stahl, Sebastian; Burkhart, Julia M.; Hinte, Florian; Tirosh, Boaz; Mohr, Hermine; Zahedi, René P.; Sickmann, Albert; Ruzsics, Zsolt; Budt, Matthias; Brune, WolframDuring viral infection, a massive demand for viral glycoproteins can overwhelm the capacity of the protein folding and quality control machinery, leading to an accumulation of unfolded proteins in the endoplasmic reticulum (ER). To restore ER homeostasis, cells initiate the unfolded protein response (UPR) by activating three ER-to-nucleus signaling pathways, of which the inositol-requiring enzyme 1 (IRE1)-dependent pathway is the most conserved. To reduce ER stress, the UPR decreases protein synthesis, increases degradation of unfolded proteins, and upregulates chaperone expression to enhance protein folding. Cytomegaloviruses, as other viral pathogens, modulate the UPR to their own advantage. However, the molecular mechanisms and the viral proteins responsible for UPR modulation remained to be identified. In this study, we investigated the modulation of IRE1 signaling by murine cytomegalovirus (MCMV) and found that IRE1-mediated mRNA splicing and expression of the X-box binding protein 1 (XBP1) is repressed in infected cells. By affinity purification, we identified the viral M50 protein as an IRE1-interacting protein. M50 expression in transfected or MCMV-infected cells induced a substantial downregulation of IRE1 protein levels. The N-terminal conserved region of M50 was found to be required for interaction with and downregulation of IRE1. Moreover, UL50, the human cytomegalovirus (HCMV) homolog of M50, affected IRE1 in the same way. Thus we concluded that IRE1 downregulation represents a previously undescribed viral strategy to curb the UPR.