Publications by authors named "Barbara Sitek"

102 Publications

Application of Label-Free Proteomics for Quantitative Analysis of Urothelial Carcinoma and Cystitis Tissue.

Methods Mol Biol 2021 ;2228:283-292

Medizinisches Proteom-Center (MPC) Medical Faculty, Ruhr-University Bochum, Bochum, Germany.

A label-free approach based on a highly reproducible and stable workflow allows for quantitative proteome analysis . Due to advantages compared to labeling methods, the label-free approach has the potential to measure unlimited samples from clinical specimen monitoring and comparing thousands of proteins. The presented label-free workflow includes a new sample preparation technique depending on automatic annotation and tissue isolation via FTIR-guided laser microdissection, in-solution digestion, LC-MS/MS analyses, data evaluation by means of Proteome Discoverer and Progenesis software, and verification of differential proteins. We successfully applied this workflow in a proteomics study analyzing human cystitis and high-grade urothelial carcinoma tissue regarding the identification of a diagnostic tissue biomarker. The differential analysis of only 1 mm of isolated tissue cells led to 74 significantly differentially abundant proteins.
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http://dx.doi.org/10.1007/978-1-0716-1024-4_20DOI Listing
January 2021

Differential Proteome Analysis Using 2D-DIGE.

Methods Mol Biol 2021 ;2228:77-84

Medizinisches Proteom-Center (MPC), Medical Faculty, Ruhr-University Bochum, Bochum, Germany.

Classical 2D-PAGE allows comparison and quantitation of proteomes by visualization of protein patterns using gel stains and comparative image analysis. The introduction of fluorescent reagents for protein labeling (difference in-gel electrophoresis or DIGE) has brought substantial improvement in this field. It provides multiplexing of up to three samples in one gel, higher sensitivity compared to normal protein staining methods, and a higher linear range for quantitation. This article gives detailed protocols for 2D-DIGE, including both minimal and saturation labeling.
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http://dx.doi.org/10.1007/978-1-0716-1024-4_7DOI Listing
January 2021

Proteome Analysis with Classical 2D-PAGE.

Methods Mol Biol 2021 ;2228:53-62

Medizinisches Proteom-Center (MPC), Medical Faculty, Ruhr-University Bochum, Bochum, Germany.

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is based on the combination of two orthogonal separation techniques. In the first dimension, proteins are separated by their isoelectric point, a technique known as isoelectric focusing (IEF). There are two important variants of IEF, which are carrier-ampholine (CA)-based IEF and immobilized pH-gradient (IPG)-based IEF. In the second dimension, proteins are further separated by their electrophoretic mobility using SDS-PAGE. Finally, proteins can be visualized and quantified by different staining procedures such as Coomassie, silver staining, or fluorescence labeling. This article gives detailed protocols for 2D-PAGE, using both CA- and IPG-based separation in the first dimension.
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http://dx.doi.org/10.1007/978-1-0716-1024-4_5DOI Listing
January 2021

Tissue-resident macrophages mediate neutrophil recruitment and kidney injury in shiga toxin-induced hemolytic uremic syndrome.

Kidney Int 2021 Apr 28. Epub 2021 Apr 28.

Department of Immunodynamics, Institute of Experimental Immunology and Imaging, University Hospital Essen, Essen, Germany. Electronic address:

Enterohaemorrhagic E. coli cause major epidemics worldwide with significant organ damage and very high percentages of death. Due to the ability of enterohaemorrhagic E. coli to produce shiga toxin these bacteria damage the kidney leading to the hemolytic uremic syndrome. A therapy against this serious kidney disease has not been developed yet and the impact and mechanism of leukocyte activation and recruitment are unclear. Tissue-resident macrophages represent the main leukocyte population in the healthy kidney, but the role of this important cell population in shiga toxin-producing E. coli-hemolytic uremic syndrome is incompletely understood. Using state of the art microscopy and mass spectrometry imaging, our preclinical study demonstrated a phenotypic and functional switch of tissue-resident macrophages after disease induction in mice. Kidney macrophages produced the inflammatory molecule TNFα and depletion of tissue-resident macrophages via the CSF1 receptor abolished TNFα levels in the kidney and significantly diminished disease severity. Furthermore, macrophage depletion did not only attenuate endothelial damage and thrombocytopenia, but also activation of thrombocytes and neutrophils. Moreover, we observed that neutrophils infiltrated the kidney cortex and depletion of macrophages significantly reduced the recruitment of neutrophils and expression of the neutrophil-attracting chemokines CXCL1 and CXCL2. Intravital microscopy revealed that inhibition of CXCR2, the receptor for CXCL1 and CXCL2, significantly reduced the infiltration of neutrophils and reduced kidney injury. Thus, our study shows activation of tissue-resident macrophages during shiga toxin-producing E. coli-hemolytic uremic syndrome leading to the production of disease-promoting TNFα and CXCR2-dependent recruitment of neutrophils.
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http://dx.doi.org/10.1016/j.kint.2021.03.039DOI Listing
April 2021

Exploiting the MUC5AC Antigen for Noninvasive Identification of Pancreatic Cancer.

J Nucl Med 2021 Mar 12. Epub 2021 Mar 12.

Memorial Sloan Kettering Cancer Center, United States.

Pancreatic cancer (PC) remains the 4th leading cause of cancer death; therefore, there is a clinically unmet need for novel therapeutics and diagnostic markers to treat this devastating disease. Physicians often rely on biopsy or CT for diagnosis, but more specific protein biomarkers are highly desired to assess the stage and severity of PC in a noninvasive manner. Serum biomarkers such as CA19.9 are of particular interest as they are commonly elevated in PC but have exhibited suboptimal performance in the clinic. MUC5AC has emerged as a useful serum biomarker that is specific for PC vs. inflammation. We developed RA96, an anti-MUC5AC antibody, to gauge its utility in PC diagnosis through immunohistochemical (IHC) analysis and whole-body PET in PC. In this study, extensive biochemical characterization determined MUC5AC as the antigen for RA96. We then determined the utility of RA96 for MUC5AC IHC on clinical PC and pre-clinical PC. Finally, we radiolabeled RA96 with zirconium-89 to assess its application as a whole-body PET radiotracer for MUC5AC quantification in PC. Immunohistochemical staining with RA96 distinguished chronic pancreatitis (CP), PanIN, and varying grades of pancreatic ductal adenocarcinoma (PDAC) in clinical samples. [Zr]Zr-DFO-RA96 was able to detect MUC5AC with high specificity in mice bearing capan-2 xenografts. Our study demonstrates that RA96 can differentiate between inflammation and PC, improving the fidelity of PC diagnosis. Our immuno-PET tracer [Zr]Zr-DFO-RA96 shows specific detection of MUC5AC+ tumors in vivo, highlighting the utility of MUC5AC targeting for diagnosis of PC.
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http://dx.doi.org/10.2967/jnumed.120.256776DOI Listing
March 2021

Proteomic and bioinformatic profiling of neutrophils in CLL reveals functional defects that predispose to bacterial infections.

Blood Adv 2021 03;5(5):1259-1272

Institute of Experimental Immunology and Imaging, University Hospital Essen, University Duisburg-Essen, Essen, Germany.

Patients with chronic lymphocytic leukemia (CLL) typically suffer from frequent and severe bacterial infections. Although it is well known that neutrophils are critical innate immune cells facilitating the early defense, the underlying phenotypical and functional changes in neutrophils during CLL remain largely elusive. Using a murine adoptive transfer model of CLL, we demonstrate aggravated bacterial burden in CLL-bearing mice upon a urinary tract infection with uropathogenic Escherichia coli. Bioinformatic analyses of the neutrophil proteome revealed increased expression of proteins associated with interferon signaling and decreased protein expression associated with granule composition and neutrophil migration. Functional experiments validated these findings by showing reduced levels of myeloperoxidase and acidification of neutrophil granules after ex vivo phagocytosis of bacteria. Pathway enrichment analysis indicated decreased expression of molecules critical for neutrophil recruitment, and migration of neutrophils into the infected urinary bladder was significantly reduced. These altered migratory properties of neutrophils were also associated with reduced expression of CD62L and CXCR4 and correlated with an increased incidence of infections in patients with CLL. In conclusion, this study describes a molecular signature of neutrophils through proteomic, bioinformatic, and functional analyses that are linked to a reduced migratory ability, potentially leading to increased bacterial infections in patients with CLL.
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http://dx.doi.org/10.1182/bloodadvances.2020002949DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7948268PMC
March 2021

Quantitative analysis of proteome dynamics in a mouse model of asthma.

Clin Exp Allergy 2021 Feb 7. Epub 2021 Feb 7.

Medizinisches Proteom-Center, Medical Faculty, Ruhr-University Bochum, Bochum, Germany.

Background: Asthma is an inflammatory disease of the respiratory system, and a major factor of increasing health care costs worldwide. The molecular actors leading to the development of chronic asthma are not fully understood and require further investigation.

Objective: The aim of this study was to monitor the proteome dynamics during asthma development from early inflammatory to late fibrotic stages.

Methods: A mouse asthma model was used to analyse the lung proteome at four time points during asthma development (0 weeks = control, 5, 8 and 12 weeks of treatment, n = 6 each). The model was analysed using lung function tests, immune cell counting and histology. Furthermore, a multi-fraction mass spectrometry-based proteome analysis was performed to achieve a comprehensive coverage and quantification of the lung proteome.

Results: At early stages, the mice showed predominant eosinophilic inflammation of the airways, which disappeared at later stages and was replaced by marked airway hyper-reactivity and fibrosis of the airways. 3325 proteins were quantified with 435 proteins found to be significantly differentially abundant between the experimental groups (ANOVA p-value ≤.05, maximum fold change ≥1.5). We applied hierarchical clustering to identify common protein abundance profiles along the asthma development and analysed these clusters using gene ontology annotation and enrichment analysis. We demonstrate the correlation of protein clusters with the course of asthma development, that is eosinophilic inflammation and fibrotic remodelling of the airways.

Conclusions And Clinical Relevance: Proteome analysis revealed proteins that were previously described to be important during asthma chronification. Moreover, we identified additional proteins previously not described in the context of asthma. We provide a comprehensive data set of a long-term mouse model of asthma that may contribute to a better understanding and allow new insights into the progression and development of chronic asthma. Data are available via ProteomeXchange with identifier PXD011159.
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http://dx.doi.org/10.1111/cea.13843DOI Listing
February 2021

Permissive HLA-DPB1 mismatches in HCT depend on immunopeptidome divergence and editing by HLA-DM.

Blood 2021 Feb;137(7):923-928

Institute for Experimental Cellular Therapy, University Hospital Essen, Essen, Germany.

In hematopoietic cell transplantation (HCT), permissive HLA-DPB1 mismatches between patients and their unrelated donors are associated with improved outcomes compared with nonpermissive mismatches, but the underlying mechanism is incompletely understood. Here, we used mass spectrometry, T-cell receptor-β (TCRβ) deep sequencing, and cellular in vitro models of alloreactivity to interrogate the HLA-DP immunopeptidome and its role in alloreactive T-cell responses. We find that permissive HLA-DPB1 mismatches display significantly higher peptide repertoire overlaps compared with their nonpermissive counterparts, resulting in lower frequency and diversity of alloreactive TCRβ clonotypes in healthy individuals and transplanted patients. Permissiveness can be reversed by the absence of the peptide editor HLA-DM or the presence of its antagonist, HLA-DO, through significant broadening of the peptide repertoire. Our data establish the degree of immunopeptidome divergence between donor and recipient as the mechanistic basis for the clinically relevant permissive HLA-DPB1 mismatches in HCT and show that permissiveness is dependent on HLA-DM-mediated peptide editing. Its key role for harnessing T-cell alloreactivity to HLA-DP highlights HLA-DM as a potential novel target for cellular and immunotherapy of leukemia.
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http://dx.doi.org/10.1182/blood.2020008464DOI Listing
February 2021

Antiplatelet Effect of Carbon Monoxide Is Mediated by NAD and ATP Depletion.

Arterioscler Thromb Vasc Biol 2020 10 13;40(10):2376-2390. Epub 2020 Aug 13.

Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Krakow, Poland.

Objectives: Carbon monoxide (CO) produced by haem oxygenases or released by CO-releasing molecules (CORM) affords antiplatelet effects, but the mechanism involved has not been defined. Here, we tested the hypothesis that CO-induced inhibition of human platelet aggregation is mediated by modulation of platelet bioenergetics. Approach and Results: To analyze the effects of CORM-A1 on human platelet aggregation and bioenergetics, a light transmission aggregometry, Seahorse XFe technique and liquid chromatography tandem-mass spectrometry-based metabolomics were used. CORM-A1-induced inhibition of platelet aggregation was accompanied by the inhibition of mitochondrial respiration and glycolysis. Interestingly, specific inhibitors of these processes applied individually, in contrast to combined treatment, did not inhibit platelet aggregation considerably. A CORM-A1-induced delay of tricarboxylic acid cycle was associated with oxidized nicotinamide adenine dinucleotide (NAD) depletion, compatible with the inhibition of oxidative phosphorylation. CORM-A1 provoked an increase in concentrations of proximal (before GAPDH [glyceraldehyde 3-phosphate dehydrogenase]), but not distal glycolysis metabolites, suggesting that CO delayed glycolysis at the level of NAD-dependent GAPDH; however, GAPDH activity was directly not inhibited. In the presence of exogenous pyruvate, CORM-A1-induced inhibition of platelet aggregation and glycolysis were lost, but were restored by the inhibition of lactate dehydrogenase, involved in cytosolic NAD regeneration, pointing out to the key role of NAD depletion in the inhibition of platelet bioenergetics by CORM-A1.

Conclusions: The antiplatelet effect of CO is mediated by inhibition of mitochondrial respiration-attributed to the inhibition of cytochrome c oxidase, and inhibition of glycolysis-ascribed to cytosolic NAD depletion.
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http://dx.doi.org/10.1161/ATVBAHA.120.314284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7505148PMC
October 2020

Deletion of Perilipin 5 Protects Against Hepatic Injury in Nonalcoholic Fatty Liver Disease via Missing Inflammasome Activation.

Cells 2020 05 28;9(6). Epub 2020 May 28.

Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry (IFMPEGKC), RWTH University Hospital Aachen, 52074 Aachen, Germany.

Nonalcoholic fatty liver disease (NAFLD) is a leading cause of chronic liver diseases with an increasing prevalence due to rising rates of obesity, metabolic syndrome and type II diabetes. Untreated NAFLD may progress to steatohepatitis (NASH) and ultimately liver cirrhosis. NAFLD is characterized by lipid accumulation, and when sufficient excess lipids are obtained, irreversible liver injury may follow. Perilipin 5 (PLIN5), a known lipid droplet coating protein and triglyceride metabolism regulator, is highly expressed in oxidatively modified tissues but it is still unclear how it affects NAFLD/NASH progress. We here studied how PLIN5 affects NAFLD development induced by a 30-week high-fat diet (HFD) administration in wild type and PLIN5 knock out () mice. The disruption of PLIN5 induced differences in lipid metabolism during HFD feeding and was associated with reduced hepatic fat accumulation. Surprisingly, mice showed mitigated activation of the NLR family pyrin domain-containing 3 (NLRP3) inflammasome, leading to minor hepatic damage. We conclude that PLIN5 is a pleiotropic regulator of hepatic homeostasis in NASH development. Targeting the PLIN5 expression appears critical for protecting the liver from inflammatory activation during chronic NAFLD.
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http://dx.doi.org/10.3390/cells9061346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7348929PMC
May 2020

C19orf66 is an interferon-induced inhibitor of HCV replication that restricts formation of the viral replication organelle.

J Hepatol 2020 09 12;73(3):549-558. Epub 2020 Apr 12.

Institute of Experimental Virology, TWINCORE Centre for Experimental and Clinical Infection Research, a joint venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany; Faculty of Medicine, Department for Molecular and Medical Virology, Ruhr University Bochum, Bochum, Germany. Electronic address:

Background & Aims: HCV is a positive-strand RNA virus that primarily infects human hepatocytes. Recent studies have reported that C19orf66 is expressed as an interferon (IFN)-stimulated gene; however, the intrinsic regulation of this gene within the liver as well as its antiviral effects against HCV remain elusive.

Methods: Expression of C19orf66 was quantified in both liver biopsies and primary human hepatocytes, with or without HCV infection. Mechanistic studies of the potent anti-HCV phenotype mediated by C19orf66 were conducted using state-of-the-art virological, biochemical and genetic approaches, as well as correlative light and electron microscopy and transcriptome and proteome analysis.

Results: Upregulation of C19orf66 mRNA was observed in both primary human hepatocytes upon HCV infection and in the livers of patients with chronic hepatitis C (CHC). In addition, pegIFNα/ribavirin therapy induced C19orf66 expression in patients with CHC. Transcriptomic profiling and whole cell proteomics of hepatoma cells ectopically expressing C19orf66 revealed no induction of other antiviral genes. Expression of C19orf66 restricted HCV infection, whereas CRIPSPR/Cas9 mediated knockout of C19orf66 attenuated IFN-mediated suppression of HCV replication. Co-immunoprecipitation followed by mass spectrometry identified a stress granule protein-dominated interactome of C19orf66. Studies with subgenomic HCV replicons and an expression system revealed that C19orf66 expression impairs HCV-induced elevation of phosphatidylinositol-4-phosphate, alters the morphology of the viral replication organelle (termed the membranous web) and thereby targets viral RNA replication.

Conclusion: C19orf66 is an IFN-stimulated gene, which is upregulated in hepatocytes within the first hours post IFN treatment or HCV infection in vivo. The encoded protein possesses specific antiviral activity against HCV and targets the formation of the membranous web. Our study identifies C19orf66 as an IFN-inducible restriction factor with a novel antiviral mechanism that specifically targets HCV replication.

Lay Summary: Interferon-stimulated genes are thought to be important to for antiviral immune responses to HCV. Herein, we analysed C19orf66, an interferon-stimulated gene, which appears to inhibit HCV replication. It prevents the HCV-induced elevation of phosphatidylinositol-4-phosphate and alters the morphology of HCV's replication organelle.
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http://dx.doi.org/10.1016/j.jhep.2020.03.047DOI Listing
September 2020

Combination immunotherapy with anti-PD-L1 antibody and depletion of regulatory T cells during acute viral infections results in improved virus control but lethal immunopathology.

PLoS Pathog 2020 03 30;16(3):e1008340. Epub 2020 Mar 30.

Institute for Virology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.

Combination immunotherapy (CIT) is currently applied as a treatment for different cancers and is proposed as a cure strategy for chronic viral infections. Whether such therapies are efficient during an acute infection remains elusive. To address this, inhibitory receptors were blocked and regulatory T cells depleted in acutely Friend retrovirus-infected mice. CIT resulted in a dramatic expansion of cytotoxic CD4+ and CD8+ T cells and a subsequent reduction in viral loads. Despite limited viral replication, mice developed fatal immunopathology after CIT. The pathology was most severe in the gastrointestinal tract and was mediated by granzyme B producing CD4+ and CD8+ T cells. A similar post-CIT pathology during acute Influenza virus infection of mice was observed, which could be prevented by vaccination. Melanoma patients who developed immune-related adverse events under immune checkpoint CIT also presented with expanded granzyme-expressing CD4+ and CD8+ T cell populations. Our data suggest that acute infections may induce immunopathology in patients treated with CIT, and that effective measures for infection prevention should be applied.
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http://dx.doi.org/10.1371/journal.ppat.1008340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7105110PMC
March 2020

Spatial proteomics revealed a CXCL1-dependent crosstalk between the urothelium and relocated macrophages through IL-6 during an acute bacterial infection in the urinary bladder.

Mucosal Immunol 2020 07 28;13(4):702-714. Epub 2020 Feb 28.

Institute of Experimental Immunology and Imaging, Department of Immunodynamics, University Hospital Essen, 45147, Essen, Germany.

The urothelium of the urinary bladder represents the first line of defense. However, uropathogenic E. coli (UPEC) damage the urothelium and cause acute bacterial infection. Here, we demonstrate the crosstalk between macrophages and the urothelium stimulating macrophage migration into the urothelium. Using spatial proteomics by MALDI-MSI and LC-MS/MS, a novel algorithm revealed the spatial activation and migration of macrophages. Analysis of the spatial proteome unravelled the coexpression of Myo9b and F4/80 in the infected urothelium, indicating that macrophages have entered the urothelium upon infection. Immunofluorescence microscopy additionally indicated that intraurothelial macrophages phagocytosed UPEC and eliminated neutrophils. Further analysis of the spatial proteome by MALDI-MSI showed strong expression of IL-6 in the urothelium and local inhibition of this molecule reduced macrophage migration into the urothelium and aggravated the infection. After IL-6 inhibition, the expression of matrix metalloproteinases and chemokines, such as CXCL1 was reduced in the urothelium. Accordingly, macrophage migration into the urothelium was diminished in the absence of CXCL1 signaling in Cxcr1 mice. Conclusively, this study describes the crosstalk between the infected urothelium and macrophages through IL-6-induced CXCL1 expression. Such crosstalk facilitates the relocation of macrophages into the urothelium and reduces bacterial burden in the urinary bladder.
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http://dx.doi.org/10.1038/s41385-020-0269-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7312419PMC
July 2020

CalibraCurve: A Tool for Calibration of Targeted MS-Based Measurements.

Proteomics 2020 06 6;20(11):e1900143. Epub 2020 Mar 6.

Medizinisches Proteom-Center, Medical Faculty, Ruhr University Bochum, Gesundheitscampus 4, Bochum, D-44 801, Germany.

Targeted proteomics techniques allow accurate quantitative measurements of analytes in complex matrices with dynamic linear ranges that span up to 4-5 orders of magnitude. Hence, targeted methods are promising for the development of robust protein assays in several sensitive areas, for example, in health care. However, exploiting the full method potential requires reliable determination of the dynamic range along with related quantification limits for each analyte. Here, a software named CalibraCurve that enables an automated batch-mode determination of dynamic linear ranges and quantification limits for both targeted proteomics and similar assays is presented. The software uses a variety of measures to assess the accuracy of the calibration, namely precision and trueness. Two different kinds of customizable graphs are created (calibration curves and response factor plots). The accuracy measures and the graphs offer an intuitive, detailed, and reliable opportunity to assess the quality of the model fit. Thus, CalibraCurve is deemed a highly useful and flexible tool to facilitate the development and control of reliable SRM/MRM-MS-based proteomics assays.
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http://dx.doi.org/10.1002/pmic.201900143DOI Listing
June 2020

Systematic Comparison of Label-Free, SILAC, and TMT Techniques to Study Early Adaption toward Inhibition of EGFR Signaling in the Colorectal Cancer Cell Line DiFi.

J Proteome Res 2020 02 14;19(2):926-937. Epub 2020 Jan 14.

Klinik für Anästhesiologie, Intensivmedizin und Schmerztherapie , Universitätsklinikum Knappschaftskrankenhaus Bochum , Bochum 44892 , Germany.

We evaluated the quantification strategies label-free (LF), stable isotope labeling by amino acids in cell culture (SILAC), and tandem mass tags (TMT) and their performance in quantification of proteins and phosphosites (p-sites) to identify the most powerful approach for monitoring cellular signaling. We analyzed the epidermal growth factor receptor (EGFR) signaling network, which plays an essential role in colorectal cancer, and studied its dynamics within 24 h upon treatment with the EGFR-blocking antibody cetuximab, representing the first cellular adaption toward therapy. LF achieved superior coverage but was outperformed by label-based approaches regarding technical variability, especially for quantification of p-sites. TMT showed the lowest coverage and most missing values. We found that its performance considerably decreases when experimental replicates are distributed over several TMT plexes. SILAC showed the highest precision and outstanding performance for quantification of p-sites, rendering it the method of choice for analyzing cellular signaling in cell culture models. On the protein level, we observed only little regulation upon cetuximab treatment, whereas a great fraction of p-sites was significantly regulated. These dynamics represented an initial downregulation of the MAPK pathway, which was partially rescued as early as 24 h after treatment. We identified upregulation and signaling via ERBB3 as well as calcium and cAMP signaling as possible mechanisms bypassing the blockage of EGFR.
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http://dx.doi.org/10.1021/acs.jproteome.9b00701DOI Listing
February 2020

Vitamin K-MK-7 improves nitric oxide-dependent endothelial function in ApoE/LDLR mice.

Vascul Pharmacol 2019 Nov - Dec;122-123:106581. Epub 2019 Aug 14.

Jagiellonian University, Jagiellonian Centre for Experimental Therapeutics (JCET), Bobrzynskiego 14, 30-348 Krakow, Poland; Jagiellonian University Medical College, Faculty of Medicine, Chair of Pharmacology, Grzegorzecka 16, 31-531 Krakow, Poland. Electronic address:

Although, vitamin K displays vasoprotective effects, it is still not known whether K treatment improves endothelial function. In ApoE/LDLR mice at the stage prior to atherosclerosis development, four-week treatment with K-MK-7, given at a low dose (0.05 mg/kg), improved acetylcholine- and flow-induced, endothelium-dependent vasodilation in aorta or in femoral artery, as assessed by MRI in vivo. This effect was associated with an increased NO production, as evidenced by EPR measurements in ex vivo aorta. Treatment with higher doses of K-MK-7 (0.5; 5 mg/kg) resulted in a dose-dependent increase in plasma K-MK-7 and K-MK-4 concentration, without further improvement in endothelial function. In ApoE/LDLR mice with developed atherosclerotic plaques, treatment with a low (0.03 mg/kg) or high (10 mg/kg) dose of K-MK-7 resulted in a similar degree of endothelium-dependent vasodilation improvement and increase in plasma nitrate concentration, what was not associated with changes in thrombin generation as measured by CAT. Both doses of K-MK-7 also reduced media thickness in the brachiocephalic artery, but did not modify atherosclerotic plaque size. In conclusion, K-MK-7 improves NO-dependent endothelial function in ApoE/LDLR mice. This study, identifies the endothelial profile of the pharmacological activity of vitamin K, which has not been previously described.
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http://dx.doi.org/10.1016/j.vph.2019.106581DOI Listing
May 2020

Multiorgan Development of Oxidative and Nitrosative Stress in LPS-Induced Endotoxemia in C57Bl/6 Mice: DHE-Based Approach.

Oxid Med Cell Longev 2019 22;2019:7838406. Epub 2019 May 22.

Jagiellonian University, Jagiellonian Centre for Experimetal Therapeutics (JCET), Bobrzynskiego 14, 30-348 Krakow, Poland.

Detection of free radicals in tissues is challenging. Most approaches rely on incubating excised sections or homogenates with reagents, typically at supraphysiologic oxygen tensions, to finally detect surrogate, nonspecific end products. In the present work, we explored the potential of using intravenously (i.v.) injected dihydroethidine (DHE) to detect superoxide radical (O) abundance by quantification of the superoxide-specific DHE oxidation product, 2-hydroxyethidium (2-OH-E), as well as ethidium (E) and DHE in multiple tissues in a murine model of endotoxemia induced by lipopolysaccharide (LPS). LPS was injected intraperitoneally (i.p.), while DHE was delivered via the tail vein one hour before sacrifice. Tissues (kidney, lung, liver, and brain) were harvested and subjected to HPLC/fluorescent analysis of DHE and its monomeric oxidation products. In parallel, electron spin resonance (EPR) spin trapping was used to measure nitric oxide (NO) production in the aorta, lung, and liver isolated from the same mice. Endotoxemic inflammation was validated by analysis of plasma biomarkers. The concentration of 2-OH-E varied in the liver, lung, and kidney; however, the ratios of 2-OH-E/E and 2-OH-E/DHE were increased in the liver and kidney but not in the lung or the brain. An LPS-induced robust level of NO burst was observed in the liver, whereas the lung demonstrated a moderate yet progressive increase in the rate of NO production. Interestingly, endothelial dysfunction was observed in the aorta, as evidenced by decreased NO production 6 hours post-LPS injection that coincided with the inflammatory burden of endotoxemia (e.g. elevated serum amyloid A and prostaglandin E). Combined, these data demonstrate that systemic delivery of DHE affords the capacity to specifically detect O production . Furthermore, the ratio of 2-OH-E/E oxidation products in tissues provides a tool for comparative insight into the oxidative environments in various organs. Based on our findings, we demonstrate that the endotoxemic liver is susceptible to both O-mediated and nonspecific oxidant stress as well as nitrosative stress. Oxidant stress in the lung was detected to a lesser extent, thus underscoring a differential response of liver and lung to endotoxemic injury induced by intraperitoneal LPS injection.
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http://dx.doi.org/10.1155/2019/7838406DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6556324PMC
December 2019

Protein Biomarker Discovery Using Human Blood Plasma Microparticles.

Methods Mol Biol 2019 ;1959:51-64

Medizinisches Proteom-Center, Ruhr-Universität Bochum, Bochum, Germany.

Cells shed into the extracellular space a population of membranous vesicles of plasma membrane origin called microparticles (MP). Given the fact that MP are abundantly present in body fluids including plasma, rich in cell-type or disease-specific proteins and formed in conditions of stress and injury, they have been extensively investigated as biomarkers in various diseases. With the advancement in the mass spectrometry-based proteome analysis, the knowledge of the protein composition of plasma MP (PMP) has been intensively expanded, which aids the discovery of novel diagnostic target proteins. However, the lack of standardized and accurate protocols for PMP isolation limits the implementation of PMP as biomarkers in clinical settings. Here, we describe in detail a robust protocol for PMP isolation from human blood plasma via ultracentrifugation followed by label-free quantitative proteome analysis of PMP.
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http://dx.doi.org/10.1007/978-1-4939-9164-8_4DOI Listing
July 2019

The PD-1/PD-L1 Pathway Affects the Expansion and Function of Cytotoxic CD8 T Cells During an Acute Retroviral Infection.

Front Immunol 2019 5;10:54. Epub 2019 Feb 5.

Institute for Virology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.

Cytotoxic CD8 T lymphocytes (CTL) efficiently control acute virus infections but can become exhausted when a chronic infection develops. The checkpoint receptor PD-1 suppresses the functionality of virus-specific CD8 T cells during chronic infection. However, the role of the PD-L1/PD-1 pathway during the acute phase of infections has not been well characterized. In the current study the effects of PD-1 or PD-L1 deficiency on the CD8 T cell response against Friend retroviral (FV) infection of knockout mice was analyzed during acute infection. We observed an enhanced proliferation, functional maturation, and reduced apoptosis of effector CD8 T cells in the absence of PD-1 or PD-L1. The knockout of PD-L1 had a stronger effect on the functionality of CD8 T cells than that of PD-1. Augmented CTL responses were associated with an improved control of FV replication. The strong phenotype of FV-infected PD-L1 knockout mice was independent of the interaction with CD80 as an additional receptor for PD-L1. Furthermore, we performed a detailed analysis of the production of different granzymes in virus-specific CD8 T cells and observed that especially the simultaneous production of multiple granzymes in individual T cells (multifunctionality) was under the control of the PD-1/PD-L1 pathway. The findings from this study allow for a better understanding of the development of antiviral cytotoxic immunity during acute viral infections.
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http://dx.doi.org/10.3389/fimmu.2019.00054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6370637PMC
December 2019

Integrated Fourier Transform Infrared Imaging and Proteomics for Identification of a Candidate Histochemical Biomarker in Bladder Cancer.

Am J Pathol 2019 03 12;189(3):619-631. Epub 2019 Feb 12.

Department of Biophysics, Ruhr University Bochum, Bochum, Germany. Electronic address:

Histopathological differentiation between severe urocystitis with reactive urothelial atypia and carcinoma in situ (CIS) can be difficult, particularly after a treatment that deliberately induces an inflammatory reaction, such as intravesical instillation of Bacillus Calmette-Guèrin. However, precise grading in bladder cancer is critical for therapeutic decision making and thus requires reliable immunohistochemical biomarkers. Herein, an exemplary potential biomarker in bladder cancer was identified by the novel approach of Fourier transform infrared imaging for label-free tissue annotation of tissue thin sections. Identified regions of interest are collected by laser microdissection to provide homogeneous samples for liquid chromatography-tandem mass spectrometry-based proteomic analysis. This approach afforded label-free spatial classification with a high accuracy and without interobserver variability, along with the molecular resolution of the proteomic analysis. Cystitis and invasive high-grade urothelial carcinoma samples were analyzed. Three candidate biomarkers were identified and verified by immunohistochemistry in a small cohort, including low-grade urothelial carcinoma samples. The best-performing candidate AHNAK2 was further evaluated in a much larger independent verification cohort that also included CIS samples. Reactive urothelial atypia and CIS were distinguishable on the basis of the expression of this newly identified and verified immunohistochemical biomarker, with a sensitivity of 97% and a specificity of 69%. AHNAK2 can differentiate between reactive urothelial atypia in the setting of an acute or chronic cystitis and nonmuscle invasive-type CIS.
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http://dx.doi.org/10.1016/j.ajpath.2018.11.018DOI Listing
March 2019

A Mass Spectrometry-Based Profiling of Interactomes of Viral DDB1- and Cullin Ubiquitin Ligase-Binding Proteins Reveals NF-κB Inhibitory Activity of the HIV-2-Encoded Vpx.

Front Immunol 2018 19;9:2978. Epub 2018 Dec 19.

Institute for Virology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.

Viruses and hosts are situated in a molecular arms race. To avoid morbidity and mortality, hosts evolved antiviral restriction factors. These restriction factors exert selection pressure on the viruses and drive viral evolution toward increasingly efficient immune antagonists. Numerous viruses exploit cellular DNA damage-binding protein 1 (DDB1)-containing Cullin RocA ubiquitin ligases (CRLs) to induce the ubiquitination and subsequent proteasomal degradation of antiviral factors expressed by their hosts. To establish a comprehensive understanding of the underlying protein interaction networks, we performed immuno-affinity precipitations for a panel of DDB1-interacting proteins derived from viruses such as mouse cytomegalovirus (MCMV, Murid herpesvirus [MuHV] 1), rat cytomegalovirus Maastricht MuHV2, rat cytomegalovirus English MuHV8, human cytomegalovirus (HCMV), hepatitis B virus (HBV), and human immunodeficiency virus (HIV). Cellular interaction partners were identified and quantified by mass spectrometry (MS) and validated by classical biochemistry. The comparative approach enabled us to separate unspecific interactions from specific binding partners and revealed remarkable differences in the strength of interaction with DDB1. Our analysis confirmed several previously described interactions like the interaction of the MCMV-encoded interferon antagonist pM27 with STAT2. We extended known interactions to paralogous proteins like the interaction of the HBV-encoded HBx with different Spindlin proteins and documented interactions for the first time, which explain functional data like the interaction of the HIV-2-encoded Vpr with Bax. Additionally, several novel interactions were identified, such as the association of the HIV-2-encoded Vpx with the transcription factor RelA (also called p65). For the latter interaction, we documented a functional relevance in antagonizing NF-κB-driven gene expression. The mutation of the DDB1 binding interface of Vpx significantly impaired NF-κB inhibition, indicating that Vpx counteracts NF-κB signaling by a DDB1- and CRL-dependent mechanism. In summary, our findings improve the understanding of how viral pathogens hijack cellular DDB1 and CRLs to ensure efficient replication despite the expression of host restriction factors.
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http://dx.doi.org/10.3389/fimmu.2018.02978DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6305766PMC
October 2019

Label-Free Proteomic Analysis of Exosomes Secreted from THP-1-Derived Macrophages Treated with IFN-α Identifies Antiviral Proteins Enriched in Exosomes.

J Proteome Res 2019 03 3;18(3):855-864. Epub 2019 Jan 3.

Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences , Shanghai Medical College of Fudan University , Shanghai 200032 , China.

Exosomes are extracellular vesicles that function in intercellular communication. We have previously reported that exosomes play an important role in the transmission of antiviral molecules during interferon-α (IFN-α) treatment. In this study, the protein profiles of THP-1-derived macrophages with or without interferon-α treatment and the exosomes secreted from these cells were analyzed by label-free liquid chromatography-tandem mass spectrometry quantitation technologies. A total of 1845 and 1550 protein groups were identified in the THP-1 macrophages and the corresponding exosomes, respectively. Treating the cells with IFN-α resulted in the differential abundance of 94 proteins in cells and 67 proteins in exosomes (greater than 2.0-fold), among which 23 proteins were up-regulated in both the IFN-α treated cells and corresponding exosomes, while 14 proteins were specifically up-regulated in exosomes but not in the donor cells. GO and KEGG analysis of the identified proteins suggested that IFN-α promoted the abundance of proteins involved in the "defense response to virus" and "type I interferon signaling pathway" in both exosomes and cells. Functional analysis further indicated that exosomes from IFN-α-treated cells exhibited potent antiviral activity that restored the impaired antiviral response of IFN-α in hepatitis B virus-replicating hepatocytes. These results have deepened the understanding of the exosome-mediated transfer of IFN-α-induced antiviral molecules and may provide a new basis for therapeutic strategies to control viral infection.
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http://dx.doi.org/10.1021/acs.jproteome.8b00514DOI Listing
March 2019

Direct-acting antivirals-based therapy decreases hepatic fibrosis serum biomarker microfibrillar-associated protein 4 in hepatitis C patients.

Clin Mol Hepatol 2019 03 19;25(1):42-51. Epub 2018 Nov 19.

Medizinisches Proteom-Center, Ruhr University Bochum, Bochum, Germany.

Background/aims: An estimated 80 million people worldwide are infected with viremic hepatitis C virus (HCV). Even after eradication of HCV with direct acting antivirals (DAAs), hepatic fibrosis remains a risk factor for hepatocarcinogenesis. Recently, we confirmed the applicability of microfibrillar-associated protein 4 (MFAP4) as a serum biomarker for the assessment of hepatic fibrosis. The aim of the present study was to assess the usefulness of MFAP4 as a biomarker of liver fibrosis after HCV eliminating therapy with DAAs.

Methods: MFAP4 was measured using an immunoassay in 50 hepatitis C patients at baseline (BL), the end-of-therapy (EoT), and the 12-week follow-up visit (FU). Changes in MFAP4 from BL to FU and their association with laboratory parameters including alanine aminotransferase (ALT), aspartate aminotransferase (AST), platelets, the AST to platelet ratio index (APRI), fibrosis-4 score (FIB-4), and albumin were analyzed.

Results: MFAP4 serum levels were representative of the severity of hepatic fibrosis at BL and correlated well with laboratory parameters, especially APRI (Spearman correlation, R²=0.80). Laboratory parameters decreased significantly from BL to EoT. MFAP4 serum levels were found to decrease from BL and EoT to FU with high statistical significance (Wilcoxon P<0.001 for both).

Conclusion: Our findings indicate that viral eradication resulted in reduced MFAP4 serum levels, presumably representing a decrease in hepatic fibrogenesis or fibrosis. Hence, MFAP4 may be a useful tool for risk assessment in hepatitis C patients with advanced fibrosis after eradication of the virus.
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http://dx.doi.org/10.3350/cmh.2018.0029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6435967PMC
March 2019

Correction to: Liver cancer cell lines distinctly mimic the metabolic gene expression pattern of the corresponding human tumours.

J Exp Clin Cancer Res 2018 11 2;37(1):267. Epub 2018 Nov 2.

Department of Medicine II, Molecular Hepatology Section, Medical Faculty Mannheim, Heidelberg University, Theodor-Kutzer-Ufer 1-3 (H42, Floor 4), 68167, Mannheim, Germany.

In the publication of this article (1), there is an error in Fig. 5b. This has now been updated in the original article (1).
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http://dx.doi.org/10.1186/s13046-018-0939-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6215359PMC
November 2018

Systematic analysis of synergistic proteome modulations in a drug combination of cisplatin and MLN4924.

Mol Omics 2018 12;14(6):450-457

Medizinisches Proteom-Center, Ruhr-Universität Bochum, Universitätsstr. 150, 44801 Bochum, Germany.

Chemotherapeutic treatment regimens often take advantage of synergistic effects of drug combinations. Anticipating that synergistic effects on the cell biological level likely manifest on the proteome level, the analysis of proteome modulations represents an appropriate strategy to study drug combinations on a molecular level. More specifically, the detection of single proteins exhibiting synergistic abundance changes could be helpful to shed light on key molecules, which contribute in mechanisms facilitating the synergistic interaction and therefore represent potential targets for specific therapeutic approaches. In the reported study we aimed to provide evidence for this assumption and investigated the drug combination of cisplatin and the neddylation inhibitor MLN4924 in HCT-116 cells via cell biological analyses and mass spectrometry-based quantitative proteomics. From 1789 proteins quantified with two unique peptides, activated RNA polymerase II transcriptional coactivator p15 (SUB1) was highlighted as the most synergistically regulated protein using a synergistic scoring approach. Western blotting and analyses of cellular processes associated with this protein (DNA damage, oxidative stress and apoptosis) revealed supporting evidence for the synergistic regulation. Whereas the distinct role of SUB1 in the investigated drug combination needs to be elucidated in future studies, the presented results demonstrated the benefit and feasibility of synergistic scoring of proteome alterations to highlight proteins that likely contribute to the underlying molecular mechanisms of synergistic effects. Data are available via ProteomeXchange with identifier PXD009185.
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http://dx.doi.org/10.1039/c8mo00115dDOI Listing
December 2018

Liver cancer cell lines distinctly mimic the metabolic gene expression pattern of the corresponding human tumours.

J Exp Clin Cancer Res 2018 Sep 3;37(1):211. Epub 2018 Sep 3.

Department of Medicine II, Molecular Hepatology Section, Medical Faculty Mannheim, Heidelberg University, Theodor-Kutzer-Ufer 1-3 (H42, Floor 4), 68167, Mannheim, Germany.

Background: Although metabolism is profoundly altered in human liver cancer, the extent to which experimental models, e.g. cell lines, mimic those alterations is unresolved. Here, we aimed to determine the resemblance of hepatocellular carcinoma (HCC) cell lines to human liver tumours, specifically in the expression of deregulated metabolic targets in clinical tissue samples.

Methods: We compared the overall gene expression profile of poorly-differentiated (HLE, HLF, SNU-449) to well-differentiated (HUH7, HEPG2, HEP3B) HCC cell lines in three publicly available microarray datasets. Three thousand and eighty-five differentially expressed genes in ≥2 datasets (P < 0.05) were used for pathway enrichment and gene ontology (GO) analyses. Further, we compared the topmost gene expression, pathways, and GO from poorly differentiated cell lines to the pattern from four human HCC datasets (623 tumour tissues). In well- versus poorly differentiated cell lines, and in representative models HLE and HUH7 cells, we specifically assessed the expression pattern of 634 consistently deregulated metabolic genes in human HCC. These data were complemented by quantitative PCR, proteomics, metabolomics and assessment of response to thirteen metabolism-targeting compounds in HLE versus HUH7 cells.

Results: We found that poorly-differentiated HCC cells display upregulated MAPK/RAS/NFkB signaling, focal adhesion, and downregulated complement/coagulation cascade, PPAR-signaling, among pathway alterations seen in clinical tumour datasets. In HLE cells, 148 downregulated metabolic genes in liver tumours also showed low gene/protein expression - notably in fatty acid β-oxidation (e.g. ACAA1/2, ACADSB, HADH), urea cycle (e.g. CPS1, ARG1, ASL), molecule transport (e.g. SLC2A2, SLC7A1, SLC25A15/20), and amino acid metabolism (e.g. PHGDH, PSAT1, GOT1, GLUD1). In contrast, HUH7 cells showed a higher expression of 98 metabolic targets upregulated in tumours (e.g. HK2, PKM, PSPH, GLUL, ASNS, and fatty acid synthesis enzymes ACLY, FASN). Metabolomics revealed that the genomic portrait of HLE cells co-exist with profound reliance on glutamine to fuel tricarboxylic acid cycle, whereas HUH7 cells use both glucose and glutamine. Targeting glutamine pathway selectively suppressed the proliferation of HLE cells.

Conclusions: We report a yet unappreciated distinct expression pattern of clinically-relevant metabolic genes in HCC cell lines, which could enable the identification and therapeutic targeting of metabolic vulnerabilities at various liver cancer stages.
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http://dx.doi.org/10.1186/s13046-018-0872-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6122702PMC
September 2018

Comparison of Pulmonary and Systemic NO- and PGI-Dependent Endothelial Function in Diabetic Mice.

Oxid Med Cell Longev 2018 4;2018:4036709. Epub 2018 Jun 4.

Jagiellonian Centre for Experimental Pharmacology (JCET), Jagiellonian University, Bobrzyńskiego 14, 30-348 Kraków, Poland.

Diabetes increases the risk of pulmonary hypertension and is associated with alterations in pulmonary vascular function. Still, it is not clear whether alterations in the phenotype of pulmonary endothelium induced by diabetes are distinct, as compared to peripheral endothelium. In the present work, we characterized differences between diabetic complications in the lung and aorta in db/db mice with advanced diabetes. Male, 20-week-old db/db mice displayed increased HbA1c and glucose concentration compatible with advanced diabetes. Diabetic lungs had signs of mild fibrosis, and pulmonary endothelium displayed significantly ultrastructural changes. In the isolated, perfused lung from db/db mice, filtration coefficient (K) and contractile response to TXA analogue were enhanced, while endothelial NO-dependent modulation of pulmonary response to hypoxic ventilation and cumulative production of NO were impaired, with no changes in immunostaining for eNOS expression. In turn, 6-keto-PGF release from the isolated lung from db/db mice was increased, as well as immunostaining of thrombomodulin (CD141). In contrast to the lung, NO-dependent, acetylcholine-induced vasodilation, ionophore-stimulated NO generation, and production of 6-keto-PGF were all impaired in aortic rings from db/db mice. Although eNOS immunostaining was not changed, that of CD141 was clearly lowered. Interestingly, diabetes-induced nitration of proteins in aorta was higher than that in the lungs. In summary, diabetes induced marked ultrastructural changes in pulmonary endothelium that were associated with the increased permeability of pulmonary microcirculation, impaired NO-dependent vascular function, with compensatory increase in PGI production, and increased CD141 expression. In contrast, endothelial dysfunction in the aorta was featured by impaired NO-, PGI-dependent function and diminished CD141 expression.
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http://dx.doi.org/10.1155/2018/4036709DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6008763PMC
October 2018

Vascular Nitric Oxide-Superoxide Balance and Thrombus Formation after Acute Exercise.

Med Sci Sports Exerc 2018 07;50(7):1405-1412

Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Krakow, POLAND.

Introduction: An acute bout of strenuous exercise in humans results in transient impairment of nitric oxide (NO)-dependent function, but it remains unknown whether this phenomenon is associated with increased risk of thrombotic events after exercise. This study aimed to evaluate effects of a single bout of exhaustive running in mice on the balance of vascular NO/reactive oxygen species production, and on thrombogenicity.

Methods: At different time points (0, 2, and 4 h) after exercise and in sedentary C57BL/6 mice, the production of NO and superoxide (O2) in aorta was measured by electron paramagnetic resonance spin trapping and by dihydroethidium/high-performance liquid chromatography-based method, respectively, whereas collagen-induced thrombus formation was analyzed in a microchip-based flow-chamber system (total thrombus-formation analysis system). We also measured pre- and postexercise plasma concentration of nitrite/nitrate and 6-keto-PGF1α.

Results: An acute bout of exhaustive running in mice resulted in decreased production of NO and increased production of O2 in aorta, with maximum changes 2 h after completion of exercise when compared with sedentary mice. However, platelet thrombus formation was not changed by exercise as evidenced by unaltered time to start of thrombus formation, capillary occlusion time, and total thrombogenicity (area under the flow pressure curve) as measured in a flow-chamber system. Strenuous exercise increased the plasma concentration of nitrite but did not affect nitrate and 6-keto-PGF1α concentrations.

Conclusion: An acute bout of strenuous exercise in mice reduced NO and in parallel increased O2 production in aorta. This response was most pronounced 2 h after exercise. Surprisingly, the reduced NO and increased O2 production in mice after exercise did not result in increased platelet-dependent thrombogenicity. These results show that transient reduction in NO bioavailability does not modify thromboresistance in healthy mice after exercise.
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http://dx.doi.org/10.1249/MSS.0000000000001589DOI Listing
July 2018

NHS-based Tandem Mass Tagging of Proteins at the Level of Whole Cells: A Critical Evaluation in Comparison to Conventional TMT-Labeling Approaches for Quantitative Proteome Analysis.

Anal Sci 2017 ;33(12):1387-1394

Medizinisches Proteom-Center, Ruhr-Universität Bochum, Universitätsstr.

Tandem mass tags (TMT) are usually introduced at the levels of isolated proteins or peptides. Here, for the first time, we report the labeling of whole cells and a critical evaluation of its performance in comparison to conventional labeling approaches. The obtained results indicated that TMT protein labeling using intact cells is generally possible, if it is coupled to a subsequent enrichment using anti-TMT antibody. The quantitative results were similar to those obtained after labeling of isolated proteins and both were found to be slightly complementary to peptide labeling. Furthermore, when using NHS-based TMT, no specificity towards cell surface proteins was observed in the case of cell labeling. In summary, the conducted study revealed first evidence for the general possibility of TMT cell labeling and highlighted limitations of NHS-based labeling reagents. Future studies should therefore focus on the synthesis and investigation of membrane impermeable TMTs to increase specificity towards cell surface proteins.
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http://dx.doi.org/10.2116/analsci.33.1387DOI Listing
August 2018

Development, validation and application of a micro-liquid chromatography-tandem mass spectrometry based method for simultaneous quantification of selected protein biomarkers of endothelial dysfunction in murine plasma.

J Pharm Biomed Anal 2018 Feb 10;149:465-474. Epub 2017 Nov 10.

Jagiellonian University, Jagiellonian Centre for Experimental Therapeutics, Bobrzynskiego 14, 30-348, Krakow, Poland; Jagiellonian University Medical College, Faculty of Pharmacy, Chair and Department of Toxicology, Medyczna 9, 30-688, Krakow, Poland. Electronic address:

The objective of this study was to develop and validate the method based on micro-liquid chromatography-tandem mass spectrometry (microLC/MS-MRM) for simultaneous determination of adiponectin (ADN), von Willebrand factor (vWF), soluble form of vascular cell adhesion molecule 1 (sVCAM-1), soluble form of intercellular adhesion molecule 1 (sICAM-1) and syndecan-1 (SDC-1) in mouse plasma. The calibration range was established from 2.5pmol/mL to 5000pmol/mL for ADN; 5pmol/mL to 5000pmol/mL for vWF; 0.375pmol/mL to 250pmol/mL for sVCAM-1 and sICAM-1; and 0.25pmol/mL to 250pmol/mL for SDC-1. The method was applied to measure the plasma concentration of selected proteins in mice fed high-fat diet (HFD), and revealed the pro-thrombotic status by increased concentration of vWF (1.31±0.17 nmol/mL (Control) vs 1.98±0.09 nmol/mL (HFD), p <0.05) and the dysregulation of adipose tissue metabolism by decreased concentration of ADN (0.62±0.08 nmol/mL (Control) vs 0.37±0.06 nmol/mL (HFD), p <0.05). In conclusion, the microLC/MS-MRM-based method allows for reliable measurements of selected protein biomarkers of endothelial dysfunction in mouse plasma.
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http://dx.doi.org/10.1016/j.jpba.2017.11.023DOI Listing
February 2018