Bone Allograft Acid Lysates Change the Genetic Signature of Gingival Fibroblasts.

Bone allografts are widely used as osteoconductive support to guide bone regrowth. Bone allografts are more than a scaffold for the immigrating cells as they maintain some bioactivity of the original bone matrix. Yet, it remains unclear how immigrating cells respond to bone allografts. To this end, we have evaluated the response of mesenchymal cells exposed to acid lysates of bone allografts (ALBA). RNAseq revealed that ALBA has a strong impact on the genetic signature of gingival fibroblasts, indicated by the increased expression of IL11, AREG, C11orf96, STC1, and GK-as confirmed by RT-PCR, and for IL11 and STC1 by immunoassays. Considering that transforming growth factor-β (TGF-β) is stored in the bone matrix and may have caused the expression changes, we performed a proteomics analysis, TGF-β immunoassay, and smad2/3 nuclear translocation. ALBA neither showed detectable TGF-β nor was the lysate able to induce smad2/3 translocation. Nevertheless, the TGF-β receptor type I kinase inhibitor SB431542 significantly decreased the expression of IL11, AREG, and C11orf96, suggesting that other agonists than TGF-β are responsible for the robust cell response. The findings suggest that IL11, AREG, and C11orf96 expression in mesenchymal cells can serve as a bioassay reflecting the bioactivity of the bone allografts

Cross-Omics Comparison of Stress Responses in Mesothelial Cells Exposed to Heat- versus Filter-Sterilized Peritoneal Dialysis Fluids

Recent research suggests that cytoprotective responses, such as expression of heat-shock proteins, might be inadequately induced in mesothelial cells by heat-sterilized peritoneal dialysis (PD) fluids. This study compares transcriptome data and multiple protein expression profiles for providing new insight into regulatory mechanisms. Two-dimensional difference gel electrophoresis (2D-DIGE) based proteomics and topic defined gene expression microarray-based transcriptomics techniques were used to evaluate stress responses in human omental peritoneal mesothelial cells in response to heat- or filter-sterilized PD fluids. Data from selected heat-shock proteins were validated by 2D western-blot analysis. Comparison of proteomics and transcriptomics data discriminated differentially regulated protein abundance into groups depending on correlating or noncorrelating transcripts. Inadequate abundance of several heat-shock proteins following exposure to heat-sterilized PD fluids is not reflected on the mRNA level indicating interference beyond transcriptional regulation. For the first time, this study describes evidence for posttranscriptional inadequacy of heat-shock protein expression by heat- sterilized PD fluids as a novel cytotoxic property. Cross-omics technologies introduce a novel way of understanding PDF bioincompatibility and searching for new interventions to reestablish adequate cytoprotective responses

Trichoderma G protein-coupled receptors: functional characterisation of a cAMP receptor-like protein from Trichoderma atroviride

Gα subunits act to regulate vegetative growth, conidiation, and the mycoparasitic response in Trichoderma atroviride. To extend our knowledge on G protein signalling, we analysed G protein-coupled receptors (GPCRs). As the genome sequence of T. atroviride is not publicly available yet, we carried out an in silico exploration of the genome database of the close relative T. reesei. Twenty genes encoding putative GPCRs distributed over eight classes and additional 35 proteins similar to the Magnaporthe grisea PTH11 receptor were identified. Subsequently, four T. atroviride GPCR-encoding genes were isolated and affliated to the cAMP receptor-like family by phylogenetic and topological analyses. All four genes showed lowest expression on glycerol and highest mRNA levels upon carbon starvation. Transcription of gpr3 and gpr4 responded to exogenously added cAMP and the shift from liquid to solid media. gpr3 mRNA levels also responded to the presence of fungal hyphae or cellulose membranes. Further characterisation of mutants bearing a gpr1-silencing construct revealed that Gpr1 is essential for vegetative growth, conidiation and conidial germination. Four genes encoding the first GPCRs described in Trichoderma were isolated and their expression characterized. At least one of these GPCRs is important for several cellular processes, supporting the fundamental role of G protein signalling in this fungus

The Peritoneal Surface Proteome in a Model of Chronic Peritoneal Dialysis Reveals Mechanisms of Membrane Damage and Preservation

Peritoneal dialysis (PD) fluids are cytotoxic to the peritoneum. Recent studies have shown that alanyl-glutamine (AlaGln) modulates the cellular stress response, improves mesothelial cell survival, reduces submesothelial thickening in experimental models of PD, and in clinical studies improves PD effluent cell stress and immune responses. However, the mechanisms of AlaGln-mediated membrane protection are not yet fully understood. Here, we explore those mechanisms through application of a novel proteomics approach in a clinically relevant in vivo model in rats. Experimental PD was performed for 5 weeks using conventional single-chamber bag (SCB) or neutral dual-chamber bag (DCB), PD fluid (PDF), with or without AlaGln supplementation, via a surgically implanted catheter. Rats subjected to a single dwell without catheter implantation served as controls. The peritoneal surface proteome was directly harvested by detergent extraction and subjected to proteomic analysis by two-dimensional difference gel electrophoresis (2D-DiGE) with protein identification by mass spectrometry. An integrated bioinformatic approach was applied to identify proteins significantly affected by the treatments despite biological variation and interfering high abundance proteins. From 505 of 744 common spots on 59 gels, 222 unique proteins were identified. Using UniProt database information, proteins were assigned either as high abundance plasma proteins, or as cellular proteins. Statistical analysis employed an adapted workflow from RNA-sequencing, the trimmed mean of M-values (TMM) for normalization, and a mixed model for computational identification of significantly differentially abundant proteins. The most prominently enriched pathways after 5 weeks chronic treatment with SCB or DCB, PDFs belonged to clusters reflecting tissue damage and cell differentiation by cytoskeletal reorganization, immune responses, altered metabolism, and oxidative stress and redox homeostasis. Although the AlaGln effect was not as prominent, associated enriched pathways showed mostly regression to control or patterns opposite that of the PDF effect. Our study describes the novel peritoneal surface proteome through combined proteomic and bioinformatic analyses, and assesses changes elicited by chronic experimental PD. The biological processes so identified promise to link molecular mechanisms of membrane damage and protection in the in vivo rat model to pathomechanisms and cytoprotective effects observed in vitro and in clinical PD

Targeted Metabolomic Profiling of Peritoneal Dialysis Effluents Shows Anti-oxidative Capacity of Alanyl-Glutamine

Readily available peritoneal dialysis (PD) effluents from PD patients in the course of renal replacement therapy are a potentially rich source for molecular markers for predicting clinical outcome, monitoring the therapy, and therapeutic interventions. The complex clinical phenotype of PD patients might be reflected in the PD effluent metabolome. Metabolomic analysis of PD effluent might allow quantitative detection and assessment of candidate PD biomarkers for prognostication and therapeutic monitoring. We therefore subjected peritoneal equilibration test effluents from 20 stable PD patients, obtained in a randomized controlled trial (RCT) to evaluate cytoprotective effects of standard PD solution (3.86% glucose) supplemented with 8 mM alanyl-glutamine (AlaGln) to targeted metabolomics analysis. One hundred eighty eight pre-defined metabolites, including free amino acids, acylcarnitines, and glycerophospholipids, as well as custom metabolic indicators calculated from these metabolites were surveyed in a high-throughput assay requiring only 10 μl of PD effluent. Metabolite profiles of effluents from the cross-over trial were analyzed with respect to AlaGln status and clinical parameters such as duration of PD therapy and history of previous episodes of peritonitis. This targeted approach detected and quantified 184 small molecules in PD effluent, a larger number of detected metabolites than in all previous metabolomic studies in PD effluent combined. Metabolites were clustered within substance classes regarding concentrations after a 4-h dwell. PD effluent metabolic profiles were differentiated according to PD patient sub-populations, revealing novel changes in small molecule abundance during PD therapy. AlaGln supplementation of PD fluid altered levels of specific metabolites, including increases in alanine and glutamine but not glutamate, and reduced levels of small molecule indicators of oxidative stress, such as methionine sulfoxide. Our study represents the first application of targeted metabolomics to PD effluents. The observed metabolomic changes in PD effluent associated with AlaGln-supplementation during therapy suggested an anti-oxidant effect, and were consistent with the restoration of important stress and immune processes previously noted in the RCT. High-throughput detection of PD effluent metabolomic signatures and their alterations by therapeutic interventions offers new opportunities for metabolome-clinical correlation in PD and for prescription of personalized PD therapy

Alanyl-Glutamine Restores Tight Junction Organization after Disruption by a Conventional Peritoneal Dialysis Fluid

Understanding and targeting the molecular basis of peritoneal solute and protein transport is essential to improve peritoneal dialysis (PD) efficacy and patient outcome. Supplementation of PD fluids (PDF) with alanyl-glutamine (AlaGln) increased small solute transport and reduced peritoneal protein loss in a recent clinical trial. Transepithelial resistance and 10 kDa and 70 kDa dextran transport were measured in primary human endothelial cells (HUVEC) exposed to conventional acidic, glucose degradation products (GDP) containing PDF (CPDF) and to low GDP containing PDF (LPDF) with and without AlaGln. Zonula occludens-1 (ZO-1) and claudin-5 were quantified by Western blot and immunofluorescence and in mice exposed to saline and CPDF for 7 weeks by digital imaging analyses. Spatial clustering of ZO-1 molecules was assessed by single molecule localization microscopy. AlaGln increased transepithelial resistance, and in CPDF exposed HUVEC decreased dextran transport rates and preserved claudin-5 and ZO-1 abundance. Endothelial clustering of membrane bound ZO-1 was higher in CPDF supplemented with AlaGln. In mice, arteriolar endothelial claudin-5 was reduced in CPDF, but restored with AlaGln, while mesothelial claudin-5 abundance was unchanged. AlaGln supplementation seals the peritoneal endothelial barrier, and when supplemented to conventional PD fluid increases claudin-5 and ZO-1 abundance and clustering of ZO-1 in the endothelial cell membrane.This work is part of the IMPROVE-PD project that has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under the Marie Sklodowska-Curie grant agreement number 812699. M.B. is funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)—Projektnummer 419826430. R.H. was supported by a research fellowship of the European Renal Association and European Dialysis and Transplant Association (ERA-EDTA). E.L. was supported by the ÚNKP-18-2 New National Excellence Program of the Ministry of Human Capacities, Hungary. E.L. and H.J. were supported by Jellinek-Harry scholarship. S.G.Z. acknowledges the Alexander von Humboldt Stiftung/Foundation for an Experienced Researcher Fellowship (2019–2021) and the International Peritoneal Dialysis Society (ISPD) for an International Cooperation Research Grant (2019–2021). C.P.S. has obtained funding from European Nephrology and Dialysis Institute (ENDI).Peer reviewe

Proteomics - Clinical Applications / The Extracorporeal Proteome : The Significance of Selective Protein Removal During Dialysis Therapy

Dialysis as renal replacement therapy aims excess water and waste solutes from the uremic patient while retaining proteins in the plasma. Irrespective of the dialysis modality, hemodialysis (HD) or peritoneal dialysis (PD), the amount and composition of proteins that are removed are important determinants of the biocompatibility of the therapy. Although hemodialysis membranes would ideally be biologically inert filtration tubes, they are known to adsorb proteins. The part of the plasma proteome that is thereby removed during every dialysis session may be regarded as the extracorporeal proteome, which has to be kept in balance with the plasma proteome, regarding the individual proteins biological roles and activation states. In a recent study, Ronci et al. (Proteomics Clin. Appl. 2018, e1700140) comprehensively compare two hemodialyzer membrane materials by shotgun LCMS proteomic analysis of adsorbed proteins and ultrafiltrates from four HD patients. While pathway analysis is an attractive tool to compare different proteomes on an abstract level, some challenges remain regarding the adaptation for such tools for special proteomes and the interpretation of relative changes compared absolute changes regarding their biological importance in dialysis techniques. In summary, selective protein removal may represent a yet unexploited therapeutic opportunity if the “right” proteins are removed from the blood.(VLID)342393

Proteomics analysis of the stress response of mesothelial cells after exposure to peritoneal dialysis fluid

Die Peritonealdialyse (PD) als Form der Nierenersatztherapie wird häufig durch technisches Versagen der Therapie gestört. Peritonealdialyseflüssigkeiten (PDF) verursachen eine Schädigung der peritonealen Mesothelzellschicht durch ihre Zytotoxizität. Da von den beteiligten zellulären Prozessen bisher nur einzelne Elemente untersucht wurden, sollte die mesotheliale Stressantwort auf PDF und dessen einzelne Bestandteile in dieser Studie auf einem globalen Niveau untersucht werden. Immortalisierte Mesothelzellen (MeT-5A) wurden entweder einfach oder mehrmals mit "ganzem" PDF oder mit Medien, die die einzelnen physikochemischen Eigenschaften von PDF widerspiegeln, oder mit Kontrollmedium stimuliert. Proteomics- und Bioinformatik-Methoden wurden miteinander kombiniert, um die auftretenden Effekte in den Mesothelzellen zu untersuchen. Die Proteinexpression wurde mittels zweidimensionaler Gelelektrophorese und Coomassie Blau Färbung oder Fluoreszenzmarkierung der Proteine gemessen und signifikant veränderte Protein-Spots wurden mittels MALDI-TOF und MS Techniken identifiziert. Die Listen der experimentell identifizierten Kandidaten-Proteine wurden mittels einer "next neighbor" Methode erweitert und auf biologische Prozesse, die einen statistisch signifikant überproportionalen Anteil aufweisen, überprüft. Um dem Problem der falsch-positiven Kandidaten in 2D Gelen zu engegnen, wurden in weiterer Folge nur die Kandidaten untersucht, die auch hier signifikant waren. Einzelne PDF Exposition führt zu einer Reduktion von allgemeinen zellulären Prozessen zugunsten reparativer Mechanismen, darunter Protein-Metabolismus, - Modifikation und Faltung, wobei die Chaperone die Hauptgruppe darstellten. Die beobachteten biologischen Prozesse, die von dieser PDF Exposition ausgelöst wurden, enthielten hauptsächlich funktionell verwobene "multi-tasking Proteine", die auch zur zytoskeletalen Reorganisation sowie zu Immunmechanismen beitragen. Die Analyse der Exposition von Mesothelzellen gegenüber den einzelnen toxischen Eigenschaften der PDF ergab einen Dämpfungseffekt auf Chaperone sobald Glukoseabbauprodukte im Medium vorhanden waren. Wiederholte PDF Exposition führte zu einer verzögerten Protein Regulation, die eine Inhibition der Stressantwort, durch hohe Niveaus von bereits induzierten Chaperonen, darstellt. Die identifizierten Proteine konnten in geringerem Ausmaß zu akuten Schädigungsmechanismen zugeordnet werden, waren aber öfter Bestandteil von spezialisierten zellulären Funktionen mit einer insgesamt verminderten Anzahl von "multi-tasking Proteinen". Dieses Ergebnis stimmt hervorragend mit dem Konzept der Konditionierung und Zytoprotektion überein. Diese Studie beschreibt das veränderte Proteom von Mesothelzellen während der Erholung von PDF Stress und Anpassung auf wiederholten Stress. Eine breit gefächerte Stressantwort mit einer Vielzahl von überlappenden Prozessen und "multi-tasking Proteinen" verändert sich im Zuge der repetitiven Exposition gegenüber PDF hin zu einer spezialisierten Antwort mit nur wenigen Prozessen und wenigen Proteinen, die gleichzeitig mehrere Aufgaben erfüllen. Die Dämpfung der Stressantwort wird möglicherweise durch spezifische Toxine, wie die Glukoseabbauprodukte, verursacht.Renal replacement therapy by peritoneal dialysis is frequently complicated by technical failure. Peritoneal dialysis fluids (PDF) cause injury to the peritoneal mesothelial cell layer due to their cytotoxicity. As only isolated elements of the involved cellular processes have been studied before, this study aimed at a global assessment of the mesothelial stress response to isolated and combined stressors of PDF. Immortalized mesothelial cells (MeT-5A) were stimulated by single or repeated exposure to 'full' PDF, or media containing only isolated physicochemical properties of PDF or control medium. Proteomics and bioinformatics techniques were combined to study effects in mesothelial cells. Protein expression was assessed by two-dimensional gel electrophoresis using Coomassie blue staining or fluorescent labeling techniques for protein detection, and significantly altered spots were identified by MALDI-TOF MS and MS techniques. The lists of experimentally derived candidate proteins were expanded by a next neighbor approach and analyzed for significantly enriched biological processes. To address the problem of an unknown portion of false positive spots in 2DGE, only proteins showing significant p-values on both levels were further interpreted. Single PDF exposure resulted in reduction of biological processes in favor of reparative responses, including protein metabolism, modification and folding, with chaperones as a major subgroup. The observed biological processes triggered by this acute PDF exposure mainly contained functionally interwoven multi-tasking proteins contributing as well to cytoskeletal reorganization and defense mechanisms. When analyzing the response to isolated toxic stimuli of PDF, a dampening effect on chaperones could be observed as soon as glucose degradation products were present in the treatment medium. Repeated PDF exposure resulted in attenuated protein regulation, reflecting inhibition of stress responses by high levels of pre-induced chaperones. The identified proteins were less attributable to acute cellular injury but rather to specialized functions with a reduced number of involved multi-tasking proteins. This finding agrees well with the concept of conditioning effects and cytoprotection. In conclusion, this study describes the re-programmed proteome of mesothelial cells during recovery from PDF exposure and adaption to repetitive stress. A broad stress response with a number of highly overlapping processes and multi-tasking proteins shifts towards a more specific response of only few less overlapping processes. Stress responses might be dampened by specific stressors such as glucose degradation products.Klaus KratochwillAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung, abstractwww.meduniwien.ac.at/poetWien, Med. Univ., Diss., 2009(VLID)171516