Characterizing Patients with Recurrent Urinary Tract Infections in Vesicoureteral Reflux: A Pilot Study of the Urinary Proteome

Recurrent urinary tract infections (UTIs) pose a significant burden on the health care system. Underlying mechanisms predisposing children to UTIs and associated changes in the urinary proteome are not well understood. We aimed to investigate the urinary proteome of a subset of children who have vesicoureteral reflux (VUR) and recurrent UTIs because of their risk of developing infection-related renal damage. Improving diagnostic modalities to identify UTI risk factors would significantly alter the clinical management of children with VUR. We profiled the urinary proteomes of 22 VUR patients with low grade VUR (1-3 out of 5), a history of recurrent UTIs, and renal scarring, comparing them to those obtained from 22 age-matched controls. Urinary proteins were analyzed by mass spectrometry followed by protein quantitation based on spectral counting. Of the 2,551 proteins identified across both cohorts, 964 were robustly quantified, as defined by meeting criteria with spectral count (SC) \u3e /=2 in at least 7 patients in either VUR or control cohort based on optimization of signal-to-noise ratio. Eighty proteins had differential expression between the two cohorts, with 44 proteins significantly upregulated and 36 downregulated (q \u3c 0.075, |FC| \u3e 1.2). Urinary proteins involved in inflammation, acute phase response (APR), modulation of extracellular matrix (ECM), and carbohydrate metabolism were overrepresented among the study cohort

Pharmaceutical post-marketing analytical development

During nineteen weeks of my internship in Technologie SERVIER I have had an opportunity to work with experts in the field of analytical chemistry and take up basic analytical methods used in pharmaceutical development. In my report I have described methods I have used in control batch analyses of Almitrine bismesylate salt and Indapamide spiked reference batch: High Performance Liquid Chromatography (HPLC) and Infrared Spectroscopy for drug substance identification Thin Layer Chromatography (TLC) for the purity verification Karl Fischer titration for the water content determination. After accomplishing my first objective to become self-confident and independent in using analytical instruments on my own, I was given a responsibility to test British pharmacopoeia draft for the Perindopril Monograph template. Therefore I was challenged to verify reproducibility of proposed Perindopril identification test and Perindopril tablet dissolution test suitability using infrared spectroscopy and HPLC-UV, respectively. Furthermore, rather than just point out British pharmacopoeia draft monograph’s ambiguities I also tried to give possible effective solutions to improve currently proposed methods

Pharmaceutical post-marketing analytical development

During nineteen weeks of my internship in Technologie SERVIER I have had an opportunity to work with experts in the field of analytical chemistry and take up basic analytical methods used in pharmaceutical development. In my report I have described methods I have used in control batch analyses of Almitrine bismesylate salt and Indapamide spiked reference batch: High Performance Liquid Chromatography (HPLC) and Infrared Spectroscopy for drug substance identification Thin Layer Chromatography (TLC) for the purity verification Karl Fischer titration for the water content determination. After accomplishing my first objective to become self-confident and independent in using analytical instruments on my own, I was given a responsibility to test British pharmacopoeia draft for the Perindopril Monograph template. Therefore I was challenged to verify reproducibility of proposed Perindopril identification test and Perindopril tablet dissolution test suitability using infrared spectroscopy and HPLC-UV, respectively. Furthermore, rather than just point out British pharmacopoeia draft monograph’s ambiguities I also tried to give possible effective solutions to improve currently proposed methods

The versatile role played by lysine deacetylase inhibitors in cancer-specific pathways and cell proliferation

Lysin-Acetyltransferasen (KATs) und Lysin-Deacetylasen (KDACs) sind vielseitige Enzyme, welche den Acetylierungsstatus von Histonen und anderen Proteinen regulieren. KDACs werden in verschiedenen Krebsarten überexprimiert, und ihre Hemmung kann zu Differenzierung und Apoptose der Krebszellen führen. Diese Wirkungsweise wurde für zahlreiche Krankheitsbilder wie Blutkrebs, soliden Tumoren oder viralen Infektionen nachgewiesen, wobei die bisher größte medizinische Verbesserung in der Behandlung von Blutkrebs erzielt wurde. Der therapeutische Einsatz von KDAC Inhibitoren (KDACi) ist von der FDA nur bei T-Zell Lymphomen und Multiplem Myelom als Einzel- beziehungsweise Kombinationstherapie zugelassen. Trotzdem zeigen neue Forschungsergebnisse vielversprechende Resultate für den Einsatz von KDACi bei weiteren Erkrankungen (zum Beispiel bei kardiovaskulären, neurodegenerativen Erkrankungen und Infektionskrankheiten). Der Schwerpunkt dieser Arbeit liegt darauf, unter verschiedenen Gesichtspunkten zu erforschen wie KDACi krebsspezifische Signaltransduktionswege beeinflussen können; mit besonderem Fokus auf Proteine, die in der Zellproliferation eine wichtige Rolle spielen. Um die frühe Reaktion auf eine KDACi Behandlung zu untersuchen, wurde ein systembiologischer Ansatz gewählt. Diese Herangehensweise stellt den Fokus auf unmittelbar betroffene Signaltransduktionswege sicher, und vermeidet die Auslösung zahlreicher sekundärer effekte durch Apoptose. Dadurch konnten Proteine identifiziert werden, die als Folge einer KDACi Behandlung ihre Expression und ihren Acetylierungsstatus verändern. Des Weiteren kann durch die Korrelation der Differenzen in Gen- und Proteinexpression auf eine spezifische Wechselwirkung geschlossen werden. Unsere Ergebnisse zeigen den Einfluss von KDACi auf Energiemetabolismus und mit Nukleotidsynthese in Zusammenhang stehende Signaltransduktionswege bereits bei geringer, nicht-apoptotischer Dosis. Diese Resultate umfassen unter anderem auch Proteine, die essentiell für das Überleben von Zellen sind. Die Gesamtheit der Daten wird im Überblick in Manuskript #1 erörtert. Die weiterführende Diskussion und der Ausblick dieser Arbeit beschäftigen sich vor allem mit krebsspezifischen Stoffwechselwegen und dem Potential, das die Erforschung eines durch KDACi beeinflussten Acetylierungsprofils in der Krebstherapie besitzt. Wir untersuchten den Einfluss der veränderten Acetylierung von MTHFD1 und SHMT2 auf Proteinstruktur, Oligomerisationsfähigkeit und Enzymfunktion. Eine Modifikation von Lysinresten durch Acetylierung wurde sowohl bei Histonen, als auch bei anderen Proteinen festgestellt. Zur Analyse der Histonacetylierung musste der übliche auf Massenspektrometrie (MS) basierende experimentelle Ansatz jedoch angepasst werden. Dem liegen insbesondere proteinchemische Unterschiede zugrunde, wie zum Beispiel Länge und Sequenz der betroffenen Peptide. Zu diesem Zweck entwickelten wir die FASIL-MS Strategie, die es uns ermöglichte, durch KDACi Behandlung hervorgerufene Lysinacetylierung positionsspezifisch auf Histonen zu quantifizieren. FASIL-MS kombiniert ein effizientes Probenvorbereitungsprotokoll und eine adaptierte MS Methode mit einer verbesserten bioinformatischen Auswertung der MS2-basierten Quantifizierung der Histonacetylierung. Dies kann zur Untersuchung weiterer experimenteller Fragestellungen herangezogen werden, beispielsweise zur Beobachtung von Veränderungen der Histonacetylierung in Abhängigkeit von Behandlungszeitpunkt, -dauer, oder Art des Inhibitors. Nach unserem Wissen stellt diese Arbeit die bisher umfassendste Strategie zur Erforschung der durch KDACi Behandlung ausgelösten quantitativen Änderungen in Gen- und Proteinexpression, Histon- und sonstiger Proteinacetylierung dar. Eine Kombination mit weiteren Methoden zur Untersuchung von Differenzen im Acetylierungsprofil wird zweifelsohne neue Einblicke in Alternativen für die zielgerichtete Krebstherapie gewähren.Lysine acetyl transferases (KATs) and lysine deacetylases (KDACs) are versatile enzymes that regulate the acetylation state of histone and non-histone proteins. KDACs are overexpressed in various types of cancer, and the inhibition thereof results in differentiation and apoptosis of tumour cells. This effect has been investigated in numerous diseases ranging from blood malignancies and solid tumours to viral infections. To date, however, the greatest medical benefits were observed in the treatment of blood cancer. KDAC inhibitor (KDACi) treatment is FDA-approved only for T cell lymphoma and multiple myeloma as a single agent or as a combinatorial therapy, respectively. Nevertheless, recent research shows promising results in extending the application of KDACis beyond blood malignancies to cardiovascular diseases, various inflammatory and neurodegenerative disorders. This thesis focuses on several aspects to investigate how KDACis influence cancer-specific pathways, particularly proteins associated with cell proliferation. The systems-biology approach was utilised to investigate an early response to KDACis in order to focus on the initial pathways that are altered after KDACi treatment, without triggering a plethora of secondary apoptotic events. This enabled identification of the proteins that are altered in expression and acetylation after KDACi treatment. Moreover, correlation of altered gene and protein expression denoted KDACi-specific modes-of-action. Our results revealed that the energy metabolism and nucleotide synthesis-related pathways are affected even under mild, non-apoptotic drug treatment. These also included proteins that are essential for cell survival. In manuscript #1, the global data is debated. Additional discussion and the future perspectives of this thesis focus on cancer-specific metabolic pathways, and the potential of exploiting KDACi-mediated acetylation changes to examine novel therapeutic strategies. To this end, the influence of altered acetylation of MTHFD1 and SHMT2 was investigated in the context of protein structure, oligomerisation potential, and enzyme function. Modification of lysine residues by acetylation has been identified on both histone and non-histone proteins. The analysis of altered histone acetylation, however, requires adaptation of the mass spectrometry-based (MS) approaches compared to analysis of non-histone protein acetylation. This is mostly due to differences in protein chemistry, i.e., length and sequence of the modified peptides. To this end, our FASIL-MS approach was developed to quantitate histone site-specific lysine acetylation mediated by KDACi treatment. FASIL-MS combines a streamlined sample preparation protocol, adjustment of the MS method and an improvement in the bioinformatic tool used for MS2-based quantitation of histone acetylation. This can be further employed in various experimental settings, such as monitoring drug- or time-dependent alterations in histone acetylation. Moreover, a combination of FASIL-MS with chromatin immunoprecipitation sequencing (ChIP-seq) can be utilised to localise certain acetylation changes at specific gene promoters. Multiple experimental approaches are needed to systematically assess the role of KDACis. To our knowledge, this thesis comprises the most comprehensive strategy to investigate the quantitative changes after KDACi treatment in gene and protein expression, together with altered histone and non-histone acetylation. A combination of these methods with additional tools to explore the function of acetylation changes will undoubtedly shed new light on alternative strategies for targeted cancer treatment.submitted by Dijana VitkoZusammenfassung in deutscher SpracheAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersMedizinische Universität Wien, Dissertation, 2017OeBB(VLID)220476

FASIL-MS: An Integrated Proteomic and Bioinformatic Workflow To Universally Quantitate In Vivo-Acetylated Positional Isomers

Dynamic changes in histone post-translational modifications (PTMs) regulate gene transcription leading to fine-tuning of biological processes such as DNA replication and cell cycle progression. Moreover, specific histone modifications constitute docking sites for recruitment of DNA damage repair proteins and mediation of subsequent cell survival. Therefore, understanding and monitoring changes in histone PTMs that can alter cell proliferation and thus lead to disease progression are of considerable medical interest. In this study, stable isotope labeling with <i>N</i>-acetoxy-D₃-succinimide (D₃-NAS) was utilized to efficiently derivatize unmodified lysine residues at the protein level. The sample preparation method was streamlined to facilitate buffer exchange between the multiple steps of the protocol by coupling chemical derivatization to filter-aided sample preparation (FASP). Additionally, the mass spectrometry method was adapted to simultaneously coisolate and subsequently cofragment all differentially H₃/D₃-acetylated histone peptide clusters. Combination of these multiplexed MS² spectra with the implementation of a data analysis algorithm enabled the quantitation of each and every in vivo-acetylated DMSO- and SAHA-treated H4(4–17) and H3(18–26) peptide. We have termed our new approach FASIL-MS for filter-aided stable isotopic labeling coupled to mass spectrometry. FASIL-MS enables the universal and site-specific quantitation of peptides with multiple in vivo-acetylated lysine residues. Data are available via ProteomeXchange (PXD003611)

Proteogenomic Analysis to Identify Missing Proteins from Haploid Cell Lines

Chromosome-centric Human Proteome Project aims at identifying and characterizing protein products encoded from all human protein-coding genes. As of early 2017, 19,837 protein-coding genes have been annotated in the neXtProt database including 2,691 missing proteins that have never been identified by mass spectrometry. Missing proteins may be low abundant in many cell types or expressed only in a few cell types in human body such as sperms in testis. In this study, we performed expression proteomics of two near haploid cell types such as HAP1 and KBM-7 to hunt for missing proteins. Proteomes from the two haploid cell lines were analyzed on an LTQ Orbitrap Velos, producing a total of 200 raw mass spectrometry files. After applying 1% false discovery rates at both levels of peptide-spectrum matches and proteins, more than ten thousand proteins were identified from HAP1 and KBM-7, resulting in the identification of nine missing proteins. Next, unmatched spectra were searched against protein databases translated in three frames from non-coding RNAs derived from RNA-Seq data, resulting in 6 novel protein-coding regions after careful manual inspection. This study demonstrates that expression proteomics coupled to proteogenomic analysis can be employed to identify many annotated and unannotated missing proteins

Integrated omics analysis unveils a DNA damage response to neurogenic injury.

Spinal cord injury (SCI) evokes profound bladder dysfunction. Current treatments are limited by a lack of molecular data to inform novel therapeutic avenues. Previously, we showed systemic inosine treatment improved bladder function following SCI in rats. Here, we applied multi-omics analysis to explore molecular alterations in the bladder and their sensitivity to inosine following SCI. Canonical pathways regulated by SCI included those associated with protein synthesis, neuroplasticity, wound healing, and neurotransmitter degradation. Upstream regulator analysis identified MYC as a key regulator, whereas causal network analysis predicted multiple regulators of DNA damage response signaling following injury, including PARP-1. Staining for both DNA damage (γH2AX) and PARP activity (poly-ADP-ribose) markers in the bladder was increased following SCI, and attenuated in inosine-treated tissues. Proteomics analysis suggested that SCI induced changes in protein synthesis-, neuroplasticity-, and oxidative stress-associated pathways, a subset of which were shown in transcriptomics data to be inosine-sensitive. These findings provide novel insights into the molecular landscape of the bladder following SCI, and highlight a potential role for PARP inhibition to treat neurogenic bladder dysfunction

TMBIM5 is the Ca 2+ /H + antiporter of mammalian mitochondria

Mitochondrial Ca(2+) ions are crucial regulators of bioenergetics and cell death pathways. Mitochondrial Ca(2+) content and cytosolic Ca(2+) homeostasis strictly depend on Ca(2+) transporters. In recent decades, the major players responsible for mitochondrial Ca(2+) uptake and release have been identified, except the mitochondrial Ca(2+)/H(+) exchanger (CHE). Originally identified as the mitochondrial K(+)/H(+) exchanger, LETM1 was also considered as a candidate for the mitochondrial CHE. Defining the mitochondrial interactome of LETM1, we identify TMBIM5/MICS1, the only mitochondrial member of the TMBIM family, and validate the physical interaction of TMBIM5 and LETM1. Cell‐based and cell‐free biochemical assays demonstrate the absence or greatly reduced Na(+)‐independent mitochondrial Ca(2+) release in TMBIM5 knockout or pH‐sensing site mutants, respectively, and pH‐dependent Ca(2+) transport by recombinant TMBIM5. Taken together, we demonstrate that TMBIM5, but not LETM1, is the long‐sought mitochondrial CHE, involved in setting and regulating the mitochondrial proton gradient. This finding provides the final piece of the puzzle of mitochondrial Ca(2+) transporters and opens the door to exploring its importance in health and disease, and to developing drugs modulating Ca(2+) exchange

Type I Interferon Signaling Disrupts the Hepatic Urea Cycle and Alters Systemic Metabolism to Suppress T Cell Function.

Infections induce complex host responses linked to antiviral defense, inflammation, and tissue damage and repair. We hypothesized that the liver, as a central metabolic hub, may orchestrate systemic metabolic changes during infection. We infected mice with chronic lymphocytic choriomeningitis virus (LCMV), performed RNA sequencing and proteomics of liver tissue, and integrated these data with serum metabolomics at different infection phases. Widespread reprogramming of liver metabolism occurred early after infection, correlating with type I interferon (IFN-I) responses. Viral infection induced metabolic alterations of the liver that depended on the interferon alpha/beta receptor (IFNAR1). Hepatocyte-intrinsic IFNAR1 repressed the transcription of metabolic genes, including Otc and Ass1, which encode urea cycle enzymes. This led to decreased arginine and increased ornithine concentrations in the circulation, resulting in suppressed virus-specific CD8+ T cell responses and ameliorated liver pathology. These findings establish IFN-I-induced modulation of hepatic metabolism and the urea cycle as an endogenous mechanism of immunoregulation. VIDEO ABSTRACT