Upregulated expression of ENaC in human CF nasal epithelium

AbstractCystic fibrosis (CF) is characterised by the absence of CFTR function resulting in a reduced Cl− secretion and an increase in Na+ absorption. This Na+ hyperabsorption is mediated by the human amiloride-sensitive epithelial sodium channel (ENaC), but the underlying mechanisms are still unknown. After demonstrating functional differences of the Na+ absorption in CF and non-CF epithelia in Ussing chamber experiments with human primary cultures, we compared ENaC sequences from CF and non-CF human nasal tissue (hnENaC), investigated the mRNA transcription levels via real-time PCR and studied the protein expression in Western blot analyses. We found no differences in the sequences of CF and non-CF hnENaC, but identified some polymorphisms. The real-time experiments revealed an enhanced mRNA amount of all three hnENaC subunits in CF tissue. By comparing the two groups on the protein level, we observed differences in the abundance of the Na+ channel. While the α- and β-hnENaC protein amount was increased in CF tissue the γ-hnENaC was decreased. We conclude that the Na+ hyperabsorption in CF is not caused by mutations in hnENaC, but by an increase in the transcription of the hnENaC subunits. This could be induced by a disturbed regulation of the channel in CF

A simulation-guided fluorescence correlation spectroscopy tool to investigate the protonation dynamics of cytochrome c oxidase

Fluorescence correlation spectroscopy (FCS) is a single molecule based technique to temporally resolve rate-dependent processes by correlating the fluorescence fluctuations of individual molecules traversing through a confocal volume. In addition, chemical processes like protonation or intersystem crossing can be monitored in the sub-microsecond range. FCS thereby provides an excellent tool for investigations of protonation dynamics in proton pumps like cytochrome c oxidase (CcO). To achieve this, the pH- dependent fluorescent dye fluorescein was attached as a protonation probe to the CcO surface via site-specific labeling of single reactive cysteines that are located close to the entry point of a proton input channel (K-pathway). The analysis of protonation dynamics is complicated by overlapping triplet and protonation rates of the fluorophore. A Monte Carlo simulation based algorithm was developed to facilitate discrimination of these temporally overlapping processes thus allowing for improved protonation reaction rate determination. Using this simulation-guided approach we determined precise local proton association and dissociation rates and provide information about protein surface effects, such as proton collecting antennae, on the transport properties of proton transfer channels

Subtoxic Alterations in Hepatocyte-Derived Exosomes: An Early Step in Drug-Induced Liver Injury?

Drug-induced liver injury (DILI) is a significant clinical and economic problem in the United States, yet the mechanisms that underlie DILI remain poorly understood. Recent evidence suggests that signaling molecules released by stressed hepatocytes can trigger immune responses that may be common across DILI mechanisms. Extracellular vesicles released by hepatocytes, principally hepatocyte-derived exosomes (HDEs), may constitute one such signal. To examine HDE alterations as a function of drug-induced stress, this work utilized prototypical hepatotoxicant acetaminophen (APAP) in male Sprague-Dawley (SD) rats, SD rat hepatocytes, and primary human hepatocytes. HDE were isolated using ExoQuick precipitation reagent and analyzed by quantification of the liver-specific RNAs albumin and microRNA-122 (miR-122). In vivo, significant elevations in circulating exosomal albumin mRNA were observed at subtoxic APAP exposures. Significant increases in exosomal albumin mRNA were also observed in primary rat hepatocytes at subtoxic APAP concentrations. In primary human hepatocytes, APAP elicited increases in both exosomal albumin mRNA and exosomal miR-122 without overt cytotoxicity. However, the number of HDE produced in vitro in response to APAP did not increase with exosomal RNA quantity. We conclude that significant drug-induced alterations in the liver-specific RNA content of HDE occur at subtoxic APAP exposures in vivo and in vitro, and that these changes appear to reflect selective packaging rather than changes in exosome number. The current findings demonstrate that translationally relevant HDE alterations occur in the absence of overt hepatocellular toxicity, and support the hypothesis that HDE released by stressed hepatocytes may mediate early immune responses in DILI

Changes in Alprazolam Metabolism by CYP3A43 Mutants

Alprazolam is a triazolobenzodiazepine which is most commonly used in the short-term management of anxiety disorders, often in combination with antipsychotics. The four human members of the CYP3A subfamily are mainly responsible for its metabolism, which yields the main metabolites 4-hydroxyalprazolam and α-hydroxyalprazolam. We performed a comparison of alprazolam metabolism by all four CYP3A enzymes upon recombinant expression in the fission yeast Schizosaccharomyces pombe. CYP3A4 and CYP3A5 show the highest 4-hydroxyalprazolam production rates, while CYP3A5 alone is the major producer of α-hydroxyalprazolam. For both metabolites, CYP3A7 and CYP3A43 show lower activities. Computational simulations rationalize the difference in preferred oxidation sites observed between the exemplary enzymes CYP3A5 and CYP3A43. Investigations of the alprazolam metabolites formed by three previously described CYP3A43 mutants (L293P, T409R, and P340A) unexpectedly revealed that they produce 4-hydroxy-, but not α-hydroxyalprazolam. Instead, they all also make a different metabolite, which is 5-N-O alprazolam. With respect to 4-hydroxyalprazolam, the mutants showed fourfold (T409R) to sixfold (L293P and P340A) higher production rates compared to the wild-type (CYP3A43.1). In the case of 5-N-O alprazolam, the production rates were similar for the three mutants, while no formation of this metabolite was found in the wild-type incubation

Development of an HPLC-MS/MS Method for Chiral Separation and Quantitation of (R)- and (S)-Salbutamol and Their Sulfoconjugated Metabolites in Urine to Investigate Stereoselective Sulfonation

The aim of this study was to develop and optimize a chiral HPLC-MS/MS method for quantitative analysis of (R)-/(S)-salbutamol and (R)-/(S)-salbutamol-4′-O-sulfate in human urine to allow for bioanalytical quantitation of the targeted analytes and investigations of stereoselectivity in the sulfonation pathway of human phase Ⅱ metabolism. For analytical method development, a systematic screening of columns and mobile phases to develop a separation via enantiomerically selective high performance liquid chromatography was performed. Electrospray ionization settings were optimized via multiple-step screening and a full factorial design-of-experiment. Both approaches were performed matrix-assisted and the predicted values were compared. The full factorial design was superior in terms of prediction power and knowledge generation. Performing a longitudinal excretion study in one healthy volunteer allowed for the calculation of excretion rates for all four targeted analytes. For this proof-of-concept, either racemic salbutamol or enantiopure levosalbutamol was administered perorally or via inhalation, respectively. A strong preference for sulfonation of (R)-salbutamol for inhalation and peroral application was found in in vivo experiments. In previous studies phenol sulfotransferase 1A3 was described to be mainly responsible for salbutamol sulfonation in humans. Thus, in vitro and in silico investigations of the stereoselectivity of sulfotransferase 1A3 complemented the study and confirmed these findings

Mind the Gap - Deciphering GPCR Pharmacology Using 3D Pharmacophores and Artificial Intelligence

G protein-coupled receptors (GPCRs) are amongst the most pharmaceutically relevant and well-studied protein targets, yet unanswered questions in the field leave significant gaps in our understanding of their nuanced structure and function. Three-dimensional pharmacophore models are powerful computational tools in in silico drug discovery, presenting myriad opportunities for the integration of GPCR structural biology and cheminformatics. This review highlights success stories in the application of 3D pharmacophore modeling to de novo drug design, the discovery of biased and allosteric ligands, scaffold hopping, QSAR analysis, hit-to-lead optimization, GPCR de-orphanization, mechanistic understanding of GPCR pharmacology and the elucidation of ligand–receptor interactions. Furthermore, advances in the incorporation of dynamics and machine learning are highlighted. The review will analyze challenges in the field of GPCR drug discovery, detailing how 3D pharmacophore modeling can be used to address them. Finally, we will present opportunities afforded by 3D pharmacophore modeling in the advancement of our understanding and targeting of GPCRs

Internationale Literaturrecherche zum Intensivwohnbereich

Die Versorgungssituation von erwachsenen Menschen mit geistiger Behinderung und herausforderndem Verhalten im Bereich Wohnen weist im bestehenden System der Behindertenhilfe immer noch einen Mangel an qualitativ hochwertigen und bedarfsgerechten Unterstützungs- und Eingliederungsangeboten für diesen Personenkreis auf. Die steigenden Anforderungen an Einrichtungen durch das Bayerische Pflege- und Wohnqualitätsgesetz, die wachsende Nachfrage nach Intensivplätzen bei vermehrter Schließung von Intensiv-Einrichtungen und Wohnheimplatzkündigungen sowie der Personal- und Fachkräftemangel betonen die Dringlichkeit eines nachhaltig wirkenden, innovativen Versorgungskonzepts. Im Rahmen des Pilotprojekts PINO - Projekt Intensivwohnen Netzwerk Oberbayern soll dieser Forderung nachgegangen und ein wissenschaftlich fundiertes, innovatives Gesamtkonzept des Intensivwohnens entwickelt werden. Gemeinsam mit dem Aufbau eines Intensivnetzwerkes über Oberbayern hinaus soll damit ein wichtiger Beitrag geleistet werden, um die Versorgungssituation von erwachsenen Menschen mit herausforderndem Verhalten nachhaltig sicherzustellen und ihre Teilhabesituation in der Gesellschaft zu verbessern. Diesem Vorsatz geht in einer ersten Phase die Erhebung des IST-Zustandes der Versorgungssituation für den im Fokus stehenden Personenkreis im Bereich des Intensivwohnens sowie eine Übersicht über bestehende Intensivwohn-Einrichtungen und deren methodische Konzepte voraus. Hierfür wurde eine systematische Recherche der nationalen und internationalen Fachliteratur durchgeführt, die folgende Ziele verfolgte: • Generieren einer fachlich relevanten und fundierten Datenbasis, • Kenntnisse gewinnen über Konzepte, die die Lebensbereiche Wohnen, Arbeit und Freizeit verzahnen sowie • Analyse relevanter Erkenntnisse zu Gelingensfaktoren für die Praxis. Für die systematische internationale Literaturrecherche war die folgende Hauptfragestellung leitend: • Welche Bedingungen muss ein Wohn- und Betreuungskonzept des Intensivwohnens erfüllen, um die Teilhabesituation (Lebens- und Betreuungssituation) der Personengruppe zu verbessern? Um die vorgegebene Zielsetzung umfassend abbilden zu können, wurden weiterhin spezifischere Teilfragen formuliert: • Welche aktuellen Beiträge, Erkenntnisse und Lösungsansätze liegen zu der Zielgruppe und deren Betreuungssettings mit Blick auf die Verbesserung der Teilhabesituation national & international vor? • Welche Konzepte gibt es für die Zielgruppe, die die Lebensbereiche Wohnen, Arbeit und Freizeit verzahnen? • Welche evidenzbasierten Wirkvariablen zeigen sich als förderlich für die Verbesserung der Teilhabesituation der Personengruppe im Intensivwohnen in nationalen und internationalen Studien? Der vorliegende Bericht stellt die Vorgehensweise sowie Befunde aus der systematischen Recherche der nationalen und internationalen Fachliteratur in Hinblick auf die formulierten Leitfragen vor

Global reorganization of the nuclear landscape in senescent cells.

Cellular senescence has been implicated in tumor suppression, development, and aging and is accompanied by large-scale chromatin rearrangements, forming senescence-associated heterochromatic foci (SAHF). However, how the chromatin is reorganized during SAHF formation is poorly understood. Furthermore, heterochromatin formation in senescence appears to contrast with loss of heterochromatin in Hutchinson-Gilford progeria. We mapped architectural changes in genome organization in cellular senescence using Hi-C. Unexpectedly, we find a dramatic sequence- and lamin-dependent loss of local interactions in heterochromatin. This change in local connectivity resolves the paradox of opposing chromatin changes in senescence and progeria. In addition, we observe a senescence-specific spatial clustering of heterochromatic regions, suggesting a unique second step required for SAHF formation. Comparison of embryonic stem cells (ESCs), somatic cells, and senescent cells shows a unidirectional loss in local chromatin connectivity, suggesting that senescence is an endpoint of the continuous nuclear remodelling process during differentiation

Profound Re-Organization of Cell Surface Proteome in Equine Retinal Pigment Epithelial Cells in Response to In Vitro Culturing

The purpose of this study was to characterize the cell surface proteome of native compared to cultured equine retinal pigment epithelium (RPE) cells. The RPE plays an essential role in visual function and represents the outer blood-retinal barrier. We are investigating immunopathomechanisms of equine recurrent uveitis, an autoimmune inflammatory disease in horses leading to breakdown of the outer blood-retinal barrier and influx of autoreactive T-cells into affected horses' vitrei. Cell surface proteins of native and cultured RPE cells from eye-healthy horses were captured by biotinylation, analyzed by high resolution mass spectrometry coupled to liquid chromatography (LC MS/MS), and the most interesting candidates were validated by PCR, immunoblotting and immunocytochemistry. A total of 112 proteins were identified, of which 84% were cell surface membrane proteins. Twenty-three of these proteins were concurrently expressed by both cell states, 28 proteins exclusively by native RPE cells. Among the latter were two RPE markers with highly specialized RPE functions: cellular retinaldehyde-binding protein (CRALBP) and retinal pigment epithelium-specific protein 65kDa (RPE65). Furthermore, 61 proteins were only expressed by cultured RPE cells and absent in native cells. As we believe that initiating events, leading to the breakdown of the outer blood-retinal barrier, take place at the cell surface of RPE cells as a particularly exposed barrier structure, this differential characterization of cell surface proteomes of native and cultured equine RPE cells is a prerequisite for future studies