Fuzzy virtual ligands for virtual screening

A new method to bridge the gap between ligand and receptor-based methods in virtual screening (VS) is presented. We introduce a structure-derived virtual ligand (VL) model as an extension to a previously published pseudo-ligand technique [1]: LIQUID [2] fuzzy pharmacophore virtual screening is combined with grid-based protein binding site predictions of PocketPicker [3]. This approach might help reduce bias introduced by manual selection of binding site residues and introduces pocket shape information to the VL. It allows for a combination of several protein structure models into a single "fuzzy" VL representation, which can be used to scan screening compound collections for ligand structures with a similar potential pharmacophore. PocketPicker employs an elaborate grid-based scanning procedure to determine buried cavities and depressions on the protein's surface. Potential binding sites are represented by clusters of grid probes characterizing the shape and accessibility of a cavity. A rule-based system is then applied to project reverse pharmacophore types onto the grid probes of a selected pocket. The pocket pharmacophore types are assigned depending on the properties and geometry of the protein residues surrounding the pocket with regard to their relative position towards the grid probes. LIQUID is used to cluster representative pocket probes by their pharmacophore types describing a fuzzy VL model. The VL is encoded in a correlation vector, which can then be compared to a database of pre-calculated ligand models. A retrospective screening using the fuzzy VL and several protein structures was evaluated by ten fold cross-validation with ROC-AUC and BEDROC metrics, obtaining a significant enrichment of actives. Future work will be devoted to prospective screening using a novel protein target of Helicobacter pylori and compounds from commercial providers

Prediction of Type III Secretion Signals in Genomes of Gram-Negative Bacteria

Background: Pathogenic bacteria infecting both animals as well as plants use various mechanisms to transport virulence factors across their cell membranes and channel these proteins into the infected host cell. The type III secretion system represents such a mechanism. Proteins transported via this pathway (‘‘effector proteins’’) have to be distinguished from all other proteins that are not exported from the bacterial cell. Although a special targeting signal at the N-terminal end of effector proteins has been proposed in literature its exact characteristics remain unknown. Methodology/Principal Findings: In this study, we demonstrate that the signals encoded in the sequences of type III secretion system effectors can be consistently recognized and predicted by machine learning techniques. Known protein effectors were compiled from the literature and sequence databases, and served as training data for artificial neural networks and support vector machine classifiers. Common sequence features were most pronounced in the first 30 amino acids of the effector sequences. Classification accuracy yielded a cross-validated Matthews correlation of 0.63 and allowed for genome-wide prediction of potential type III secretion system effectors in 705 proteobacterial genomes (12% predicted candidates protein), their chromosomes (11%) and plasmids (13%), as well as 213 Firmicute genomes (7%). Conclusions/Significance: We present a signal prediction method together with comprehensive survey of potential type III secretion system effectors extracted from 918 published bacterial genomes. Our study demonstrates that the analyzed signal features are common across a wide range of species, and provides a substantial basis for the identification of exported pathogenic proteins as targets for future therapeutic intervention. The prediction software is publicly accessible from our web server ( www.modlab.org )

Vaccination directed against the human endogenous retrovirus-K envelope protein inhibits tumor growth in a murine model system

Human endogenous retrovirus (HERV) genomes are chromosomally integrated in all cells of an individual. They are normally transcriptionally silenced and transmitted only vertically. Enhanced expression of HERV-K accompanied by the emergence of anti-HERV-K-directed immune responses has been observed in tumor patients and HIV-infected individuals. As HERV-K is usually not expressed and immunological tolerance development is unlikely, it is an appropriate target for the development of immunotherapies. We generated a recombinant vaccinia virus (MVA-HKenv) expressing the HERV-K envelope glycoprotein (ENV), based on the modified vaccinia virus Ankara (MVA), and established an animal model to test its vaccination efficacy. Murine renal carcinoma cells (Renca) were genetically altered to express E. coli beta-galactosidase (RLZ cells) or the HERV-K ENV gene (RLZ-HKenv cells). Intravenous injection of RLZ-HKenv cells into syngenic BALB/c mice led to the formation of pulmonary metastases, which were detectable by X-gal staining. A single vaccination of tumor-bearing mice with MVA-HKenv drastically reduced the number of pulmonary RLZ-HKenv tumor nodules compared to vaccination with wild-type MVA. Prophylactic vaccination of mice with MVA-HKenv precluded the formation of RLZ-HKenv tumor nodules, whereas wild-type MVA-vaccinated animals succumbed to metastasis. Protection from tumor formation correlated with enhanced HERV-K ENV-specific killing activity of splenocytes. These data demonstrate for the first time that HERV-K ENV is a useful target for vaccine development and might offer new treatment opportunities for diverse types of cancer

Scenario-based Failure Analysis of Product Systems and their Environment

During the usage phase, a technical product system is in permanent interaction with its environment. This interaction can lead to failures that significantly endanger the safety of the user and negatively affect the quality and reliability of the product. Conventional methods of failure analysis focus on the technical product system. The interaction of the product with its environment in the usage phase is not sufficiently considered, resulting in undetected potential failures of the product that lead to complaints. For this purpose, a methodology for failure identification is developed, which is continuously improved through product usage scenarios. The use cases are modelled according to a systems engineering approach with four views. The linking of the product system, physical effects, events and environmental factors enable the analysis of fault chains. These four parameters are subject to great complexity and must be systematically analysed using databases and expert knowledge. The scenarios are continuously updated by field data and complaints. The new approach can identify potential failures in a more systematic and holistic way. Complaints provide direct input on the scenarios. Unknown, previously unrecognized events can be systematically identified through continuous improvement. The complexity of the relationship between the product system and its environmental factors can thus be adequately taken into account in product development. Keywords: failure analysis, methodology, product development, systems engineering, scenario analysis, scenario improvement, environmental factors, product environment, continuous improvement

Reliability of fault-tolerant system architectures for automated driving systems

Automated driving functions at high levels of autonomy operate without driver supervision. The system itself must provide suitable responses in case of hardware element failures. This requires fault-tolerant approaches using domain ECUs and multicore processors operating in lockstep mode. The selection of a suitable architecture for fault-tolerant vehicle systems is currently challenging. Lockstep CPUs enable the implementation of majority redundancy or M-out-of-N ($M$oo$N$) architectures. In addition to structural redundancy, diversity redundancy in the ECU architecture is also relevant to fault tolerance. Two fault-tolerant ECU architecture groups exist: architectures with one ECU (system on a chip) and architectures consisting of multiple communicating ECUs. The single-ECU systems achieve higher reliability, whereas the multi-ECU systems are more robust against dependent failures, such as common-cause or cascading failures, due to their increased potential for diversity redundancy. Yet, it remains not fully understood how different types of architectures influence the system reliability. The work aims to design architectures with respect to CPU and sensor number, $M$oo$N$ expression, and hardware element reliability. The results enable a direct comparison of different architecture types. We calculate their reliability and quantify the effort to achieve high safety requirements. Markov processes allow comparing sensor and CPU architectures by varying the number of components and failure rates. The objective is to evaluate systems' survival probability and fault tolerance and design suitable sensor-CPU architectures. The results show that the system architecture strongly influences the reliability. However, a suitable system architecture must have a trade-off between reliability and self-diagnostics that parallel systems without majority redundancies do not provide.Comment: 12 pages, 4 figures, ESREL2022 Conferenc

Auswirkungen des Finanzausgleichs im Konvergenzprozess von Regionen. Dynamische Simulationsanalysen unter besonderer Berücksichtigung von Anpassungskosten und unvollkommenen Arbeitsmärkten

Es ist damit zu rechnen, dass noch über einen langen Zeitraum hinweg hohe Summen öffentlicher Finanztransfers von den alten in die neuen Bundesländer fließen werden. Vor diesem Hintergrund erscheint es von großer Bedeutung zu untersuchen, wie sich Maßnahmen des Finanzausgleichs auf den Verlauf interregionaler Konvergenzprozesse auswirken. Die wenigen in der Literatur zu dieser Fragestellung bisher vorhandenen theoretischen Modelle weisen eine Reihe von Schwächen auf. So bilden sie die institutionellen Gegebenheiten auf den Arbeitsmärkten nicht oder nur unzureichend ab und berücksichtigen keine Investitionsanpassungs- und Wanderungskosten. Zudem sind sie nicht geeignet, generationenspezifische Wohlfahrtswirkungen zu ermitteln. In der vorliegenden Arbeit wird deshalb stufenweise ein neues Modell entwickelt, das die genannten Schwächen behebt. Die Wirkungen eines finanzkraftorientierten Finanzausgleichs werden anschließend in mehreren Varianten dieses Modells mittels dynamischer Simulationsanalysen untersucht

Die regulierte Intramembranproteolyse des Amyloid Precursor Proteins in Drosophila melanogaster

In unserer alternden Gesellschaft stellen neurodegenerative Krankheiten eine der größten Herausforderungen für die medizinische Forschung dar. Um effektive Therapieansätze entwickeln zu können, müssen die zellulären Mechanismen der Pathogenese verstanden werden. Der entscheidende Schritt in der Entstehung der Alzheimerschen Erkrankung ist die Produktion des Abeta-Peptids durch proteolytische Spaltung aus dem Vorläuferprotein APP. In den letzten Jahren konnten die verantwortlichen Proteasen identifiziert werden. Jedoch ist noch unklar, wie diese Proteine reguliert werden, vor allem im Kontext eines intakten Gewebes. In der vorliegenden Arbeit wurde der Modellorganismus Drosophila melanogaster verwendet, um diese Mechanismen weiter aufzuklären. Im ersten Teil der Arbeit wurden die Möglichkeiten eines genetischen Screens ausgenutzt, um durch Überexpression neue Gene zu finden, die die Prozessierung von APP beeinflussen. Da die Prozessierung von APP der Prozessierung des Rezeptors Notch sehr ähnlich ist, wurden für diesen Screen P-Element Insertionen verwendet, die bekanntermaßen den Signalweg von Notch modifizieren. Diese Vorauswahl hat es erlaubt, trotz einer geringen Zahl getesteter Gene neun Regulatoren der APP Prozessierung zu identifizieren. Zwei dieser Gene wurden molekularbiologisch charakterisiert. Die Kinase Hops/Tlk induziert die vollständige Degradation von APP, ohne dass sich proteolytische Fragmente anreichern. Obwohl gezeigt werden konnte, dass dieser Effekt spezifisch für APP ist, muss der zu Grunde liegende Mechanismus noch weiter aufgeklärt werden. Das Protein dBeach1 führt durch Veränderung der subzellulären Lokalisation von APP zur Akkumulation des alpha-geschnittenen Fragments. Dafür muss das für die Endozytose verantwortliche NPTY-Motiv in der intrazellulären Domäne von APP vorhanden sein. Im weiteren Verlauf der Arbeit konnte gezeigt werden, dass die subzelluläre Lokalisation des Vorläuferproteins von entscheidender Bedeutung für die Prozessierung ist. Der zweite Teil dieser Arbeit ging der Frage nach, wie in-vivo die Freisetzung der intrazellulären Domäne durch Intramembranproteolyse reguliert wird. Durch Verwendung eines GFP-basierten in-vivo Reportersystems konnte gezeigt werden, dass die Effizienz der gamma-Sekretase vermittelten Prozessierung von APP abhängig vom Zelltyp ist. In der larvalen Augenimaginalscheibe ist sie auf neuronale Zellen beschränkt. Diese Beschränkung beruht auf direkter Regulation der gamma-Sekretase. Durch den Vergleich verschiedener Substrate konnte gezeigt werden, dass es im Gegensatz zu einer gängigen Hypothese sowohl von der Länge der extrazellulären Domäne, als auch von der Aminosäuresequenz abhängt, ob ein Typ-I Membranprotein von der gamma-Sekretase umgesetzt werden kann. Die in-vivo Rekonstitution der gamma-Sekretase ergab Hinweise auf das Vorhandensein unterschiedlicher Proteasekomplexe mit distinkten Eigenschaften. Die Identifizierung neuer Regulatoren der APP-Prozessierung bietet nun die Möglichkeit, nach neuen pharmakologischen Angriffspunkten zu suchen. Schon länger wird versucht, Inhibitoren der gamma-Sekretase als Therapeutika einzusetzen, doch bisher führte der negative Einfluss auf andere Substrate der Protease zu schweren Nebenwirkungen. Sollten sich jedoch die Aktivitäten, die für die Prozessierung der einzelnen Proteine verantwortlich sind, voneinander trennen lassen, würde sich neue Hoffnung ergeben, die Produktion von Abeta ohne die Beeinträchtigung lebenswichtiger Signalwege inhibieren zu können

Virtuelles Screening nach Inhibitoren der Protease HtrA aus Helicobacter pylori

Helicobacter pylori (H. pylori) ist ein gram-negatives, mikroaerophiles Bakterium. Es kolonisiert die menschliche Magenschleimhaut, wobei mehr als 50% der Menschheit befallen sind. Als Pathogen begünstigt es die Entstehung von Magengeschwüren und –krebs. Experimentelle Befunde deuten darauf hin, dass H. pylori während der Infektion Kontakt zu Membranproteinen der Wirtszellen aufnimmt, um ein Typ IV Sekretionssystem aufzubauen und den primären Virulenzfaktor CagA (Cytotoxin Associated Antigen A) in die Wirtszelle zu translokieren. Diese Integrine genannten Membranproteine werden bei polaren Epithelzellen allerdings bevorzugt basolateral expremiert. Außerdem können extrazellulär geschnittene E-Cadherinfragmente im Medium mit H. pylori infizierter Zellkulturen nachgewiesen werden. Beide Beobachtungen legen den Schluss nahe, dass eine Protease von H. pylori sekretiert wird und die Zell-Zell-Kontakte degradiert, um H. pylori den Zugang zur basolateralen Seite der Wirtszellen zu ermöglichen. Das vom Gen hp1019 des Stammes H. pylori 26695 codierte Protein HtrA konnte im Rahmen einer Kooperation mit dem Paul-Ehrlich-Institut in Langen im Überstand von H. pylori mit proteolytischer Aktivität nachgewiesen werden. Um den Einfluss dieser extrazellulären Protease auf die Infektion von Kulturzellen mir H. pylori zu untersuchen, sollte ein niedermolekularer Inhibitor für HtrA gefunden werden. Ein Homologiemodell als Grundlage für ein strukturbasiertes virtuelles Screening wurde berechnet, wobei die aktive Konformation der Protease DegP von Escherichia coli als Vorlage diente (PDB Identifikation 3cs0). Für einen neue, im Rahmen dieser Untersuchung entwickelten Methode wurde PocketPicker eingesetzt, um Größe und Form der die Bindetaschen auf der Proteinoberfläche vorherzusagen. Durch die komplementäre Projektion von Proteinatomtypen auf diese definierte Volumen kann so für eine von PocketPicker vorgesagte Bindetasche ein potentielles Pharmakophormodell berechnet und für Datenbanksuchen eingesetzt werden. In retrospektiven Studien konnte die Funktion dieser Berechungen für eine Auswahl an pharmakologisch wichtigen Proteinen aus verschiedenen Strukturklassen validiert werden. Dabei stellte sich vor allem eine Abhängigkeit der Güte der Modelle von der Güte der Vorhersage von PocketPicker heraus, was den Schluss zulässt, dass eine möglichst genaue Definition der Bindetasche für das Gelingen eines strukturbasierten virtuellen Screening unerlässlich ist. Für die Protease HtrA von H. pylori konnten erfolgreich drei strukturabgeleitete Pharmakophormodelle berechnet werden, wobei jeweils verschiedene von PocketPicker vorhergesagte Bindetaschen einbezogen wurden. Die Molekülkataloge der Firmen Asinex und Specs wurden nach Ähnlichkeit zu diesen Modellen sortiert und nach Begutachtung der jeweils ähnlichsten 100 Substanzen wurden 26 Substanzen ausgewählt und bestellt. In einem in vitro Assay mit der rekombinanten Protease HtrA inhibierten 6 Substanzen den Verdau eines rekombinanten Substrats. Die beste Verbindung erreichte in dem Assay eine maximale Inhibition von ca. 77 % bei einer mittleren inhibitorischen Konzentration bei halbmaximaler Inhibition (IC50) von ca. 26 µM.Helicobacter pylori (H. pylori) is a Gram negative, microaerophilic bacterium. It colonizes the human gastric mucosa and more than 50 % of the human population is infected. Experimental results hint that H. pylori needs to interact with certain membrane proteins of the host cells in order to build a type IV secretion system and translocate the primary virulence factor Cytotoxin Associated Antigen A (CagA) into the cytoplasm of the host cell. These membrane proteins are called integrins and are expressed mainly at the basloateral surface of polarized epithelium cells. Also E-cadherin fragments can be found in the medium of culture cells infected by H. pylori. Both observations suggest that H. pylori actively secretes a protease in order to degrade the cell-cell contacts, allowing access to the basolateral surface of the host cells. The gene hp1019 of H. pylori strain 26695 codes for the secreted protein HtrA, which was shown to be an active protease and is located in the supernatant of H. pylori cultures. The aim of this work is to find a small molecule inhibitor for the protease HtrA in order to study the influence of this protease on the pathogenesis of H. pylori infections. A homology model of HtrA based on the template DegP of Escherichia coli (PDB identifier 3CS0) is used to perform a structure based virtual screening. Within this task a novel method for structure based virtual screening was developed. The software PocketPicker is used to predict shape and size of potential binding sites on protein surfaces. By projecting complementary interaction features of the protein atoms inside this defined space a structure based pharmacophore model is calculated and used for database screenings. In retrospective studies this approach was validated for a range of protein targets of pharmaceutical interest. The performance of the screening was dependent on the quality of the PocketPicker predictions. An overestimation of the size of the binding site may lead to a decreased enrichment. Three different structured based pharmacophore models were calculated for the protease HtrA, each including a different set of predicted binding sites. The compound databases of the commercial providers Asinex and Specs were screened using this models and a total of 26 compounds were picked from the result lists. Six compounds inhibit the proteolytical activity on a recombinant substrate in an in-vitro assay. The best performing compound has a maximal inhibition of about 77%. The half maximum inhibition (IC50) is achieved at a concentration of about 26 microM. This molecule can serve as a starting point for a further optimization towards a lead compound

Validation of the smooth step model by particle-in-cell/Monte Carlo collisions simulations

Bounded plasmas are characterized by a rapid but smooth transition from quasi-neutrality in the volume to electron depletion close to the electrodes and chamber walls. The thin non-neutral region, the boundary sheath, comprises only a small fraction of the discharge domain but controls much of its macroscopic behavior. Insights into the properties of the sheath and its relation to the plasma are of high practical and theoretical interest. The recently proposed smooth step model provides a closed analytical expression for the electric field in a planar, radio-frequency modulated sheath. It represents (i) the space charge field in the depletion zone, (ii) the generalized Ohmic and ambipolar field in the quasi-neutral zone, and (iii) a smooth interpolation for the transition in between. This investigation compares the smooth step model with the predictions of a more fundamental particle-in-cell/Monte Carlo collisions simulation and finds good quantitative agreement when the assumed length and time scale requirements are met. A second simulation case illustrates that the model remains applicable even when the assumptions are only marginally fulfilled

Adaptive model to increase resilience for emerging supply chains within the circular economy : "Zirkelmesser" an innovative case study

Variations in quantity, quality and time availability of input materials pose a major risk to circular supply chains (CSC) and require new models for creating and evaluating adaptive and resilient CSC in the circular economy (CE). This can be achieved through consistent modelling of the overarching relationship between resource input- and output streams, without neglecting the associated risks. The model proposed below consists of five components based on five resilience requirements for supply-chains (SCs). It provides a data-based recommended course of action for managers with a low entry-barrier. It consists of a CSC visualization, safety stock calculation, risk monitoring for each SC node, reporting logic, and a measurement catalogue. The inspiration for this model came from an innovative case study ("Zirkelmesser") in the metal processing industry, where secondary products and materials are used to produce new products. Here, the problem of maintaining the resource supply arose and led to resilience issues. The mentioned case study serves as an application example for the model application and contributes to making emerging circular supply chains predictable and more controllable, thus increasing their resilience