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FRET-based dynamic structural biology: Challenges, perspectives and an appeal for open-science practices
Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever-increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among different labs using various procedures. These multi-lab studies have led to the development of smFRET procedures and documentation, which are important when submitting entries into the archiving system for integrative structure models, PDB-Dev. This position paper describes the current âstate of the artâ from different perspectives, points to unresolved methodological issues for quantitative structural studies, provides a set of âsoft recommendationsâ about which an emerging consensus exists, and lists openly available resources for newcomers and seasoned practitioners. To make further progress, we strongly encourage âopen scienceâ practices
Three-Dimensional Hydrogel Bioprinting Technology as a Scaffold of Novel Drug Delivery and Biomedical Devices: A Comprehensive Review
Polymer hydrogel used as computer-aided, non-biological arsenal utilize as a drug delivery vehicle overthe past few years.New advances in three-dimensional (3D) bioprinting technology have created new opportunitiesfor the use of hydrogel polymer-based medication delivery systems. 3D printing can deliver the ideal shapes or changecapabilities under specific circumstances which have a better adaptation to physiological function. The accuracy of 3Dprinting technology was significantly higher than that of conventional production techniques.A model bioink acquireproper physicochemical characteristics (mechanical and rheological) and biological properties important for proper functioning.It acts as additive manufacturing with complex spatial structure in biomedical research. In this review, we outlined the currentdevelopments in 3D printed polymer hydrogels as delivery and other platforms
Optimizing Excipient Properties to Prevent Aggregation in Biopharmaceutical Formulations
Excipients are included within protein biotherapeutic solution formulations to improve colloidal and conformational stability but are generally not designed for the specific purpose of preventing aggregation and improving cryoprotection in solution. In this work, we have explored the relationship between the structure and antiaggregation activity of excipients by utilizing coarse-grained molecular dynamics modeling of protein-excipient interaction. We have studied human serum albumin as a model protein, and we report the interaction of 41 excipients (polysorbates, fatty alcohol ethoxylates, fatty acid ethoxylates, phospholipids, glucosides, amino acids, and others) in terms of the reduction of solvent accessible surface area of aggregation-prone regions, proposed as a mechanism of aggregation prevention. Polyoxyethylene sorbitan had the greatest degree of interaction with aggregation-prone regions, decreasing the solvent accessible surface area of APRs by 20.7 nm2 (40.1%). Physicochemical descriptors generated by Mordred are employed to probe the structure-property relationship using partial least-squares regression. A leave-one-out cross-validated model had a root-mean-square error of prediction of 4.1 nm2 and a mean relative error of prediction of 0.077. Generally, longer molecules with a large number of alcohol-terminated PEG units tended to interact more, with qualitatively different protein interactions, wrapping around the protein. Shorter or less ethoxylated compounds tend to form hemimicellar clusters at the protein surface. We propose that an improved design would feature many short chains of 5 to 10 PEG units in many distinct branches and at least some hydrophobic content in the form of medium-length or greater aliphatic chains (i.e., six or more carbon atoms). The combination of molecular dynamics simulation and quantitative modeling is an important first step in an all-purpose protein-independent model for the computer-aided design of stabilizing excipients
Specificity Determining Features at the Interface of Biomolecular Complexes as Regulators of Biological Functions
Amino acid residues at the biomolecular interface play essential roles in many biological and cellular processes; relevant to this thesis, protein-protein interactions regulate signaling pathways and enzymatic activity, whereas protein-DNA interactions control gene expression, and protein-peptide interactions are central to the immune system. Biomolecular recognition and binding stability are largely determined by residues at the molecular interface. In this thesis, we focused on three biological datasets that are related to humans and human health: 1) dysregulated citrullination in the inflamed joints of rheumatoid arthritis patients, 2) a novel family of PRD-like transcription factors critical to the first few cell divisions in human life, and 3) epitopes that likely activate a cytotoxic T cell-mediated immune response against SARS-CoV-2 infection. For each dataset, in order to study the structural and functional consequences of molecular interactions, we applied a wide range of bioinformatics techniques to analyze sequences, structures and biological data retrieved from various databases, as well as taking into account experimental results from collaborators and from the literature.
In rheumatoid arthritis, normally cytoplasmic peptidylarginine deiminase (PAD) enzymes citrullinate arginine residues in extracellular matrix (ECM) proteins. To examine specificity determining features that regulate the citrullination activity, we analyzed the sequence and structure data of the ECM proteins that were found citrullinated in chronically inflamed human joints. For citrullination, we found that an arginine side chain needs to be exposed to solvent but can arise from ÎČ-strands, α-helices, loops and ÎČ-turns. Moreover, there is no sequence motif linked to enzymatic activity. In addition, we studied the effect of citrullination on proteins important for a normal ECM, focusing on integrin binding to fibronectin and transforming growth factor-ÎČ (TGF-ÎČ). Citrullination of these proteins was found to inhibit cell attachment and spreading since PAD-treatment of the isoDGR motif in fibronectin and the RGD motif in TGF-ÎČ significantly reduced their binding with integrin αVÎČ3 and αVÎČ6, respectively.
The expression of the human paired (PRD)-like transcription factors (TFs) are limited to the period of embryonic genome activation up to the 8-cell stage. We identified that one of these PRD-like TFs, LEUTX, binds to a TAATCC sequence motif. Sequence comparisons revealed that LEUTX protein is comprised of two domains: the DNA-binding homeodomain and a Leutx domain containing a transactivation domain. We identified specificity determining residues in the LEUTX homeodomain that are important for recognition of the TAATCC-containing 36 bp DNA motif enriched in genes involved in embryonic genome activation. We demonstrated using molecular models why a heterozygotic missense mutation A54V at the DNA-specificity determining position of LEUTX has significantly reduced overall transcriptional activity, as well as why the double mutant â I47T and A54V â form of LEUTX restores binding to the DNA motif similarly to that seen in the I47T mutation alone.
At the onset of the COVID-19 pandemic we sought to understand the molecular factors that trigger the cytotoxic T cell-mediated immune response against the SARS-CoV-2 virus, taking advantage of binding data and 3D structures for related viruses and other pathogenic organisms. We first predicted the MHC class I (MHC-I)-specific immunogenic epitopes of length 8- to 11 amino acids from the SARS-CoV-2 proteins. Next, we predicted that the 9-mer epitopes would have the highest potential to elicit a strong immune response. For experimental validation, the predicted 9-mer epitopes were matched with the SARS-CoV-derived epitopes that are known to elicit an effective T cell response in vitro. Furthermore, our observations provide a structural explanation for the binding of SARS-CoV-2 epitopes to MHC-I molecules, identifying conserved immunogenic epitopes essential for understanding the pathogenesis of COVID-19.
The three investigated datasets were made in concert with collaborative experimental studies and/or considering publicly available experimental data. The experimental studies generally provided the starting point for the in silico studies, which in turn had the objective of providing a detailed explanation of the experimental results. Furthermore, the in silico results could be used to devise novel and focused experiments, suggesting that bioinformatics predictions and wet-laboratory experimental investigations optimally take place with multiple advantages. Overall, this thesis demonstrates the synergy that is possible by applying this interdisciplinary approach to understanding the consequences of molecular interactions.Aminosyror i kontaktytan mellan olika biomolekyler spelar en viktig roll i mÄnga biologiska och cellulÀra processer; relevanta interaktioner för den hÀr avhandlingen Àr protein-protein interaktioner som reglerar signaleringsrutter och enzymatisk aktivitet, protein-DNA interaktioner som kontrollerar genexpression, samt protein-peptid interaktioner som har en central roll i immunförsvaret. BiomolekylÀr igenkÀnning och bindningsstabilitet beror till stor del pÄ de aminosyror som finns i den molekylÀra kontaktytan. I den hÀr avhandlingen fokuserade vi pÄ tre biologiska dataset som Àr relaterade till mÀnniskor och mÀnniskors hÀlsa: 1) felreglerad citrullinering i inflammerade leder hos patienter med reumatoid artrit, 2) en nyupptÀckt familj av PRD (human paired)-lika transkriptionsfaktorer som Àr nödvÀndiga för de första celldelningarna i mÀnniskolivet, och 3) epitoper som troligen aktiverar en cytotoxisk T-cell-förmedlad immunrespons mot SARS-CoV-2 infektioner. För att studera de strukturella och funktionella konsekvenserna av de molekylÀra interaktionerna i varje dataset, anvÀndes en mÀngd olika bioinformatiska tekniker för att analysera sekvenser, strukturer och biologiska data frÄn olika databaser och dessutom beaktades experimentella resultat frÄn samarbetspartners och frÄn litteraturen.
I reumatoid artrit citrullinerar vanligen PAD (cytoplasmatiska peptidyl arginin deiminas)-enzymer arginin-aminosyror i proteiner i det extracellulĂ€ra matrixet (ECM). För att undersöka egenskaper som avgör specificiteten hos citrullineringsaktiviteten analyserade vi sekvens- och strukturdata för ECM-proteiner som blir citrullinerade i kroniskt inflammerade leder hos mĂ€nniskor. Vi upptĂ€ckte att en argininsidokedja mĂ„ste vara i kontakt med det omgivande lösningsmedlet för att kunna citrullineras, att de kan finnas i beta-strĂ€ngar, alfa-helixar och beta-svĂ€ngar, samt att det inte finns nĂ„gra sekvensmotiv som Ă€r kopplade till enzymatisk aktivitet. Utöver detta studerade vi effekten av citrullinering pĂ„ proteiner som Ă€r viktiga för normal extracellulĂ€r matrix, med fokus pĂ„ integrinbinding till fibronektin och TGF-ÎČ (transforming growth factor-ÎČ). Citrullinering av dessa proteiner upptĂ€cktes inhibera cellvidhĂ€ftning och spridning eftersom PAD-behandling av isoDGR-motivet i fibronektin och RGD-motivet i TGF-ÎČ ordentligt reducerar deras bindning till integrin αVÎČ3 och αVÎČ6, respektive.
ExpressionsnivÄerna av PRD-lika transkriptionsfaktorer (TF) Àr begrÀnsade till perioden av zygotens genomaktivering upp till 8-cells stadiet. Vi identifierade att en av dessa PRD-lika transkriptionsfaktorer, LEUTX, binder till ett TAATCC sekvensmotiv. SekvensjÀmförelser avslöjade att LEUTX proteinet bestÄr av tvÄ domÀner, det DNA-bindande homeodomÀnet och en leutx-domÀn som innehÄller en transaktiveringsdomÀn. Vi identifierade specificitetsbestÀmmande aminosyror i LEUTX homeodomÀnen som Àr viktiga för igenkÀnning av TAATCC-innehÄllande 36 baspars DNA-motivet som Àr berikad med gener involverade i zygotens genomaktivering. Vi anvÀnde molekylÀra modeller för att visa varför en heterozygotisk missense-mutation, A54V, i DNA-specificitetsbestÀmmande positionen i LEUTX har ordentligt minskad generell transkriptionsaktivitet, och varför dubbelmutanten I47T och A54V ÄterstÀller bindning till DNA-motivet pÄ samma sÀtt som observerats i enbart I47T mutationen.
NÀr COVID-19 pandemin inleddes försökte vi förstÄ de molekylÀra faktorer som startar den cytotoxiska T-cell-förmedlade immunresponsen mot SARS-CoV-2 viruset, genom att utnyttja bindningsdata och 3D strukturer för relaterade virus och andra patogena organismer. Vi förutspÄdde först MHC klass I (MHC-I)-specifika immunogena epitoper av lÀngden 8 till 11 aminosyror frÄn SARS-CoV-2 proteiner. DÀrefter förutspÄdde vi att epitoper bestÄende av 9 aminosyror hade den högsta potentialen att orsaka en stark immunrespons. För experimentell validering matchades de 9 aminosyror lÄnga epitoperna med epitoper frÄn SARS-CoV som man vet att orsakar en effektiv T-cell respons in vitro. VÄra observationer bidrar ocksÄ med en strukturell förklaring för bindningen av SARS-CoV-2 epitoper till MHC-I molekyler, vilket identifierar konserverade immunogena epitoper som Àr nödvÀndiga för att förstÄr patogenesen hos COVID-19.
De tre undersökta dataseten gjordes i samarbete med experimentella studier och/eller genom att ta allmĂ€nt tillgĂ€ngliga experimentella data i beaktande. De experimentella studierna gav en startpunkt för in silico-studierna, vilka i sin tur hade som mĂ„l att ge en detaljerad förklaring till de experimentella resultaten. In silico-resultaten kan ocksĂ„ anvĂ€ndas för att utveckla nya och fokuserade experiment, vilket indikerar att bioinformatiska förutspĂ„elser och experimentella studier optimalt sker med mĂ„nga fördelar. Ăver lag visar denna avhandling synergin som Ă€r möjlig genom att anvĂ€nda detta interdisciplinĂ€ra arbetssĂ€tt för att förstĂ„ konsekvenserna av molekylĂ€ra interaktioner
Stem Cells in Domestic Animals
Stem cells are an attractive tool for cell-based therapies in regenerative medicine, both for humans and animals. The research and review articles published in this first book of the Collection âStem Cells in Domestic Animals: Applications in Health and Productionâ are excellent examples of the recent advances made in the field of stem/stromal cell research in veterinary medicine. In this field, sophisticated and new treatments are now required for improving patientsâ quality of life; in livestock animals, the goal of regenerative medicine is to improve not only animal welfare but also the quality of production, aiming to preserve human health. The contributions collected in this book concern both laboratory research and clinical applications of mesenchymal stem/stromal cells. The increasing knowledge of cell-based therapies may constitute an opportunity for researchers, veterinary practitioners, and animal owners to contribute to animal and human health and well-being
Rational development of stabilized cyclic disulfide redox probes and bioreductive prodrugs to target dithiol oxidoreductases
Countless biological processes allow cells to develop, survive, and proliferate. Among these, tightly balanced regulatory enzymatic pathways that can respond rapidly to external impacts maintain dynamic physiological homeostasis. More specifically, redox homeostasis broadly affects cellular metabolism and proliferation, with major contributions by thiol/disulfide oxidoreductase systems, in particular, the Thioredoxin Reductase Thioredoxin (TrxR/Trx) and the Glutathione Reductase-Glutathione-Glutaredoxin (GR/GSH/Grx) systems.
These cascades drive vital cellular functions in many ways through signaling, regulating other proteins' activity by redox switches, and by stoichiometric reductant transfers in metabolism and antioxidant systems. Increasing evidence argues that there is a persistent alteration of the redox environment in certain pathological states, such as cancer, that heavily involve the Trx system: upregulation and/or overactivity of the Trx system may support or drive cancer progression, making both TrxR and Trx promising targets for anti-cancer drug development.
Understanding the biochemical mechanisms and connections between certain redox cascades requires research tools that interact with them. The state-of-the-art genetic tools are mostly ratiometric reporters that measure reduced:oxidized ratios of selected redox pairs or the general thiol pool. However, the precise cellular roles of the central oxidoreductase systems, including TrxR and Trx, remain inaccessible due to the lack of probes to selectively measure turnover by either of these proteins. However, such probes would allow measuring their effective reductive activity apart from expression levels in native systems, including in cells, animals, or patient samples. They are also of high interest to identify chemical inhibitors for TrxR/Trx in cells and to validate their potential use as anti-cancer agents (to date, there is no selective cellular Trx inhibitor, and most known TrxR inhibitors were not comprehensively evaluated considering selectivity and potential off-targets). However, small molecule redox imaging tools are underdeveloped: their protein specificity, spectral properties, and applicability remain poorly precedented.
This work aimed to address this opportunity gap and develop novel, small molecule diagnostic and therapeutic tools to selectively target the Trx system based on a modular trigger cargo design: artificial cyclic disulfide substrates (trigger) for oxidoreductases are tethered to molecular agents (cargo) such that the cargoâs activity is masked and is re-established only through reduction by a target protein.
The rational design of these novel reduction sensors to target the cell's strongest disulfide-reducing enzymes was driven by the following principles: (i) cyclic disulfide triggers with stabilized ring systems were used to gain low reduction potentials that should resist reduction except by the strongest cellular reductases, such as Trx; and (ii) the cyclic topology also offers the potential for kinetic reversibility that should select for dithiol-type redox proteins over the cellular monothiol background. Creating imaging agents based on such two-component designs to selectively measure redox protein activity in native cells required to combine the correct trigger reducibility, probe activation kinetics, and imaging modalities and to consider the overall molecular architecture.
The major prior art in this field has applied cyclic 5-membered disulfides (1,2 dithiolanes) as substrates for TrxR in a similar way to create such tools. However, this motif was described elsewhere as thermodynamically instable and was due to widely used for dynamic covalent cascade reactions. By comparing a novel 1,2 dithiolane-based probe to the state-of-the-art probes, including commercial TrxR sensors, by screening a conclusive assay panel of cellular TrxR modulations, I clarified that 1,2 dithiolanes are not selective substrates for TrxR in biological settings (Nat Commun 2022).
Instead, aiming for more stable ring systems and thus more robust redox probes, during this work, I developed bicyclic 6 membered disulfides (piperidine fused 1,2 dithianes) with remarkably low reduction potentials. I showed that molecular probes using them as reduction sensors can be mostly processed by thioredoxins while being stable against reduction by GSH. The thermodynamically stabilized decalin like topology of the cis-annelated 1,2 dithianes requires particularly strong reductants to be cleaved. They also select for dithiol type redox proteins, like Trx, based on kinetic reversibility and offer fast cyclization due to the preorganization by annelation (JACS 2021).
This work further expanded the systemâs modularity with structural cores based on piperazine-fused 1,2 dithianes with the two amines allowing independent derivatization. Diagnostic tools using them as reduction sensors proved equally robust but with highly improved activation kinetics and were thus cellularly activated. Cellular studies evolved that they are substrates for both Trxs and their protein cousins Grxs, so measuring the cellular dithiol protein pool rather than solely Trx activity (preprint 2023).
Finally, a trigger based on a slightly adapted reduction sensor, a desymmetrized 1,2 thiaselenane, was designed for selective reduction by TrxRâs selenol/thiol active site, then combined with a precipitating large Stokesâ shift fluorophore and a solubilizing group, to evolve the first selective probe RX1 to measure cellular TrxR activity, which even allowed high throughput inhibitor screening (Chem 2022).
The central principle of this work was further advanced to therapeutic prodrugs based on the duocarmycin cargo (CBI) with tunable potency (JACS Au 2022) that can be used to create off-to-on therapeutic prodrugs. Such CBI prodrugs employing stabilized 1,2 dichalcogenide triggers proved to be cytotoxins that depend on Trx system activity in cells. They could further be exploited for cell-line dependent reductase activity profiling by screening their redox activation indices, the reduction-dependent part of total prodrug activation, in 177 cell lines. Beyond that, these prodrugs were well-tolerated in animals and showed anti-cancer efficacy in vivo in two distinct mouse tumor models (preprint 2022).
Taken together, I introduced unique monothiol-resistant reducible motifs to target the cellular Trx system with chemocompatible units for each for TrxR and Trx/Grx, where the cyclic nature of the dichalcogenides avoids activation by GSH. By using them with distinct molecular cargos, I developed novel selective fluorescent reporter probes; and introduced a new class of bioreductive therapeutic constructs based on a common modular design. These were either applied to selectively measure cellular reductase activity or to deliver cytotoxic anti cancer agents in vivo. Ongoing work aims to differentiate between the two major redox effector proteins Trx and Grx, requiring additional layers of selectivity that may be addressed by tuned molecular recognition. The flexible use of various molecular cargos allows harnessing the same cellular redox machinery by either probes or prodrugs. This allows predictive conclusions from diagnostics to be directly translated into therapy and offers great potential for future adaptation to other enzyme classes and therapeutic venues.Die zellulĂ€re Redox-Homöostase hĂ€ngt von Thiol/Disulfid-Oxidoreduktasen ab, die den Stoffwechsel, die Proliferation und die antioxidative Antwort von Zellen beeinflussen. Die wichtigsten Netzwerke sind die Thioredoxin Reduktase-Thioredoxin (TrxR/Trx) und Glutathion Reduktase-Glutathion-Glutaredoxin (GR/GSH/Grx) Systeme, die ĂŒber Redox-Schalter in Substratproteinen lebenswichtige zellulĂ€re Funktionen steuern und so an der Redox-Regulation und -SignalĂŒbertragung beteiligt sind. Persistente VerĂ€nderungen des Redoxmilieus in pathologischen ZustĂ€nden, wie z. B. bei Krebs, sind in hohem MaĂe mit dem Trx-System verbunden. Eine Hochregulierung und/oder ĂberaktivitĂ€t des Trx-Systems, die bei vielen Krebsarten auftreten, unterstĂŒtzt zudem das Fortschreiten des Krebswachstums, was TrxR/Trx zu vielversprechenden Zielproteinen fĂŒr die Entwicklung neuer Krebsmedikamente macht.
Um die biochemischen Prozesse dahinter zu erforschen, sind spezielle Techniken zur Visualisierung und Messung enzymatischer AktivitĂ€t nötig. Die hierzu geeigneten, meist genetischen Sensoren messen ratiometrisch das VerhĂ€ltnis reduzierter/oxidierter Spezies in zellulĂ€rem Umfeld oder spezifisch ausgewĂ€hlte Redoxpaare. Die weitere Erforschung der exakten Funktion von TrxR/Trx und deren Substrate ist jedoch durch mangelnde Nachweismethoden limitiert. Diese sind auĂerdem zur Validierung chemischer Hemmstoffe fĂŒr TrxR/Trx in Zellen und deren potenziellen Verwendung als Krebsmittel von groĂem Interesse. Bislang gibt es keinen selektiven zellulĂ€ren Trx-Inhibitor und potenzielle Off-Target-Effekte der bekannten TrxR-Inhibitoren wurden nicht abschlieĂend bewertet.
Ziel dieser Arbeit ist die Entwicklung niedermolekularer, diagnostischer und therapeutischer Werkzeuge, die selektiv auf das Trx-System abzielen und auf einem modularen Trigger-Cargo Design basieren. Hierzu werden zyklische Disulfid-Substrate (Trigger) fĂŒr Oxidoreduktasen so mit molekularen Wirkstoffen (Cargo) verknĂŒpft, dass dabei die WirkstoffaktivitĂ€t maskiert, und erst nach Reduktion durch ein Zielprotein wiederhergestellt wird. Diese neuartigen, synthetischen Reduktionssensoren basieren auf den folgenden Grundprinzipien: (i) Zyklische Disulfide sind thermodynamisch stabilisiert und können nur durch die stĂ€rksten Reduktasen gespalten werden; und (ii) die zyklische Topologie ermöglicht die kinetische ReversibilitĂ€t der zwei Thiol-Disulfid-Austauschreaktionen, die eine erste Reaktion mit Monothiolen, wie z. B. GSH, sofort umkehrt und so eine vollstĂ€ndige Reduktion verhindert.
Die meisten frĂŒheren Arbeiten auf diesem Gebiet verwendeten ein zyklisches, fĂŒnfgliedriges Disulfid (1,2 Dithiolan) als Substrat fĂŒr TrxR. Das gleiche Strukturmotiv wurde jedoch an anderer Stelle als thermodynamisch instabil beschrieben und aufgrund dieser Eigenschaft explizit fĂŒr dynamische Kaskadenreaktionen verwendet. Deshalb vergleicht diese Arbeit zu Beginn einen neuen 1,2 Dithiolan basierten fluorogenen Indikator mit bestehenden, z. T. kommerziellen, Redox Sonden fĂŒr TrxR in einer Reihe von Zellkultur-Experimenten unter Modulation der zellulĂ€ren TrxR AktivitĂ€t und stellt so einen Widerspruch in der Literatur klar: 1,2 Dithiolane eignen sich nicht als selektive Substrate fĂŒr TrxR, da sie labil sowohl gegen die Reduktion durch andere Redoxproteine, als auch gegen den Monothiol Hintergrund in Zellen sind (Nat. Commun. 2022).
Als alternatives Strukturmotiv wird in dieser Arbeit ein bizyklisches sechsgliedriges Disulfid (anneliertes 1,2 Dithian) etabliert. Durch sein niedriges Reduktionspotenzial, also seine hohe Resistenz gegen Reduktion, werden molekulare Sonden basierend auf diesem 1,2 Dithian als Reduktionssensor fast ausschlieĂlich von Trx aktiviert, nicht aber von TrxR oder GSH (JACS 2021). Dieses Kernmotiv bestimmt dabei die Reduzierbarkeit, und damit die EnzymspezifitĂ€t, durch seine zyklische Natur und die Annelierung, auch unter Verwendung unterschiedlicher Farb-/Wirkstoffe. Auf dieser Grundlage konnte die molekulare Struktur durch einen weiteren Modifikationspunkt fĂŒr die flexible Verwendung weiterer funktioneller Einheiten ergĂ€nzt werden. Obwohl zellulĂ€re Studien ergaben, dass diese neuartigen 1,2 Dithian Einheiten in Zellen sowohl Trx als auch das strukturell verwandte Grx adressieren, sind die daraus resultierenden diagnostischen MolekĂŒle wertvoll, um den katalytischen Umsatz zellulĂ€rer Dithiol-Reduktasen, der sogenannten Trx Superfamilie, selektiv anzuzeigen (Preprint 2023).
BegĂŒnstigt durch das modulare MolekĂŒldesign stellt diese Arbeit zudem das erste Reportersystem RX1 zum selektiven Nachweis der TrxR-AktivitĂ€t in Zellen vor. Es basiert auf der Verwendung eines zyklischen, unsymmetrischen Selenenylsulfid-Sensors (1,2 Thiaselenan), der selektiv von dem einzigartigen Selenolat der TrxR angegriffen wird, und dadurch letztlich nur von TrxR reduziert werden kann. RX1 eignete sich zudem fĂŒr eine Hochdurchsatz-Validierung bestehender TrxR Inhibitoren und unterstreicht dadurch den kommerziellen Nutzen derartiger Diagnostika (Chem 2022).
Das zentrale Trigger-Cargo Konzept dieser Arbeit wurde fĂŒr therapeutische Zwecke weiterentwickelt und nutzt dabei den einzigartigen Wirkmechanismus der Duocarmycin-Naturstoffklasse (CBI) (JACS Au 2022) zur Entwicklung reduktiv aktivierbarer Therapeutika. CBI Prodrugs basierend auf stabilisierten Redox-Schaltern (1,2 Dithiane fĂŒr Trx; 1,2 Thiaselenan fĂŒr TrxR) reagierten signifikant auf TrxR-Modulation in Zellen. Sie wurden darĂŒber hinaus durch das Referenzieren ihrer AktivitĂ€t gegenĂŒber nicht-reduzierbaren KontrollmolekĂŒle fĂŒr die Erstellung zelllinienabhĂ€ngiger Profile der ReduktaseaktivitĂ€t in 177 Zelllinien genutzt. SchlieĂlich waren diese neuen Krebsmittel im Tiermodell gut vertrĂ€glich und zeigten in zwei verschiedenen Mausmodellen eine krebshemmende Wirkung (Preprint 2022b).
Zusammenfassend prĂ€sentiert diese Dissertation monothiol-resistente reduzierbare Trigger-Einheiten fĂŒr das zellulĂ€re Trx-System zur Entwicklung neuartiger, selektiver Reporter-Sonden, sowie eine neue Klasse reduktiv aktivierbarer Krebsmittel auf Basis eines adaptierbaren Trigger-Cargo Designs. Diese fanden entweder zur selektiven Messung zellulĂ€rer ProteinaktivitĂ€t oder zum Einsatz als Antikrebsmittel Verwendung. Es wurden chemokompatible Motive sowohl fĂŒr TrxR als auch fĂŒr Trx/Grx identifiziert, wobei deren zyklische Natur eine Aktivierung durch GSH verhindert. Eine weitere Differenzierung zwischen den beiden Redox-Proteinen Trx und Grx und anderen Proteinen der Trx-Superfamilie erfordert eine zusĂ€tzliche Ebene der Selektierung, z. B. durch molekulare Erkennung, und ist Gegenstand laufender Arbeiten.
Die flexible Verwendung verschiedener molekularer Wirkstoffe ermöglicht dabei die âPipeline-Entwicklungâ von Diagnostika und Therapeutika, die von der zellulĂ€ren Redox-Maschinerie analog umgesetzt werden, und dadurch Schlussfolgerungen aus der Diagnostik direkt auf eine Therapie ĂŒbertragbar machen. Dies birgt groĂes Potenzial fĂŒr kĂŒnftige Entwicklungen bei einer potenziellen Ăbertragung des modularen Konzepts auf andere Enzymklassen und therapeutische Einsatzgebiete
A computational view on nanomaterial intrinsic and extrinsic features for nanosafety and sustainability
In recent years, an increasing number of diverse Engineered Nano-Materials (ENMs), such as nanoparticles and nanotubes, have been included in many technological applications and consumer products. The desirable and unique properties of ENMs are accompanied by potential hazards whose impacts are difficult to predict either qualitatively or in a quantitative and predictive manner.
Alongside established methods for experimental and computational characterisation, physics-based modelling tools like molecular dynamics are increasingly considered in Safe and Sustainability-by-design (SSbD) strategies that put user health and environmental impact at the centre of the design and development of new products. Hence, the further development of such tools can support safe and
sustainable innovation and its regulation.
This paper stems from a community effort and presents the outcome of a four-year-long discussion on the benefits, capabilities and limitations of adopting physics-based modelling for computing suitable features of nanomaterials that can be used for toxicity assessment of nanomaterials in combination with data-based models and experimental assessment of toxicity endpoints. We review modern multiscale physics-based models that generate advanced system-dependent (intrinsic) or timeand
environment-dependent (extrinsic) descriptors/features of ENMs (primarily, but not limited to nanoparticles, NPs), with the former being related to the bare NPs and the latter to their dynamic fingerprinting upon entering biological media. The focus is on (i) effectively representing all nanoparticle attributes for multicomponent nanomaterials, (ii) generation and inclusion of intrinsic nanoform properties, (iii) inclusion of selected extrinsic properties, (iv) the necessity of considering distributions of structural advanced features rather than only averages. This review enables us to identify and highlight a number of key challenges associated with ENMsâ data generation, curation,
representation and use within machine learning or other advanced data-driven models to ultimately enhance toxicity assessment. Finally, the set up of dedicated databases as well as the development of grouping and read-across strategies based on the mode of action of ENMs using omics methods are identified as emerging methodologies for safety assessment and reduction of animal testing
Theory of Soft Centrifugation of Structured Fluids Based on Hydrotropes
Centrifugation is a versatile and powerful technique used in analytical or preparative procedures.
In countless variants - low-speed/high-speed/ultracentrifugation, in batch process or continuously operated - it is indispensable in industrial applications and the laboratory.
However, some phenomena that have been observed over the years in connection with centrifugation have not yet been conclusively clarified not to mention reliably predicted.
The aim of this work is to gain deeper knowledge through the theoretical description of different systems in centrifugal fields.
For this purpose, a general description of systems in external fields was developed as a classical density functional theory.
The energy of a system is composed of internal (thermodynamic) and external (gravitational) contributions.
This theoretical framework is universally applicable to all systems, but requires an adequate description of the internal force component.
In the context of this work, several systems were considered with the method.
On the one hand, specific composition profiles can be determined as a function of the radius, or more general Centrifugation Maps can be calculated for visualisation.
The latter show the local composition changes in the entire phase diagram with the help of steam lines.
The strength of this visualisation method is that it is completely independent of specific external fields, because only the magnitude of the gradients - but not the direction - is influenced by the external field.
In this way, regions in the ternary phase diagram can be directly identified that react particularly strongly (or weakly) to centrifugation.
The theory was applied to a simple, regular (water/ethanol/ethyl acetate) and a more complex, aggregating (water/ethanol/octanol) system.
In both systems, it was shown consistently that compositions close to the phase boundary are significantly more influenced by the external field.
Ethanol-rich compositions far in the single-phase range react almost not at all to centrifugation in the first system and only slightly in the second.
Close to the phase boundary and especially around the critical point, centrifugation is clearly more efficient, which manifests itself in phase separations and extremely high composition gradients.
As a third system, a binary mixture of a pair of enantiomers was investigated.
It was shown that small compositional dependencies of the density can be used to separate racemates.
Although these density changes have been described in detail in crystals, their occurrence in fluids is much less documented.
The possibility of racemate resolution without any additives is nevertheless theoretically possible, albeit with increased equipment requirements.
The last part of this work deals with the molecular dynamics study of a derivative of the surfactant AOT.
In combination with small angle scattering, the aggregation behaviour was investigated.
The increased critical micelle concentration and the absence of a liquid crystalline phase oppose the classification as a classical surfactant, the aggregation and scattering already in the binary aqueous mixture oppose classical hydrotropes, which is why the molecule is identified as a link between the two classes.
The molecular dynamic investigation is only a first step towards a better understanding of progressively aggregating systems, which should ultimately enable the application of centrifugation theory to this and similar systems
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