4,780 research outputs found

    Investigating the effects of palmitoylation on the dopamine 1 receptor (D1)

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    The dopamine D1 receptor (D1) is a G protein-coupled receptor (GPCR) which regulates various key brain functions like attention, movement, reward, and memory. Understanding D1 signalling may open the horizon for novel treatments for neurological disorders. Upon agonist activation, the heterotrimeric G proteins Gαs activate adenylyl cyclase to increase cAMP/PKA signalling. D1 also engages β-arrestin proteins leading to β-arrestin dependent signalling. The D1 has two palmitoylation sites on cysteines 347&351 in its C-tail domain. However, the distinct roles and implications of palmitoylation on the D1 signalling, trafficking and β-arrestins recruitment are still largely unexplored. A palmitoylation D1 mutant was generated and luminescent based techniques such as BRET and split-Nanoluc complementation assay were employed, to delineate D1 palmitoylation effects on its pharmacology and signalling. The D1 agonists induced 50% less cAMP production in the mutant compared to wildtype (WT) and WT showed a more efficient dissociation of its Gαs. Moreover, the mutant receptor failed to recruit β-arrestin1&2, induced less ERK1/2 activation and internalises in an agonist-independent process while showing an altered intracellular Golgi trafficking. Also, in β-arrestin 1&2 KO HEK 293 cells similar cAMP production levels were reported for D1 WT and palmitoylation mutant. β-arrestin 1&2 KO blocked agonist-induced WT D1 plasma membrane trafficking, indicating that these β-arrestins are driving the differences between WT and the palmitoylation mutant D1. Taken together, our studies indicate that Gαs is the main transducer for D1 cAMP and ERK1/2 signalling and that palmitoylation is essential for its β-arrestin 1&2 interactions and modulating D1 signalling cascades in a drug-dependant process

    Gasdermins: a dual role in pyroptosis and tumor immunity

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    The gasdermin (GSDM) protein family plays a pivotal role in pyroptosis, a process critical to the body’s immune response, particularly in combatting bacterial infections, impeding tumor invasion, and contributing to the pathogenesis of various inflammatory diseases. These proteins are adept at activating inflammasome signaling pathways, recruiting immune effector cells, creating an inflammatory immune microenvironment, and initiating pyroptosis. This article serves as an introduction to the GSDM protein-mediated pyroptosis signaling pathways, providing an overview of GSDMs’ involvement in tumor immunity. Additionally, we explore the potential applications of GSDMs in both innovative and established antitumor strategies

    From oral infection to autoimmunity : studies of antibodies and B cells on the path towards rheumatoid arthritis

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    Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by inflammation of the synovial joints, which can lead to irreversible joint destruction and disability if not treated properly. The majority of patients are seropositive, defined by presence of autoantibodies, i.e., rheumatoid factor (RF) and/or anti-citrullinated protein antibodies (ACPA). My thesis work focuses on ACPA+ RA, which is known for its more severe disease course. ACPA can be detected in the blood years before clinical signs of RA and were for a long time suggested to contribute to pathology, an hypothesis which is currently under debate. Still, the presence of ACPA, and the successful use of B-cell depleting therapies in RA, points to an important role for autoantibodies and B cells in the development of RA. Notably, most known risk factors for RA, in particular smoking and HLA-DRB1 shared epitope (SE) alleles, are specifically linked to onset of ACPA+ RA. My studies have investigated another potential risk factor for RA, namely the oral pathogen Porphyromonas gingivalis (Pg), one of the main drivers of periodontitis (PD). PD is a common disease that is driven by dysbiosis in the oral cavity triggering gingivitis and eventually leads to destruction of the jawbone and toothsupporting surrounding soft tissues. PD has a higher prevalence in RA than in the general population. Interestingly, Pg has the unique characteristic to express its own citrullinating enzyme and has therefore been suggested to contribute to the generation of RA autoantigens, break of tolerance and systemic ACPA production. The overall aims of my thesis were: 1) to determine if presence of antibodies against the Pg virulence factor RgpB could serve as biomarker to identify patients with PD at increased risk for systemic autoimmunity and onset of RA; 2) to explore the gingiva as a site for ACPA production, and Pg as a driver of the ACPA response; and 3) to phenotypically characterise peripheral blood B cells in the risk-RA phase, to understand B-cell dysregulation prior to RA onset. Investigating the anti-Rgp IgG response in two PD cohorts showed that this antibody response could only poorly discriminate PD from controls. However, elevated anti-Rgp IgG levels defined a subset of PD patients with active gingivitis and advanced marginal jawbone loss. We also showed a higher prevalence of ACPA+ individuals in PD versus controls, and higher anti-Rgp IgG levels in ACPA+ versus ACPA- individuals. Moreover, in a prospective study of ACPA+ individuals at increased risk for RA we found significantly higher anti-Rgp IgG levels compared to controls, but antibody levels did not differ between those who progressed to arthritis and those who remained arthritis free. Generation of monoclonal antibodies derived from RA gingival tissue B cells demonstrated the presence of citrulline-reactive clones binding epitopes on both Pg and self-proteins, and this cross-reactivity was also shown for an RA peripheral blood-derived ACPA+ clone. Investigating the serum polyclonal response, 11% of patients with early RA were positive for antibodies targeting a citrullinated Pg peptide. When assessing peripheral blood B cells in ACPA+ Risk-RA individuals, we detected dysregulation of B-cell subsets already before clinical onset, specifically showing loss of CD27 on class-switched IgG+ memory B cells, a feature previously described in autoimmunity. Collectively, these studies can link anti-Pg antibodies – as a proxy for Pg infection – to severe forms of PD and to the ACPA response, but not to arthritis onset. Thus, suggesting Pg-infection could be an early event in the natural history of RA, possibly triggering ACPA production in the gum mucosa by mechanisms of molecular mimicry. Moreover, the detection of B-cell changes already in the at-risk phase, supports the use of immune monitoring to capture individuals at risk of RA onset, that may benefit the most from pre-clinical intervention

    Dissecting Extracellular Matrix Internalisation Mechanisms using Functional Genomics

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    Breast and ovarian malignancies account for one third of female cancers. The role of the stroma in supporting invasive growth in breast cancer has become clear. Breast cancer cells interact and respond to the cues from the surrounding extracellular matrix (ECM). Integrins are main cell adhesion receptors and key players in invasive migration by linking the ECM to the actin cytoskeleton. In addition, integrins mediate distinctive biochemical and biomechanical signals to support cancer invasion. The role of matrix proteases in promoting ECM degradation and cancer dissemination has been extensively studied; however, cancer cells possess additional means to support those processes, such as integrin-mediated ECM endocytosis and consequent degradation in the lysosomes. Internalisation of the extracellular matrix is upregulated in invasive breast cancer. Nonetheless, the mechanisms by which cancer cells regulate this process are poorly understood. We developed a high throughput pH sensitive system to detect ECM uptake. Here, we show that MDA-MB-231 breast cancer cells converge in macropinocytosis to internalise diverse ECM components and we confirm that this process is modulated by PAK1. To unravel which ECM components breast cancer cells internalise in a complex environment (namely, cell derived matrices), we performed mass spectrometry. Proteomic analysis identified Annexin A6, Collagen VI, Tenascin C and fibronectin, among other matrisome proteins, to be internalised by invasive breast cancer cells. Following ECM endocytosis, ECM is targeted for lysosomal degradation. To unravel the molecular mechanisms behind this process, we performed a trafficking screen and identified the AP3 complex, VAMP7, Arf1 and ARFGEF2. Our results suggest that the AP3 complex may regulate ECM-integrin delivery to lysosomes. To gain more insight on the signalling pathways governing macropinocytosis in breast cancer cells, we performed a kinase and phosphatase screen that unravelled MAP3K1 and PPP2R1A, a subunit of protein phosphatase 2A (PP2A) as relevant regulators of ECM endocytosis. Furthermore, our data suggests that p38 mitogen-activated protein kinase (MAPK) activation upon binding to the ECM is required for ECM macropinocytosis. Outstandingly, inhibiting p38 MAPK led to profound changes in the ability of breast cancer cells to migrate in cell derived matrices. Previous work from the Rainero lab focused on characterising the receptors involved in ECM internalisation; α2β1 integrin was identified as the main regulator of ECM uptake in MDA-MB-231 cells. In particular, α2β1 integrin has been shown to activate p38 MAPK pathway. Taken together, we hypothesise that binding of ECM to α2β1 integrin results in the activation of PAK1 and MAP3K1, which in turn leads to ECM endocytosis. p38 MAPK activity may induce changes in actin polymerisation via PPP2R1A and/or focal adhesion turnover, which consequently promotes ECM macropinocytosis and invasive migration

    The effects of anthracyclines on calcium handling and contractility in sheep ventricular myocytes; role of oxidative stress

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    Anthracyclines such as doxorubicin (DOX) and daunorubicin (DAUN) are effective chemotherapeutics and contribute to improved cancer survival rates in children and adults. However, anthracyclines exhibit acute and chronic cardiotoxicity which can produce heart failure in cancer survivors. While the cellular basis remains unclear, limited previous studies show DOX perturbs certain aspects of excitation-contraction coupling and increases production of reactive oxygen species (ROS). Fewer studies have investigated the effects of DAUN and the effects of either anthracycline on excitation-contraction coupling (ECC) in a large animal model has yet to be demonstrated. Furthermore, the extent to which altered ECC is dependent on anthracycline-induced ROS production remains ambiguous. This is compounded by the fact that no studies have investigated whether elevated ROS production produces oxidative stress in cardiac myocytes. To address these gaps in our understanding, we performed the first integrative investigation of the effects of DOX and DAUN in sheep ventricular myocytes. We also measured the effect of DOX and DAUN on oxidative stress in these cells and further elucidated the underlying sources of ROS. Furthermore, we investigated the dependence of perturbed ECC on ROS thence oxidative stress elevations.Sheep ventricular myocytes were enzymatically isolated in accordance with the Animals (Scientific Procedures) Act, UK, 1986 and used for all experiments. Intracellular calcium and contractility dynamics were measured using epi-fluorescent photometry and video sarcomere detection simultaneously. Cells were field stimulated at 0.5 Hz then acutely exposed to 1 nM DOX or DAUN. Rapid application of 10 mM caffeine was used to measure SR Ca content. Oxidative stress was measured using CellROX red. Fluorescent images were captured using the cytation imaging system and cell fluorescence determined using ImageJ software. DOX reduced the activity of SERCA and increased the activity of NCX resulting in a reduction in SR Ca content. DAUN also reduced SR Ca content however due to an interaction with caffeine the mechanism could not be fully elucidated. The decrease in SR Ca content accounted for a decrease in systolic Ca which underpinned a decrease in systolic shortening. Both DOX and DAUN increased myofilament sensitivity to Ca, potentially offsetting the effect on contractility. DOX increased oxidative stress in a concentration and time-dependent manner. DAUN also increased oxidative stress, but only at relatively high concentrations (10 mM). Removal of oxidative stress by n-acetylcysteine (NAC) attenuated the effects of DOX on the majority of Ca handling and contractility parameters. For example, the effect of DOX on SR Ca content and Ca transient amplitude were reduced by approximately 50 %. Inhibition of the ROS producing enzymes NADPH oxidase (NOX) and xanthine oxidase (XO) reduced DOX-mediated oxidative stress by ~50 % and ~20 % respectively and attenuated the effects on ECC.In cells from sheep with heart failure, DOX reduced SR Ca content thence systolic Ca and contractility but had no effect on SERCA and NCX. These findings suggest that in a large animal model, DOX and DAUN decrease SR Ca content leading to a reduction in systolic Ca thence contractility. In the case of DOX, decreased SERCA and increased NCX activity likely contribute to the decrease of SR Ca content. However, that this isn’t the case in heart failure suggests a role for other mechanisms. These findings are also the first to show that DOX and DAUN increase intracellular oxidative stress in the heart and that NOX and XO are key enzymatic sources of ROS. Furthermore, these findings show this increase in oxidative stress is pathologically important as it accounts for approximately half of the effects of DOX on ECC. Collectively, these findings further elucidate the effects of anthracyclines on ECC and make important contributions to the understanding of the cellular basis of anthracycline-mediated cardiotoxicity. Furthermore, the dependence on and sources of oxidative stress reveal clinically relevant therapeutic targets

    Rational development of stabilized cyclic disulfide redox probes and bioreductive prodrugs to target dithiol oxidoreductases

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    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 cargo’s 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 system’s 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 TrxR’s selenol/thiol active site, then combined with a precipitating large Stokes’ 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 zelluläre Redox-Homöostase hängt 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 über Redox-Schalter in Substratproteinen lebenswichtige zelluläre Funktionen steuern und so an der Redox-Regulation und -Signalübertragung beteiligt sind. Persistente Veränderungen des Redoxmilieus in pathologischen Zuständen, wie z. B. bei Krebs, sind in hohem Maße mit dem Trx-System verbunden. Eine Hochregulierung und/oder Überaktivität des Trx-Systems, die bei vielen Krebsarten auftreten, unterstützt zudem das Fortschreiten des Krebswachstums, was TrxR/Trx zu vielversprechenden Zielproteinen für die Entwicklung neuer Krebsmedikamente macht. Um die biochemischen Prozesse dahinter zu erforschen, sind spezielle Techniken zur Visualisierung und Messung enzymatischer Aktivität nötig. Die hierzu geeigneten, meist genetischen Sensoren messen ratiometrisch das Verhältnis reduzierter/oxidierter Spezies in zellulärem Umfeld oder spezifisch ausgewählte Redoxpaare. Die weitere Erforschung der exakten Funktion von TrxR/Trx und deren Substrate ist jedoch durch mangelnde Nachweismethoden limitiert. Diese sind außerdem zur Validierung chemischer Hemmstoffe für TrxR/Trx in Zellen und deren potenziellen Verwendung als Krebsmittel von großem Interesse. Bislang gibt es keinen selektiven zellulären Trx-Inhibitor und potenzielle Off-Target-Effekte der bekannten TrxR-Inhibitoren wurden nicht abschließend 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) für Oxidoreduktasen so mit molekularen Wirkstoffen (Cargo) verknüpft, dass dabei die Wirkstoffaktivität 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 stärksten Reduktasen gespalten werden; und (ii) die zyklische Topologie ermöglicht die kinetische Reversibilität der zwei Thiol-Disulfid-Austauschreaktionen, die eine erste Reaktion mit Monothiolen, wie z. B. GSH, sofort umkehrt und so eine vollständige Reduktion verhindert. Die meisten früheren Arbeiten auf diesem Gebiet verwendeten ein zyklisches, fünfgliedriges Disulfid (1,2 Dithiolan) als Substrat für TrxR. Das gleiche Strukturmotiv wurde jedoch an anderer Stelle als thermodynamisch instabil beschrieben und aufgrund dieser Eigenschaft explizit für dynamische Kaskadenreaktionen verwendet. Deshalb vergleicht diese Arbeit zu Beginn einen neuen 1,2 Dithiolan basierten fluorogenen Indikator mit bestehenden, z. T. kommerziellen, Redox Sonden für TrxR in einer Reihe von Zellkultur-Experimenten unter Modulation der zellulären TrxR Aktivität und stellt so einen Widerspruch in der Literatur klar: 1,2 Dithiolane eignen sich nicht als selektive Substrate für 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 ausschließlich von Trx aktiviert, nicht aber von TrxR oder GSH (JACS 2021). Dieses Kernmotiv bestimmt dabei die Reduzierbarkeit, und damit die Enzymspezifität, durch seine zyklische Natur und die Annelierung, auch unter Verwendung unterschiedlicher Farb-/Wirkstoffe. Auf dieser Grundlage konnte die molekulare Struktur durch einen weiteren Modifikationspunkt für die flexible Verwendung weiterer funktioneller Einheiten ergänzt werden. Obwohl zelluläre 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 Moleküle wertvoll, um den katalytischen Umsatz zellulärer Dithiol-Reduktasen, der sogenannten Trx Superfamilie, selektiv anzuzeigen (Preprint 2023). Begünstigt durch das modulare Moleküldesign stellt diese Arbeit zudem das erste Reportersystem RX1 zum selektiven Nachweis der TrxR-Aktivität 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 für eine Hochdurchsatz-Validierung bestehender TrxR Inhibitoren und unterstreicht dadurch den kommerziellen Nutzen derartiger Diagnostika (Chem 2022). Das zentrale Trigger-Cargo Konzept dieser Arbeit wurde für 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 für Trx; 1,2 Thiaselenan für TrxR) reagierten signifikant auf TrxR-Modulation in Zellen. Sie wurden darüber hinaus durch das Referenzieren ihrer Aktivität gegenüber nicht-reduzierbaren Kontrollmoleküle für die Erstellung zelllinienabhängiger Profile der Reduktaseaktivität in 177 Zelllinien genutzt. Schließlich waren diese neuen Krebsmittel im Tiermodell gut verträglich und zeigten in zwei verschiedenen Mausmodellen eine krebshemmende Wirkung (Preprint 2022b). Zusammenfassend präsentiert diese Dissertation monothiol-resistente reduzierbare Trigger-Einheiten für das zelluläre 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 zellulärer Proteinaktivität oder zum Einsatz als Antikrebsmittel Verwendung. Es wurden chemokompatible Motive sowohl für TrxR als auch für 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 zusätzliche Ebene der Selektierung, z. B. durch molekulare Erkennung, und ist Gegenstand laufender Arbeiten. Die flexible Verwendung verschiedener molekularer Wirkstoffe ermöglicht dabei die „Pipeline-Entwicklung“ von Diagnostika und Therapeutika, die von der zellulären Redox-Maschinerie analog umgesetzt werden, und dadurch Schlussfolgerungen aus der Diagnostik direkt auf eine Therapie übertragbar machen. Dies birgt großes Potenzial für künftige Entwicklungen bei einer potenziellen Übertragung des modularen Konzepts auf andere Enzymklassen und therapeutische Einsatzgebiete

    Role of cytokine in malignant T-cell metabolism and subsequent alternation in T-cell tumor microenvironment

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    T cells are an important component of adaptive immunity and T-cell-derived lymphomas are very complex due to many functional sub-types and functional elasticity of T-cells. As with other tumors, tissues specific factors are crucial in the development of T-cell lymphomas. In addition to neoplastic cells, T- cell lymphomas consist of a tumor micro-environment composed of normal cells and stroma. Numerous studies established the qualitative and quantitative differences between the tumor microenvironment and normal cell surroundings. Interaction between the various component of the tumor microenvironment is crucial since tumor cells can change the microenvironment and vice versa. In normal T-cell development, T-cells must respond to various stimulants deferentially and during these courses of adaptation. T-cells undergo various metabolic alterations. From the stage of quiescence to attention of fully active form T-cells undergoes various stage in terms of metabolic activity. Predominantly quiescent T-cells have ATP-generating metabolism while during the proliferative stage, their metabolism tilted towards the growth-promoting pathways. In addition to this, a functionally different subset of T-cells requires to activate the different metabolic pathways, and consequently, this regulation of the metabolic pathway control activation and function of T-cells. So, it is obvious that dynamic, and well-regulated metabolic pathways are important for the normal functioning of T-cells and their interaction with the microenvironment. There are various cell signaling mechanisms of metabolism are involved in this regulation and more and more studies have suggested the involvement of additional signaling in the development of the overall metabolic phenotype of T cells. These important signaling mediators include cytokines and hormones. The impact and role of these mediators especially the cytokines on the interplay between T-cell metabolism and the interaction of T-cells with their micro-environments in the context of T-cells lymphomas are discussed in this review article

    Re-evaluation of the risks to public health related to the presence of bisphenol A (BPA) in foodstuffs

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    Publisher Copyright: © 2023 European Food Safety Authority. EFSA Journal published by Wiley-VCH GmbH on behalf of European Food Safety Authority.In 2015, EFSA established a temporary tolerable daily intake (t-TDI) for BPA of 4 μg/kg body weight (bw) per day. In 2016, the European Commission mandated EFSA to re-evaluate the risks to public health from the presence of BPA in foodstuffs and to establish a tolerable daily intake (TDI). For this re-evaluation, a pre-established protocol was used that had undergone public consultation. The CEP Panel concluded that it is Unlikely to Very Unlikely that BPA presents a genotoxic hazard through a direct mechanism. Taking into consideration the evidence from animal data and support from human observational studies, the immune system was identified as most sensitive to BPA exposure. An effect on Th17 cells in mice was identified as the critical effect; these cells are pivotal in cellular immune mechanisms and involved in the development of inflammatory conditions, including autoimmunity and lung inflammation. A reference point (RP) of 8.2 ng/kg bw per day, expressed as human equivalent dose, was identified for the critical effect. Uncertainty analysis assessed a probability of 57–73% that the lowest estimated Benchmark Dose (BMD) for other health effects was below the RP based on Th17 cells. In view of this, the CEP Panel judged that an additional uncertainty factor (UF) of 2 was needed for establishing the TDI. Applying an overall UF of 50 to the RP, a TDI of 0.2 ng BPA/kg bw per day was established. Comparison of this TDI with the dietary exposure estimates from the 2015 EFSA opinion showed that both the mean and the 95th percentile dietary exposures in all age groups exceeded the TDI by two to three orders of magnitude. Even considering the uncertainty in the exposure assessment, the exceedance being so large, the CEP Panel concluded that there is a health concern from dietary BPA exposure.Peer reviewe

    An Investigation into the Impact of Culture Method on Biofilm Recalcitrance from the Perspective of Chronic Ischemic Wounds

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    Antimicrobial resistance is a global crisis that requires urgent attention. Although there is significant investment into drug development few of these drugs actually make it to market. There are shortcomings with some of the currently available testing methods, as these do not often take into account the in vivo environment in which the infections form. This requires a better understanding of the parameters that affect resistance and susceptibility, such as hypoxia or the recalcitrance of biofilms. Biofilms (communities of surface associated microorganisms) present a significant challenge for researchers and clinicians alike, as they are reported to be up to 1000 times more resistant to antimicrobials (Van Acker et al, 2014). These complex communities have a wealth of factors that induce increased recalcitrance to antimicrobials, and are reported to be present in up to 80% of chronic infections (Uruén et al., 2021). Yet, antimicrobial testing of biofilms is not yet widely adopted, and the use of biofilm testing in drug development is often not reported. This work aims to investigate these parameters to gain insight on how antimicrobial discovery and testing can be refined. The first aim of this project was to determine the influence of culture method and hypoxia on S aureus susceptibility in several in vitro antimicrobial susceptibility testing methods. This compared broth microdilution methods (BMM), the Calgary Biofilm device (CBD), and the biofilm microtiter assay (MTA). Hypoxia was revealed to impact S aureus susceptibility in the BMM and CBD methods, and there was a large difference in the density of biofilms formed in the CBD and MTA. The second aim of this project was to set up an ex vivo ovine wounded skin model infected with Staphylococcus aureus, which is simple, cost-effective, high throughput, and reproducible. The establishment of wound infection was confirmed by an increase in viable bacterial counts compared to the inoculum. This enabled the final aim to assess if the ex vivo model formed biofilms and if hypoxia would have an impact on the susceptibility of the biofilms within the ex vivo model. The presence of biofilms was confirmed through scanning electron microscopy, histology, and antimicrobial challenge of the biofilms. Hypoxia elicited an impact on the susceptibility of the ex vivo associated biofilms in an antimicrobial specific manner. This work provides insight into the complex environment that is an ischemic infected wound, and has given insight into the parameters that can affect antimicrobial drug development in the preclinical stages
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