710 research outputs found
Treatment of unruptured intracranial aneurysms : a surgical long-term evaluation for preoperative predictive analytics
No full textAuthor Dr.med.univ. Nico Henrique Stroh-HollyDissertation Johannes Kepler Universität Linz 202
Funktionelle Analyse von NaOCl-sensitiven Thiol-Schaltern und deren Einfluss auf das Bacillithiol-Redoxpotential in Staphylococcus aureus
No full textThe Gram-positive bacterium Staphylococcus aureus colonises asymptomatically ca. 30% of the human population. However, S. aureus is also a major human pathogen and can cause a wide range of live-threatening diseases, such as soft tissue infections, systemic and invasive diseases. In addition, S. aureus has acquired resistance to multiple antibiotics. The pathogen is well adapted to its host, which enables the bacterium to evade the host immune system. Under infections or in its ecological niche, S. aureus is exposed to reactive oxygen species and reactive chlorine species (ROS, RCS), such as H2O2 and HOCl, which are produced by macrophages, neutrophils or competitive bacteria. Therefore, S. aureus has evolved mechanisms to defend itself against oxidative stress, including antioxidant enzymes or low molecular weight (LMW) thiols.
Eukaryotes and Gram-negative bacteria utilize the tripeptide glutathione (GSH), while Actinomycetes use mycothiol (MSH) as their major LMW thiol. However, S. aureus lacks GSH and MSH biosynthetic genes. Instead, S. aureus uses bacillithiol (BSH) and coenzyme A (CoASH) as an alternative LMW thiol. These small molecules play an essential role in detoxification of ROS and RCS as well as in maintenance of the reduced redox homeostasis inside the cell under different kinds of stress. An overview of the large functional diversity of BSH in Bacillus subtilis and S. aureus is presented in chapter 1.
LMW thiols are important for the redox homeostasis. Thus, redox biology research often focuses on the function of LMW thiols on the cellular redox potential. During the last decades, genetically encoded redox-sensitive green fluorescent protein (roGFP2)-fused biosensors were established as tools for real-time monitoring of dynamic changes of the redox potential. Glutaredoxins (Grx), mycoredoxins (Mrx) and bacilliredoxins (Brx) were fused to roGFP2 to monitor changes of the redox potential in high spatiotemporal resolution in eukaryotes and bacteria. The applications of several roGFP2-fused biosensors in pathogenic bacteria under oxidative stress and infection conditions are summarized in chapter 2.
Under oxidative stress and infections, BSH and CoASH can function as redox modifications of protein thiols, leading to S-thiolations. These mixed protein disulfides are termed as S-bacillithiolations or CoAlations. S-thiolations function in redox regulation of proteins and protect proteins against overoxidation to sulfonic acids. S-bacillithiolations can be reduced by bacilliredoxins (Brx), resulting in S-bacillithiolated Brx, which are reduced by another molecule of BSH, leading to bacillithiol disulfide (BSSB). For a long time, it was postulated that the NADPH-dependent flavin oxidoreductase YpdA might function as a BSSB reductase. Thus, the investigation of the physiological role of YpdA and the Brx/BSH/YpdA redox pathway under oxidative stress and infections in S. aureus was a subject of this PhD thesis and is presented in chapter 3. Our results demonstrated that YpdA acts as the BSSB reductase in vitro and in vivo. The enzymatic activity of YpdA was shown to depend on the conserved Cys14 residue. Using genetically encoded Brx-roGFP2 and Tpx-roGFP2 biosensors and HPLC metabolomics, we revealed that the S. aureus ΔypdA mutant has significantly higher BSSB levels and is impaired in the regeneration of the reduced BSH redox potential (EBSH). These results indicated that YpdA is important to maintain the redox homeostasis and to restore the reduced EBSH after oxidative stress. Phenotype analyses showed that YpdA improves the survival after oxidative stress and under infection conditions and thus, it is involved in the virulence of S. aureus. In addition, we demonstrated that YpdA acts together with BSH and BrxA in the BrxA/BSH/YpdA redox pathway. Moreover, we showed that BrxA contributes also to the fitness of S. aureus under oxidative stress and infections.
S. aureus rapidly acquires resistance to multiple antibiotics, resulting in methicillin-resistant Staphylococcus aureus (MRSA) strains. To combat S. aureus infections, the development of new antimicrobial compounds is required. Quinones are potent antimicrobial substances because of their bivalent mode of action as electrophiles and oxidants. In this doctoral thesis, we investigated the antimicrobial effect and mode of action of the plant-derived 1,4-naphthoquinone lapachol in S. aureus (chapter 4). Phenotype analyses, using growth and survival assays, demonstrated that lapachol is growth-inhibitory and lethal for S. aureus. The antimicrobial effect of lapachol in S. aureus depends strongly on oxygen availability, since the toxicity of lapachol was decreased in survival assays under microaerophilic conditions compared to aerobic conditions. As revealed by RNA-seq, lapachol induces a strong quinone-specific and oxidative stress response in S. aureus. Furthermore, applications of the Brx- roGFP2 and Tpx-roGFP2 biosensors showed that S. aureus exhibits an increased EBSH and enhanced intracellular H2O2 levels after lapachol stress, indicating ROS-production by lapachol. ROS-induction by lapachol can induce S-bacillithiolations of GapDH in S aureus in vitro and in vivo. Moreover, the addition of the ROS scavenger N-acetyl cysteine to lapachol-stressed S. aureus cells improves the survival of the bacteria. The H2O2-scavenging catalase (KatA) and the BrxA/BSH/YpdA redox pathway were further shown to be essential for survival under lapachol treatment. In addition, no protein aggregation has been detected in vitro and in vivo after lapachol stress, supporting that lapachol does not act via the S-alkylating mode.
In conclusion, the results of this PhD thesis provided new insights in the maintenance of the BSH redox homeostasis in S. aureus under oxidative stress, infection conditions and antibiotics treatment. The BSSB reductase YpdA was shown to play a crucial role in redox homeostasis and is a part of the BrxA/BSH/YpdA redox pathway, which contributes to the virulence of S. aureus. Furthermore, the BrxA/BSH/YpdA redox pathway has been shown to protect S. aureus against ROS, produced in the oxidative mode of lapachol stress. In the future, YpdA could be a novel drug target. Also, lapachol and its derivatives could be applied as new antimicrobials to combat life-threatening MRSA infections.Das grampositive Bakterium Staphylococcus aureus kolonisiert asymptomatisch ca. 30 % der menschlichen Bevölkerung. Jedoch ist S. aureus auch ein wichtiger humanpathogener Infektionserreger und verursacht viele lebensbedrohliche, invasive und systemische Erkrankungen. S. aureus hat zahlreiche Antibiotikaresistenzen erworben und ist gut an seinem Wirt angepasst, wodurch es dem Bakterium ermöglicht wird, das Immunsystem des Wirts zu umgehen. Unter Infektionsbedingungen oder in seiner ökologischen Nische ist S. aureus verschiedenen reaktiven Sauerstoff- und Chlor-Spezies (ROS, RCS) ausgesetzt, wie zum Beispiel H2O2 und HOCl, die von Makrophagen, Neutrophilen oder konkurrierenden Bakterien produziert werden. Daher hat S. aureus viele Mechanismen zur Abwehr gegen oxidativem Stress entwickelt, wie z. B. antioxidative Enzyme oder niedermolekulare Thiolverbindungen.
Eukaryoten und gramnegative Bakterien verwenden das Tripeptid Glutathion (GSH) und Actinomyceten nutzen Mycothiol (MSH) als niedermolekulare Thiolverbindungen. Allerdings fehlen S. aureus die Gene für die Biosynthese von GSH und MSH. Stattdessen nutzt S. aureus Bacillithiol (BSH) und Coenzym A (CoASH) als alternative niedermolekulare Thiolverbindungen. Diese Metabolite spielen eine essentielle Rolle bei der Entgiftung von ROS und RCS sowie bei der Aufrechterhaltung der reduzierten Redox-Homöostase in der Zelle bei verschiedenen Arten von Stress. Ein Überblick über die Biosynthese und Funktionen von BSH in Bacillus subtilis und S. aureus wird im Kapitel 1 gegeben.
Niedermolekulare Thiolverbindungen spielen eine wichtige Rolle in der Redox-Homöostase aller Zellen. Deshalb ist die Untersuchung des zellulären Redoxpotentials ein Schwerpunkt in der Forschung der Redoxbiologie. In den letzten Jahrzehnten wurden neue genetisch-kodierte Redox-Biosensoren entwickelt, basierend auf dem redox-sensitiven grün-fluoreszierenden Protein (roGFP2). Dadurch konnte die Messung dynamischer Veränderungen des zellulären Redoxpotentials in Echtzeit durchgeführt werden. Dabei werden Redoxine, wie z. B. Glutaredoxin (Grx), Mycoredoxin (Mrx) oder Bacilliredoxin (Brx), an roGFP2 gekoppelt, um in hoher räumlich-zeitlicher Auflösung Veränderungen des Redoxpotentials in Eukaryoten und Prokaryoten zu messen. Die Anwendungen einiger roGFP2-fusionierter Biosensoren in pathogenen Bakterien unter oxidativen Stress und Infektionsbedingungen sind im Kapitel 2 zusammengefasst.
Bei oxidativen Stress und Infektionen können BSH und CoASH Proteine durch S-Thiolierungen posttranslational modifizieren. Diese gemischten Protein-Disulfide werden in S. aureus als S-Bacillithiolierungen oder CoA-Thiolierungen bezeichnet. Durch S-Thiolierungen wird die Aktivität von Proteinen reguliert und ein Schutz der Proteine vor Überoxidation zur irreversiblen Cystein-Sulfonsäure vermittelt. S-Bacillithiolierungen können durch Bacilliredoxine (Brx) reduziert werden, wodurch Brx selbst S-bacillithioliert wird. Brx kann durch ein weiteres BSH-Molekül reduziert werden, wobei Bacillithiol-Disulfid (BSSB) entsteht. Lange Zeit wurde angenommen, dass die NADPH-abhängige Flavin-Oxidoreduktase YpdA als BSSB-Reduktase fungiert. Die Untersuchung der physiologischen Rolle von YpdA und des Brx/BSH/YpdA-Weges in S. aureus unter oxidativen Stress und Infektionsbedingungen wird in Kapitel 3 als ein Hauptteil der Promotionsarbeit beschrieben. Wir konnten zeigen, dass YpdA in vitro und in vivo als BSSB-Reduktase fungiert. In vitro hängt die enzymatische Aktivität von YpdA vom konservierten Cystein-14 ab. Mittels genetisch-kodierter Brx-roGFP2 und Tpx-roGFP2 Biosensoren und HPLC-Metabolomanalysen konnte ich zeigen, dass die S. aureus ΔypdA-Mutante deutlich höhere BSSB-Mengen aufweist und bei der Regeneration des reduzierten BSH-Redoxpotentials (EBSH) beeinträchtigt ist. Somit besitzt YpdA eine wichtige Funktion bei der Aufrechterhaltung der Redox-Homöostase und zur Regeneration des reduzierten EBSH nach oxidativem Stress. Phänotyp-Analysen zeigten, dass YpdA wichtig ist für das Überleben von S. aureus nach oxidativem Stress und unter Infektionsbedingungen und damit in die Virulenz involviert ist. Zusätzlich konnte gezeigt werden, dass YpdA zusammen mit BSH und BrxA im BrxA/BSH/YpdA-Redoxweg interagiert. Zusätzlich ist BrxA von Bedeutung für die Fitness von S. aureus unter oxidativem Stress und Infektionsbedingungen.
S. aureus kann schnell Antibiotikaresistenzen erwerben, wodurch sich unter anderem Methicillin-resistente Staphylococcus aureus (MRSA) Stämme entwickelt haben. Um S. aureus-Infektionen erfolgreich bekämpfen zu können, ist die Entwicklung neuer antimikrobieller Substanzen notwendig. Chinone sind aufgrund ihrer beiden Wirk-mechanismen als Elektrophile und Oxidantien starke antimikrobielle Substanzen. In der vorliegenden Dissertation habe ich weiterhin in Kapitel 4 die antimikrobielle Wirkung und den Wirkmechanismus des natürlichen 1,4-Naphthochinons Lapachol in S. aureus untersucht. In Phänotyp-Analysen mittels Wachstums- und Überlebensversuchen zeigte Lapachol eine Wachstums-inhibierende und letale Wirkung in S. aureus. Dabei ist die antimikrobielle Wirkung von Lapachol in S. aureus von Sauerstoff abhängig, da die toxische Wirkung von Lapachol in Überlebensversuchen unter mikroaerophillen Bedingungen im Vergleich zu aeroben Bedingungen deutlich vermindert war. Durch RNA-Seq wurde gezeigt, dass Lapachol sowohl eine Chinon-spezifische Antwort als auch eine oxidative Stressantwort in S. aureus auslöst. Des Weiteren konnte durch die Brx roGFP2 und Tpx-roGFP2 Biosensoren gezeigt werden, dass S. aureus ein oxidiertes EBSH und eine erhöhte intrazelluläre H2O2-Menge nach Lapachol-Stress aufweist, was den oxidativen Wirkmechanismus der ROS-Produktion bestätigt. Lapachol-induziertes ROS bewirkt die S-Bacillithiolierung von GapDH in vitro und in S. aureus in vivo. Das aerobe Wachstum von S. aureus konnte durch N-Acteylcystein nach Lapachol-Stress verbessert werden. Phänotyp-Untersuchungen zeigten wichtige Funktionen der H2O2-entgiftenden Katalase (KatA) und des BrxA/BSH/YpdA-Redoxweges im Wachstum und Überleben von S. aureus nach Lapachol-Stress. Außerdem konnte keine Proteinaggregation in vitro und in vivo nach Lapachol-Stress nachgewiesen werden, was die Wirkung von Lapachol über S-Alkylierung und Aggregation von Proteinen widerlegt.
Zusammenfassend wurden in der Dissertation neue Erkenntnisse zur Aufrecht-erhaltung der BSH-Redoxbalance in S. aureus unter oxidativen Stress, Infektionsbedingungen und Antibiotika-Stress erzielt. Dabei spielt YpdA eine entscheidende Rolle und ist ein Teil des BrxA/BSH/YpdA-Redoxweges, der an der Virulenz von S. aureus beteiligt ist. Des Weiteren konnten wir ebenfalls eine Rolle des BrxA/BSH/YpdA-Weges beim Schutz von S. aureus unter Lapachol-Stress, welcher ROS-Produktion und oxidativen Stress in S. aureus verursacht, zeigen. Zukünftig könnte YpdA ein neues Ziel für die Entwicklung von Antibiotika sein und Lapachol und dessen Derivate als neue antimikrobielle Substanzen angewendet werden, um MRSA-Infektionen zu bekämpfen
Effective Inhibitor Removal from Wastewater Samples Increases Sensitivity of RT-dPCR and Sequencing Analyses and Enhances the Stability of Wastewater-Based Surveillance
No full textWastewater-based surveillance (WBS) is a proven tool for monitoring population-level infection events. Wastewater contains high concentrations of inhibitors, which contaminate the total nucleic acids (TNA) extracted from these samples. We found that TNA extracts from raw influent of Berlin wastewater treatment plants contained highly variable amounts of inhibitors that impaired molecular analyses like dPCR and next-generation sequencing (NGS). By using dilutions, we were able to detect inhibitory effects. To enhance WBS sensitivity and stability, we applied a combination of PCR inhibitor removal and TNA dilution (PIR+D). This approach led to a 26-fold increase in measured SARS-CoV-2 concentrations, practically reducing the detection limit. Additionally, we observed a substantial increase in the stability of the time series. We define suitable stability as a mean absolute error (MAE) below 0.1 log10 copies/L and a geometric mean relative absolute error (GMRAE) below 26%. Using PIR+D, the MAE could be reduced from 0.219 to 0.097 and the GMRAE from 65.5% to 26.0%, and even further in real-world WBS. Furthermore, PIR+D improved SARS-CoV-2 genome alignment and coverage in amplicon-based NGS for low to medium concentrations. In conclusion, we strongly recommend both the monitoring and removal of inhibitors from samples for WBS
Timely Monitoring of SARS-CoV-2 RNA Fragments in Wastewater Shows the Emergence of JN.1 (BA.2.86.1.1, Clade 23I) in Berlin, Germany
No full textThe importance of COVID-19 surveillance from wastewater continues to grow since case-based surveillance in the general population has been scaled back world-wide. In Berlin, Germany, quantitative and genomic wastewater monitoring for SARS-CoV-2 is performed in three wastewater treatment plants (WWTP) covering 84% of the population since December 2021. The SARS-CoV-2 Omicron sublineage JN.1 (B.2.86.1.1), was first identified from wastewater on 22 October 2023 and rapidly became the dominant sublineage. This change was accompanied by a parallel and still ongoing increase in the notification-based 7-day-hospitalization incidence of COVID-19 and COVID-19 ICU utilization, indicating increasing COVID-19 activity in the (hospital-prone) population and a higher strain on the healthcare system. In retrospect, unique mutations of JN.1 could be identified in wastewater as early as September 2023 but were of unknown relevance at the time. The timely detection of new sublineages in wastewater therefore depends on the availability of new sequences from GISAID and updates to Pango lineage definitions and Nextclade. We show that genomic wastewater surveillance provides timely public health evidence on a regional level, complementing the existing indicators
Enough of 'tough': Youth Justice in Scotland
No full textHow can we build an effective youth justice system that enjoys public confidence while recognising that children must be redeemable?Nico Juetten presents a view from Scotland, considering the challenges to the children's hearings system Copyright (c) 2009 The Author. Journal compilation (c) 2009 ippr.
Data_Sheet_1_The Antimicrobial Activity of the AGXX® Surface Coating Requires a Small Particle Size to Efficiently Kill Staphylococcus aureus.PDF
No full textMethicillin-resistant Staphylococcus aureus (MRSA) isolates are often resistant to multiple antibiotics and pose a major health burden due to limited treatment options. The novel AGXX® surface coating exerts strong antimicrobial activity and successfully kills multi-resistant pathogens, including MRSA. The mode of action of AGXX® particles involves the generation of reactive oxygen species (ROS), which induce an oxidative and metal stress response, increased protein thiol-oxidations, protein aggregations, and an oxidized bacillithiol (BSH) redox state in S. aureus. In this work, we report that the AGXX® particle size determines the effective dose and time-course of S. aureus USA300JE2 killing. We found that the two charges AGXX®373 and AGXX®383 differ strongly in their effective concentrations and times required for microbial killing. While 20–40 μg/ml AGXX®373 of the smaller particle size of 1.5–2.5 μm resulted in >99.9% killing after 2 h, much higher amounts of 60–80 μg/ml AGXX®383 of the larger particle size of >3.2 μm led to a >99% killing of S. aureus USA300JE2 within 3 h. Smaller AGXX® particles have a higher surface/volume ratio and therefore higher antimicrobial activity to kill at lower concentrations in a shorter time period compared to the larger particles. Thus, in future preparations of AGXX® particles, the size of the particles should be kept at a minimum for maximal antimicrobial activity.</p
Ciò che resta della terza dimensione
No full textThe s.c. «third dimension» is one of the first definitions given in the sociological arena to define the development of a ate element that was not identifiable with generalist instances of the state or with those particularistic of the market. Deriving its conceptualization from the Husserlian theory, Ardigò through the use this approach emphasizing the need to reconstruct the crisis through new forms of transaction between the social system and life-worlds. The use of this conceptualization led to the identification of these intermediary organizations between different systems. The text takes a brief overview of the third dimension organizations and the way how they operate the reconciliation between inter-subjectivity and super-ordination present in the systemic sense. It is proposed a scheme of interpretation, drawn from the author in question, through which classifying not merely the organization but the possible interconnections between micro and macro and their consequences. It is concluded that the abandon of the analytical perspective proposed by Ardigò, primarily for the affirmation of a vision of an economic nature, was determined both by the theoretical vulgate established around of the 90, and the difficulty in operationalizing a definition that owed much to the knowledge from the phenomenology of the Bolognese scientist
The plant-derived naphthoquinone lapachol causes an oxidative stress response in Staphylococcus aureus.
No full textLinzner N, Fritsch VN, Busche T, et al. The plant-derived naphthoquinone lapachol causes an oxidative stress response in Staphylococcus aureus. Free radical biology & medicine. 2020;158:126-136.Staphylococcus aureus is a major human pathogen, which causes life-threatening systemic and chronic infections and rapidly acquires resistance to multiple antibiotics. Thus, new antimicrobial compounds are required to combat infections with drug resistant S. aureus isolates. The 2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone lapachol was previously shown to exert antimicrobial effects. In this study, we investigated the antimicrobial mode of action of lapachol in S. aureus using RNAseq transcriptomics, redox biosensor measurements, S-bacillithiolation assays and phenotype analyses of mutants. In the RNA-seq transcriptome, lapachol caused an oxidative and quinone stress response as well as protein damage as revealed by induction of the PerR, HypR, QsrR, MhqR, CtsR and HrcA regulons. Lapachol treatment further resulted in up-regulation of the SigB and GraRS regulons, which is indicative for cell wall and general stress responses. The redox-cycling mode of action of lapachol was supported by an elevated bacillithiol (BSH) redox potential (EBSH), higher endogenous ROS levels, a faster H2O2 detoxification capacity and increased thiol-oxidation of GapDH and the HypR repressor in vivo. The ROS scavenger N-acetyl cysteine and microaerophilic growth conditions improved the survival of lapachol-treated S. aureus cells. Phenotype analyses revealed an involvement of the catalase KatA and the Brx/BSH/YpdA pathway in protection against lapachol-induced ROS-formation in S. aureus. However, no evidence for irreversible protein alkylation and aggregation was found in lapachol-treated S. aureus cells. Thus, the antimicrobial mode of action of lapachol in S. aureus is mainly caused by ROS formation resulting in an oxidative stress response, an oxidative shift of the EBSH and increased protein thiol-oxidation. As ROS-generating compound, lapachol is an attractive alternative antimicrobial to combat multi-resistant S. aureus isolates. Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved
Alla ricerca della sovranità nello studio di Carlo Lavagna sulle figure giuridiche soggettive
No full textIl presente contributo ripercorre il saggio di Carlo Lavagna, Basi per uno studio delle figure giuridiche soggettive contenute nella Costituzione italiana, allo scopo di ricercare il filo conduttore della sovranità in tutte le figure giuridiche soggettive sia che esse siano solo raccomandate, sia che siano garantite o anche tutelate dalla Costituzione, secondo la classificazione dell’Autore. Tale analisi è stata preceduta da un breve richiamo al metodo di indagine dell’Autore e alla sua concezione quadrangolare della democrazia.This contribution reinterprets the essay by Carlo Lavagna, Basis for a study of the subjective juridical figures contained in the Italian Constitution, in order to search for the sphere of sovereignty in all subjective legal figures whether they are only recommended, whether they are guaranteed or also protected by the Constitution, according to the classification of the Author. This analysis was preceded by a brief recall of the Author's method of investigation and its quadrangular concept of democracy
The Understood Author: A hermeneutical exploration of audiences interpretation of the author as productive practices behind a text
No full textin English This project builds on the tradition of audience research that theoretically and empirically established that meaning is formed in the negotiation between the text and the reader, that the audiences are agentic, their interpretation of media is contextual and situated, and that they have the potential to resist the media. It argues that it is important to explore people's attention to different sources: who is listening, to whom, and why - and asks how people understand the notion of an author that they encounter in the interpretation of factual media texts, and how this interpretation is brought back into the interpretative act. The thesis subscribes to philosophical hermeneutics as an overarching theoretical as well as methodological framework to devise a concept of an understood author that is a result of the interpretative act, where the author imagined and anticipated by the reader (in the form of prior knowledge and prejudices) is encountered and actualised by the author inscribed into the text. I carried out 28 in-depth face-to-face interviews with Czechs born between 1948-1963 to explore participants' experiences and relationship with media and media authors. Employing hermeneutical analysis, the focus is not to capture what is an author, but how it happened to be so. The..
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