DaNa2.0 – Daten und Wissen zu Nanomaterialien: Aufbereitung gesellschaftlich relevanter naturwissenschaftlicher Fakten - Schlussbericht DaNa2.0 - Einzelantrag KIT

Das Projekt DaNa2.0 hatte zum Ziel, in einem interdisziplinären Ansatz mit Wissenschaftlern aus Humantoxikologie, Ökotoxikologie, Biologie, Physik, Chemie und Pharmazie Forschungsergebnisse zu Nanomaterialien und deren Auswirkungen auf den Menschen und die Umwelt für interessierte Verbraucher verständlich aufzubereiten. Diese Daten wurden auf der Internetplattform www.nanopartikel.info und auch durch andere Medien (Info-Flyer, Broschüren) der Öffentlichkeit zugänglich gemacht. Die wissenschaftlichen Ergebnisse der Projekte der BMBF-Fördermaßnahmen NanoCare, NanoNature, NanoCare4.0 und ERANET SIINN wurden als erweiterte Basis dieses Informationsangebots aufbereitet. Zudem wurde Literatur zu Human- und Umwelttoxikologie von Nanomaterialien auch anderer wissenschaftlicher Gruppen zur Erweiterung der Wissensplattform evaluiert. Die Bewertung der aktuellen Forschungsergebnisse für die DaNa-Wissensbasis erfolgt auf der Grundlage sorgfältiger wissenschaftlicher Vorgehensweise anhand des DaNa Literatur-Kriterienkatalogs

The DaNa2.0 Knowledge Base Nanomaterials - An Important Measure Accompanying Nanomaterials Development

Nanotechnology is closely related to the tailored manufacturing of nanomaterials for a huge variety of applications. However, such applications with newly developed materials are also a reason for concern. The DaNa2.0 project provides information and support for these issues on the web in condensed and easy-to-understand wording. Thus, a key challenge in the field of advanced materials safety research is access to correct and reliable studies and validated results. For nanomaterials, there is currently a continuously increasing amount of publications on toxicological issues, but criteria to evaluate the quality of these studies are necessary to use them e.g., for regulatory purposes. DaNa2.0 discusses scientific results regarding 26 nanomaterials based on actual literature that has been selected after careful evaluation following a literature criteria checklist. This checklist is publicly available, along with a selection of standardized operating protocols (SOPs) established by different projects. The spectrum of information is rounded off by further articles concerning basics or crosscutting topics in nanosafety research. This article is intended to give an overview on DaNa2.0 activities to support reliable toxicity testing and science communication alik

Eight Weeks Later - The Unprecedented Rise of 3D Printing during the COVID-19 Pandemic : a Case Study, Lessons Learned, and Implications on the Future of Global Decentralized Manufacturing

The eruption of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (corona virus disease, COVID-19) in Wuhan, China, and its global spread has led to an exponentially growing number of infected patients, currently exceeding over 6.6 million and over 390,000 deaths as of the 5th of June 2020. In this pandemic situation, health systems have been put under stress, and the demand for personal protective equipment (PPE) exceeded the delivery capabilities of suppliers. To address this issue, 3D printing was identified as a possible solution to quickly produce PPE items such as face shields, mask straps, masks, valves, and ear savers. Around the world, companies, universities, research institutions, and private individuals/hobbyists stepped into the void, using their 3D printers to support hospitals, doctors, nursing homes, and even refugee camps by providing them with PPE. In Germany, the makervsvirus movement took up the challenge and connected thousands of end users, makers, companies, and logistic providers for the production and supply of face shields, protective masks, and ear savers. The Karlsruhe Institute of Technology (KIT) also joined the makervsvirus movement and used its facilities to print headbands for face shield assemblies and ear savers. Within this paper, the challenges and lessons learned from the quick ramp up of a research laboratory to a production site for medium-sized batches of PPE, the limitations in material supply, selection criteria for suitable models, quality measures, and future prospects are reported and conclusions drawn

Cerebrospinal fluid findings in adults with acute Lyme neuroborreliosis

Presence of BB-specific antibodies in the cerebrospinal fluid (CSF) with evidence of their intrathecal production in conjunction with the white cell count in the CSF and typical clinical symptoms is the traditional diagnostic gold standard of Lyme neuroborreliosis (LNB). Few data are available on the CSF lactate concentration in European adults with the diagnosis of acute LNB. The objective of the study was to investigate the CSF changes during acute LNB. Routine CSF parameters [leukocyte count, protein, lactate and albumin concentrations, CSF/serum quotients of albumin (QAlb), IgG, IgA and IgM, and oligoclonal IgG bands] and the Borrelia burgdorferi (BB)-specific antibody index were retrospectively studied in relation to the clinical presentation in patients diagnosed with acute LNB. A total of 118 patients with LNB were categorized into the following groups according to their symptoms at presentation; group 1: polyradiculoneuritis (Bannwarth’s syndrome), group 2: isolated facial palsy and group 3: predominantly meningitic course of the disease. In addition to the CSF of patients with acute LNB, CSF of 19 patients with viral meningitis (VM) and 3 with neurolues (NL) were analyzed. There were 97 patients classified with definite LNB, and 21 as probable LNB. Neck stiffness and fever were reported by 15.3% of patients. Most of these patients were younger than 50 years. Polyradiculoneuritis was frequently found in patients older than 50 years. Lymphopleocytosis was found in all patients. Only 5 patients had a CSF lactate ≥3.5 mmol/l, and the mean CSF lactate level was not elevated (2.1 ± 0.6 mmol/l). The patients with definite LNB had significantly higher lactate levels than patients with probable LNB. Elevated lactate levels were accompanied by fever and headache. In the Reiber nomograms, intrathecal immunoglobulin synthesis was found for IgM in 70.2% followed by IgG in 19.5%. Isoelectric focussing detected an intrathecal IgG synthesis in 83 patients (70.3%). Elevated BB AIs in the CSF were found in 97 patients (82.2%). Patients with VM showed lower CSF protein concentration and CSF/serum quotients of albumin than LNB patients. In acute LNB, all patients had elevated cerebrospinal fluid (CSF) leukocyte counts. In contrast to infections by other bacteria, CSF lactate was lower than 3.5 mmol/l in all but 5 patients. The CSF findings did not differ between polyradiculoneuritis, facial palsy, and meningitis. The CSF in LNB patients strongly differed from CSF in VM patients with respect to protein concentration and the CSF/serum albumin quotient

Identification and functional analysis of peroxisomal proteins

0 Titelblatt und Inhalt 1 Einleitung 1 2 Material und Methoden 15 3 Ergebnisse 38 4 Diskussion 113 5 Zusammenfassung 137 6 Abstract 139 7 Literatur 141 8 Anhang 152Identifizierung In dieser Arbeit wurden unter Verwendung des ursprünglichen reversen genetischen Ansatzes zur Identifizierung peroxisomaler Proteine 78 Proteinbanden massenspektrometrisch analysiert und dabei 59 verschiedene Proteine identifiziert. Neunzehn der Proteine erwiesen sich als peroxisomal, darunter auch die neu identifizierten Proteine Eci1p (Enoyl-CoA Isomerase), Dci1p (Dienoyl-CoA Isomerase), welchen eine akzessorische Funktion beim Abbau ungesättigter Fettsäuren zukommt, und die peroxisomale Thioesterase Tes1p. Für elf weitere Proteine dieses Ansatzes konnte eine potentielle peroxisomale Lokalisation angenommen werden. Im Rahmen dieser Studie wurde ein zweiter, modifizierter Ansatz zur Analyse des peroxisomalen Proteoms etabliert, der auf dem kombinatorischen Einsatz von 1D-SDS-Gelelektrophorese und MALDI-MS und LC- ESI-MS basierte. Hierbei konnten 66 Proteine identifiziert werden, darunter befanden sich 25 peroxisomale. 17 Proteine, deren Lokalisation in der Zelle noch nicht festgestellt worden war, wurden daraufhin untersucht. Funktionsanalyse Die subzelluläre Lokalisation unbekannter Proteine wurde mit Hilfe von Doppel-Fluoreszenzmikroskopie unter Verwendung von GFP- Fusionsproteinen in Verbindung mit einem rot-fluoreszierenden peroxisomalen Markerprotein (PTS2DsRed) und Zellfraktionierungsanalysen untersucht. Zur Klärung der potentiellen peroxisomalen Funktion eines neuen Proteins, wurde das Wachstum der entsprechenden Deletionsmutante auf verschiedenen Kohlenstoffquellen analysiert. Es konnte gezeigt werden, dass Yor084wp eine ölsäureinduzierbare, potentielle peroxisomale Lipase/Esterase ist, die Pex5p- abhängig in die Peroxisomenmatrix importiert wird. Die Deletion des entsprechenden Gens hat keinen Einfluss auf die Degradation von Fettsäuren. Die potentielle 2-Hydroxyphytanoyl-CoA-Lyase Yel020cp kann zu den peroxisomalen Matrixproteinen gezählt werden, die Pex5p-abhängig importiert werden. Sie verfügt außerdem über eine Thiamin-Diphosphat-Bindestelle. Für die Biogenese von Peroxisomen ist das Protein nicht essentiell. Ygl184cp ist eine mögliche Cystathionin-b-Lyase und konnte in dieser Arbeit in der Membran von Peroxisomen nachgewiesen werden. Für die Biogenese der Organellen wird dieses Protein nicht benötigt. Yir034cp ist ein peroxisomales Matrixprotein, das aufgrund seiner PTS1-Sequenz in die Organellen gelangt. Seine Rolle in der Biosynthese des Lysins führte zu der Hypothese, dass dieser Aminosäurestoffwechsel in Hefe in Peroxisomen stattfindet. Für die Acetyl-CoA- Hydrolase Ybl015wp konnte gezeigt werden, dass sie aufgrund ihrer potentiellen N-terminalen peroxisomalen (PTS2) als auch mitochondrialen Zielsteuerungssequenz (MTS) in beide Organellen importiert wird. Ycr091wp wurde ebenfalls in diesen beiden Organellen nachgewiesen und sorgt dort als potentielle Serin/Threonin-Kinase vermutlich für entsprechende posttranslationale Modifikationen. Die mitochondriale Lokalisation des unbekannten Proteins Yor228cp konnte gezeigt werden. Computergestützte Analysen weisen außerdem auf drei transmembrane Bereiche hin, so dass es sich um ein mitochondriales Membranprotein handeln könnte. Der Succinat-Fumarat- Transporter Sfc1p/Yjr095wp gehört zur Familie der mitochondrialen Transporter, der auch peroxisomale Proteine angehören. Eine zusätzliche peroxisomale Lokalisation konnte für dieses Protein nicht bestimmt werden. Das unbekannte Yhr199cp wurde ebenfalls in den Mitochondrien detektiert, die zelluläre Funktion bleibt allerdings unbekannt. Die vermutete peroxisomale Lokalisation des Yal054cp konnte nicht bestätigt werden. Dagegen wurde eine mikrosomale Lokalisation vermutet. Für weitere 22 untersuchte Proteine konnte ebenfalls keine peroxisomale Lokalisation festgestellt werden, so dass diese Proteine als Kontaminationen bei der Isolierung peroxisomaler Proteine betrachtet werden müssen. Pex11 Im Rahmen dieser Arbeit konnte mittels elektrophysiologischer Messungen nicht nachgewiesen werden, ob Pex11p einen spannungsgesteuerten Kanal in der Peroxisomenmembran bildet. ScPex11p wurde in Form eines TAP-Fusionsproteins erfolgreich exprimiert und kann so zur affinitätschromatographischen Isolierung und weiterführenden Funktionsanalysen verwendet werden.Identification In this study, a reverse genetic screen for peroxisomal proteins was used to analyze 78 protein bands of SDS-PAGE by mass spectrometry. 59 different proteins could be identified. 19 of the proteins proved to be peroxisomal, among them the 3 newly identified proteins Eci1p (enoyl-CoA isomerase), Dci1p (dienoyl-CoA isomerase) which play an accessory role in degradation of unsaturated fatty acids and Tes1p (thioesterase). 11 unknown proteins were suspected to be peroxisomal. In this study, an alternative approach for the analysis of the peroxisomal proteome was established, which combines protein separation during a 1-D SDS-PAGE with MALDI-MS and LC-ESI-MS. It was possible to identify 66 proteins, 25 peroxisomal and 17 proteins of not yet known localization Functional analysis The localization of newly identified proteins was confirmed by double- fluorescence microscopy using GFP fusion proteins in conjunction with a peroxisomal red fluorescent protein marker (PTS2DsRed) as well as by subcellular fractionation studies. To reveal the function of newly identified proteins, corresponding knockout strains were analyzed phenotypically for growth on various carbon sources. It was shown, that Yor084wp functions as an oleic acid-inducible putative peroxisomal lipase/esterase. Import in the peroxisomal membrane is Pex5p-dependent. The observed phenotype of the analyzed mutant allows the conclusions that the corresponding gene has no effect of fatty acid degradation. The putative 2-hydroxyphytanoyl CoA lyase Yel020cp belongs to the peroxisomal, Pex5p-dependent imported matrix proteins. The protein possesses a thiamin-pyrophosphate-binding site. The gene seems to be not essential for peroxisomal biogenesis. Ygl184cp functions as a putative cystathionine b-lyase and in this study it was detected at the peroxisomal membrane. Yir034cp is a protein of the peroxisomal matrix that is imported in the organelle because of its PTS1-sequence. Its role in the biosynthesis of lysine leads to the hypothesis, that this kind of amino acid pathway is localized in yeast peroxisomes. For the acetyl-CoA hydrolase Ybl015wp, it was shown that it is imported in peroxisomes and in mitochondria because of the potential N-terminal peroxisomal (PTS2) as well as the mitochondrial targeting signal (MTS). Ycr091wp is also present in mitochondria and peroxisomes and functions presumably as a putative serine/threonine kinase in posttranslational modifications. In this study, the mitochondrial localization of the unknown protein Yor228cp was shown and three transmembrane domains were detected by in silico analysis, that led to the assumption that it is localized in the mitochondrial membrane. Sfc1p/Yjr095wp is a member of the mitochondrial carrier family (MCF) and works as a succinate-fumarate transporter. A second, peroxisomal localization was not found. Yhr199cp was also found in mitochondria because of a mitochondria targeting signal, but its cellular role was not analyzed. The suspected peroxisomal localization of the Yal054cp could be disproved. The observed subcellular localization gave rise to the suspicion that it is localized in ER. 22 proteins were not peroxisomal, indicating that minor contaminations of the preparation were also detected due to the sensitivity of the method. Pex11p In this study, the putative function of Pex11p as a voltage-dependent channel in the peroxisomal membrane could not be demonstrated. The expression of the yeast Pex11p as a TAP-fusion protein was successful and the protein can be purified by affinity columns and than used for further functional analysis

The DaNa2.0 Knowledge Base Nanomaterials—An Important Measure Accompanying Nanomaterials Development

Nanotechnology is closely related to the tailored manufacturing of nanomaterials for a huge variety of applications. However, such applications with newly developed materials are also a reason for concern. The DaNa2.0 project provides information and support for these issues on the web in condensed and easy-to-understand wording. Thus, a key challenge in the field of advanced materials safety research is access to correct and reliable studies and validated results. For nanomaterials, there is currently a continuously increasing amount of publications on toxicological issues, but criteria to evaluate the quality of these studies are necessary to use them e.g., for regulatory purposes. DaNa2.0 discusses scientific results regarding 26 nanomaterials based on actual literature that has been selected after careful evaluation following a literature criteria checklist. This checklist is publicly available, along with a selection of standardized operating protocols (SOPs) established by different projects. The spectrum of information is rounded off by further articles concerning basics or crosscutting topics in nanosafety research. This article is intended to give an overview on DaNa2.0 activities to support reliable toxicity testing and science communication alike