Lipokonjugate als neues Werkzeug zur mikrostrukturierten Funktionalisierung von Oberflächen in biochemischen und zellulären Assays

Lipokonjugate wurden als neue Strategie für eine mikrostrukturierte Funktionalisierung von Oberflächen etabliert. Niedermolekulare Bausteine, wie Haptene und Peptide werden kovalent an eine Pam3Cys (N-Palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cystein) Ankergruppe gekoppelt. Wässrige Lösungen dieser mit drei Acylresten modifizierten Lipoaminosäure werden auf hydrophob silanisierte Glasträger aufpipettiert. Durch den Einsatz dieses mit drei Acylresten substituierten Lipides wird der Abstand zwischen den funktionellen Endgruppen erhöht. Auf diese Weise wird eine optimale Zugänglichkeit für die Erkennung durch Biomoleküle wie z. B. Antikörper gewährleistet, ohne dass weitere nicht-funktionalisierte Lipopeptide zugesetzt werden müssen. Die Immobilisierung der Lipokonjugate auf dem Substrat erfolgt nicht-kovalent. Im Gegensatz zu Strategien, bei denen die Anbindung kovalent erfolgt, besteht kein Risiko, dass durch Verlust reaktiver Gruppen die biologische Aktivität der Testsubstanz beeinflusst werden könnte. Trotz des Fehlens einer kovalenten Anknüpfung ist die Funktionalisierung mit Lipokonjugaten stabil gegenüber den Bedingungen biochemischer Bindungsassays und sogar gegenüber Zellwachstum. Die Anwendbarkeit im Screening von Molekülkollektionen auf Proteinbindung wurde anhand der Bindung von fluoreszent markiertem Streptavidin und indirekte Immunfluoreszenz mit Antikörpern gegen Peptid-Lipokonjugate gezeigt (Abb. 1). In Hinblick auf die Anwendung in zellbiologischen Microarrays wurde die Biokompatibilität der Oberflächen für das Wachstum von adhärenten Zellkulturzellen demonstriert (Abb. 2). Ferner konnte durch Funktionalisierung mit Antikörpern, die Oberflächenmoleküle von Zellen in Suspension erkennen, eine spezifische Anheftung der Zellen an die Oberflächen erreicht werden

Investigating nucleo-cytoplasmic shuttling of the human DEAD-box helicase DDX3

The human DEAD-box helicase DDX3 is a multi-functional protein involved in the regulation of gene expression and additional non-conventional roles as signalling adaptor molecule that are independent of its enzymatic RNA remodeling activity. It is a nucleo-cytoplasmic shuttling protein and it has previously been suggested that dysregulation of its subcellular localization could contribute to tumourigenesis. Indeed, both tumour suppressor and oncogenic functions have been attributed to DDX3. In this study, we investigated the regulation of DDX3's nucleocytoplasmic shuttling. We confirmed that an N-terminal conserved Nuclear Export Signal (NES) is required for export of human DDX3 from the nucleus, and identified three regions within DDX3 that can independently facilitate its nuclear import. We also aimed to identify conditions that alter DDX3's subcellular localisation. Viral infection, cytokine treatment and DNA damage only induced minor changes in DDX3's subcellular distribution as determined by High Content Analysis. However, DDX3's nuclear localization increased in early mitotic cells (during prophase) concomitant with an increase in DDX3 expression levels. Our results are likely to have implications for the proposed use of (nuclear) DDX3 as a prognostic biomarker in cancer

Peptide microarrays for the profiling of cytotoxic T-lymphocyte activity using minimum numbers of cells

The identification of epitopes that elicit cytotoxic T-lymphocyte activity is a prerequisite for the development of cancer-specific immunotherapies. However, especially the parallel characterization of several epitopes is limited by the availability of T cells. Microarrays have enabled an unprecedented miniaturization and parallelization in biological assays. Here, we developed peptide microarrays for the detection of CTL activity. MHC class I-binding peptide epitopes were pipetted onto polymer-coated glass slides. Target cells, loaded with the cell-impermeant dye calcein, were incubated on these arrays, followed by incubation with antigen-expanded CTLs. Cytotoxic activity was detected by release of calcein and detachment of target cells. With only 200,000 cells per microarray, CTLs could be detected at a frequency of 0.5% corresponding to 1,000 antigen-specific T cells. Target cells and CTLs only settled on peptide spots enabling a clear separation of individual epitopes. Even though no physical boundaries were present between the individual spots, peptide loading only occurred locally and cytolytic activity was confined to the spots carrying the specific epitope. The peptide microarrays provide a robust platform that implements the whole process from antigen presentation to the detection of CTL activity in a miniaturized format. The method surpasses all established methods in the minimum numbers of cells required. With antigen uptake occurring on the microarray, further applications are foreseen in the testing of antigen precursors that require uptake and processing prior to presentation

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Enhanced sediment purification techniques for the <10 µm fraction of diatom silica and a comparison of contamination assessment before analysing oxygen isotopes

The analysis of oxygen isotopes of biogenic silica as a paleolimnological-proxy is of growing interest. In lacustrine environments the main particle and diatom size is often to be found in the 95 %, n=6). The detection limit for this analysis is comparably high (app. 0.1 %) but still suitable for this purpose to verify samples having an SiO2 content greater than app. 97 %. The preparation process for the ICP-OES is timeconsuming but the method proved to give more accurate results (1&#963; 95 %, n=4). No significant difference could be detected when comparing the analysis of 50 mg to 10 mg of the same material using ICP-OES. The optical methods do not provide exact, quantified results for the lesser than 10 &#956;m fraction. The EDX can be combined with the optical SEM for detail shots and is the most recommended analysis after this comparison: It is the quickest method and needs less than 0.5 mg weighted sample. Still, its precision is good enough for the purpose of assessing the degree of contamination to implement potential correction factors. The updated protocol for removing contaminants in the <10 &#956;m fraction is able to gain a SiO2 content greater than 97% (analysis by EDX). When looking at the development of the isotopic composition an asymptotic approximation towards &#948;18O values not being influenced by contaminants is expected. First results will be presented at the Paleolimnology Symposium

3.Advertising

Advertising information used for promoting the BQ4 main study

2.Presentations

Presentations used for promoting the BQ4 pilot experiments

Overview_pilots

Overview of all the pilot experiments

III Data Retrieval

Scripts to retrieve data from the Big Question 4 Frinex test