Crosslinking of Dam methyltransferase with S-adenosyl-methionine

AbstractHighly purified DNA-adenine methyltransferase was irradiated in the presence of different concentrations of radiolabelled S-adenosyl-methionine (AdoMet) with a conventional Mineralight UV-lamp from several minutes up to 1 h while incubating in ice. Incorporation of radioactivity was monitored by electrophoresis of the crosslink between S-adenosyl-methionine and Dam methylase on SDS-polyacrylamide gels followed by fluorography. Crosslinking reached a maximum in presence of 10 μM S-adenosyl-methionine; it was inhibited in the presence of substates which competitively inhibit methylation of DNA by Dam methylase, like sinefungin or S-adenosyl-homocysteine, but not in the presence of non-inhibitors like ATP or S-isobutyl-adenosine. The crosslink obtained was resistant against a wide range of even drastic conditions commonly used in protein and peptide chemistry. Proteins which do not bind S-adenosyl-methionine, as well as heat activated Dam methylase were not photolabelled. After limited proteolysis the radioactive label appeared only in certain of the peptides obtained. From Western blots carried out with polyclonal antibodies produced against a synthetic peptide corresponding in its sequence to amino acids 92-106 of the Dam methylase, the crosslinking of AdoMet could be tentatively mapped at a position after amino acid 106

Entwicklung und Darstellung des Online-Kurses : „DNA-Schnupperkurs – Knochenmarkspende & Leukämie“

Dieser Beitrag thematisiert eine Möglichkeit, Schüler*innen der 9. und 10. Jahrgangsstufe einen kleinen Einblick in das Thema der Leukämie und der Stammzellenspende zu geben. Der experimentelle Fokus wird dabei auf die DNA-Extraktion gelegt, ohne diese im Labor selbst durchzuführen. Die Thematik  wird während des Online-Kurses hauptsächlich durch interaktive Inhalte vermittelt. Der Kurs lässt sich sowohl in der Schule synchron mit der gesamten Klasse, als auch asynchron – in Form von Homeschooling oder einer individuellen Fördermaßnahme – bearbeiten

Hole dynamics in a photovoltaic donor-acceptor couple revealed by simulated time-resolved X-ray absorption spectroscopy

Theoretical and experimental methodologies that can characterize electronic and nuclear dynamics, and the coupling between the two, are needed to understand photoinduced charge transfer in molecular building blocks used in organic photovoltaics. Ongoing developments in ultrafast pump-probe techniques such as time-resolved X-ray absorption spectroscopy, using an X-ray free electron laser in combination with an ultraviolet femtosecond laser, present desirable probes of coupled electronic and nuclear dynamics. In this work, we investigate the charge transfer dynamics of a donor-acceptor pair, which is widely used as a building block in low bandgap block copolymers for organic photovoltaics. We simulate the dynamics of the benzothiadiazole-thiophene molecule upon photoionization with a vacuum ultraviolet (VUV) pulse and study the potential of probing the subsequent charge dynamics using time-resolved X-ray absorption spectroscopy. The photoinduced dynamics are calculated using on-the-fly nonadiabatic molecular dynamics simulations based on Tully's Fewest Switches Surface Hopping approach. We calculate the X-ray absorption spectrum as a function of time after ionization at the Hartree-Fock level. The changes in the time-resolved X-ray absorption spectrum at the sulfur K-edge reveal the ultrafast charge carrier dynamics in the molecule occurring on a femtosecond time scale. These theoretical findings anticipate that ultrafast time-resolved X-ray absorption spectroscopy using an X-ray probe in combination with a VUV pump offers a new approach to investigate the detailed dynamics of organic photovoltaic materials

Seasonal Variations in the Metabolome and Bioactivity Profile of Fucus vesiculosus Extracted by an Optimised, Pressurised Liquid Extraction Protocol

The metabolism of seaweeds depends on environmental parameters, the availability of nutrients, and biotic/abiotic stresses; therefore, their chemical composition fluctuates throughout the year. This study investigated seasonal variations in the metabolome of the Baltic Sea brown alga Fucus vesiculosus and its potential relation to the bioactivity profile. By using a definitive screening design (DSD) combined with pressurised liquid extraction (PLE), an optimised protocol was developed to extract algal biomass monthly for a full calendar year. An untargeted metabolomics approach using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MSn)-based molecular networking and manual dereplication was employed. The extracts were simultaneously screened for their in vitro antimicrobial, anticancer/apoptotic, and free radical scavenging activities. 44 compounds were putatively dereplicated in the metabolome. Many compounds were found to vary with the sampling month; phlorotannin total ion count (TIC) was highest in summer, whilst chlorophylls, lipids, and carotenoids peaked in winter and spring. The greatest radical scavenging and apoptotic activities against pancreas cancer cells observed in the summer months were attributed to high phlorotannin TIC. Methicillin-resistant Staphylococcus aureus (MRSA) inhibitory activity was produced year-round without a clear seasonal trend. This is the first study applying DSD-based optimised PLE extraction combined with a metabolome analysis of F. vesiculosus for the identification of seasonal variations in both metabolome and bioactivity

Incidence of central retinal artery occlusion peaks in winter season

IntroductionStroke incidence exhibits seasonal trends, with the highest occurrences observed during winter. This study investigates the incidence of central retinal artery occlusion (CRAO), a stroke equivalent of the retina, and explores its monthly and seasonal variations, as well as potential associations with weather and ambient air pollutants.MethodsA retrospective search of medical records spanning 15 years (January 2008–December 2022) was conducted at the University Eye Hospital Tübingen, Germany, focusing on diagnosed cases of CRAO. Incidences were evaluated on a monthly and seasonal basis (winter, spring, summer, fall). Weather data (temperature, precipitation, atmospheric pressure) and concentrations of ambient air pollutants [fine particulate matter (PM2.5), coarse particulate matter (PM10), nitrogen dioxide (NO2), and ozone (O3)], were analyzed for a potential association with CRAO incidence.ResultsOut of 432 patients diagnosed with CRAO between 2008 and 2022, significantly varying incidences were observed monthly (p = 0.025) and seasonally (p = 0.008). The highest rates were recorded in February and winter, with the lowest rates in June and summer. Concentrations of NO2, PM2.5 and lower ambient air temperature (average, minimum, maximum) showed significant correlations with CRAO incidence.DiscussionThis comprehensive 15-year analysis reveals a pronounced winter peak in CRAO incidence, with the lowest occurrences in summer. Potential associations between CRAO incidence and ambient air pollutants and temperature underscore the importance of considering seasonal trends and call for further investigations to elucidate contributing factors, potentially leading to targeted preventive strategies and public health interventions

Tissue Viability of Free Flaps after Extracorporeal Perfusion Using a Modified Hydroxyethyl Starch Solution

Background: In free flap surgery, tissue is stored under hypothermic ischemia. Extracorporeal perfusion (EP) has the potential to extend storage time and the tissue's perspective of survival. In the present study, the aim is to improve a recently established, simplified extracorporeal perfusion system. Methods: Porcine musculus rectus abdominis were stored under different conditions. One group was perfused continuously with a simplified one-way perfusion system for six hours, while the other received only a single flush but no further treatment. A modified hydroxyethyl starch solution was used as a perfusion and flushing solution. Vitality, functionality, and metabolic activity of both groups were analyzed. Results: Perfused muscles, in contrast to the ischemically stored ones, showed no loss of vitality and significantly less functionality loss, confirming the superiority of storage under continuous perfusion over ischemic storage. Furthermore, in comparison to a previous study, the results were improved even further by using a modified hydroxyethyl starch solution. Conclusion: The use of EP has major benefits compared to the clinical standard static storage at room temperature. Continuous perfusion not only maintains the oxygen and nutrient supply but also removes toxic metabolites formed due to inadequate storage conditions

Engraftment of engineered ES cell–derived cardiomyocytes but not BM cells restores contractile function to the infarcted myocardium

Cellular cardiomyoplasty is an attractive option for the treatment of severe heart failure. It is, however, still unclear and controversial which is the most promising cell source. Therefore, we investigated and examined the fate and functional impact of bone marrow (BM) cells and embryonic stem cell (ES cell)–derived cardiomyocytes after transplantation into the infarcted mouse heart. This proved particularly challenging for the ES cells, as their enrichment into cardiomyocytes and their long-term engraftment and tumorigenicity are still poorly understood. We generated transgenic ES cells expressing puromycin resistance and enhanced green fluorescent protein cassettes under control of a cardiac-specific promoter. Puromycin selection resulted in a highly purified (>99%) cardiomyocyte population, and the yield of cardiomyocytes increased 6–10-fold because of induction of proliferation on purification. Long-term engraftment (4–5 months) was observed when co-transplanting selected ES cell–derived cardiomyocytes and fibroblasts into the injured heart of syngeneic mice, and no teratoma formation was found (n = 60). Although transplantation of ES cell–derived cardiomyocytes improved heart function, BM cells had no positive effects. Furthermore, no contribution of BM cells to cardiac, endothelial, or smooth muscle neogenesis was detected. Hence, our results demonstrate that ES-based cell therapy is a promising approach for the treatment of impaired myocardial function and provides better results than BM-derived cells