Changes of Protein Expression after CRISPR/Cas9 Knockout of miRNA-142 in Cell Lines Derived from Diffuse Large B-Cell Lymphoma

Background: As microRNA-142 (miR-142) is the only human microRNA gene where mutations have consistently been found in about 20% of all cases of diffuse large B-cell lymphoma (DLBCL), we wanted to determine the impact of miR-142 inactivation on protein expression of DLBCL cell lines. Methods: miR-142 was deleted by CRISPR/Cas9 knockout in cell lines from DLBCL. Results: By proteome analyses, miR-142 knockout resulted in a consistent up-regulation of 52 but also down-regulation of 41 proteins in GC-DLBCL lines BJAB and SUDHL4. Various mitochondrial ribosomal proteins were up-regulated in line with their pro-tumorigenic properties, while proteins necessary for MHC-I presentation were down-regulated in accordance with the finding that miR-142 knockout mice have a defective immune response. CFL2, CLIC4, STAU1, and TWF1 are known targets of miR-142, and we could additionally confirm AKT1S1, CCNB1, LIMA1, and TFRC as new targets of miR-142-3p or -5p. Conclusions: Seed-sequence mutants of miR-142 confirmed potential targets and novel targets of miRNAs can be identified in miRNA knockout cell lines. Due to the complex contribution of miRNAs within cellular regulatory networks, in particular when miRNAs highly present in RISC complexes are replaced by other miRNAs, primary effects on gene expression may be covered by secondary layers of regulation

Influence of medicinal plants on vascular smooth muscle cell proliferation

Arteriosklerose ist die zugrundeliegende Ursache für Erkrankungen, die weltweit die meisten Todesfälle verursachen. Zu diesen zählen koronare Herzkrankheiten und Schlaganfälle. Die Akkumulation und Einwanderung von vaskulären glatten Muskelzellen (VSMC) in die Intima, sowie deren starke Produktion an extrazellulärer Matrix trägt zum Prozess der Angiostenose (Gefäßverengung) bei. Eine Strategie zur Entwicklung neuer Medikamente und Therapien ist die Zellzyklusregulation von glatten Muskelzellen. Das primäre Ziel dieser Arbeit war, die Identifizierung und Charakterisierung von Naturstoffen, die die Proliferation von VSMC inhibieren. Diese Arbeit wurde als Teil des nationalen Forschungsnetzwerkes „Drugs from Nature Targeting Inflammation“ (DNTI), welches vom FWF (Wissenschaftsfond) finanziert wird, durchgeführt. Dieses interdisziplinäre Netzwerk verbindet das Wissen und Können verschiedener Bereiche aus Pharmazie, Chemie, Veterinär- und Humanmedizin und verknüpft Universitäten in ganz Österreich. Im Zuge des Projektes wurden Pflanzen einerseits durch „Pharmacophore modelling“, also durch einen computergestützten Ansatz ausgewählt. Andererseits basierte die Auswahl auf einem ethnopharmakologischen Ansatz, welcher auf der Auswertung traditionellen Wissens beruht. Das Screening von 150 verschiedenen Pflanzenextrakten mittels ‚Resazurin Conversion assay‘ ergab einige interessante Ergebnisse. Folgende Pflanzenextrakte konnten das VSMC Wachstum hemmen; Sideritis hyssopifolia, Aristolochia debilis, Stephania tetrandra, Melia toosendan und Peucedanum ostruthium. In einer parallelen Studie wurde von unseren Partnern eine quantitative und qualitative Analyse der Cumarine von Peucedanum ostruthium durchgeführt und die 7 bedeutendsten Cumarine identifiziert. Diese Cumarine wurden uns zur Verfügung gestellt und via Resazurin Assay überprüft. Dabei wurde Ostruthin als die aktive Substanz identifiziert. Die Untersuchung von strutkurell ähnlichen Furanocumarinen führte zum Schluss dass die Geranyl–Gruppe an der C-6 Position für den inhibierenden Effekt notwendig ist. Somit konnten wir die antiproliferative Wirkung des Peucedanum ostruthium Extraktes identifizieren und eine neue Bioaktivität von Ostruthin enthüllen.Atherosclerosis is the underlying cause of the diseases responsible for the most deaths worldwide, including ischemic heart disease and cerebrovascular disease. The accumulation of vascular smooth muscle cells (VSMC) within the intima of the arterial walls and the increase in extracellular matrix they produce, contributes to vessel narrowing in the pathogenesis of atherosclerosis and restenosis. One strategy for the treatment of vascular disease is to target cell cycle regulation of smooth muscle cells. The primary aim of this work is the identification and characterization of compounds which derive from natural sources and are capable to inhibit VSMC proliferation. This work was done as part of the national research network ‘drugs from nature targeting inflammation (DNTI)’, project an interdisciplinary network, which interlinks the knowledge and the skills of members of pharmacy, chemistry, veterinarian and human medicine from different universities in Austria. The plants were selected either by a computational approach using pharmacophore models of relevant target proteins or an ethnopharmacological approach using traditional knowledge. The screening of about 150 different plant extracts via the resazurin conversion assay yielded several interesting candidate plant extracts, which inhibited the growth of VSMC, including Sideritis hyssopifolia, Aristolochia debilis, Stephania tetrandra, Melia toosendan and Peucedanum ostruthium. Furthermore, in a parallel study, a comprehensive qualitative and quantitative analysis of the main coumarins in extracts from P. ostruthium was performed by our collaboration partners at the University of Vienna and led to the identification of 7 major coumarins in those extracts. These coumarins were further forwarded to our group and testing via the resazurin conversion method revealed that the active principle inhibiting the growth of VSMC is ostruthin. The investigation of structurally similar linear furanocoumarins led to the conclusion that the geranyl group at the C-6 position is necessary for the inhibitory effect. Thus, we identified the anti-proliferative principle of the Peucedanum ostruthium extract and uncovered a novel bioactivity for ostruthin

Selective egg cell polyspermy bypasses the triploid block

Mao Y, Gabel A, Nakel T, et al. Selective egg cell polyspermy bypasses the triploid block. eLife. 2020;9: e52976.Polyploidization, the increase in genome copies, is considered a major driving force for speciation. We have recently provided the first direct in planta evidence for polyspermy induced polyploidization. Capitalizing on a novel sco1-based polyspermy assay, we here show that polyspermy can selectively polyploidize the egg cell, while rendering the genome size of the ploidy-sensitive central cell unaffected. This unprecedented result indicates that polyspermy can bypass the triploid block, which is an established postzygotic polyploidization barrier. In fact, we here show that most polyspermy-derived seeds are insensitive to the triploid block suppressor admetos. The robustness of polyspermy-derived plants is evidenced by the first transcript profiling of triparental plants and our observation that these idiosyncratic organisms segregate tetraploid offspring within a single generation. Polyspermy-derived triparental plants are thus comparable to triploids recovered from interploidy crosses. Our results expand current polyploidization concepts and have important implications for plant breeding.</jats:p

Long time persistence and evolution of carbapenemase-producing Enterobacterales in the wastewater of a tertiary care hospital in Germany

Background: Worldwide observations revealed increased frequencies of multi-resistant Enterobacterales and resistance genes in hospital wastewater compared to any other type of wastewater. Despite the description of clonal lineages possibly adapted to hospital wastewater, little is known about long term persistence as well as evolution of these lineages. Methods: In this study, wastewater isolates of different Enterobacterales species from a tertiary care hospital were investigated with 2.5 years distance. Whole Genome Sequencing (WGS) and resistance gene identification were performed for E. coli, C. freundii, S. marcescens, K. pneumoniae, K. oxytoca, and E. cloacae isolates (n = 59), isolated in 2022 and compared with strains isolated from the same wastewater pipeline in 2019 (n = 240). Results: Individual clonal lineages with highly related isolates could be identified in all species identified more than once in 2022 that appear to persist in the wastewater drainage. A common motif of all persistent clonal lineages was the carriage of mobile genetic elements encoding carbapenemase genes with hints for horizontal gene transfer in persistent clones in this environment observed over the 2.5-year period. Multiple plasmid replicons could be detected in both years. In 2022 isolates blaVIM-1 replaced blaOXA-48 as the most common carbapenemase gene compared to 2019. Interestingly, despite a similar abundance of carbapenemase genes (>80% of all isolates) at both time points genes encoding extended spectrum β-lactamases decreased over time. Conclusions: This data indicates that hospital wastewater continuously releases genes encoding carbapenemases to the urban wastewater system. The evolution of the resident clones as well as the reasons for the selection advantage in this specific ecological niche needs to be further investigated in the future

Identification of Ostruthin from <i>Peucedanum ostruthium</i> Rhizomes as an Inhibitor of Vascular Smooth Muscle Cell Proliferation

Inhibition of vascular smooth muscle cell (VSMC) proliferation is of substantial interest in combating cardiovascular disease. A dichloromethane extract from the rhizomes of <i>Peucedanum ostruthium</i>, a traditionally used Austrian medicinal plant with anti-inflammatory properties, was examined for a putative antiproliferative activity in rat aortic VSMC. This extract inhibited serum (10%)-induced VSMC proliferation concentration dependently. Further identification and biological testing of its major constituents revealed that the coumarin ostruthin (<b>7</b>) is the major antiproliferative substance. In summary, a new bioactivity of <i>P. ostruthium</i> rhizomes is described, and <b>7</b> has been identified as the responsible compound