Strategies to optimize cationic polymers for gene delivery via endosomal escape

Cationic polymers with different backbones were characterized regarding their performance at different steps of the gene delivery process. The interaction with plasmid DNA (pDNA), cytotoxicity, uptake of pDNA, endosomal escape and protein expression in different cell lines were investigated using methods like fluorometric assays, dynamic light scattering, flow cytometry and confocal laser scanning microscopy. Starting with homopolymers of different degrees of polymerization and different cationic moieties, a positive correlation between toxicity and endosomal escape was demonstrated. By combination with hydrophobic functionalities either within block copolymers resulting in cationic micelles or within statistic copolymers, the transfection efficiency was increased even in hard-to-transfect leukemia cells. To reduce the accompanying toxicity, further hydrophilic neutral and/or anionic functionalities were introduced either within block copolymers or as an additional layer outside the preformed cationic micelles. Different factors influenced the efficiency-toxicity ratio of the polymers, such as the ratio, type and combination method of cationic and anionic/hydrophilic moieties. The introduction of only anionic moieties was most promising and led to an increase in viability while maintaining high transfection efficiencies comparable to the cationic micelle. The combination of cationic micelle and anionic-hydrophilic block copolymer exhibited the highest shielding efficiency with decreased but still moderate transfection efficiency, rendering it suitable for long circulation times and targeted delivery. This thesis illustrates the high suitability of cationic polymers for gene delivery and their optimization in different parameters towards high cell viability while maintaining superior gene delivery efficiency. Hence, polymers represent promising tools for future gene therapy and further (in vivo) studies are required to transfer these results to humans

Not just by means alone: why the evolution of distribution shapes matters for understanding opinion dynamics. The case of the French reaction to the war in Ukraine

Understanding the dynamics of citizens' opinions, preferences, perceptions, and attitudes is pivotal in political science and essential for informed policymaking. Although highly sophisticated tools have been developed for analyzing these dynamics through surveys, outside the field of polarization, these analyses often focus on average responses, thereby missing important information embedded in other parameters of data distribution. Our study aims to fill this gap by illustrating how analyzing the evolution of both the mean and the distribution shape of responses can offer complementary insights into opinion dynamics. Specifically, we explore this through the French public's perception of defense issues, both before and after the onset of the war in Ukraine. Our findings underscore how routinely combining classical approaches with the use of existing tools for measuring distribution shapes can provide valuable perspectives for researchers and policymakers alike, by highlighting the nuanced shifts in public opinion that traditional methods might overlook

Tracking the Endosomal Escape: A Closer Look at Calcein and Related Reporters

Crossing the cellular membrane and delivering active pharmaceuticals or biologicals into the cytosol of cells is an essential step in the development of nanomedicines. One of the most important intracellular processes regarding the cellular uptake of biologicals is the endolysosomal pathway. Sophisticated nanocarriers are developed to overcome a major hurdle, the endosomal entrapment, and delivering their cargo to the required site of action. In parallel, in vitro assays are established analyzing the performance of these nanocarriers. Among them, the release of the membrane‐impermeable dye calcein has become a popular and straightforward method. It is accessible for most researchers worldwide, allows for rapid conclusions about the release potential, and enables the study of release mechanisms. This review is intended to provide an overview and guidance for scientists applying the calcein release assay. It comprises a survey of several applications in the study of endosomal escape, considerations of potential pitfalls, challenges, and limitations of the assay, and a brief summary of complementary methods. Based on this review, it is hoped to encourage further research groups to take advantage of the calcein release assay for their own purposes and help to create a database for more efficient cross‐correlations between nanocarriers

The SAF-box domain of chromatin protein DEK

DEK is an abundant chromatin protein in metazoans reaching copy numbers of several millions/nucleus. Previous work has shown that human DEK, a protein of 375 amino acids, has two functional DNA-binding domains, of which one resides in a central part of the molecule and contains sequences corresponding to the scaffold attachment factor-box (SAF-box) domain as found in a growing number of nuclear proteins. Isolated SAF-box peptides (amino acids 137–187) bind weakly to DNA in solution, but when many SAF-box peptides are brought into close proximity on the surface of Sephadex beads, cooperative effects lead to a high affinity to DNA. Furthermore, a peptide (amino acids 87–187) that includes a sequence on the N-terminal side of the SAF-box binds efficiently to DNA. This peptide prefers four-way junction DNA over straight DNA and induces supercoils in relaxed circular DNA just like the full-length DEK. Interestingly, however, the 87–187 amino acid peptide introduces negative supercoils in contrast to the full-length DEK, which is known to introduce positive supercoils. We found that two adjacent regions (amino acids 68–87 and 187–250) are necessary for the formation of positive supercoils. Our data contribute to the ongoing characterization of the abundant and ubiquitous DEK chromatin protein

Behandlungseffekte der repetitiven transkraniellen Magnetstimulation (rTMS) bei Patienten mit chronischem Tinnitus aurium: Erste Ergebnisse der Behandlung mit niederfrequenter rTMS

Einleitung: In der Bundesrepublik Deutschland erkranken jährlich über 350.000 Patienten behandlungsbedürftig an Tinnitus. Die repetitive transkranielle Magnetstimulation (rTMS) wird in den letzten Jahren in verschiedenen Kliniken innerhalb einer Multicenterstudie als eine innovative technische Behandlungsmethode bei chronischen Ohrgeräuschen erprobt. Methode: An einer Stichprobe von 28 ambulanten Patienten mit chronischem Tinnitus aurium wurde die Wirksamkeit der niederfrequenten Magnetstimulation (1 Hz) bei Tinnitus im Rahmen der Routineversorgung überprüft. Ergebnisse: Im Prä-Post-Vergleich zeigte sich im Stichprobendurchschnitt eine 7 Punkte umfassende Reduktion des Tinnitus-Wertes, erfasst anhand des Tinnitus-Fragebogens (TF, Göbel und Hiller) sowie eine signifikante Reduktion der depressiv gefärbten Stimmung der Patienten im Fremd- (MÅDRS) wie im Selbsturteil (BDI II). 57% der mit rTMS Behandelten konnten als Responder eingestuft werden (Response-Kriterium: Abnahme des TF-Scores um mindestens 5 Punkte). Schlussfolgerung: Die Ergebnisse erlauben mit Einschränkungen einen positiven Ausblick in die Zukunft der rTMS in der Tinnitus-Behandlung

Editorial: Intracellular Mechanisms of α-Synuclein Processing

Editorial on the Research Topic Intracellular Mechanisms of α-Synuclein Processin

Treatment of Focal Cartilage Defects in Minipigs with Zonal Chondrocyte/Mesenchymal Progenitor Cell Constructs

Despite advances in cartilage repair strategies, treatment of focal chondral lesions remains an important challenge to prevent osteoarthritis. Articular cartilage is organized into several layers and lack of zonal organization of current grafts is held responsible for insufficient biomechanical and biochemical quality of repair-tissue. The aim was to develop a zonal approach for cartilage regeneration to determine whether the outcome can be improved compared to a non-zonal strategy. Hydrogel-filled polycaprolactone (PCL)-constructs with a chondrocyte-seeded upper-layer deemed to induce hyaline cartilage and a mesenchymal stromal cell (MSC)-containing bottom-layer deemed to induce calcified cartilage were compared to chondrocyte-based non-zonal grafts in a minipig model. Grafts showed comparable hardness at implantation and did not cause visible signs of inflammation. After 6 months, X-ray microtomography (_CT)-analysis revealed significant bone-loss in both treatment groups compared to empty controls. PCL-enforcement and some hydrogel-remnants were retained in all defects, but most implants were pressed into the subchondral bone. Despite important heterogeneities, both treatments reached a significantly lower modified O’Driscoll-score compared to empty controls. Thus, PCL may have induced bone-erosion during joint loading and misplacement of grafts in vivo precluding adequate permanent orientation of zones compared to surrounding native cartilage. © 2019 by the authors. Licensee MDPI, Basel, Switzerland

On the state of implementation of the provisions in the National Action Plan for the Sustainable Use of Plant Protection Products in the field of Minor Uses

Im Nationalen Aktionsplan zur nachhaltigen Anwendung von Pflanzenschutzmitteln (NAP) hat die Bundesregierung für den Bereich Lückenindikationen/Sonderkulturen und für den Vorratsschutz zur Verbesserung der Verfügbarkeit von Pflanzenschutzmittel folgendes Ziel formuliert: „in 80% aller relevanten Anwendungsgebiete stehen bis zum Jahr 2023 min­destens 3 Wirkstoffgruppen zur Verfügung“. Im Rahmen der Bund-Länder Arbeitsgruppe Lückenindikationen (BLAG-LÜCK) führten die zugehö­rigen Unterarbeitsgruppen Lückenindikationen (UAG) und das Julius Kühn-Institut (JKI) im Jahr 2017 eine Analyse zum Ist-Stand für alle Sparten der UAG durch. Als Grund­lage für die Analyse wurden von den UAG insgesamt 194 relevante Anwendungsgebiete ausgewählt, für die das JKI zum Stichtag, dem 3. Oktober 2016, ins­gesamt 1.058 Zulassungen ermittelte. Diese Zulassungen wurden hinsichtlich ihrer „Passfähigkeit“ zu den Anwendungsgebieten bezüglich Kultur, Schadorganismus und Anwendungsbedingungen eingeteilt. Nachfolgend schätz­ten die UAG aufgrund ihrer fachlichen Expertise ein, ob mit den ausgewiesenen Zulassungen und den damit vorhandenen Wirkstoffklassen die „Lücken geschlos­sen“ sind oder nicht. Das Ergebnis der Analyse belegt, dass die Zielquote des NAP „mindestens 3 Wirkstoffgruppen stehen zur Verfügung“ derzeit bei keiner Sparte erreicht wird.In the National Action Plan for the Sustainable Use of Plant Protection Products (NAP), the following goal was formulated by the Federal Government for the area of minor uses/speciality crops and for storage protection to improve the availability of pesticides: “in 80% of all relevant uses at least 3 groups of active substances are available by 2023”. Within the scope of the German Bund-Länder Working Group on Minor Uses, the associated Sub-Working Groups Minor Uses (UAG) and the Julius Kühn-Institute (JKI) conducted an analysis of the current status in all crop sectors in 2017. For this purpose, a total of 194 relevant uses were designated by the UAG, for which the JKI identified 1,058 different authorisations at a key date, the 3rd of October 2016. These approvals were grouped according to their suitability for the selected uses regarding crop, pest and application conditions. Subsequently, based on their technical expertise, the UAG assessed whether or not the “gaps are closed” with the approved registrations and the classes of active substances they contain. The result proves that the target ratio of the NAP “at least 3 active ingredient groups available” is currently not achieved in any crop sector

Comparative study of virus and lymphocyte distribution with clinical data suggests early high dose immunosuppression as potential key factor for the therapy of patients with BoDV-1 infection

Borna disease virus 1 (BoDV-1) was just recently shown to cause predominantly fatal encephalitis in humans. Despite its rarity, bornavirus encephalitis (BVE) can be considered a model disease for encephalitic infections caused by neurotropic viruses and understanding its pathomechanism is of utmost relevance. Aim of this study was to compare the extent and distribution pattern of cerebral inflammation with the clinical course of disease, and individual therapeutic procedures. For this, autoptic brain material from seven patients with fatal BVE was included in this study. Tissue was stained immunohistochemically for pan-lymphocytic marker CD45, the nucleoprotein of BoDV-1, as well as glial marker GFAP and microglial marker Iba1. Sections were digitalized and counted for CD45-positive and BoDV-1-positive cells. For GFAP and Iba1, a semiquantitative score was determined. Furthermore, detailed information about the individual clinical course and therapy were retrieved and summarized in a standardized way. Analysis of the distribution of lymphocytes shows interindividual patterns. In contrast, when looking at the BoDV-1-positive glial cells and neurons, a massive viral involvement in the brain stem was noticeable. Three of the seven patients received early high-dose steroids, which led to a significantly lower lymphocytic infiltration of the central nervous tissue and a longer survival compared to the patients who were treated with steroids later in the course of disease. This study highlights the potential importance of early high-dose immunosuppressive therapy in BVE. Our findings hint at a promising treatment option which should be corroborated in future observational or prospective therapy studies

Long-Term Stability of Polymer-Coated Surface Transverse Wave Sensors for the Detection of Organic Solvent Vapors

Arrays with polymer-coated acoustic sensors, such as surface acoustic wave (SAW) and surface transverse wave (STW) sensors, have successfully been applied for a variety of gas sensing applications. However, the stability of the sensors’ polymer coatings over a longer period of use has hardly been investigated. We used an array of eight STW resonator sensors coated with different polymers. This sensor array was used at semi-annual intervals for a three-year period to detect organic solvent vapors of three different chemical classes: a halogenated hydrocarbon (chloroform), an aliphatic hydrocarbon (octane), and an aromatic hydrocarbon (xylene). The sensor signals were evaluated with regard to absolute signal shifts and normalized signal shifts leading to signal patterns characteristic of the respective solvent vapors. No significant time-related changes of sensor signals or signal patterns were observed, i.e., the polymer coatings kept their performance during the course of the study. Therefore, the polymer-coated STW sensors proved to be robust devices which can be used for detecting organic solvent vapors both qualitatively and quantitatively for several years