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Cytokine-induced killer (CIK) cells are an ex vivo expanded heterogeneous cell population with an enriched NK-T phenotype (CD3+CD56+). Due to the convenient and relatively inexpensive expansion capability, together with low incidence of graft versus host disease (GVHD) in allogeneic cancer patients, CIK cells are a promising candidate for immunotherapy. It is well known that natural killer group 2D (NKG2D) plays an important role in CIK cell-mediated antitumor activity; however, it remains unclear whether its engagement alone is sufficient or if it requires additional co-stimulatory signals to activate the CIK cells. Likewise, the role of 2B4 has not yet been identified in CIK cells. Herein, we investigated the individual and cumulative contribution of NKG2D and 2B4 in the activation of CIK cells. Our analysis suggests that (a) NKG2D (not 2B4) is implicated in CIK cell (especially CD3+CD56+ subset)-mediated cytotoxicity, IFN-γ secretion, E/T conjugate formation, and degranulation; (b) NKG2D alone is adequate enough to induce degranulation, IFN-γ secretion, and LFA-1 activation in CIK cells, while 2B4 only provides limited synergy with NKG2D (e.g., in LFA-1 activation); and (c) NKG2D was unable to costimulate CD3. Collectively, we conclude that NKG2D engagement alone suffices to activate CIK cells, thereby strengthening the idea that targeting the NKG2D axis is a promising approach to improve CIK cell therapy for cancer patients. Furthermore, CIK cells exhibit similarities to classical invariant natural killer (iNKT) cells with deficiencies in 2B4 stimulation and in the costimulation of CD3 with NKG2D. In addition, based on the current data, the divergence in receptor function between CIK cells and NK (or T) cells can be assumed, pointing to the possibility that molecular modifications (e.g., using chimeric antigen receptor technology) on CIK cells may need to be customized and optimized to maximize their functional potential.
In thyroid carcinoma cells, the soluble βgalactosidespecific lectin, galectin3, is extra and intracellularly expressed and plays a significant role in thyroid cancer diagnosis. The functional relevance of this molecule, particularly in its extracellular environment however, warrants further elucidation. To gain insight into this topic, the present study characterized principal functional properties of galectin3 in 3 commonly used thyroid carcinoma cell lines (BCPAP, Cal62 and FTC133) that express the molecule intra and extracellulary. Cellintrinsic galectin3 harbors a functional carbohydrate recognition domain as determined by affinity purification. Moreover, cell surface expressed galectin3 can be partially removed by treatment with lactose or asialofetuin, but not with sucrose. Thyroid carcinoma cells adhere to substratebound galectin3 in a βgalactosidespecific manner, whereby only cell adhesion, but not cell migration is promoted. Thus, thyroid tumor cells harbor functional active galectin3 that, inter alia, specifically interacts with cell surfaceexpressed molecular ligands in a βgalactosidedependent manner, whereby the molecule can at least interfere with cell adhesion. The modulation of galectin3 expression level or its ligands in such tumor cells could be of therapeutic interest and needs further experimental clarification.
The synthesis and characterization of a new class of 1,2,4-oxadiazolylpyridinium as a cationic scaffold for fluorinated ionic liquid crystals is herein described. A series of 12 fluorinated heterocyclic salts based on a 1,2,4-oxadiazole moiety, connected through its C(5) or C(3) to an N-alkylpyridinium unit and a perfluoroheptyl chain, differing in the length of the alkyl chain and counterions, has been synthesized. As counterions iodide, bromide and bis(trifluoromethane)sulfonimide have been considered. The synthesis, structure, and liquid crystalline properties of these compounds are discussed on the basis of the tuned structural variables. The thermotropic properties of this series of salts have been investigated by differential scanning calorimetry and polarized optical microscopy. The results showed the existence of an enantiotropic mesomorphic smectic liquid crystalline phase for six bis(trifluoromethane)sulfonimide salts.
New sustainable, environmentally friendly materials for thermal insulation of buildings are necessary to reduce their carbon footprints. In this study, Miscanthus fiber-reinforced geopolymer composites, foamed with sodium dodecyl sulfate (SDS), were developed using fly ash as a geopolymer precursor. The effects of fiber content, fiber size, curing temperature, foaming agent content, fumed silica specific surface area and fumed silica content on thermal conductivity and compressive strength were evaluated using a Plackett-Burman design of experiment. Furthermore, the microstructure of geopolymer composites was investigated using X-ray diffraction (XRD), X-ray micro-computed tomography (μCT) and scanning electron microscopy (SEM). The measured characteristic values were in the following ranges: Thermal conductivity 0.057 W (m K)−1 to 0.127 W (m K)−1, compressive strength 0.007 MPa–0.719 MPa and porosity 49 vol% to 76 vol%. The results reveal an enhancement of thermal conductivity by elevated fiber size and foaming agent content. In contrast, the compressive strength is enhanced by high fiber content. Additionally, SEM images indicate a good interaction between the fibers and the geopolymer matrix, because nearly the whole fiber surface is covered by the geopolymer.
Aim: To understand how transcriptional factors Pdr1 and Pdr3, belonging to the pleiotropic drug resistance system, are activated, and regulated after introducing chemical toxins to the cell in the model organism Saccharomyces cerevisiae.
Methods: Series of molecular methods were applied using different strains of S. cerevisiae over-expressing proteins of interest as a eukaryotic cell model. The chemical stress introduced to the cell is represented by menadione. Results were obtained performing protein detection and analysis. Additionally, the regulation of the DNA binding of the transcriptional activators after stimulation is quantified using chromatin immunoprecipitation, employing epitope-tagged factors and real-time qPCR.
Results: Our results indicated higher expression levels of the Pdr1 transcriptional factor, compared to its homologous Pdr3 after treatment with menadione. The yeast-cell defence system was tested against various organic solvents to exclude the possibility of their presence potentially affecting the results. The results indicate that Pdr1 is most abundant after 30 minutes from the beginning of the treatment, compared with 240 minutes after the treatment when the function of the transcription factor is faded. It appears that Pdr1 binding to the PDR5 and SNQ2 promoters, which are both activated by Pdr1, peaks around the same time, or more precisely after 40 minutes from the start of the treatment.
Conclusion: The tendency of Pdr1 reduction after its activation by menadione is detected. One possibility is that Pdr1, after recognizing the xenobiotic menadione, is removed by a degradation mechanism. Given the fact that Pdr1 directly binds the xenobiotic molecule, its destruction might help the cells to remove toxic levels of menadione. It is possible that overexpressing the part of Pdr1 which recognizes menadione alone was sufficient to detoxify and hence produce a tolerance towards menadione.
Intimate swabs taken for examination in sexual assault cases typically yield mixtures of sperm and epithelial cell types. While powerful, differential extraction protocols to overcome such cell type mixtures by separate lysis of epithelial cells and spermatozoa can still prove ineffective, in particular if only few sperm cells are present or if swabs contain sperm from more than one individual leading to complex low level DNA mixtures. A means to avoid such mixtures consists in the analysis of single micromanipulated sperm cells. However, the quantity of DNA from single sperm cells is not sufficient for conventional STR analysis. Here, we describe a simple method for micromanipulating individual sperm cells from intimate swabs and show that whole genome amplification can generate sufficient amounts of DNA from single cells for subsequent DNA profiling. We recovered over 80% of alleles of haploid autosomal STR profiles from the majority of individual sperm cells. Furthermore, we demonstrate that in mixtures of sperm from two contributors, Y-STR and X-STR profiles of individual sperm cells can be used to sort the haploid autosomal profiles to develop the diploid consensus STR profiles of the individual donors. Finally, by analysing single sperm cells from mock sexual assault swabs with one or two sperm donors, we showed that our protocols enabled the identification of the unknown male contributors.
A series of reactive binaphthyl‐diimine‐based dopants is prepared and investigated with respect to their potential for the chiral induction of structural coloration in nematic liquid crystal mixture E7 and the selective photonic sensing of nitrogen dioxide (NO2). Studies of the helical twisting power (HTP) in 4‐cyano‐4′‐pentylbiphenyl (5CB) reveal HTP values as high as 375 µm‐1 and the tremendous impact of structural compatibility and changes of the dihedral binaphthyl angle on the efficiency of the chiral transfer. Detailed investigation of the sensing capabilities of the systems reveals an extraordinarily high selectivity for NO2 and a response to concentrations as low as 100 ppm. The systems show a direct response to the analyte gas leading to a concentration‐dependent shift of the reflectance wavelength of up to several hundred nanometers. Incorporation of copper ions remarkably improves the sensor's properties in terms of sensitivity and selectivity, enabling the tailored tweaking of the system's properties.
Dental stem cells have been isolated from the medical waste of various dental tissues. They have been characterized by numerous markers, which are evaluated herein and differentiated into multiple cell types. They can also be used to generate cell lines and iPSCs for long-term in vitro research. Methods for utilizing these stem cells including cellular systems such as organoids or cell sheets, cell-free systems such as exosomes, and scaffold-based approaches with and without drug release concepts are reported in this review and presented with new pictures for clarification. These in vitro applications can be deployed in disease modeling and subsequent pharmaceutical research and also pave the way for tissue regeneration. The main focus herein is on the potential of dental stem cells for hard tissue regeneration, especially bone, by evaluating their potential for osteogenesis and angiogenesis, and the regulation of these two processes by growth factors and environmental stimulators. Current in vitro and in vivo publications show numerous benefits of using dental stem cells for research purposes and hard tissue regeneration. However, only a few clinical trials currently exist. The goal of this review is to pinpoint this imbalance and encourage scientists to pick up this research and proceed one step further to translation.
The ability to discriminate between different ionic species, termed ion selectivity, is a key feature of ion channels and forms the basis for their physiological function. Members of the degenerin/epithelial sodium channel (DEG/ENaC) superfamily of trimeric ion channels are typically sodium selective, but to a surprisingly variable degree. While acid-sensing ion channels (ASICs) are weakly sodium selective (sodium:potassium ratio ∼10:1), ENaCs show a remarkably high preference for sodium over potassium (>500:1). This discrepancy may be expected to originate from differences in the pore-lining second transmembrane segment (M2). However, these show a relatively high degree of sequence conservation between ASICs and ENaCs, and previous functional and structural studies could not unequivocally establish that differences in M2 alone can account for the disparate degrees of ion selectivity. By contrast, surprisingly little is known about the contributions of the first transmembrane segment (M1) and the preceding pre-M1 region. In this study, we used conventional and noncanonical amino acid-based mutagenesis in combination with a variety of electrophysiological approaches to show that the pre-M1 and M1 regions of mASIC1a channels are major determinants of ion selectivity. Mutational investigations of the corresponding regions in hENaC show that these regions contribute less to ion selectivity, despite affecting ion conductance. In conclusion, our work suggests that the remarkably different degrees of sodium selectivity in ASICs and ENaCs are achieved through different mechanisms. These results further highlight how M1 and pre-M1 are likely to differentially affect pore structure in these related channels.
The solvent exchange as one of the most important steps during the manufacturing process of organic aerogels was investigated. This step is crucial as a preparatory step for the supercritical drying, since the pore solvent must be soluble in supercritical carbon dioxide to enable solvent extraction. The development and subsequent optimization of a suitable system with a peristaltic pump for automatic solvent exchange proved to be a suitable approach. In addition, the influence of zeolites on the acceleration of the process was found to be beneficial. To investigate the process, the water content in acetone was determined at different times using Karl Fischer titration. The shrinkage, densities, as well as the surface areas of the aerogels were analyzed. Based on these, the influence of various process parameters on the final structure of the obtained aerogels was investigated and evaluated. Modeling on diffusion in porous materials completes this study.