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- cytokine-induced killer cells (2)
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Silicon carbide and graphene possess extraordinary chemical and physical properties. Here, these different systems are linked and the changes in structural and dynamic properties are investigated. For the simulations performed a classical molecular dynamic (MD) approach was used. In this approach, a graphene layer (N = 240 atoms) was grafted at different distances on top of a 6H-SiC structure (N = 2400 atoms) and onto a 3C-SiC structure (N = 1728 atoms). The distances between the graphene and the 6H are 1.0, 1.3 and 1.5 Å and the distances between the graphene layer and the 3C-SiC are 2.0, 2.3, and 2.5 Å. Each system has been equilibrated at room temperature until no further relaxation was observed. The 6H-SiC structure in combination with graphene proves to be more stable compared to the combination with 3C-SiC. This can be seen well in the determined energies. Pair distribution functions were influenced slightly by the graphene layer due to steric and energetic changes. This becomes clear from the small shifts of the C-C distances. Interactions as well as bonds between graphene and SiC lead to the fact that small shoulders of the high-frequency SiC-peaks are visible in the spectra and at the same time the high-frequency peaks of graphene are completely absent.
Modeling of Creep Behavior of Particulate Composites with Focus on Interfacial Adhesion Effect
(2022)
Evaluation of creep compliance of particulate composites using empirical models always provides parameters depending on initial stress and material composition. The effort spent to connect model parameters with physical properties has not resulted in success yet. Further, during the creep, delamination between matrix and filler may occur depending on time and initial stress, reducing an interface adhesion and load transfer to filler particles. In this paper, the creep compliance curves of glass beads reinforced poly(butylene terephthalate) composites were fitted with Burgers and Findley models providing different sets of time-dependent model parameters for each initial stress. Despite the finding that the Findley model performs well in a primary creep, the Burgers model is more suitable if secondary creep comes into play; they allow only for a qualitative prediction of creep behavior because the interface adhesion and its time dependency is an implicit, hidden parameter. As Young’s modulus is a parameter of these models (and the majority of other creep models), it was selected to be introduced as a filler content-dependent parameter with the help of the cube in cube elementary volume approach of Paul. The analysis led to the time-dependent creep compliance that depends only on the time-dependent creep of the matrix and the normalized particle distance (or the filler volume content), and it allowed accounting for the adhesion effect. Comparison with the experimental data confirmed that the elementary volume-based creep compliance function can be used to predict the realistic creep behavior of particulate composites.
Telogene Einzelhaare sind häufig vorkommende Spurentypen an Tatorten. Derzeit werden sie zumeist von der STR-Typisierung ausgeschlossen, weil ihre STR-Profile aufgrund geringer DNA-Mengen und starker DNA-Degradierung in vielen Fällen unvollständig und schwierig zu interpretieren sind. In der vorliegenden Arbeit wurde eine systematische Vorgehensweise angewandt, um Korrelationen zwischen der DNA-Menge und DNA-Degradierung zu dem Erfolg der STR-Typisierung aufzuweisen und darauf basierend den Typisierungs-Erfolg von DNA aus Haaren vorhersagen zu können.
Zu diesem Zweck wurde ein human- (RiboD) und ein canin-spezifischer (RiboDog) qPCR-basierter Assay zur Messung der DNA-Menge und Bewertung der DNA-Integrität mittels eines Degradierungswerts (D-Wert) entwickelt. Aufgrund der Lage der genutzten Primer, welche auf ubiquitär vorkommende ribosomale DNA-Sequenzen abzielen, ist das Funktionsprinzip schnell und kostengünstig auf unterschiedliche Spezies anzuwenden. Die Funktionsweise der Assays wurde mittels seriell degradierter DNA bestätigt und der humane Assay wurde im Vergleich zum kommerziellen Quantifiler? Trio DNA Quantification Kit validiert. Schließlich wurde mit den Assays an DNA aus telogenen und katagenen Einzelhaaren von Menschen und Hunden der Zusammenhang zwischen DNA-Menge und DNA-Integrität zu der Vollständigkeit der STR-Allele (Allel Recovery) von DNA-Profilen untersucht, die mittels kapillarelektrophoretischer (CE) STR-Kits erhaltenen wurde. Es zeigte sich, dass bei humanen Einzelhaaren die Allel-Recovery sowohl von der DNA-Menge als auch der DNA-Integrität abhängt. Dagegen war die DNA-Degradierung bei einzelnen Hundehaaren durchweg geringer und die Allel-Recovery hing allein von der extrahierten DNA-Menge ab.
Um die STR-Analytik degradierter humaner DNA-Proben weiter zu verbessern, wurde ein neuartiger NGS-basierter Assay (maSTR, Mini-Amplikon-STR) etabliert, der die 16 forensischen STR-Loci des European Standard Sets und Amelogenin als sehr kurze Amplikons (76-296 bp) parallel amplifiziert. Mit intakter DNA generierte der maSTR-Assay im Mengenbereich von 200 pg eingesetzter DNA reproduzierbare, vollständige Profile ohne Allelic Drop-ins. Bei niedrigeren DNA-Mengen traten vereinzelt Allelic Drop-ins auf, wobei unter Verwendung von mindestens 43 pg DNA vollständige Profile erhalten wurden.
Die kombinierte Strategie aus RiboD-Messungen der DNA-Menge und -Integrität und daraus resultierendem STR-Typisierungserfolg des maSTR-Assays wurde an degradierter DNA validiert. Anschließend wurde die Strategie auf DNA aus telogenen und katagenen Einzelhaaren angewandt und mit den Ergebnissen des CE-basierten PowerPlex? ESX 17-Kits verglichen, das dasselbe STR-Marker-Set analysiert. Dabei zeigte sich, dass der Erfolg der STR-Typisierung beider STR-Assays sowohl von der optimalen Menge der Template-DNA als auch von der DNA-Integrität abhängt. Mit dem maSTR-Assay wurden vollständige Profile mit ungefähr 50 pg Input-DNA für leicht degradierte DNA aus Einzelhaaren nachgewiesen, sowie mit ungefähr 500 pg stark degradierter DNA. Aufgrund der geringen DNA-Mengen von telogenen Einzelhaaren schwankte die Reproduzierbarkeit der maSTR-Ergebnisse, war jedoch stets dem PowerPlex? ESX 17-Kit in Bezug auf die Allel-Recovery überlegen.
Ein Vergleich mit zwei, hinsichtlich der Längenverteilung der Amplikons komplementären CE-basierten STR-Kits (PowerPlex? ESX 17 und ESI 17 Fast), sowie mit einem kommerziellen NGS-Kit (ForenSeq? DNA Signature Prep) ergab, dass nicht die Technik der NGS, sondern die Kürze der Amplikons der wichtigste Faktor zur Typisierung degradierter DNA ist. Der maSTR-Assay wies in allen Vergleichen mit den genutzten kommerziellen Kits jedoch eine höhere Anzahl an Allelic Drop-ins auf. Diese traten umso häufiger auf, je geringer die verwendete DNA-Menge und je stärker degradiert diese war.
Weil Profile mit Allelic Drop-ins Mischprofilen entsprechen, wurden die per maSTR-Assay generierten STR-Profile mit Verfahren zur Interpretation von Mischspuren untersucht. Bei der Composite-Interpretation werden alle vorkommenden Allele von Replikaten gezählt, bei der Consensus-Interpretation lediglich die reproduzierbaren Allele. Dabei stellte sich heraus, dass im Fall von wenigen Allelic Drop-ins (PowerPlex? ESX 17-generierte Profile) die Composite-Interpretation und bei Allelic Drop-in-haltigen Profilen (maSTR-generierte Profile) die Consensus-Interpretation am besten geeignet ist.
Schließlich wurde mittels der GenoProof Mixture 3-Software untersucht, inwieweit semi- und vollständig kontinuierliche probabilistische Verfahren bei der biostatistischen Bewertung der DNA-Profile aus Einzelhaaren geeignet sind. Dabei zeigte sich, dass der maSTR-Assay aufgrund der hohen Anzahl an Allelic Drop-ins den CE-basierten Methoden nur in Fällen von DNA leicht überlegen ist, die in ausreichender Menge und gering degradiert vorliegt. In diesem Bereich gelingt die Zuordnung des Profils aus Haaren zum Referenzprofil jedoch ebenfalls mittels CE-basierten Methoden.
Aus allen Ergebnissen wurde eine Empfehlung für die Handhabung von DNA aus ausgefallenen Einzelhaaren abgeleitet, die auf dem DNA-Degradierungsgrad in Kombination mit der DNA-Menge basiert. Die vorliegende Arbeit schafft somit eine Grundlage, um ausgefallene Einzelhaare in der Routine-Arbeit von kriminaltechnischen Ermittlungen nutzbar zu machen, sowie gegebenenfalls auf andere Spurentypen mit degradierter DNA geringer Menge anzuwenden. Dadurch könnte die Nutzbarkeit solcher Spurentypen für die forensische Kriminalistik erhöht werden, insbesondere wenn die standardmäßig verwendeten CE-basierten Methoden versagen. Perspektivisch ist die Technik der NGS aufgrund der großen Multiplexierbarkeit uniformer, kurzer Marker generell der CE-basierten Technik bei der Typisierung degradierter DNA überlegen.
The human enzymes GLYAT (glycine N-acyltransferase), GLYATL1 (glutamine N-phenylacetyltransferase) and GLYATL2 (glycine N-acyltransferase-like protein 2) are not only important in the detoxification of xenobiotics via the human liver, but are also involved in the elimination of acyl residues that accumulate in the form of their coenzyme A (coA) esters in some rare inborn errors of metabolism. This concerns, for example, disorders in the degradation of branched-chain amino acids, such as isovaleric acidemia or propionic acidemia. In addition, they also assist in the elimination of ammonium, which is produced during the transamination of amino acids and accumulates in urea cycle defects. Sequence variants of the enzymes have also been investigated, which may provide evidence of impaired enzyme activities, from which therapy adjustments can potentially be derived. A modified Escherichia coli strain was chosen for the overexpression and partial biochemical characterization of the enzymes, which may allow solubility and proper folding. Since post-translational protein modifications are very limited in bacteria, we also attempted to overexpress the enzymes in HEK293 cells (human-derived). In addition to characterization via immunoblots and activity assays, intracellular localization of the enzymes was also performed using GFP coupling and confocal laser scanning microscopy in transfected HEK293 cells. The GLYATL2 enzyme may have tasks beyond detoxification and metabolic defects and the preliminary molecular biology work has been performed as part of this project - the enzyme activity determinations were outsourced to a co-supervised bachelor thesis. The enzyme activity determinations with purified recombinant human enzyme from Escherichia coli provided a threefold higher activity of the sequence variant p.(Asn156Ser) for GLYAT, which should be considered as the probably authentic wild type of the enzyme. In addition, a reduced activity of the GLYAT variant p.(Gln61Leu), which is very common in South Africa, was shown, which could be of particular importance in the treatment of isovaleric acidemia, which is also common in South Africa. Intracellularly, GLYAT and GLYATL1 could be localized mitochondrially. As the analyses have shown, sequence variations of GLYAT and GLYATL1 influence their enzyme activity. As an example, the GLYAT variant p.(Gln61Leu) is frequently found in South Africa. In the case of reduced GLYAT activity, patients could be increasingly treated with L-carnitine in the sense of an individualized therapy, since the conjugation of the toxic isovaleryl-coA with glycine is restricted by the GLYAT sequence variation. Activity-reducing variants identified in this project are of particular interest, as they may influence the treatment of certain metabolic defects.
SLC6A14 (ATB0,+) is unique among SLC proteins in its ability to transport 18 of the 20 proteinogenic (dipolar and cationic) amino acids and naturally occurring and synthetic analogues (including anti-viral prodrugs and nitric oxide synthase (NOS) inhibitors). SLC6A14 mediates amino acid uptake in multiple cell types where increased expression is associated with pathophysiological conditions including some cancers. Here, we investigated how a key position within the core LeuT-fold structure of SLC6A14 influences substrate specificity. Homology modelling and sequence analysis identified the transmembrane domain 3 residue V128 as equivalent to a position known to influence substrate specificity in distantly related SLC36 and SLC38 amino acid transporters. SLC6A14, with and without V128 mutations, was heterologously expressed and function determined by radiotracer solute uptake and electrophysiological measurement of transporter-associated current. Substituting the amino acid residue occupying the SLC6A14 128 position modified the binding pocket environment and selectively disrupted transport of cationic (but not dipolar) amino acids and related NOS inhibitors. By understanding the molecular basis of amino acid transporter substrate specificity we can improve knowledge of how this multi-functional transporter can be targeted and how the LeuT-fold facilitates such diversity in function among the SLC6 family and other SLC amino acid transporters.
While many proteins are known clients of heat shock protein 90 (Hsp90), it is unclear whether the transcription factor, thyroid hormone receptor beta (TRb), interacts with Hsp90 to control hormonal perception and signaling. Higher Hsp90 expression in mouse fibroblasts was elicited by the addition of triiodothyronine (T3). T3 bound to Hsp90 and enhanced adenosine triphosphate (ATP) binding of Hsp90 due to a specific binding site for T3, as identified by molecular docking experiments. The binding of TRb to Hsp90 was prevented by T3 or by the thyroid mimetic sobetirome. Purified recombinant TRb trapped Hsp90 from cell lysate or purified Hsp90 in pull-down experiments. The affinity of Hsp90 for TRb was 124 nM. Furthermore, T3 induced the release of bound TRb from Hsp90, which was shown by streptavidin-conjugated quantum dot (SAv-QD) masking assay. The data indicate that the T3 interaction with TRb and Hsp90 may be an amplifier of the cellular stress response by blocking Hsp90 activity.
Microarray-based experiments revealed that thyroid hormone triiodothyronine (T3) enhanced the binding of Cy5-labeled ATP on heat shock protein 90 (Hsp90). By molecular docking experiments with T3 on Hsp90, we identified a T3 binding site (TBS) near the ATP binding site on Hsp90. A synthetic peptide encoding HHHHHHRIKEIVKKHSQFIGYPITLFVEKE derived from the TBS on Hsp90 showed, in MST experiments, the binding of T3 at an EC50 of 50 μM. The binding motif can influence the activity of Hsp90 by hindering ATP accessibility or the release of ADP.
Cathepsin K (CatK) is a target for the treatment of osteoporosis, arthritis, and bone metastasis. Peptidomimetics with a cyanohydrazide warhead represent a new class of highly potent CatK inhibitors; however, their binding mechanism is unknown. We investigated two model cyanohydrazide inhibitors with differently positioned warheads: an azadipeptide nitrile Gü1303 and a 3-cyano-3-aza-β-amino acid Gü2602. Crystal structures of their covalent complexes were determined with mature CatK as well as a zymogen-like activation intermediate of CatK. Binding mode analysis, together with quantum chemical calculations, revealed that the extraordinary picomolar potency of Gü2602 is entropically favoured by its conformational flexibility at the nonprimed-primed subsites boundary. Furthermore, we demonstrated by live cell imaging that cyanohydrazides effectively target mature CatK in osteosarcoma cells. Cyanohydrazides also suppressed the maturation of CatK by inhibiting the autoactivation of the CatK zymogen. Our results provide structural insights for the rational design of cyanohydrazide inhibitors of CatK as potential drugs.
The epithelial sodium channel (ENaC) is a heterotrimeric ion channel that plays a key role in sodium and water homeostasis in tetrapod vertebrates. In the aldosterone-sensitive distal nephron, hormonally controlled ENaC expression matches dietary sodium intake to its excretion. Furthermore, ENaC mediates sodium absorption across the epithelia of the colon, sweat ducts, reproductive tract, and lung. ENaC is a constitutively active ion channel and its expression, membrane abundance, and open probability (PO) are controlled by multiple intracellular and extracellular mediators and mechanisms [9]. Aberrant ENaC regulation is associated with severe human diseases, including hypertension, cystic fibrosis, pulmonary edema, pseudohypoaldosteronism type 1, and nephrotic syndrome [9].
Mobiles Laser-Schneidsystem zur Unterstützung der USBV-Entschärfung und Beweissicherung (mobiLaS)
(2022)
P30 - Das Elektrospinnen von halbleitenden Zinndioxidfasern für die Detektion von Wasserstoff
(2022)
Das Ziel dieser Arbeit ist die Entwicklung von dünnen keramischen Fasern als halbleitendes Sensormaterial zum Nachweis von Wasserstoff, möglichst bei Zimmertemperatur. Die elektrische Leitfähigkeit halbleitender Metalloxide ändert sich durch die Einwirkung von oxidierenden und reduzierenden Gasen auf die Oberfläche des Metalloxids. Dieser Effekt kann zur Messung der Gaskonzentration genutzt werden. Die Reaktion von Zinn(IV)-oxid mit Wasserstoff basiert auf der Reduktion des Zinn(IV)-oxids zum Zinn, wobei die Elektronen des Zinn(IV)-oxids im metallischen Zinn verbleiben und dort im nicht gebundenen Zustand zu einer Leitfähigkeitserhöhung beitragen. Die Reaktion des Wasserstoffes kann sowohl mit den Sauerstoffatomen des Oxids als auch mit adsorbierten Sauerstoffatomen an der Oxidoberfläche stattfinden.[ 6] Da die Reaktionen an der Oberfläche des Oxids stattfinden, sollten Sensoren mit einer großen Oberfläche im Vergleich zu metalloxidischen Bulkmaterialien eine höhere Empfindlichkeit aufweisen. [3] Die Verwendung von Fasern anstelle von Dünn- oder Dickschichten führt dabei zu einer besseren Sensitivität gegenüber Gasen.
Process-induced changes in the morphology of biodegradable polybutylene adipate terephthalate (PBAT) and polylactic acid (PLA) blends modified with various multifunctional chainextending cross-linkers (CECLs) are presented. The morphology of unmodified and modified films produced with blown film extrusion is examined in an extrusion direction (ED) and a transverse direction (TD). While FTIR analysis showed only small peak shifts indicating that the CECLs modify the molecular weight of the PBAT/PLA blend, SEM investigations of the fracture surfaces of blown extrusion films revealed their significant effect on the morphology formed during the processing. Due to the combined shear and elongation deformation during blown film extrusion, rather spherical PLA islands were partly transformed into long fibrils, which tended to decay to chains of elliptical islands if cooled slowly. The CECL introduction into the blend changed the thickness of the PLA fibrils, modified the interface adhesion, and altered the deformation behavior of the PBAT matrix from brittle to ductile. The results proved that CECLs react selectively with PBAT, PLA, and their interface. Furthermore, the reactions of CECLs with PBAT/PLA induced by the processing depended on the deformation directions (ED and TD), thus resulting in further non-uniformities of blown extrusion films.
Background: Cancer heterogeneity poses a serious challenge concerning the toxicity and adverse effects of therapeutic inhibitors, especially when it comes to combinatorial therapies that involve multiple targeted inhibitors. In particular, in non-small cell lung cancer (NSCLC), a number of studies have reported synergistic effects of drug combinations in the preclinical models, while they were only partially successful in the clinical setup, suggesting those alternative clinical strategies (with genetic background and immune response) should be considered. Herein, we investigated the antitumor effect
of cytokine-induced killer (CIK) cells in combination with ALK and PD-1 inhibitors in vitro on genetically variable NSCLC cell lines.
Methods: We co-cultured the three genetically different NSCLC cell lines NCI-H2228 (EML4-ALK), A549 (KRAS mutation), and HCC-78 (ROS1 rearrangement) with and without nivolumab (PD-1 inhibitor) and crizotinib (ALK inhibitor). Additionally, we profiled the variability of surface expression multiple immune checkpoints, the concentration of absolute dead cells, intracellular granzyme B on CIK cells using flow cytometry as well as RT-qPCR. ELISA and Western blot were performed to verify the activation of CIK cells.
Results: Our analysis showed that (a) nivolumab significantly weakened PD-1 surface expression on CIK cells without impacting other immune checkpoints or PD-1 mRNA expression, (b) this combination strategy showed an effective response on cell viability, IFN-g production, and intracellular release of granzyme B in CD3+ CD56+ CIK cells, but solely in NCI-H2228, (c) the intrinsic expression of Fas ligand (FasL) as a T-cell activation marker in CIK cells was upregulated by this additive effect, and (d) nivolumab induced Foxp3 expression in CD4+CD25+ subpopulation of CIK cells significantly increased. Taken together, we could show that CIK cells in combination with crizotinib and nivolumab can enhance the anti-tumor immune response through FasL activation, leading to increased IFN-g and granzyme B, but only in NCI-H2228 cells with EML4-ALK rearrangement. Therefore, we hypothesize that CIK therapy may be a potential alternative in NSCLC patients harboring EML4-ALK rearrangement, in addition, we support the idea that combination therapies offer significant potential when they are optimized on a patient-by-patient basis.
Cytokine-induced killer cells (CIK) in combination with dendritic cells (DCs) have shown favorable outcomes in renal cell carcinoma (RCC), yet some patients exhibit recurrence or no response to this therapy. In a broader perspective, enhancing the antitumor response of DC-CIK cells may help to address this issue. Considering this, herein, we investigated the effect of anti-CD40 and anti-CTLA-4 antibodies on the antitumor response of DC-CIK cells against RCC cell lines. Our analysis showed that, a) anti-CD40 antibody (G28.5) increased the CD3+CD56+ effector cells of CIK cells by promoting the maturation and activation of DCs, b) G28.5 also increased CTLA-4 expression in CIK cells via DCs, but the increase could be hindered by the CTLA-4 inhibitor (ipilimumab), c) adding ipilimumab was also able to significantly increase the proportion of CD3+CD56+ cells in DC-CIK cells, d) anti-CD40 antibodies predominated over anti-CTLA-4 antibodies for cytotoxicity, apoptotic effect and IFN-g secretion of DC-CIK cells against RCC cells, e) after ipilimumab treatment, the population of Tregs in CIK cells remained unaffected, but ipilimumab combined with G28.5 significantly reduced the expression of CD28 in CIK cells. Taken together, we suggest that the agonistic anti-CD40 antibody rather than CTLA-4 inhibitor may improve the antitumor response of DC-CIK cells, particularly in RCC. In addition, we pointed towards the yet to be known contribution of CD28 in the crosstalk between anti-CTLA-4 and CIK cells.
Introduction of Matrix-Filler Adhesion to Modelling of Elastic Moduli of Particulate Composites
(2022)
Cube in cube elementary volume (EV) concept serves to predict a filler-content dependent Young´s moduli of particle filled composites using moduli of a matrix EM and a filler EF. Paul and Ishai-Cohen derived formulas for composites moduli considering different load transfer boundaries in the EV assuming a complete filler-matrix adhesion. In this paper it is confirmed that their models represent the upper and lower bounds, respectively, with the respect to the experimental data. However, in vast majority of composites a filler-matrix adhesion is not complete. Therefore, an adhesion factor kadh gaining values between 0 and 1 was introduced into Paul´s model to consider the reduced adhesion as the reduction of the filler-matrix contact area for glass beads filled in polar and unpolar thermoplastic matrices as well as elastomer. The evaluation of these composite systems provides reasonable adhesion coefficients of PA66 > PBT > PP > PE-LD >> BR. It was also found that stiffening only occurs if kadh exceeds the minimum value adhesion of root square of E(M) divided by E(F). The determined kadh correspond to scanning electron microscopy observations of the composites fracture surfaces. Additionally, finite element analysis of the cubic and hexagonal arrangements of the EV show that the stress distributions are different, but they affect the calculated moduli only for the filler volume contents exceeding 20 %. The introduction of the filler-matrix adhesion provides more reliable predictions of Young´s moduli of particulate composites.
The utilization of simulation procedures is gaining increasing attention in the product development of extrusion blow molded parts. However, some simulation steps, like the simulation of shrinkage and warpage, are still associated with uncertainties. The reason for this is on the one hand a lack of standardized interfaces for the transfer of simulation data between different simulation tools, and on the other hand the complex time-, temperature- and process-dependent material behavior of the used semi crystalline polymers. Using a new vendor neutral interface standard for the data transfer, the shrinkage analysis of a simple blow molded part is investigated and compared to experimental data. A linear viscoelastic material model in combination with an orthotropic process- and temperature-dependent thermal expansion coefficient is used for the shrinkage prediction. A good agreement is observed. Finally, critical parameters in the simulation models that strongly influence the shrinkage analysis are identified by a sensitivity study.
Jet engines of airplanes are designed such that in some components damage occurs and accumulates in service without being critical up to a certain level of damage. Since maintenance, repair, and component exchange are very cost-intensive, it is necessary to predict efficiently the component lifetime with high accuracy. A former developed lifetime model, based on interpolated results of aerodynamic and structural mechanics simulations, uses material parameters estimated from literature values of standard creep experiments. For improved accuracy, an experimental procedure is developed for the characterization of the short-time creep behavior, which is relevant for the operation of turbine blades of jet engines. To consider microstructural influences resulting from the manufacturing of thin-walled single crystal turbine blades, small-scale specimens from used turbine blades are extracted and tested in short- and medium-time creep experiments. Based on experimental results and literature values, a creep model, which describes the fracture behavior for a wide range of creep loads, is calibrated and is now used for the lifetime prediction of turbine blades under real loading conditions.
Cathepsin K (CatK) is a target for the treatment of osteoporosis, arthritis, and bone metastasis. Peptidomimetics with a cyanohydrazide warhead represent a new class of highly potent CatK inhibitors; however, their binding mechanism is unknown. We investigated two model cyanohydrazide inhibitors with differently positioned warheads: an azadipeptide nitrile Gü1303 and a 3-cyano-3-aza-β-amino acid Gü2602. Crystal structures of their covalent complexes were determined with mature CatK as well as a zymogen-like activation intermediate of CatK. Binding mode analysis, together with quantum chemical calculations, revealed that the extraordinary picomolar potency of Gü2602 is entropically favoured by its conformational flexibility at the nonprimed-primed subsites boundary. Furthermore, we demonstrated by live cell imaging that cyanohydrazides effectively target mature CatK in osteosarcoma cells. Cyanohydrazides also suppressed the maturation of CatK by inhibiting the autoactivation of the CatK zymogen. Our results provide structural insights for the rational design of cyanohydrazide inhibitors of CatK as potential drugs.
Discrimination of Stressed and Non-Stressed Food-Related Bacteria Using Raman-Microspectroscopy
(2022)
As the identification of microorganisms becomes more significant in industry, so does the utilization of microspectroscopy and the development of effective chemometric models for data analysis and classification. Since only microorganisms cultivated under laboratory conditions can be identified, but they are exposed to a variety of stress factors, such as temperature differences, there is a demand for a method that can take these stress factors and the associated reactions of the bacteria into account. Therefore, bacterial stress reactions to lifetime conditions (regular treatment, 25 °C, HCl, 2-propanol, NaOH) and sampling conditions (cold sampling, desiccation, heat drying) were induced to explore the effects on Raman spectra in order to improve the chemometric models. As a result, in this study nine food-relevant bacteria were exposed to seven stress conditions in addition to routine cultivation as a control. Spectral alterations in lipids, polysaccharides, nucleic acids, and proteins were observed when compared to normal growth circumstances without stresses. Regardless of the involvement of several stress factors and storage times, a model for differentiating the analyzed microorganisms from genus down to strain level was developed. Classification of the independent training dataset at genus and species level for Escherichia coli and at strain level for the other food relevant microorganisms showed a classification rate of 97.6%.