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Forensic DNA profiles are established by multiplex PCR amplification of a set of highly variable short tandem repeat (STR) loci followed by capillary electrophoresis (CE) as a means to assign alleles to PCR products of differential length. Recently, CE analysis of STR amplicons has been supplemented by high-throughput next generation sequencing (NGS) techniques that are able to detect isoalleles bearing sequence polymorphisms and allow for an improved analysis of degraded DNA. Several such assays have been commercialised and validated for forensic applications. However, these systems are cost-effective only when applied to high numbers of samples. We report here an alternative, cost-efficient shallow-sequence output NGS assay called maSTR assay that, in conjunction with a dedicated bioinformatics pipeline called SNiPSTR, can be implemented with standard NGS instrumentation. In a back-to-back comparison with a CE-based, commercial forensic STR kit, we find that for samples with low DNA content, with mixed DNA from different individuals, or containing PCR inhibitors, the maSTR assay performs equally well, and with degraded DNA is superior to CE-based analysis. Thus, the maSTR assay is a simple, robust and cost-efficient NGS-based STR typing method applicable for human identification in forensic and biomedical contexts.
Die Freiheit in Forschung und Lehre ist zusammen mit der Wissenschaftsfreiheit eines der bürgerlichen Grundrechte in Deutschland. Aber die Forschungsfreiheit kann nicht grenzenlos sein. Industrie wie akademische Institute müssen ihre Entwicklungen dokumentieren. Nur so ist ein Stoff später für die Vermarktung zulassungsfähig und nur so lässt sich belegen, wer die Urheberrechte daran hat.
Mutations in SELENBP1, encoding a novel human methanethiol oxidase, cause extraoral halitosis
(2017)
The application of Raman and infrared (IR) microspectroscopy is leading to hyperspectral data containing complementary information concerning the molecular composition of a sample. The classification of hyperspectral data from the individual spectroscopic approaches is already state-of-the-art in several fields of research. However, more complex structured samples and difficult measuring conditions might affect the accuracy of classification results negatively and could make a successful classification of the sample components challenging. This contribution presents a comprehensive comparison in supervised pixel classification of hyperspectral microscopic images, proving that a combined approach of Raman and IR microspectroscopy has a high potential to improve classification rates by a meaningful extension of the feature space. It shows that the complementary information in spatially co-registered hyperspectral images of polymer samples can be accessed using different feature extraction methods and, once fused on the feature-level, is in general more accurately classifiable in a pattern recognition task than the corresponding classification results for data derived from the individual spectroscopic approaches.
Durch Dotierung eines nematischen Flüssigkristalles mit einer chiralen Substanz wird eine helikal strukturierte Phase induziert, die in der Lage ist, einfallendes Licht wellenlängenselektiv zu reflektieren. Bei der Reaktion des Dotiermittels mit einem gasförmigen Analyten verändern sich die Ganghöhe dieser Struktur und damit die reflektierte Wellenlänge. Liegt diese im Bereich des sichtbaren Lichts, ist eine Farbänderung mit dem menschlichen Auge zu beobachten. Es ist dabei sinnvoll den Flüssigkristall z.B. in einem Polymer einzukapseln, um ihn vor mechanischen Einflüssen und Umwelteinflüssen zu schützen. Eine Möglichkeit zur Einkapselung ist das koaxiale Elektrospinnen. Vorteile sind unter anderem die Realisierung einer großen Oberfläche und einer sehr geringen Wanddicke der schützenden Schale, die die Diffusion von Gasen durch die Wand hindurch ermöglicht. Um die Funktionsfähigkeit eines solchen Sensors zu testen, wurde ein CO2-sensitiver Flüssigkristall verwendet. Dieser wurde in eine Schale aus Polyvinylpyrrolidon (PVP) versponnen und die Reaktion mit CO2 spektroskopisch analysiert.
Optical gas sensors based on chiral-nematic liquid crystals (N* LCs) forming one-dimensional photonic crystals do not require electrical energy and have a considerable potential to supplement established types of sensors. A chiral-nematic phase with tunable selective reflection is induced in a nematic host LC by adding reactive chiral dopants. The selective chemical reaction between dopant and analyte is capable to vary the pitch length (the lattice constant) of the soft, self-assembled, one-dimensional photonic crystal. The progress of the ongoing chemical reaction can be observed even by naked eye because the color of the samples varies. In this work, we encapsulate the responsive N* LC in microscale polyvinylpyrrolidone (PVP) fibers via coaxial electrospinning. The sensor is, thus, given a solid form and has an improved stability against nonavoidable environmental influences. The reaction behavior of encapsulated and nonencapsulated N* LC toward a gaseous analyte is compared, systematically. Making use of the encapsulation is an important step to improve the applicability.
The analysis of Δ9-tetrahydrocannabinol (THC) and its metabolites 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC), and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH) from blood serum is a routine task in forensic toxicology laboratories. For examination of consumption habits, the concentration of the phase I metabolite THC-COOH is used. Recommendations for interpretation of analysis values in medical-psychological assessments (regranting of driver’s licenses, Germany) include threshold values for the free, unconjugated THC-COOH. Using a fully automated two-step liquid-liquid extraction, THC, 11-OH-THC, and free, unconjugated THC-COOH were extracted from blood serum, silylated with N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA), and analyzed by GC/MS. The automation was carried out by an x-y-z sample robot equipped with modules for shaking, centrifugation, and solvent evaporation. This method was based on a previously developed manual sample preparation method. Validation guidelines of the Society of Toxicological and Forensic Chemistry (GTFCh) were fulfilled for both methods, at which the focus of this article is the automated one. Limits of detection and quantification for THC were 0.3 and 0.6 μg/L, for 11-OH-THC were 0.1 and 0.8 μg/L, and for THC-COOH were 0.3 and 1.1 μg/L, when extracting only 0.5 mL of blood serum. Therefore, the required limit of quantification for THC of 1 μg/L in driving under the influence of cannabis cases in Germany (and other countries) can be reached and the method can be employed in that context. Real and external control samples were analyzed, and a round robin test was passed successfully. To date, the method is employed in the Institute of Legal Medicine in Giessen, Germany, in daily routine. Automation helps in avoiding errors during sample preparation and reduces the workload of the laboratory personnel. Due to its flexibility, the analysis system can be employed for other liquid-liquid extractions as well. To the best of our knowledge, this is the first publication on a comprehensively automated classical liquid-liquid extraction workflow in the field of forensic toxicological analysis.
DT-13 attenuates inflammation by inhibiting NLRP3-inflammasome related genes in RAW264.7 macrophages
(2024)
Plant derived saponins or other glycosides are widely used for their anti-inflammatory, antioxidant, and anti-viral properties in therapeutic medicine. In this study, we focus on understanding the function of the less known steroidal saponin from the roots of Liriope muscari L. H. Bailey – saponin C (also known as DT-13) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages in comparison to the well-known saponin ginsenoside Rk1 and anti-inflammatory drug dexamethasone. We proved that DT-13 reduces LPS-induced inflammation by inhibiting nitric oxide (NO) production, interleukin-6 (IL-6) release, cycloxygenase-2 (COX-2), tumour necrosis factor-alpha (TNF-α) gene expression, and nuclear factor kappa-B (NFκB) translocation into the nucleus. It also inhibits the inflammasome component NOD-like receptor family pyrin domain containing protein 3 (NLRP3) regulating the inflammasome activation. This was supported by the significant inhibition of caspase-1 and interleukin-1 beta (IL-1β) expression and release. This study demonstrates the anti-inflammatory effect of saponins on LPS-stimulated macrophages. For the first time, an in vitro study shows the attenuating effect of DT-13 on NLRP3-inflammasome activation. In comparison to the existing anti-inflammatory drug, dexamethasone, and triterpenoid saponin Rk1, DT-13 more efficiently inhibits inflammation in the applied cell culture model. Therefore, DT-13 may serve as a lead compound for the development of new more effective anti-inflammatory drugs with minimised side effects.
Matrix metalloproteinases (MMPs) are matrix-degrading enzymes that are over-expressed in joints of rheumatoid arthritis (RA) patients. However, the contribution of specific MMPs for the development of arthritic joints is unknown. This study is aimed at studying the role of matrix metalloproteinase-9 (MMP-9) in mice, using the K/BxN serum-transfer model of RA. Arthritis was induced in Balb/c mice by injecting K/BxN serum. Development of arthritis was followed in these mice by measuring ankle thickness and clinical index score. MMP-9 expression in the joints of mice killed at various time points during the disease progression was determined by gelatin zymography using ankle lysates. We found that MMP-9 expression increased with the severity of arthritis. Importantly MMP-9 deficient mice injected with K/BxN serum showed a milder form of arthritis in comparison to the control C57BL/6 mice injected with K/BxN serum. We therefore conclude that MMP-9 promotes arthritis in mice.
Background: Migration of mature and immature leukocytes in response to chemokines is not only essential during inflammation and host defense, but also during development of the hematopoietic system. Many molecules implicated in migratory polarity show uniform cellular distribution under non-activated conditions, but acquire a polarized localization upon exposure to migratory cues.
Methodology/Principal Findings: Here, we present evidence that raft-associated endocytic proteins (flotillins) are preassembled in lymphoid, myeloid and primitive hematopoietic cells and accumulate in the uropod during migration. Furthermore, flotillins display a polarized distribution during immunological synapse formation. Employing the membrane lipid-order sensitive probe Laurdan, we show that flotillin accumulation in the immunological synapse is concomittant with membrane ordering in these regions.
Conclusions: Together with the observation that flotillin polarization does not occur in other polarized cell types such as polarized epithelial cells, our results suggest a specific role for flotillins in hematopoietic cell polarization. Based on our results, we propose that in hematopoietic cells, flotillins provide intrinsic cues that govern segregation of certain microdomain-associated molecules during immune cell polarization.
Process-dependent thermo-mechanical viscoelastic properties and the corresponding morphology of HDPE extrusion blow molded (EBM) parts were investigated. Evaluation of bulk data showed that flow direction, draw ratio, and mold temperature influence the viscoelastic behavior significantly in certain temperature ranges. Flow induced orientations due to higher draw ratio and higher mold temperature lead to higher crystallinities. To determine the local viscoelastic properties, a new microindentation system was developed by merging indentation with dynamic mechanical analysis. The local process-structure-property relationship of EBM parts showed that the cross-sectional temperature distribution is clearly reflected by local crystallinities and local complex moduli. Additionally, a model to calculate three-dimensional anisotropic coefficients of thermal expansion as a function of the process dependent crystallinity was developed based on an elementary volume unit cell with stacked layers of amorphous phase and crystalline lamellae. Good agreement of the predicted thermal expansion coefficients with measured ones was found up to a temperature of 70 °C.
This study presents a microindentation system which allows spatially resolved local as well as bulk viscoelastic material information to be obtained within one instrument. The microindentation method was merged with dynamic mechanical analysis (DMA) for a tungsten cone indenter. Three tungsten cone indenters were investigated: tungsten electrode, tungsten electrode + 2% lanthanum, and tungsten electrode + rare earth elements. Only the tungsten electrode + 2% lanthanum indenter showed the sinusoidal response, and its geometry remained unaffected by the repeated indentations. Complex moduli obtained from dynamic microindentation for high-density polyethylene, polybutylene terephthalate, polycarbonate, and thermoplastic polyurethane are in agreement with the literature. Additionally, by implementing a specially developed x-y-stage, this study showed that dynamic microindentation with a tungsten cone indenter was an adequate method to determine spatially resolved local viscoelastic surface properties.