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Pyrolysis–Gas Chromatography
(2022)
The methodology of analytical pyrolysis-GC/MS has been known for several years, but is seldom used in research laboratories and process control in the chemical industry. This is due to the relative difficulty of interpreting the identified pyrolysis products as well as the variety of them. This book contains full identification of several classes of polymers/copolymers and biopolymers that can be very helpful to the user. In addition, the practical applications can encourage analytical chemists and engineers to use the techniques explored in this volume.
The French–Italian Concordia Research Station, situated on the Antarctic Polar Plateau at an elevation of 3233 m above sea level, offers a unique opportunity to study the presence and variation of microbes introduced by abiotic or biotic vectors and, consequently, appraise the amplitude of human impact in such a pristine environment. This research built upon a previous work, which explored microbial diversity in the surface snow surrounding the Concordia Research Station. While that study successfully characterized the bacterial assemblage, detecting fungal diversity was hampered by the low DNA content. To address this knowledge gap, in the present study, we optimized the sampling by increasing ice/snow collected to leverage the final DNA yield. The V4 variable region of the 16S rDNA and Internal Transcribed Spacer (ITS1) rDNA was used to evaluate bacterial and fungal diversity. From the sequencing, we obtained 3,352,661 and 4,433,595 reads clustered in 930 and 3182 amplicon sequence variants (ASVs) for fungi and bacteria, respectively. Amplicon sequencing revealed a predominance of Basidiomycota (49%) and Ascomycota (42%) in the fungal component; Bacteroidota (65.8%) is the main representative among the bacterial phyla. Basidiomycetes are almost exclusively represented by yeast-like fungi. Our findings provide the first comprehensive overview of both fungal and bacterial diversity in the Antarctic Polar Plateau’s surface snow/ice near Concordia Station and to identify seasonality as the main driver of microbial diversity; we also detected the most sensitive microorganisms to these factors, which could serve as indicators of human impact in this pristine environment and aid in planetary protection for future exploration missions.
The non-filarial and non-communicable disease podoconiosis affects around 4 million people and is characterized by severe leg lymphedema accompanied with painful intermittent acute inflammatory episodes, called acute dermatolymphangioadenitis (ADLA) attacks. Risk factors have been associated with the disease but the mechanisms of pathophysiology remain uncertain. Lymphedema can lead to skin lesions, which can serve as entry points for bacteria that may cause ADLA attacks leading to progression of the lymphedema. However, the microbiome of the skin of affected legs from podoconiosis individuals remains unclear. Thus, we analysed the skin microbiome of podoconiosis legs using next generation sequencing. We revealed a positive correlation between increasing lymphedema severity and non-commensal anaerobic bacteria, especially Anaerococcus provencensis, as well as a negative correlation with the presence of Corynebacterium, a constituent of normal skin flora. Disease symptoms were generally linked to higher microbial diversity and richness, which deviated from the normal composition of the skin. These findings show an association of distinct bacterial taxa with lymphedema stages, highlighting the important role of bacteria for the pathogenesis of podoconiosis and might enable a selection of better treatment regimens to manage ADLA attacks and disease progression.
Spektroskopische Qualifizierung und Quantifizierung von Hyaluronsäure in Nahrungsergänzungsmitteln
(2023)
Host-derived succinate accumulates in the airways during bacterial infection. Here, we show that luminal succinate activates murine tracheal brush (tuft) cells through a signaling cascade involving the succinate receptor 1 (SUCNR1), phospholipase Cβ2, and the cation channel transient receptor potential channel subfamily M member 5 (TRPM5). Stimulated brush cells then trigger a long-range Ca2+ wave spreading radially over the tracheal epithelium through a sequential signaling process. First, brush cells release acetylcholine, which excites nearby cells via muscarinic acetylcholine receptors. From there, the Ca2+ wave propagates through gap junction signaling, reaching also distant ciliated and secretory cells. These effector cells translate activation into enhanced ciliary activity and Cl- secretion, which are synergistic in boosting mucociliary clearance, the major innate defense mechanism of the airways. Our data establish tracheal brush cells as a central hub in triggering a global epithelial defense program in response to a danger-associated metabolite.
The Potential of Sustainable Antimicrobial Additives for Food Packaging from Native Plants in Benin
(2019)
Microbiome analyses are essential for understanding microorganism composition and diversity, but interpretation is often challenging due to biological and technical variables. DNA extraction is a critical step that can significantly bias results, particularly in samples containing a high abundance of challenging-to-lyse microorganisms. Taking into consideration the distinctive microenvironments observed in different bodily locations, our study sought to assess the extent of bias introduced by suboptimal bead-beating during DNA extraction across diverse clinical sample types. The question was whether complex targeted extraction methods are always necessary for reliable taxonomic abundance estimation through amplicon sequencing or if simpler alternatives are effective for some sample types. Hence, for four different clinical sample types (stool, cervical swab, skin swab, and hospital surface swab samples), we compared the results achieved from extracting targeted manual protocols routinely used in our research lab for each sample type with automated protocols specifically not designed for that purpose. Unsurprisingly, we found that for the stool samples, manual extraction protocols with vigorous bead-beating were necessary in order to avoid erroneous taxa proportions on all investigated taxonomic levels and, in particular, false under- or overrepresentation of important genera such as Blautia, Faecalibacterium, and Parabacteroides. However, interestingly, we found that the skin and cervical swab samples had similar results with all tested protocols. Our results suggest that the level of practical automation largely depends on the expected microenvironment, with skin and cervical swabs being much easier to process than stool samples. Prudent consideration is necessary when extending the conclusions of this study to applications beyond rough estimations of taxonomic abundance.
Transdermal therapeutic systems (TTS) represent an up-to-day medication applied to human skin, which consists of a drug-containing pressure-sensitive adhesive (PSA) and a flexible backing layer. The development of a reliable TTS requires precise knowledge of the viscoelastic tack behavior of PSA in terms of adhesion and detaching. Tailoring of a PSA can be achieved by altering the resin content or modifying the chemical properties of the macromolecules. In this study, three different resin content of two silicone-based PSA – non-amine compatible, and less tack, amine-compatible – were investigated with the help of recently developed RheoTack method to characterize the retraction speed dependent tack behavior for various geometries of the testing rods. The obtained force-retraction displacement-curves clearly depict the effect of the chemical structure as well as the resin content. Decreasing the resin content shifts the start of fibril fracture to larger deformations states and significantly enhances the stretchability of the fibrils. To compare various rod geometries precisely, the force-retraction displacement curves were normalized to account for effective contact areas. The flat and spherical rods led to completely different failure and tack behaviors. Furthermore, the adhesion formation between TTS with flexible backing layers and rods during the dwell phase happens in a different manner compared to rigid plates, in particular for flat rods, where maximum compression stresses occur at the edges and not uniformly over the cross-section. Thus, the approach to follow ASTM D2949 has to be reconsidered for tests of these materials.
This work presents an open source database with suitable retention parameters for prediction and simulation of GC separations and gives a short introduction to three common retention models. Useful computer simulations play an important role to save resources and time in method development in GC. Thermodynamic retention parameters for the ABC model and the K-centric model are determined by isothermal measurements. This standardized procedure of measurements and calculations, presented in this work, have a useful benefit for all chromatographers, analytical chemists, and method developers because it can be used in their own laboratories to simplify the method development. The main benefits as simulations of temperature-programed GC separations are demonstrated and compared to measurements. The observed deviations of predicted retention times are in most cases less than 1%. The database includes more than 900 entries with a large range of compounds such as VOCs, PAHs, FAMEs, PCBs, or allergenic fragrances over 20 different GC columns.
The development of whole-genome amplification (WGA) techniques has opened up new avenues for genetic analysis and genome research, in particular by facilitating the genome-wide analysis of few or even single copies of genomic DNA, such as from single cells (prokaryotic or eukaryotic) or virions. Using WGA, the few copies of genomic DNA obtained from such entities are unspecifically amplified using PCR or PCR-related processes in order to obtain higher DNA quantities that can then be successfully analysed further.
The epithelial sodium channel (ENaC) is a key regulator of sodium homeostasis that contributes to blood pressure control. ENaC open probability is adjusted by extracellular sodium ions, a mechanism referred to as sodium self-inhibition (SSI). With a growing number of identified ENaC gene variants associated with hypertension, there is an increasing demand for medium- to high-throughput assays allowing the detection of alterations in ENaC activity and SSI. We evaluated a commercially available automated two-electrode voltage-clamp (TEVC) system that records transmembrane currents of ENaC-expressing Xenopus oocytes in 96-well microtiter plates. We employed guinea pig, human and Xenopus laevis ENaC orthologs that display specific magnitudes of SSI. While demonstrating some limitations over traditional TEVC systems with customized perfusion chambers, the automated TEVC system was able to detect the established SSI characteristics of the employed ENaC orthologs. We were able to confirm a reduced SSI in a gene variant, leading to C479R substitution in the human α-ENaC subunit that has been reported in Liddle syndrome. In conclusion, automated TEVC in Xenopus oocytes can detect SSI of ENaC orthologs and variants associated with hypertension. For precise mechanistic and kinetic analyses of SSI, optimization for faster solution exchange rates is recommended.
Cyanobacteria are gaining considerable interest as a method of supporting the long-term presence of humans on the Moon and settlements on Mars due to their ability to produce oxygen and their potential as bio-factories for space biotechnology/synthetic biology and other applications. Since many unknowns remain in our knowledge to bridge the gap and move cyanobacterial bioprocesses from Earth to space, we investigated cell division resumption on the rehydration of dried Chroococcidiopsis sp. CCMEE 029 accumulated DNA damage while exposed to space vacuum, Mars-like conditions, and Fe-ion radiation. Upon rehydration, the monitoring of the ftsZ gene showed that cell division was arrested until DNA damage was repaired, which took 48 h under laboratory conditions. During the recovery, a progressive DNA repair lasting 48 h of rehydration was revealed by PCR-stop assay. This was followed by overexpression of the ftsZ gene, ranging from 7.5- to 9-fold compared to the non-hydrated samples. Knowing the time required for DNA repair and cell division resumption is mandatory for deep-space experiments that are designed to unravel the effects of reduced/microgravity on this process. It is also necessary to meet mission requirements for dried-sample implementation and real-time monitoring upon recovery. Future experiments as part of the lunar exploration mission Artemis and the lunar gateway station will undoubtedly help to move cyanobacterial bioprocesses beyond low Earth orbit. From an astrobiological perspective, these experiments will further our understanding of microbial responses to deep-space conditions.
ESKAPEE Pathogen Biofilm Control on Surfaces with Probiotic Lactobacillaceae and Bacillus species
(2023)
Combatting the rapidly growing threat of antimicrobial resistance and reducing prevalence and transmission of ESKAPEE pathogens in healthcare settings requires innovative strategies, one of which is displacing these pathogens using beneficial microorganisms. Our review comprehensively examines the evidence of probiotic bacteria displacing ESKAPEE pathogens, with a focus on inanimate surfaces. A systematic search was conducted using the PubMed and Web of Science databases on 21 December 2021, and 143 studies were identified examining the effects of Lactobacillaceae and Bacillus spp. cells and products on the growth, colonization, and survival of ESKAPEE pathogens. While the diversity of study methods limits evidence analysis, results presented by narrative synthesis demonstrate that several species have the potential as cells or their products or supernatants to displace nosocomial infection-causing organisms in a variety of in vitro and in vivo settings. Our review aims to aid the development of new promising approaches to control pathogen biofilms in medical settings by informing researchers and policymakers about the potential of probiotics to combat nosocomial infections. More targeted studies are needed to assess safety and efficacy of different probiotic formulations, followed by large-scale studies to assess utility in infection control and medical practice.
Several species of (poly)saccharides and organic acids can be found often simultaneously in various biological matrices, e.g., fruits, plant materials, and biological fluids. The analysis of such matrices sometimes represents a challenging task. Using Aloe vera (A. vera) plant materials as an example, the performance of several spectroscopic methods (80 MHz benchtop NMR, NIR, ATR-FTIR and UV-Vis) for the simultaneous analysis of quality parameters of this plant material was compared. The determined parameters include (poly)saccharides such as aloverose, fructose and glucose as well as organic acids (malic, lactic, citric, isocitric, acetic, fumaric, benzoic and sorbic acids). 500 MHz NMR and high-performance liquid chromatography (HPLC) were used as the reference methods.
UV-VIS data can be used only for identification of added preservatives (benzoic and sorbic acids) and drying agent (maltodextrin) and semiquantitative analysis of malic acid. NIR and MIR spectroscopies combined with multivariate regression can deliver more informative overview of A. vera extracts being able to additionally quantify glucose, aloverose, citric, isocitric, malic, lactic acids and fructose. Low-field NMR measurements can be used for the quantification of aloverose, glucose, malic, lactic, acetic, and benzoic acids. The benchtop NMR method was successfully validated in terms of robustness, stability, precision, reproducibility and limit of detection (LOD) and quantification (LOQ), respectively.
All spectroscopic techniques are useful for the screening of (poly)saccharides and organic acids in plant extracts and should be applied according to its availability as well as information and confidence required for the specific analytical goal. Benchtop NMR spectroscopy seems to be the most feasible solution for quality control of A. vera products.
Monitoring the content of dissolved ozone in purified water is often mandatory to ensure the appropriate levels of disinfection and sanitization. However, quantification bears challenges as colorimetric assays require laborious off-line analysis, while commercially available instruments for electrochemical process analysis are expensive and often lack the possibility for miniaturization and discretionary installation. In this study, potentiometric ionic polymer metal composite (IPMC) sensors for the determination of dissolved ozone in ultrapure water (UPW) systems are presented. Commercially available polymer electrolyte membranes are treated via an impregnation-reduction method to obtain nanostructured platinum layers. By applying 25 different synthesis conditions, layer thicknesses of 2.2 to 12.6 µm are obtained. Supporting radiographic analyses indicate that the platinum concentration of the impregnation solution has the highest influence on the obtained metal loading. The sensor response behavior is explained by a Langmuir pseudo-isotherm model and allows the quantification of dissolved ozone to trace levels of less than 10 µg L−1. Additional statistical evaluations show that the expected Pt loading and radiographic blackening levels can be predicted with high accuracy and significance (R2adj. > 0.90, p < 10−10) solely from given synthesis conditions.
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.
Analytical Chemistry I
(2023)
This workbook takes you through the successful work Harris, Textbook of Quantitative Analysis and is designed primarily for self-study. In five parts, the lecture content of analytical chemistry is summarized and explained using selected examples. Basic concepts of analytical chemistry are presented as well as the principle and various techniques of dimensional analysis and chromatography. UV/VIS, infrared and Raman spectroscopy are used to explain the investigation of molecularly present compounds, and selected techniques of atomic spectroscopy conclude the introduction to the fundamentals of analysis. The textbook's essential sections and illustrations are repeatedly referred to, which facilitates independent learning of the fundamentals of analytical chemistry.
ENaC channels
(2023)
Im Rahmen dieser Arbeit wurden zunächst neuartige ionische Agarosederivate synthetisiert und anschließend umfassend charakterisiert. Anionische Agarosesulfate mit einer regioselektiven Derivatisierung in Position G6 wurden durch homogene Umsetzung in ionischer Flüssigkeit erhalten. Kationische Agarosecarbamate mit einstellbarem Funktionalisierungsgrad waren durch einen zweistufigen Syntheseansatz zugänglich. Hierzu wurden zunächst Agarosephenylcarbonate in einer homogenen Synthese hergestellt, im Anschluss folgte eine Aminolyse zu den gewünschten funktionalen Agarosederivaten. Die ionischen Agarosederivate waren bereits bei geringen Funktionalisierungsgraden vollständig löslich in Wasser. Damit war es möglich, Alginatmikrokapseln polyelektrolytisch zu beschichten und diese als Träger für eine kontrollierte Wirkstofffreisetzung zu verwenden. Ebenfalls konnten Kompositgele aus Agarose, Hydroxyapatit und Agarosederivaten hergestellt und charakterisiert werden. Im zweiten Teil wurden sowohl die Kompositträgermaterialien als auch die Alginatmikrokapseln mit vier verschiedenen Modellwirkstoffen (ATP, Suramin, Methylenblau und A740003) beladen und die Wirkstofffreisetzung über einen Zeitraum von zwei Wochen untersucht. Für die ionischen Modellwirkstoffe erwiesen sich Kompositträgermaterialien mit ionischem Agarosederivat, die beschichteten Mikrokapseln sowie die Kombination aus Komposit und Kapseln als effektiv, um die Freisetzung auf bis zu 40% zu verlangsamen. Für die schlecht wasserlösliche Substanz A740003, ein Rezeptorligand für die osteogene Differenzierung von Stammzellen, wurde eine stark verzögerte Freisetzung aus Polyelektrolytemikrokapseln festgestellt. Mithilfe von literaturbekannten und neu entwickelten Anpassungsmodellen gelang es, die Diffusion als Hauptmechanismus der Wirkstofffreisetzung zu identifizieren und die Freisetzungskurven mathematisch akkurat zu beschreiben und daraus Rückschlüsse über die einzelnen Phasen der Freisetzung zu ziehen.
Pitfalls of using sequence databases for heterologous expression studies - a technical review
(2023)
Synthesis of DNA fragments based on gene sequences available in public resources has become an efficient and affordable method that gradually replaced traditional cloning efforts such as PCR cloning from cDNA. However, database entries based on genome sequencing results are prone to errors which can lead to false sequence information and, ultimately, errors in functional characterization of proteins such as ion channels and transporters in heterologous expression systems. We have identified five common problems that repeatedly appear in public resources: 1) Not every gene has yet been annotated; 2) Not all gene annotations are necessarily correct; 3) Transcripts may contain automated corrections; 4) There are mismatches between gene, mRNA, and protein sequences; and 5) Splicing patterns often lack experimental validation. This technical review highlights and provides a strategy to bypass these issues in order to avoid critical mistakes that could impact future studies of any gene/protein of interest in heterologous expression systems. Abstract figure legend Projects involving heterologous gene expression are often characterised by similar steps. Initially, database research (A) is necessary to retrieve information of full of partial sequences of a gene of interest. A multitude of genome assemblies are annotated and deposited in public databases or that are available for refined search options using individual sequence information. The search results need to be scrutinised and compared to already available information (B). Once the sequence has been determined, DNA synthesis (C) by PCR or commercial synthesis are necessary for further cloning procedures (D). Eventually, the DNA needs to be transfected (E) and expressed in, e.g., eukaryotic cells (F). Finally, the expression of the gene of interest needs to be documented and its function analysed (G). This article is protected by copyright. All rights reserved.
A biodegradable blend of PBAT—poly(butylene adipate-co-terephthalate)—and PLA—poly(lactic acid)—for blown film extrusion was modified with four multi-functional chain extending cross-linkers (CECL). The anisotropic morphology introduced during film blowing affects the degradation processes. Given that two CECL increased the melt flow rate (MFR) of tris(2,4-di-tert-butylphenyl)phosphite (V1) and 1,3-phenylenebisoxazoline (V2) and the other two reduced it (aromatic polycarbodiimide (V3) and poly(4,4-dicyclohexylmethanecarbodiimide) (V4)), their compost (bio-)disintegration behavior was investigated. It was significantly altered with respect to the unmodified reference blend (REF). The disintegration behavior at 30 and 60 °C was investigated by determining changes in mass, Young’s moduli, tensile strengths, elongations at break and thermal properties. In order to quantify the disintegration behavior, the hole areas of blown films were evaluated after compost storage at 60 °C to calculate the kinetics of the time dependent degrees of disintegration. The kinetic model of disintegration provides two parameters: initiation time and disintegration time. They quantify the effects of the CECL on the disintegration behavior of the PBAT/PLA compound. Differential scanning calorimetry (DSC) revealed a pronounced annealing effect during storage in compost at 30 °C, as well as the occurrence of an additional step-like increase in the heat flow at 75 °C after storage at 60 °C. The disintegration consists of processes which affect amorphous and crystalline phase of PBAT in different manner that cannot be understood by a hydrolytic chain degradation only. Furthermore, gel permeation chromatography (GPC) revealed molecular degradation only at 60 °C for the REF and V1 after 7 days of compost storage. The observed losses of mass and cross-sectional area seem to be attributed more to mechanical decay than to molecular degradation for the given compost storage times.
Background: the potency of drugs that interfere with glucose metabolism, i.e., glucose transporters (GLUT) and nicotinamide phosphoribosyltransferase (NAMPT) was analyzed in neuroendocrine tumor (NET, BON-1, and QPG-1 cells) and small cell lung cancer (SCLC, GLC-2, and GLC-36 cells) tumor cell lines. (2) Methods: the proliferation and survival rate of tumor cells was significantly affected by the GLUT-inhibitors fasentin and WZB1127, as well as by the NAMPT inhibitors GMX1778 and STF-31. (3) Results: none of the NET cell lines that were treated with NAMPT inhibitors could be rescued with nicotinic acid (usage of the Preiss–Handler salvage pathway), although NAPRT expression could be detected in two NET cell lines. We finally analyzed the specificity of GMX1778 and STF-31 in NET cells in glucose uptake experiments. As previously shown for STF-31 in a panel NET-excluding tumor cell lines, both drugs specifically inhibited glucose uptake at higher (50 μM), but not at lower (5 μM) concentrations. (4) Conclusions: our data suggest that GLUT and especially NAMPT inhibitors are potential candidates for the treatment of NET tumors.
PURPOSE
Cervical cancer (CC) is caused by a persistent high-risk human papillomavirus (hrHPV) infection. The cervico-vaginal microbiome may influence the development of (pre)cancer lesions. Aim of the study was (i) to evaluate the new CC screening program in Germany for the detection of high-grade CC precursor lesions, and (ii) to elucidate the role of the cervico-vaginal microbiome and its potential impact on cervical dysplasia.
METHODS
The microbiome of 310 patients referred to colposcopy was determined by amplicon sequencing and correlated with clinicopathological parameters.
RESULTS
Most patients were referred for colposcopy due to a positive hrHPV result in two consecutive years combined with a normal PAP smear. In 2.1% of these cases, a CIN III lesion was detected. There was a significant positive association between the PAP stage and Lactobacillus vaginalis colonization and between the severity of CC precursor lesions and Ureaplasma parvum.
CONCLUSION
In our cohort, the new cervical cancer screening program resulted in a low rate of additional CIN III detected. It is questionable whether these cases were only identified earlier with additional HPV testing before the appearance of cytological abnormalities, or the new screening program will truly increase the detection rate of CIN III in the long run. Colonization with U. parvum was associated with histological dysplastic lesions. Whether targeted therapy of this pathogen or optimization of the microbiome prevents dysplasia remains speculative.
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.
Isovaleric acidemia (IVA), due to isovaleryl-CoA dehydrogenase (IVD) deficiency, results in the accumulation of isovaleryl-CoA, isovaleric acid and secondary metabolites. The increase in these metabolites decreases mitochondrial energy production and increases oxidative stress. This contributes to the neuropathological features of IVA. A general assumption in the literature exists that glycine N-acyltransferase (GLYAT) plays a role in alleviating the symptoms experienced by IVA patients through the formation of N-isovalerylglycine. GLYAT forms part of the phase II glycine conjugation pathway in the liver and detoxifies excess acyl-CoA’s namely benzoyl-CoA. However, very few studies support GLYAT as the enzyme that conjugates isovaleryl-CoA to glycine. Furthermore, GLYATL1, a paralogue of GLYAT, conjugates phenylacetyl-CoA to glutamine. Therefore, GLYATL1 might also be a candidate for the formation of N-isovalerylglycine. Based on the findings from the literature review, we proposed that GLYAT or GLYATL1 can form N-isovalerylglycine in IVA patients. To test this hypothesis, we performed an in-silico analysis to determine which enzyme is more likely to conjugate isovaleryl-CoA with glycine using AutoDock Vina. Thereafter, we performed in vitro validation using purified enzyme preparations. The in-silico and in vitro findings suggested that both enzymes could form N-isovaleryglycine albeit at lower affinities than their preferred substrates. Furthermore, an increase in glycine concentration does not result in an increase in N-isovalerylglycine formation. The results from the critical literature appraisal, in-silico, and in vitro validation, suggest the importance of further investigating the reaction kinetics and binding behaviors between these substrates and enzymes in understanding the pathophysiology of IVA.
This research studies in detail four different assays, namely DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), FRAP (ferric ion reducing antioxidant potential) and FC (Folin-Ciocalteu), to determine the antioxidant capacity of standard substances as well as 50 organosolv lignins, and two kraft lignins. The coefficient of variation was determined for each method and was lowest for ABTS and highest for DPPH. The best correlation was found for FRAP and FC, which both rely on a single electron transfer mechanism. A good correlation between ABTS, FRAP and FC, respectively, could be observed, even though ABTS relies on a more complex reaction mechanism. The DPPH assay merely correlates with the others, implying that it reflects different antioxidative attributes due to a different reaction mechanism. Lignins obtained from paulownia and silphium have been investigated for the first time regarding their antioxidant capacity. Paulownia lignin is in the same range as beech wood lignin, while silphium lignin resembles wheat straw lignin. Miscanthus lignin is an exception from the grass lignins and possesses a significantly higher antioxidant capacity. All lignins possess a good antioxidant capacity and thus are promising candidates for various applications, e. g. as additives in food packaging or for biomedical purposes.
Indoor spaces exhibit microbial compositions that are distinctly dissimilar from one another and from outdoor spaces. Unique in this regard, and a topic that has only recently come into focus, is the microbiome of hospitals. While the benefits of knowing exactly which microorganisms propagate how and where in hospitals are undoubtedly beneficial for preventing hospital-acquired infections, there are, to date, no standardized procedures on how to best study the hospital microbiome. Our study aimed to investigate the microbiome of hospital sanitary facilities, outlining the extent to which hospital microbiome analyses differ according to sample-preparation protocol. For this purpose, fifty samples were collected from two separate hospitals—from three wards and one hospital laboratory—using two different storage media from which DNA was extracted using two different extraction kits and sequenced with two different primer pairs (V1–V2 and V3–V4). There were no observable differences between the sample-preservation media, small differences in detected taxa between the DNA extraction kits (mainly concerning Propionibacteriaceae), and large differences in detected taxa between the two primer pairs V1–V2 and V3–V4. This analysis also showed that microbial occurrences and compositions can vary greatly from toilets to sinks to showers and across wards and hospitals. In surgical wards, patient toilets appeared to be characterized by lower species richness and diversity than staff toilets. Which sampling sites are the best for which assessments should be analyzed in more depth. The fact that the sample processing methods we investigated (apart from the choice of primers) seem to have changed the results only slightly suggests that comparing hospital microbiome studies is a realistic option. The observed differences in species richness and diversity between patient and staff toilets should be further investigated, as these, if confirmed, could be a result of excreted antimicrobials.
The Concordia Research Station provides a unique location for preparatory activities for future human journey to Mars, to explore microbial diversity at subzero temperatures, and monitor the dissemination of human-associated microorganisms within the pristine surrounding environment. Amplicon sequencing was leveraged to investigate the microbial diversity of surface snow samples collected monthly over a two-year period, at three distances from the Station (10, 500, and 1000 m). Even when the extracted total DNA was below the detection limit, 16S rRNA gene sequencing was successfully performed on all samples, while 18S rRNA was amplified on 19 samples out of 51. No significant relationships were observed between microbial diversity and seasonality (summer or winter) or distance from the Concordia base. This suggested that if present, the anthropogenic impact should have been below the detectable limit. While harboring low microbial diversity, the surface snow samples were characterized by heterogeneous microbiomes. Ultimately, our study corroborated the use of DNA sequencing-based techniques for revealing microbial presence in remote and hostile environments, with implications for Planetary Protection during space missions and for life-detection in astrobiology relevant targets.
Thermo-chemical conversion of cucumber peel waste for biobased energy and chemical production
(2022)
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.
Bonding wires made of aluminum are the most used materials for the transmission of electrical signals in power electronic devices. During operation, different cyclic mechanical and thermal stresses can lead to fatigue loads and a failure of the bonding wires. A prediction or prevention of the wire failure is not yet possible by design for all cases. The following work presents meaningful fatigue tests in small wire dimensions and investigates the influence of the R-ratio on the lifetime of two different aluminum wires with a diameter of 300 μm each. The experiments show very reproducible fatigue results with ductile failure behavior. The endurable stress amplitude decreases linearly with an increasing stress ratio, which can be displayed by a Smith diagram, even though the applied maximum stresses exceed the initial yield stresses determined by tensile tests. A scaling of the fatigue results by the tensile strength indicates that the fatigue level is significantly influenced by the strength of the material. Due to the very consistent findings, the development of a generalized fatigue model for predicting the lifetime of bonding wires with an arbitrary loading situation seems to be possible and will be further investigated.
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.
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.
The Chemotype of Chromanones as a Privileged Scaffold for Multineurotarget Anti-Alzheimer Agents
(2022)
The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel and the epithelial Na+ channel (ENaC) play essential roles in transepithelial ion and fluid transport in numerous epithelial tissues. Inhibitors of both channels have been important tools for defining their physiological role in vitro. However, two commonly used CFTR inhibitors, CFTRinh-172 and GlyH-101, also inhibit non-CFTR anion channels, indicating they are not CFTR specific. However, the potential off-target effects of these inhibitors on epithelial cation channels has to date not been addressed. Here, we show that both CFTR blockers, at concentrations routinely employed by many researchers, caused a significant inhibition of store-operated calcium entry (SOCE) that was time-dependent, poorly reversible and independent of CFTR. Patch clamp experiments showed that both CFTRinh-172 and GlyH-101 caused a significant block of Orai1-mediated whole cell currents, establishing that they likely reduce SOCE via modulation of this Ca2+ release-activated Ca2+ (CRAC) channel. In addition to off-target effects on calcium channels, both inhibitors significantly reduced human αβγ-ENaC-mediated currents after heterologous expression in Xenopus oocytes, but had differential effects on δβγ-ENaC function. Molecular docking identified two putative binding sites in the extracellular domain of ENaC for both CFTR blockers. Together, our results indicate that caution is needed when using these two CFTR inhibitors to dissect the role of CFTR, and potentially ENaC, in physiological processes.
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.
The following work presents algorithms for semi-automatic validation, feature extraction and ranking of time series measurements acquired from MOX gas sensors. Semi-automatic measurement validation is accomplished by extending established curve similarity algorithms with a slope-based signature calculation. Furthermore, a feature-based ranking metric is introduced. It allows for individual prioritization of each feature and can be used to find the best performing sensors regarding multiple research questions. Finally, the functionality of the algorithms, as well as the developed software suite, are demonstrated with an exemplary scenario, illustrating how to find the most power-efficient MOX gas sensor in a data set collected during an extensive screening consisting of 16,320 measurements, all taken with different sensors at various temperatures and analytes.