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Metabolomics: Metabolites - a new generation of biomarkers for the detection of fatal traumatic brain injury
No full textDas SHT ist eine ubiquitär vorkommende, häufige Unfallfolge mit einer jährlichen Prävalenz von mehreren Millionen Fällen weltweit und massiven Kosten für die Gesundheitssysteme. Auch in vielen rechtsmedizinischen Obduktionen stellt es eine potenzielle Todesursache dar, welche hierbei bestätigt oder ausgeschlossen werden muss. Trotz der hohen Fallzahlen im Arbeitsalltag existieren neben der Autopsie bisher kaum laboranalytische Methoden zur Identifikation eines SHTs. Die vorliegende Dissertation befasst sich daher mit der Frage, ob ein massenspektrometrischer Nachweis gewisser Metabolite in der CSF geeignet ist, um als zuverlässiger Surrogatmarker im Rahmen einer Obduktion postmortal ein SHT als Todesursache zu identifizieren - beziehungsweise dieses von der Kontrollgruppe des akuten Herztodes zu unterscheiden. Die Untersuchung diente zur Prüfung der Hypothese, ob der Quotient einer bestimmten CSF-Metaboliten-Kombination ein SHT zuverlässig von der genannten Kontrollgruppe abgrenzen und somit als Biomarker verwendet werden kann. Die LC/MS-Analyse der autoptisch gewonnenen CSF mittels MeOH/H2O- und modifizierter Bligh/Dyer (B/D)-Extraktion, die anschließende statistische und graphische Auswertung und die Bildung des SHT-Herztod-Quotienten zeigten hierbei vielversprechende Ergebnisse. So konnten 212 Metabolite identifiziert werden, die eine signifikante Konzentrationsänderung bei letal verlaufenem SHT im Vergleich zur Kontrollgruppe aufwiesen. Des Weiteren konnte ein Quotient aus insgesamt zehn Metaboliten gebildet werden, der nach Festlegung des Cut-off-Wertes auf ≥ 8,626 93,3% der SHTs und 100% der akuten Herztodesfälle richtig positiv erkannt hat (SHT-Sensitivität: 93,3%, SHT-Spezifität: 100%). Viele Metaboliten-Messwerte nach letal verlaufenem SHT können anhand wissenschaftlicher Publikationen und physiologischer Modelle erklärt und begründet werden. Die vorliegende Arbeit stellt hierzu aber auch einige neue Theorien auf, die spezifische posttraumatisch-metabolische Abläufe des menschlichen Körpers zu erklären versuchen. Die LC/MS-Analyse des Metaboloms kann zukünftig in der Rechtsmedizin als innovative und erfolgversprechende Methode zur Detektion von posttraumatischen und postmortalen Stoffwechselabläufen angesehen werden. Darüber hinaus kann sie zu einem verbesserten Grundverständnis physiologischer Abläufe beitragen und hierbei auch zur Entwicklung neuer klinischer Therapiemethoden eines SHTs führen.TBI is a ubiquitous and frequent consequence of accidents with an annual prevalence of several million cases worldwide and massive costs for healthcare systems. It is also a potential cause of death in many forensic autopsies, which must be confirmed or ruled out. Despite the high number of cases in everyday work, there are hardly any laboratory analysis methods for identifying a TBI apart from autopsies. This dissertation therefore deals with the question of whether mass spectrometric detection of certain metabolites in CSF is suitable as a reliable surrogate marker in the context of a post-mortem autopsy to identify a TBI as the cause of death - or to distinguish it from the control group of acute cardiac death. The study served to test the hypothesis as to whether the quotient of a specific CSF metabolite combination can reliably differentiate a TBI from the aforementioned control group and thus be used as a biomarker. The LC/MS analysis of the autopsy-derived CSF using MeOH/H2O and modified Bligh/Dyer (B/D) extraction, the subsequent statistical and graphical evaluation and the formation of the SHT cardiac death quotient showed promising results. 212 metabolites were identified that showed a significant change in concentration in lethal TBI compared to the control group. Furthermore, a quotient was formed from a total of ten metabolites, which, after setting the cut-off value at ≥ 8.626, correctly recognised 93.3% of TBIs and 100% of acute cardiac deaths (TBI sensitivity: 93.3%, TBI specificity: 100%). Many metabolite measurements after lethal TBI can be explained and justified on the basis of scientific publications and physiological models. However, this study also presents some new theories that attempt to explain specific post-traumatic metabolic processes in the human body. In future, LC/MS analysis of the metabolome can be seen as an innovative and promising method for detecting post-traumatic and post-mortem metabolic processes in forensic medicine. In addition, it can contribute to an improved basic understanding of physiological processes and also lead to the development of new clinical treatment methods for TBI
Investigation on the function of LASP1 in macrophages in respect of atherosclerosis
No full textIm Rahmen der Krebsforschung ist LASP1 in Aktin-reichen Strukturen des Zytoskeletts (Invadopodien, Pseudopodien) nachgewiesen worden und beeinflusst nachweislich zelluläre Eigenschaften wie Migration, Proliferation und Adhäsion. In MDAMB-231 Zellen ist bekannt, dass LASP1 die Expression und Sekretion von MMP1, -2, -3 und -9 beeinflusst.
Die nachweislich von LASP1 veränderten zellulären Eigenschaften sind auch für Immunzellen im Rahmen der Pathogenese der Atherosklerose relevant. Um die Effekte von LASP1 auf die Atherosklerose zu analysieren, wurde ein LASP1-/- LDLR-/- Doppelknockout Mausmodell für in vitro und zukünftige in vivo-Studien etabliert.
Aus dem Mausmodell gewonnene M-mBMDM zeigen nach mLASP1 Knockout eine verringerte Proliferation und sind damit konkordant zu humanen Befunden. Ein Einfluss von mLASP1 auf die Adhäsion konnte in M-mBMDM nicht nachgewiesen werden. Die mRNA-Expression von MMPs in M-mBMDM wurde ausführlich durch qPCR untersucht. Hierbei zeigte sich lediglich eine signifikante Steigerung der mRNA-Expression von MMP12 nach mLASP1 Knockout. Als mögliche Ursache für die fehlende Regulation von MMP2 und -9 durch mLASP1 konnte ein verändertes Bindemotiv für LASP1 im murinen ZO-2 Homolog identifiziert werden. ZO-2 ist essenziell für die Translokation von hLASP1 in den Zellkern.
Das im Western Blot gemessene Proteinlevel von mMMP12 zeigte sich nach LASP1 Knockout nicht signifikant verändert. Da hLASP1 aber auch nachweislich die Sekretion von MMPs beeinflusst, wurde zur Messung der mMMP12-Aktivität ein Gelatine-Degradationsassay etabliert. Da M-mBMDM jedoch nur eine sehr geringe Gelatinedegradation zeigten, wurde der Assay schließlich mit GM-mBMDM durchgeführt. So konnte nach LASP1 Knockout eine verringerte Gelatinedegradation beobachtet werden.
Die Vermittlung pro- oder anti-atherogener Effekte in Makrophagen ist auch vom Mikromilieau abhängig. Nach LASP1 Knockout zeigte sich keine Veränderung der mRNA-Expression der Markergene iNOS (M1) bzw. Arg1 (M2) in M-mBMDM nach Stimulation mit LPS bzw. IL-4.In the context of cancer research, LASP1 has been detected in actin-rich structures of the cytoskeleton (invadopodia, pseudopodia), and has been shown to significantly influence cellular properties such as migration, proliferation and adhesion. It is known that LASP1 affects the expression and secretion of MMP1, -2, -3, and -9 in MDAMB-231 cells.
The cellular attributes evidently altered by LASP1 are also relevant for immune cells in the pathogenesis of atherosclerosis. To investigate the effects of LASP1 on atherosclerosis, a LASP1-/- LDLR-/- double knockout mouse model has been established for in vitro and subsequent in vivo studies.
M-mBMDMs obtained from this mouse model showed reduced proliferation following LASP1 knockout, in agreement with findings from human studies. An influence of LASP1 on the adhesion of M-mBMDM could not be obtained. The mRNA expression of MMPs in M-mBMDM was examined extensively by qPCR. Here only MMP12 showed a significant increase in mRNA expression following LASP1 knockout. As a possible cause for the lack of regulation of MMP2 and -9 by mLASP1, a modified binding motif for LASP1 was identified in the murine ZO-2 homolog. ZO-2 is essential for the translocation of hLASP1 into the nucleus.
Western Blot studies did not find a corresponding alteration in protein level of MMP12. Since hLASP1 evidently also affects the secretion of MMPs, a gelatin degradation assay was used to measure mMMP12 activity. However, M-mBMDM demonstrated only a low level of gelatin degradation. Therefore, the degradation assay was conducted using GM-mBMDM. So, following LASP1 knockout the assay showed a reduced degradation of gelatin.
The mediation of pro- or anti-atherogenic effects in macrophages also depends on the microenvironment. No change in the mRNA expression of the marker genes iNOS (M1) or Arg1 (M2) in M-mBMDM was observed after stimulation with LPS or IL-4 following LASP1 knockout
Mental health of nursing home staff during the COVID-19 pandemic
No full textDie COVID-19-Pandemie zeigt ihre Auswirkungen nicht ausschließlich durch direkte Infektionsgeschehen, sondern auch indirekt als Krise mentaler Gesundheit. Als besonders betroffen wurden hierbei Gesundheitsfachkräfte identifiziert. Obwohl das Coronavirus Einrichtungen der stationären Langzeitpflege in besonderer Härte traf, mangelt es an Studien zum psychischen Wohlergehen von Altenpflegepersonal, insbesondere unter Berücksichtigung langfristiger Entwicklungen. Diese Arbeit zielte daher darauf ab, den Verlauf von psychischen Belastungen unter Pflegeheimmitarbeitenden zu erfassen und signifikante Einflussfaktoren auf deren Manifestation herauszuarbeiten. Hierfür wurde ein umfassender Fragebogen eingesetzt, welcher psychische und physische Belastungen, COVID-spezifische, protektive und berufliche Faktoren durch COVID-19 beinhaltete und von Mitarbeitenden aus zehn langzeitpflegerischen Einrichtungen in Unterfranken beantwortet wurde. Die Ergebnisse der Analyse über das psychische Befinden über einen Zeitraum von zwei Jahren offenbarten stagnierend hohe Werte des PHQ-2 und GAD-2, eine Abnahme der IES-R-Werte sowie eine Zunahme des MBI. Diese sollten bezüglich ihrer Ausprägung und Dynamik als beunruhigend bewertet werden. Die Untersuchung potenzieller Einflussfaktoren ergab u.a. eine deutlich hohe Korrelation zwischen PHQ-2, GAD-2, IES-R und MBI, was die generelle psychische Vulnerabilität von Individuen in Krisensituationen betont und die Notwendigkeit ihrer Identifikation hervorhebt. Als besonders ausgeprägt erwies sich zusätzlich der Zusammenhang zwischen dem MBI-Wert und weiteren arbeitsplatzbezogenen Faktoren wie einer erheblichen Beeinträchtigung durch muskuloskelettale Beschwerden bzw. Kopfschmerzen, einer reduzierten subjektiven Arbeitsfähigkeit sowie einer intensiven Arbeitsbelastung in der Woche vor der Befragung. Dies erfordert eine Entlastung des Personals bezüglich qualitativer und quantitativer Arbeitsanforderungen. Vor dem Hintergrund der anhaltenden Dynamik und hohen Prävalenzen psychischer Belastungen ist einerseits eine fortlaufende Reevaluation der Gesamtsituation geboten. Andererseits erscheint eine gezielte therapeutische Unterstützung aktuell betroffener Mitarbeitenden sowie die Initiierung frühzeitiger präventiver Interventionen im Setting der Langzeitpflege für zukünftige Krisensituationen unabdingbar.The Covid-19 pandemic has manifested not only through direct infection incidents but also indirectly as a mental health crisis. Concerning this, health care workers have been identified as particularly affected. Despite the disproportional impact of the coronavirus on long-term care facilities, there is a notable lack of studies on mental health and the psychological distress of their staff, especially with regard to long-term effects. Thus, this study aimed to assess themental health of staff in nursing homes during the course of the pandemic and to identify risk and protective factors. Herefore, between November 2020 and December 2022, an exploratory observational study using an online questionnaire was conducted in five waves across ten long-term nursing facilities in Bavaria. A total of 87 questionnaires were included in the analysis. The study compared mean scores over time for mental burden assessed by symptoms of depression (PHQ-2), anxiety (GAD-2), PTSD (IES-R), the burnout syndrome (MBI), subjective work ability (WAS), and subjective prognosis of work capacity (SPE-scale) as well as for physical strains. Additionally, data on COVID-specific and protective factors and occupational effects of COVID-19 was collected and analyzed using bivariate correlation analysis to investigate potential associations with mental health. Longitudinal analysis of nursing home staff’s mental burden showed persistently high rates of depressive (PHQ-2 in May 2021: M=1.77 (SEM=0.24)) and anxiety (GAD-2 in May 2021: M=1.62 (SEM=0.27)) symptoms, a decrease in the mean IES-R scores regarding post-traumatic stress symptoms (from M=7.6 (SEM=0.7) in May 2021 to M=6.4 (SEM=0.8) in May 2022) and an increase in burnout symptoms (MBI from M=2.1 (SEM=1.0) in November 2020 to M=6.8 (SEM=1.2) in November 2022). Subjective prognosis of work capacity also worsened over time: in the last point of data collection, for two-thirds of the nursing home staff a premature retirement within the next two to three years was likely according to the SPE-scale. Bivariate correlation analysis revealed significant correlations between PHQ-2 and GAD-2 (ρ=0.76, p<0.001), IES-R (ρ=0.71, p<0.001) and MBI (ρ=0.64, p=0.002) as well as between IES-R and GAD-2 (ρ=0.76, p<0.001) or MBI (ρ=0.68, p<0.001). This emphasizes a general vulnerability of some individuals to the development of psychological symptoms. Increased incidences of burnout symptoms were associated with significant impairment due to musculoskeletal complaints or headaches (ρ=0.65, p=0.002) and intensively perceived job stress in the week preceding the survey (ρ=0.60, p=0.012). This means, nursing home staff have been identified as a comparably heavily burdened group throughout the pandemic. Due to the persistently high prevalence of mental health issues, the integration of preventive and therapeutic measures into the professional routine is required. Concerning potential future pandemics, early preventive interventions in the setting of long-term care are crucial to avert severe impacts on the mental health of nursing home staff. These measures are urgently necessary not only for the individual, but they also are of societal importance due to the potential loss of working capacity and the subsequent exacerbation of nursing shortage
Reversible formation of tetraphenylpentalene, a room temperature stable antiaromatic hydrocarbon†
No full text1,3,4,6-Tetraphenylpentalene (PhPn) has been synthesised by chemical oxidation of the corresponding pentalenide complex Mg[PhPn] with iodine. PhPn is a rare example of a room-temperature stable hydrocarbon that is antiaromatic by Hückel's rule and has been fully characterised by NMR and UV-vis spectroscopy, mass spectrometry as well as single-crystal X-ray diffraction. Quantum chemical studies including nucleus-independent chemical shift (NICS) and anisotropy of the induced current density (ACID) calculations showed the existence of an 8π antiaromatic core decorated with four independent 6π aromatic substituents. The formation of PhPn is reversible and it can be reduced back to the 10π aromatic PhPn with potassium
An insight into the structure of acebutolol tetraphenylborate: crystal structure and quantum chemical calculations
No full textReaction between Acebutolol hydrochloride and sodium tetraphenylborate in water afforded an Acebutolol-tetraphenyl-borate complex. This ion-pair was characterized by a variety of analytical and spectroscopic tools including X-ray crystallographic analysis. Comprehensive theoretical studies including ground-state geometry optimization, Mulliken atomic charges, and vibrational analysis were executed to get an insight into the nature of the charge transfer between the donor and acceptor ions. The crystal structure demonstrated that three inter- and intramolecular hydrogen-bonds stabilize the molecular packing in the solid state. Natural bond orbital analysis confirmed the presence of many interactions between B(Ph)− and particular Acebutolol functional groups, such as NH and OH groups. The positive Mulliken atomic charges of the acidic protons of the NH and OH groups of Acebutolol were increased upon the formation of the ion-pair. This has been experimentally confirmed by IR and NMR spectroscopies. Through the use of frontier molecular orbital models, time-dependent density functional theory calculations have provided more insight into the existence of two electronic transitions, beginning at B(Ph)− and terminating at the π* system of mono-protonated Acebutolol ions
Antimicrobial triazinedione inhibitors of the translocase MraY–protein E interaction site: synergistic effects with bacitracin imply a new mechanism of action
No full textEscherichia coli translocase MraY is the target for bacteriolytic protein E from bacteriophage ϕX174, interacting at a site close to Phe-288 on helix 9, on the extracellular face of the protein. A peptide motif Arg-Trp-x-x-Trp from protein E was used to design a set of triazinedione peptidomimetics, which inhibit particulate MraY (6d IC 48 μM), and show antimicrobial activity against Gram-negative and Gram-positive antibiotic-resistant clinical strains (7j MIC Acinetobacter baumannii 16 μg mL, Staphyloccoccus aureus MRSA 2–4 μg mL). Docking against a predicted structure for E. coli MraY revealed two possible binding sites close to helix 9, the binding site for protein E. Antimicrobial activity of analogue 6j was found to be synergistic with bacitracin in Micrococcus flavus, consistent with a link between this inhibition site and undecaprenyl phosphate uptake. Alkaloid michellamine B, also predicted to bind in the cleft adjacent to helix 9, was also found to be synergistic with bacitracin. These data provide experimental evidence that the unusual hydrophobic cleft adjacent to helix 9 in MraY is involved in uptake of undecaprenyl phosphate, in addition to recently identified transporters UptA and PopT, and that this process can be targeted by small molecules as a novel antibacterial mechanism
Cytotoxicity of Pd(II) and Pt(II) complexes of 2′,6′-di(thiazol-2-yl)-2,4′-bipyridine: insights into the mode of cell death and cell cycle arrest
No full textSquare-planar complexes were synthesized by the reaction of 2′,6′-di(thiazol-2-yl)-2,4′-bipyridine with either Na[PdCl] or K[PtCl], and these were thoroughly structurally characterized using some analytical and spectroscopic techniques. Density functional theory computations, including natural bond orbital analysis, were used to complement the experimental work to gain insight into the natural charge and electronic arrangement of the metal ion, as well as the strength of the metal–ligand bonds. The Pd(ii) complex exhibited exceptional cytotoxicity against the A549 and HCT-116 cell lines with IC values of 60.1 ± 3.45 and 23.8 ± 1.48 μM, respectively. Unfortunately, the Pd(ii) complex was harmful to the Vero normal cell line with an IC value of 24.5 ± 2.13 μM. The Pt(ii) complex is unstable and has a high likelihood of exchanging the chlorido ligand for solvent molecules such as DMSO. The fluorescent-stain photos of the treated HCT-116 cells with the Pd(ii) complex showed increased apoptotic bodies, indicating both early (18%) and late apoptosis (32%), as well as a necrosis ratio of about 10%. Flow cytometric analysis demonstrated that a cell arrest was induced by the Pd(ii) complex on HCT-116 cells in the G/M phase
In vivo vascularization of cell-supplemented spider silk-based hydrogels in the arteriovenous loop model
No full textThe goal of reconstructive surgery in treating tissue defects is to achieve a stable reconstructive outcome while minimizing donor site morbidity. As a result, tissue engineering has emerged as a key focus in the pursuit of this goal. One approach is to create a tissue container that can be preconditioned and later transplanted into the defect area. The characteristics of the matrices used in the tissue container are critical to this approach’s success. Matrices generated with recombinant, functionalized spider silk (eADF4(C16)-RGD) have been reported to be biocompatible and easy to vascularize. However, the effect of exogenously added proangiogenic cells, such as endothelial cells (T17b), on the vascularization process of matrices generated with this hydrogel in vivo has not been described yet. In this study, we implanted arteriovenous (AV) loop containers filled with a spider silk hydrogel consisting of an eADF4(C16)-RGD matrix and encapsulated, differentiated endothelial T17b cells producing the reporter protein TNFR2-Fc-Flag-GpL. The histological and µCT analyses revealed spontaneous angiogenesis and fibrovascular tissue formation in the container at 2 and 4 weeks post-implantation. The reporter protein was detected after 4 weeks. No severe immune response was observed. Altogether, this study demonstrates that cell-supplemented recombinant spider silk is a highly promising hydrogel to produce matrices for tissue engineering applications
Fluoroquinolone-mediated tendinopathy and tendon rupture
No full textThe fluoroquinolone (FQ) class of antibiotics includes the world’s most prescribed antibiotics such as ciprofloxacin, levofloxacin, and ofloxacin that are known for their low bacterial resistance. This is despite their potential to trigger severe side effects, such as myopathy, hearing loss, tendinopathy, and tendon rupture. Thus, healthcare organizations around the world have recommended limiting the prescription of FQs. Tendinopathy is a common name for maladies that cause pain and degeneration in the tendon tissue, which can result in tendon rupture. Whilst there are several identified effects of FQ on tendons, the exact molecular mechanisms behind FQ-mediated tendon rupture are unclear. Previous research studies indicated that FQ-mediated tendinopathy and tendon rupture can be induced by changes in gene expression, metabolism, and function of tendon resident cells, thus leading to alterations in the extracellular matrix. Hence, this review begins with an update on FQs, their mode of action, and their known side effects, as well as summary information on tendon tissue structure and cellular content. Next, how FQs affect the tendon tissue and trigger tendinopathy and tendon rupture is explored in detail. Lastly, possible preventative measures and promising areas for future research are also discussed. Specifically, follow-up studies should focus on understanding the FQ-mediated tendon changes in a more complex manner and integrating in vitro with in vivo models. With respect to in vitro systems, the field should move towards three-dimensional models that reflect the cellular diversity found in the tissue
Lasting benefits of a web-based training in understanding informal arguments
No full textBeing able to comprehend informal arguments in scientific texts is important for scientific literacy in higher education. Successful intervention studies demonstrating that these skills can be trained in university students have not yet provided evidence that gains of explicit training can be maintained beyond immediate post-training assessment. In this study, we tested whether the gains in argument structure comprehension achieved using a self-directed, web-based training intervention could be maintained over several weeks as an indication of sustained improvement in scientific literacy. We also explored characteristics of students and their engagement with the training intervention that resulted in significant and sustained improvements of their argument structure comprehension skills. One hundred students took part in a voluntary supplement to their university courses, completing an online pretest, a 45-minute training session, a posttest (n = 88), and a follow-up test (n = 31). Training effects at posttest were compared with an active control group. The results suggest that the training group exhibited significant gains in argument structure comprehension. These gains were maintained across a four-week period. Students with low starting ability profited the most from the training and gains in argument comprehension were greatest for complex arguments. Training results were positively related to student motivation and this effect was fully mediated by their engagement with the training exercises. The results demonstrate that training gains can be maintained after immediate post-training assessment and suggest that training is particularly effective for low-performing students, for complex arguments, and when students are motivated and engage with the training exercises