610 Medizin und Gesundheit
Filtern
Erscheinungsjahr
Dokumenttyp
- Wissenschaftlicher Artikel (5887)
- Dissertation (1939)
- Preprint (268)
- Ausgabe (Heft) zu einer Zeitschrift (243)
- Konferenzveröffentlichung (178)
- Buch (Monographie) (175)
- Beitrag zu einer (nichtwissenschaftlichen) Zeitung oder Zeitschrift (167)
- Rezension (46)
- Teil eines Buches (Kapitel) (39)
- Arbeitspapier (17)
Sprache
- Englisch (6039)
- Deutsch (2883)
- Latein (26)
- mis (13)
- Mehrsprachig (13)
- Französisch (11)
- Portugiesisch (1)
- Rumänisch (1)
- Russisch (1)
- Spanisch (1)
Volltext vorhanden
- ja (8990) (entfernen)
Schlagworte
- inflammation (94)
- COVID-19 (67)
- SARS-CoV-2 (54)
- apoptosis (40)
- cancer (40)
- Inflammation (39)
- glioblastoma (38)
- breast cancer (34)
- autophagy (32)
- Cancer (29)
Institut
- Medizin (7168)
- Pharmazie (297)
- Biowissenschaften (264)
- Präsidium (250)
- Biochemie, Chemie und Pharmazie (227)
- Biochemie und Chemie (213)
- Frankfurt Institute for Advanced Studies (FIAS) (154)
- Sportwissenschaften (130)
- Georg-Speyer-Haus (125)
- Zentrum für Arzneimittelforschung, Entwicklung und Sicherheit (90)
Highligthts
• Marburg virus infects and replicates in primary human proximal tubular cells (PTC).
• Transcriptome analyses at multiple time points revealed a profound inflammatory response by IFNα, -y and TNFα signaling.
• Among the strongly downregulated gene sets were targets of the transcription factors MYC and E2F, the G2M checkpoint, as well as oxidative phosphorylation.
• Importantly, the downregulated factors comprise PGC-1α, a key factor in mitochondrial biogenesis and renal energy homeostasis, to be substantially downregulated in MARV-infected PTC.
• Our results suggest inflammation-induced changes in tubular energy metabolism as a possible factor in MARV-associated tubular dysfunction.
Abstract
Marburg virus, a member of the Filoviridae, is the causative agent of Marburg virus disease (MVD), a hemorrhagic fever with a case fatality rate of up to 90 %. Acute kidney injury is common in MVD and is associated with increased mortality, but its pathogenesis in MVD remains poorly understood. Interestingly, autopsies show the presence of viral proteins in different parts of the nephron, particularly in proximal tubular cells (PTC). These findings suggest a potential role for the virus in the development of MVD-related kidney injury. To shed light on this effect, we infected primary human PTC with Lake Victoria Marburg virus and conducted transcriptomic analysis at multiple time points. Unexpectedly, infection did not induce marked cytopathic effects in primary tubular cells at 20 and 40 h post infection. However, gene expression analysis revealed robust renal viral replication and dysregulation of genes essential for different cellular functions. The gene sets mainly downregulated in PTC were associated with the targets of the transcription factors MYC and E2F, DNA repair, the G2M checkpoint, as well as oxidative phosphorylation. Importantly, the downregulated factors comprise PGC-1α, a well-known factor in acute and chronic kidney injury. By contrast, the most highly upregulated gene sets were those related to the inflammatory response and cholesterol homeostasis. In conclusion, Marburg virus infects and replicates in human primary PTC and induces downregulation of processes known to be relevant for acute kidney injury as well as a strong inflammatory response.
The human immune system is determined by the functionality of the human lymph node. With the use of high-throughput techniques in clinical diagnostics, a large number of data is currently collected. The new data on the spatiotemporal organization of cells offers new possibilities to build a mathematical model of the human lymph node - a virtual lymph node. The virtual lymph node can be applied to simulate drug responses and may be used in clinical diagnosis. Here, we review mathematical models of the human lymph node from the viewpoint of cellular processes. Starting with classical methods, such as systems of differential equations, we discuss the values of different levels of abstraction and methods in the range from artificial intelligence techniques formalism.
Resilienz als Schutzfaktor vor der Entwicklung psychischer Erkrankungen ist angesichts der zunehmenden gesellschaftlichen Belastung durch chronischen Stress und stressassoziierte Folgeerkrankungen ein hochaktuelles Forschungsthema. Obwohl bereits zahlreiche Erhebungen zur Resilienz existieren, herrscht kein Konsens über die konkrete Operationalisierung des Konzepts. In der Folge differieren vorliegende Studienergebnisse stark voneinander, sind nur schwer miteinander vergleichbar und fraglich auf die Gesellschaft zu übertragen. Die vorliegende Arbeit untersucht daher, ob Haarcortisol einen geeigneten Biomarker in der Resilienzforschung darstellt und einen Beitrag zur Vereinheitlichung und Objektivierung dieses Forschungsgebietes leisten kann.
Zur Beantwortung dieser Frage wurde auf die Daten des aktuell laufenden Langzeitforschungsprojekts „Longitudinal Resilience Assessment“ der Universitätskliniken Frankfurt und Mainz aus den Jahren 2017 bis 2019 zurückgegriffen. Alle Proband*innen der psychisch gesunden Stichprobe (N= 192, 18-50 Jahre) füllten zu insgesamt drei Erhebungszeitpunkten nach Studieneinschluss und im Abstand von je drei Monaten Onlinefragebögen zur Ermittlung der aktuell vorherrschenden Stressexposition, der allgemeinen psychischen Gesundheit sowie des subjektiven Stresserlebens aus. Gleichzeitig fand die Entnahme von Haarproben für die Analyse der Haarcortisolkonzentration statt. Zum Studieneinschluss erfolgte ergänzend die Erhebung der Traumatisierung und negativen Erfahrungen im Kindesalter.
Mittels linearer multivariater Regression wurde ein Effekt möglicher anthropometrischer und soziodemographischer Einflussfaktoren auf die Cortisolkonzentration im Haar ausgeschlossen. Ferner wurde die Auswirkung der Lagerungszeit auf die Höhe der Cortisolwerte im Haar untersucht. Es zeigte sich, dass Haarproben mit einer kürzeren Lagerungszeit signifikant höhere Cortisolwerte aufwiesen als solche mit einer längeren Lagerungszeit. Die Stabilität der Cortisolkonzentrationen über die einzelnen Messzeitpunkte hinweg erwies sich dabei insgesamt nur als mäßig hoch. Zur Untersuchung des Zusammenhangs zwischen subjektivem und objektivem Stress, Traumatisierung und negativen kindlichen Erfahrungen, allgemeiner psychischer Gesundheit, Resilienz und HCC wurden Spearman-Rang-Korrelationen angewandt. Auch hier ließen sich keine statistisch signifikanten Zusammenhänge feststellen. Dennoch konnten bei Personen mit einer Veränderung des Stressniveaus während des Beobachtungszeitraumes eine insgesamt deskriptiv erhöhte Stressexposition sowie statistisch signifikant höhere Mengen an Haarcortisol beobachtet werden. Bei weiteren explorativen Datenanalysen innerhalb einer kleinen Subpopulation der Studienstichprobe konnten während des Einwirkens eines intensiven spezifischen Stressors erhöhte HCC-Werte in Verbindung mit schlechteren psychischen Gesundheitsergebnissen gebracht werden.
Auf Grundlage dieser Arbeit lässt sich kein relevanter Einfluss verschiedener Stressvariablen auf die Cortisolkonzentration im Haar ableiten. Auch die individuelle Resilienz steht in keinem Zusammenhang zum HCC. Die Ergebnisse der explorativen Untersuchungen deuten in Zusammenschau mit einer mäßig hohen Stabilität des HCC jedoch darauf hin, dass Haarcortisol ein veränderungssensitiver Biomarker sein kann. Bei intensiver spezifischer Stresseinwirkung steht es in Zusammenhang mit schlechteren Gesundheitsergebnissen und damit indirekt mit der individuellen Resilienz. In zukünftigen Arbeiten sollte untersucht werden, auf welche Weise die Höhe des Haarcortisols Hinweise auf die Auswirkungen einer spezifischen Stressexposition und damit auf den psychischen Gesundheitszustand eines Individuums liefern kann, um so Risikogruppen identifizieren und diese durch frühzeitige Interventionen vor der Entwicklung psychischer Erkrankungen schützen zu können.
Background/Objective: Evidence-based clinical pathways can be a useful tool for guideline implementation. However, there seem to be barriers to the use of clinical pathways. The aim of the present questionnaire survey was to assess the perceived usability of the clinical pathway “Overweight/obesity in children and adolescents at primary care level” and to identify factors promoting and hindering the use of the clinical pathway.
Methods: In January 2020, an online questionnaire survey was sent out to 3,916 general practitioners and 470 pediatricians in Austria. The data collected were analysed descriptively.
Results: A total of 148 people took part in the questionnaire survey (response rate 3.7 %). The majority of respondents indicated that they, in general, perceive evidence-based clinical pathways as helpful (90 %) and also make use of them (57 %). Few respondents (9 %) felt well-informed about new clinical pathways developed in Austria. Most of the respondents considered the clinical pathway “Overweight/obesity in children and adolescents at primary care level” as a useful support (60 %), as a reference work (72 %) or as a facilitator for justifying their approach to their patients (68 %). However, a large proportion of the respondents stated that the clinical pathway is not easily applicable in everyday practice. The three most frequently cited barriers to using the clinical pathway were lack of time resources, lack of structures and lack of financial incentives. Other display and access options (e. g., individualisation, integration into practice software) were most frequently cited as factors that might promote the use of the pathway.
Conclusion: Although the majority of the respondents had positive expectations regarding the use of the clinical pathway “Overweight/obesity in children and adolescents at primary care level”, many of them still perceived its usability in everyday clinical practice as difficult. The necessary next steps to improve the use of evidence-based clinical pathways seem to be: an economic and practicable design, easy accessibility of clinical pathways and the creation of framework conditions that facilitate their use in everyday practice.
Although exercise guidelines now recommend exercise for patients with MCI, the long-term effects of exercise in patients with MCI has not been reviewed systematically. The aim was to assess (1) the effectiveness of exercise and physical activity (EXPA) interventions in improving long-term patient-relevant cognitive and non-cognitive outcomes in people with mild cognitive impairment, (2) how well the included trials reported details of the intervention, and (3) the extent to which reported endpoints were in line with patient preferences that were assessed in patient workshops. Following PRISMA guidelines, we performed a systematic review and meta-analysis including randomized controlled trials. A total of ten studies were included after searching in six electronic sources from 1995 onwards. There is a trend that 6 + -month EXPA interventions improve global cognition 12 months after initiation. Evidence on long-term effects of EXPA interventions on non-cognitive health outcomes could not be meaningfully pooled and the individual studies reported mixed results. Workshop participants considered freedom from pain and stress, mood, motivation and self-efficacy to be important, but these outcomes were rarely addressed. Too little information is available on intervention details for EXPA programs to be replicated and confidently recommended for patients with MCI. PROSPERO registration in December, 2021 (CRD42021287166).
Objectives: Patient-level factors that influence compliance with a recommendation for CBT in nursing home residents diagnosed with depression were identified.
Methods: Within a cluster-randomized trial on stepped care for depression in nursing homes (DAVOS-study, Trial registration: DRKS00015686), participants received an intake interview administered by a licensed psychotherapist. If psychotherapy was required, patients were offered a referral for CBT. Sociodemographic characteristics, severity of depression, loneliness, physical health, antidepressant medication, prior experience with psychotherapy, and attitudes towards own aging were assessed. A binary regression determined predictors of compliance with referral.
Results: Of 123 residents receiving an intake interview, 80 were recommended a CBT. Forty-seven patients (58.8 %) followed the recommendation. The binary logistic regression model on compliance with recommended CBT was significant, χ2(9) = 21.64, p = .010. Significant predictors were age (Odds Ratio (OR) = 0.9; 95 % Confidence Interval (CI) = 0.82, 0.99; p = .024) and depression (OR = 1.33; 95 % CI = 1.08, 1.65; p = .008).
Conclusion: Within the implemented setting compliance rate was comparable to other age groups. Future interventions should include detailed psychoeducation on the benefits of psychotherapy on mild depressive symptoms in older age and evidence-based interventions to address the stigma of depression. Interventions such as reminiscence-based methods or problem-solving could be useful to increase compliance with referral, especially in very old patients (80+). Language barriers and a culturally sensitive approach should be considered when screening residents.
Inner world and milieu : art, madness, and Brazilian psychiatry in the work of Nise da Silveira
(2024)
This short essay focuses on the work of Brazilian doctor Nise da Silveira, a pioneer in psychiatry who introduced artistic tools to work with psychiatric patients, especially those diagnosed as psychotic. She founded the Museum of Images from the Unconscious in 1952 inside an asylum in Rio de Janeiro to assemble and exhibit the works produced by her patients. As an iconoclast who did not systematize her theory, she engaged with several European psychiatrists, psychoanalysts, and thinkers to produce a very innovative reflection and practical clinical work. Her work resonates in particular with French Institutional Psychotherapy, as well as with Frantz Fanon's psychiatric work in Algeria, but, differently from the former, places art at the core of its clinical method and proposes a radical positioning against every form of medicalized approach.
Association of mortality and early tracheostomy in patients with COVID-19: a retrospective analysis
(2022)
COVID-19 adds to the complexity of optimal timing for tracheostomy. Over the course of this pandemic, and expanded knowledge of the disease, many centers have changed their operating procedures and performed an early tracheostomy. We studied the data on early and delayed tracheostomy regarding patient outcome such as mortality. We performed a retrospective analysis of all tracheostomies at our institution in patients diagnosed with COVID-19 from March 2020 to June 2021. Time from intubation to tracheostomy and mortality of early (≤ 10 days) vs. late (> 10 days) tracheostomy were the primary objectives of this study. We used mixed cox-regression models to calculate the effect of distinct variables on events. We studied 117 tracheostomies. Intubation to tracheostomy shortened significantly (Spearman’s correlation coefficient; rho = − 0.44, p ≤ 0.001) during the course of this pandemic. Early tracheostomy was associated with a significant increase in mortality in uni- and multivariate analysis (Hazard ratio 1.83, 95% CI 1.07–3.17, p = 0.029). The timing of tracheostomy in COVID-19 patients has a potentially critical impact on mortality. The timing of tracheostomy has changed during this pandemic tending to be performed earlier. Future prospective research is necessary to substantiate these results.
Aim: To evaluate the influence of the width of keratinized tissue (KT) on the prevalence of peri-implant diseases, and soft- and hard-tissue stability.
Materials and methods: Clinical studies reporting on the prevalence of peri-implant diseases (primary outcome), plaque index (PI), modified plaque index (mPI), bleeding index (mBI), bleeding on probing (BOP), probing pocket depths (PD), mucosal recession (MR), and marginal bone loss (MBL) and/or patient-reported outcomes (PROMs; secondary outcomes) were searched. The weighted mean differences (WMD) were estimated for the assessed clinical and radiographic parameters by employing a random-effect model that considered different KT widths (i.e., <2 and ≥2 mm).
Results: Twenty-two articles describing 21 studies (15 cross-sectional, five longitudinal comparative studies, and one case series with pre–post design) with an overall high to low risk of bias were included. Peri-implant mucositis and peri-implantitis affected 20.8% to 42% and at 10.5% to 44% of the implants with reduced or absent KT (i.e., <2 mm or 0 mm). The corresponding values at the implant sites with KT width of ≥2 mm or >0 mm were 20.5% to 53% and 5.1% to 8%, respectively. Significant differences between implants with KT < 2 mm and those with KT ≥ 2 mm were revealed for WMD for BOP, mPI, PI, MBL, and MR all favoring implants with KT ≥ 2 mm.
Conclusion: Reduced KT width is associated with an increased prevalence of peri-implantitis, plaque accumulation, soft-tissue inflammation, mucosal recession, marginal bone loss, and greater patient discomfort.
Compressive knee joint contact force during walking is thought to be related to initiation and progression of knee osteoarthritis. However, joint loading is often evaluated with surrogate measures, like the external knee adduction moment, due to the complexity of computing joint contact forces. Statistical models have shown promising correlations between medial knee joint contact forces and knee adduction moments in particularly in individuals with knee osteoarthritis or after total knee replacements (R2 = 0.44–0.60). The purpose of this study was to evaluate how accurately model-based predictions of peak medial and lateral knee joint contact forces during walking could be estimated by linear mixed-effects models including joint moments for children and adolescents with and without valgus malalignment. Peak knee joint moments were strongly correlated (R2 > 0.85, p < 0.001) with both peak medial and lateral knee joint contact forces. The knee flexion and adduction moments were significant covariates in the models, strengthening the understanding of the statistical relationship between both moments and medial and lateral knee joint contact forces. In the future, these models could be used to evaluate peak knee joint contact forces from musculoskeletal simulations using peak joint moments from motion capture software, obviating the need for time-consuming musculoskeletal simulations.
Objective: To assess predictive factors for poststroke pneumonia (PSP) in patients with acute ischemic stroke (AIS) due to large vessel occlusion (LVO) of the anterior circulation, with special regard to the impact of intravenous thrombolysis (IVT) and endovascular treatment (EVT) on the risk of PSP. As a secondary goal, the validity of the A2DS2, PNEUMONIA, and ISAN scores in LVO will be determined.
Methods: Analysis was based on consecutive data for the years 2017 to 2019 from the prospective inpatient stroke registry covering the entire federal state of Hesse, Germany, using the Kruskal-Wallis test and binary logistic regression.
Results: Data from 4,281 patients with LVO were included in the analysis (54.8% female, median age = 78 years, range = 18–102), of whom 66.4% (n = 2,843) received recanalization therapy (RCT). In total, 19.4% (n = 832) of all LVO patients developed PSP. Development of PSP was associated with an increase in overall in-hospital mortality of 32.1% compared with LVO patients without PSP (16.4%; p < 0.001). Incidence of PSP was increased in 2132 patients with either EVT (n = 928; 25.9% PSP incidence) or combined EVT plus IVT (n = 1,204; 24.1%), compared with 2,149 patients with IVT alone (n = 711; 15.2%) or conservative treatment only (n = 1,438; 13.5%; p < 0.001). Multivariate analysis identified EVT (OR 1.5) and combined EVT plus IVT (OR 1.5) as significant independent risk factors for PSP. Furthermore, male sex (OR 1.9), age ≥ 65 years (OR 1.7), dysphagia (OR 3.2) as well as impaired consciousness at arrival (OR 1.7) and the comorbidities diabetes (OR 1.4) and atrial fibrillation (OR 1.3) were significantly associated risk factors (each p < 0.001). Minor stroke (NIHSS ≤ 4) was associated with a significant lower risk of PSP (OR 0.5). Performance of risk stratification scores varied between A2DS2 (96.1% sensitivity, 20.7% specificity), PNEUMONIA (78.2% sensitivity and 45.1% specificity) and ISAN score (98.0% sensitivity, 20.0% specificity).
Conclusion: Nearly one in five stroke patients with LVO develops PSP during acute care. This risk of PSP is further increased if an EVT is performed. Other predictive factors are consistent with those previously described for all AIS patients. Available risk stratification scores proved to be sensitive tools in LVO patients but lack specificity.
The increasing incidence of infected skin wounds poses a major challenge in clinical practice, especially when conventional antibiotic therapy fails. In this context, bacteriophages emerged as promising alternatives for the treatment of antibiotic-resistant bacteria. However, clinical implementation remains hampered by the lack of efficient delivery approaches to infected wound tissue. In this study, bacteriophage-loaded electrospun fiber mats were successfully developed as next-generation wound dressings for the treatment of infected wounds. We employed a coaxial electrospinning approach, creating fibers with a protective polymer shell, enveloping bacteriophages in the core while maintaining their antimicrobial activity. The novel fibers exhibited a reproducible fiber diameter range and morphology, while the mechanical fiber properties were ideal for application onto wounds. Further, immediate release kinetics for the phages were confirmed as well as the biocompatibility of the fibers with human skin cells. Antimicrobial activity was demonstrated against Staphylococcus aureus and Pseudomonas aeruginosa and the core/shell formulation maintained the bacteriophage activity for 4 weeks when stored at − 20 °C. Based on these promising characteristics, our approach holds great potential as a platform technology for the encapsulation of bioactive bacteriophages to enable the translation of phage therapy into clinical application.
Das CD-44-Molekül ist ein membranständiger Oberflächenrezeptor, der als Adhäsionselement von Tumorzellen im Rahmen der Metastasierung genutzt wird. Berichte verweisen auf eine direkte Korrelation zwischen CD-44-Expression eines Tumors und der klinischen Prognose. Darüber hinaus kann der CD-44-Rezeptor auch intrazelluläre Signalwege aktivieren, und als solches Signalelement in die Regulation des Zellzyklus eingreifen.
Im Rahmen der vorliegenden Arbeit wurde postuliert, dass der CD-44-Rezeptor zellzyklusabhängige Veränderungen erfährt und sich daraus Modifikationen des Adhäsionsverhaltens von Tumorzellen ergeben.
Repräsentativ wurde an der Magenkarzinom-Zell-Linie MKN-45 die CD-44-Expression bzw. dessen Splice-Varianten während des Zellzyklus fluorometrisch untersucht (FACS-Analyse, konfokale Laserscan-Mikroskopie). Parallel wurde das Adhäsionsverhalten an isolierten und kultivierten humanen Endothelzellen evaluiert. Die Tumorzellen wurden zuvor mittels Aphidicolin synchronisiert. Als Kontrolle dienten nicht-synchronisierte Zellen.
Die Untersuchungen verdeutlichten eine spezifische Expression der CD-44-Varianten CD44v4, CD44v5, CD44v7. Die Inkubation der Tumorzellen mit Aphidicolin bewirkte eine ausgeprägte Akkumulation von MKN-45-Zellen in der S-Phase. Nach Absetzen des Aphidicolins und Freisetzung in den Zellzyklus kam es zur signifikanten zyklusabhängigen Modulation der Rezeptorexpression. CD44v4, CD44v5 und CD44v7 waren in der G2/MPhase gegenüber der G0/G1- und S-Phase deutlich vermehrt auf der Membran detektierbar. In der G2/M-Phase erhöhte sich zudem signifikant die Adhäsionskapazität der MKN-45-Zellen. Blockadestudien mit gegen die CD-44-Varianten gerichteten monoklonalen Antikörpern belegten die CD-44-abhängige Tumorzell-Endothelzell-Interaktion.
Die Studien belegen zumindest am in vitro Zellkulturmodell die zellzyklus-gesteuerte CD-44-Expression und CD-44-abhängige Invasionseigenschaften von Tumorzellen. Es lässt sich daraus ableiten, dass eine anti-tumorale Therapie an zuvor synchronisierten Tumorzellen womöglich besonders effektiv sein kann. Auch die pharmakologische Blockade des CD-44-Rezeptors könnte einen anti-tumoralen Effekt besitzen.
A novel approach to measure brain-to-brain spatial and temporal alignment during positive empathy
(2022)
Empathy is defined as the ability to vicariously experience others’ suffering (vicarious pain) or feeling their joy (vicarious reward). While most neuroimaging studies have focused on vicarious pain and describe similar neural responses during the observed and the personal negative affective involvement, only initial evidence has been reported for the neural responses to others’ rewards and positive empathy. Here, we propose a novel approach, based on the simultaneous recording of multi-subject EEG signals and exploiting the wavelet coherence decomposition to measure the temporal alignment between ERPs in a dyad of interacting subjects. We used the Third-Party Punishment (TPP) paradigm to elicit the personal and vicarious experiences. During a positive experience, we observed the simultaneous presence in both agents of the Late Positive Potential (LPP), an ERP component related to emotion processing, as well as the existence of an inter-subject ERPs synchronization in the related time window. Moreover, the amplitude of the LPP synchronization was modulated by the presence of a human-agent. Finally, the localized brain circuits subtending the ERP-synchronization correspond to key-regions of personal and vicarious reward. Our findings suggest that the temporal and spatial ERPs alignment might be a novel and direct proxy measure of empathy.
Background: Dialectical behaviour therapy for posttraumatic stress disorder (DBT-PTSD), which is tailored to treat adults with PTSD and co-occurring emotion regulation difficulties, has already demonstrated its efficacy, acceptance and safety in an inpatient treatment setting. It combines elements of DBT with trauma-focused cognitive behavioural interventions.
Objective: To investigate the feasibility, acceptance and safety of DBT-PTSD in an outpatient treatment setting by therapists who were novice to the treatment, we treated 21 female patients suffering from PTSD following childhood sexual abuse (CSA) plus difficulties in emotion regulation in an uncontrolled clinical trial.
Method: The Clinician Administered PTSD Symptom Scale (CAPS), the Davidson Trauma Scale (DTS), the Borderline Section of the International Personality Disorder Examination (IPDE) and the Borderline Symptom List (BSL-23) were used as primary outcomes. For secondary outcomes, depression and dissociation were assessed. Assessments were administered at pretreatment, post-treatment and six-week follow-up.
Results: Improvement was significant for PTSD as well as for borderline personality symptomatology, with large pretreatment to follow-up effect sizes for completers based on the CAPS (Cohens d = 1.30), DTS (d = 1.50), IPDE (d = 1.60) and BSL-23 (d = 1.20).
Conclusion: The outcome suggests that outpatient DBT-PTSD can safely be used to reduce PTSD symptoms and comorbid psychopathology in adults who have experienced CSA.
Two-person neuroscience (2 PN) is a recently introduced conceptual and methodological framework used to investigate the neural basis of human social interaction from simultaneous neuroimaging of two or more subjects (hyperscanning). In this study, we adopted a 2 PN approach and a multiple-brain connectivity model to investigate the neural basis of a form of cooperation called joint action. We hypothesized different intra-brain and inter-brain connectivity patterns when comparing the interpersonal properties of joint action with non-interpersonal conditions, with a focus on co-representation, a core ability at the basis of cooperation. 32 subjects were enrolled in dual-EEG recordings during a computerized joint action task including three conditions: one in which the dyad jointly acted to pursue a common goal (joint), one in which each subject interacted with the PC (PC), and one in which each subject performed the task individually (Solo).
A combination of multiple-brain connectivity estimation and specific indices derived from graph theory allowed to compare interpersonal with non-interpersonal conditions in four different frequency bands. Our results indicate that all the indices were modulated by the interaction, and returned a significantly stronger integration of multiple-subject networks in the joint vs. PC and Solo conditions. A subsequent classification analysis showed that features based on multiple-brain indices led to a better discrimination between social and non-social conditions with respect to single-subject indices. Taken together, our results suggest that multiple-brain connectivity can provide a deeper insight into the understanding of the neural basis of cooperation in humans.
Highlights
• The goal was to assess the intra- and inter-scanner reproducibility of qMRI data.
• Mean scan-rescan variations were not exceeding 2.14%.
• Mean inter-scanner model deviations were not exceeding 5.21%.
• Provided that identical acquisition sequences are used, discrepancies between qMRI data acquired with different scanner models are low.
Abstract
Background: Quantitative MRI (qMRI) techniques allow assessing cerebral tissue properties. However, previous studies on the accuracy of quantitative T1 and T2 mapping reported a scanner model bias of up to 10% for T1 and up to 23% for T2. Such differences would render multi-centre qMRI studies difficult and raise fundamental questions about the general precision of qMRI. A problem in previous studies was that different methods were used for qMRI parameter mapping or for measuring the transmitted radio frequency field B1 which is critical for qMRI techniques requiring corrections for B1 non-uniformities.
Aims: The goal was to assess the intra- and inter-scanner reproducibility of qMRI data at 3 T, using two different scanner models from the same vendor with exactly the same multiparametric acquisition protocol.
Methods: Proton density (PD), T1, T2* and T2 mapping was performed on healthy subjects and on a phantom, performing each measurement twice for each of two scanner models. Although the scanners had different hardware and software versions, identical imaging sequences were used for PD, T1 and T2* mapping, adapting the codes of an existing protocol on the older system line by line to match the software version of the newer scanner. For T2-mapping, the respective manufacturer’s sequence was used which depended on the software version. However, system-dependent corrections were carried out in this case. Reproducibility was assessed by average values in regions of interest.
Results: Mean scan-rescan variations were not exceeding 2.14%, with average values of 1.23% and 1.56% for the new and old system, respectively. Inter-scanner model deviations were not exceeding 5.21% with average values of about 2.2–3.8% for PD, 2.5–3.0% for T2*, 1.6–3.1% for T1 and 3.3–5.2% for T2.
Conclusions: Provided that identical acquisition sequences are used, discrepancies between qMRI data acquired with different scanner models are low. The level of systematic differences reported in this work may help to interpret multi-centre data.
Highlights
• Increased values in SVD, suggesting reduced oxygen extraction fraction (OEF).
• Vascular dysfunction and microstructural impairment limit OEF capacity.
• Association between prolonged and more alkaline intracellular pH.
• Adaptation of intracellular energy metabolism compensates for reduced OEF.
Abstract
Background: We aimed to investigate whether combined phosphorous (31P) magnetic resonance spectroscopic imaging (MRSI) and quantitative T′2 mapping are able to detect alterations of the cerebral oxygen extraction fraction (OEF) and intracellular pH (pHi) as markers the of cellular energy metabolism in cerebral small vessel disease (SVD).
Materials and methods: 32 patients with SVD and 17 age-matched healthy control subjects were examined with 3-dimensional 31P MRSI and oxygenation-sensitive quantitative T′2 mapping (1/T′2 = 1/T2* - 1/T2) at 3 Tesla (T). PHi was measured within the white matter hyperintensities (WMH) in SVD patients. Quantitative T′2 values were averaged across the entire white matter (WM). Furthermore, T′2 values were extracted from normal-appearing WM (NAWM) and the WMH and compared between patients and controls.
Results: Quantitative T′2 values were significantly increased across the entire WM and in the NAWM in patients compared to control subjects (149.51 ± 16.94 vs. 138.19 ± 12.66 ms and 147.45 ± 18.14 vs. 137.99 ± 12.19 ms, p < 0.05). WM T′2 values correlated significantly with the WMH load (ρ=0.441, p = 0.006). Increased T′2 was significantly associated with more alkaline pHi (ρ=0.299, p < 0.05). Both T′2 and pHi were significantly positively correlated with vascular pulsatility in the distal carotid arteries (ρ=0.596, p = 0.001 and ρ=0.452, p = 0.016).
Conclusions: This exploratory study found evidence of impaired cerebral OEF in SVD, which is associated with intracellular alkalosis as an adaptive mechanism. The employed techniques provide new insights into the pathophysiology of SVD with regard to disease-related consequences on the cellular metabolic state.
Background and Objectives: Proteins of the coagulation system contribute to autoimmune inflammation in patients with multiple sclerosis (MS). On blood-brain barrier (BBB) disruption, fibrinogen enters the CNS and is rapidly converted to fibrin, unfolding pleiotropic autoimmune mechanisms. Fibrin accumulation leads to subsequent proteolytic degradation that results in D-dimer generation. The primary objective of this study was to determine intrathecal levels of D-dimer in CSF as a measure of intrathecal coagulation cascade activation and to evaluate its diagnostic utility in patients with MS in contrast to healthy subjects. Key secondary objectives included analysis of CSF D-dimer in differential diagnoses of MS and its relation to routine clinical markers of disease activity.
Methods: Patients admitted for the assessment of suspected MS were prospectively recruited from October 2017 to December 2020. Blood plasma and citrated CSF samples were analyzed using a highly sensitive luminescent oxygen channeling immunoassay. Intrathecal generation of D-dimer was analyzed by adjusting for CSF/serum albumin (Qalb) and CSF/plasma D-dimer quotients (QD-dimer), and corresponding CSF fibrinogen levels were determined. Final diagnoses after full evaluation and clinical data were recorded.
Results: Of 187 patients, 113 patients received a diagnosis of MS or clinically/radiologically isolated syndrome. We found increased intrathecal CSF D-dimer generation levels (QD-dimer/Qalb-index) for patients with relapsing-remitting MS (RRMS; n = 71, median 4.7, interquartile range [IQR] 2.5–8.0) when compared with those for disease controls (n = 22, median 2.6, IQR 2.1–4.8, p = 0.031). Absolute CSF D-dimer values correlated with CSF fibrinogen levels (r = 0.463; p < 0 .001) and CSF leukocytes (r = 0.273; p = 0.003) and were elevated in MS patients with contrast enhancement (CE) compared with MS patients without CE on MRI (n = 48, median 6 ng/mL, and IQR 3–15.25 vs n = 41, median 4 ng/mL, and IQR 2–7; p = 0.026). Exploratory subgroup analyses indicated a correlation of intrathecal inflammatory activity and CSF D-dimer levels.
Discussion: D-dimer in CSF can be reliably determined and correlates with markers of CNS inflammation and CSF fibrinogen levels. Adjusted for BBB dysfunction, CSF D-dimer may allow the identification of intrathecal coagulation cascade activation in patients with MS.
Classification of Evidence: This study provides Class I evidence that CSF D-dimer levels are elevated in patients with RRMS.
Nanoplastics affect the inflammatory cytokine release by primary human monocytes and dendritic cells
(2022)
So far, the human health impacts of nano- and microplastics are poorly understood. Thus, we investigated whether nanoplastics exposure induces inflammatory processes in primary human monocytes and monocyte-derived dendritic cells. We exposed these cells in vitro to nanoplastics of different shapes (irregular vs. spherical), sizes (50–310 nm and polydisperse mixtures) and polymer types (polystyrene; polymethyl methacrylate; polyvinyl chloride, PVC) using concentrations of 30–300 particles cell−1. Our results show that irregular PVC particles induce the strongest cytokine release of these nanoplastics. Irregular polystyrene triggered a significantly higher pro-inflammatory response compared to spherical nanoplastics. The contribution of chemicals leaching from the particles was minor. The effects were concentration-dependent but varied markedly between cell donors. We conclude that nanoplastics exposure can provoke human immune cells to secrete cytokines as key initiators of inflammation. This response is specific to certain polymers (PVC) and particle shapes (fragments). Accordingly, nanoplastics cannot be considered one homogenous entity when assessing their health implications and the use of spherical polystyrene nanoplastics may underestimate their inflammatory effects.
There has been a renewed interest in the potential use of psychedelics for the treatment of psychiatric conditions. Nevertheless, little is known about the mechanism of action and molecular pathways influenced by ayahuasca use in humans. Therefore, for the first time, our study aims to investigate the human metabolomics signature after consumption of a psychedelic, ayahuasca, and its connection with both the psychedelic-induced subjective effects and the plasma concentrations of ayahuasca alkaloids.
Plasma samples of 23 individuals were collected both before and after ayahuasca consumption. Samples were analysed through targeted metabolomics and further integrated with subjective ratings of the ayahuasca experience (i.e., using the 5-Dimension Altered States of Consciousness Rating Scale [ASC]), and plasma ayahuasca-alkaloids using integrated network analysis. Metabolic pathways enrichment analysis using diffusion algorithms for specific KEGG modules was performed on the metabolic output.
Compared to baseline, the consumption of ayahuasca increased N-acyl-ethanolamine endocannabinoids, decreased 2-acyl-glycerol endocannabinoids, and altered several large-neutral amino acids (LNAAs). Integrated network results indicated that most of the LNAAs were inversely associated with 9 out of the 11 subscales of the ASC, except for tryptophan which was positively associated. Several endocannabinoids and hexosylceramides were directly associated with the ayahuasca alkaloids. Enrichment analysis confirmed dysregulation in several pathways involved in neurotransmission such as serotonin and dopamine synthesis.
In conclusion, a crosstalk between the circulating LNAAs and the subjective effects is suggested, which is independent of the alkaloid concentrations and provides insights into the specific metabolic fingerprint and mechanism of action underlying ayahuasca experiences.
Pathologies associated with tissue ischemia/reperfusion (I/R) in highly metabolizing organs such as the brain and heart are leading causes of death and disability in humans. Molecular mechanisms underlying mitochondrial dysfunction during acute injury in I/R are tissue-specific, but their details are not completely understood. A metabolic shift and accumulation of substrates of reverse electron transfer (RET) such as succinate are observed in tissue ischemia, making mitochondrial complex I of the respiratory chain (NADH:ubiquinone oxidoreductase) the most vulnerable enzyme to the following reperfusion. It has been shown that brain complex I is predisposed to losing its flavin mononucleotide (FMN) cofactor when maintained in the reduced state in conditions of RET both in vitro and in vivo. Here we investigated the process of redox-dependent dissociation of FMN from mitochondrial complex I in brain and heart mitochondria. In contrast to the brain enzyme, cardiac complex I does not lose FMN when reduced in RET conditions. We proposed that the different kinetics of FMN loss during RET is due to the presence of brain-specific long 50 kDa isoform of the NDUFV3 subunit of complex I, which is absent in the heart where only the canonical 10 kDa short isoform is found. Our simulation studies suggest that the long NDUFV3 isoform can reach toward the FMN binding pocket and affect the nucleotide affinity to the apoenzyme. For the first time, we demonstrated a potential functional role of tissue-specific isoforms of complex I, providing the distinct molecular mechanism of I/R-induced mitochondrial impairment in cardiac and cerebral tissues. By combining functional studies of intact complex I and molecular structure simulations, we defined the critical difference between the brain and heart enzyme and suggested insights into the redox-dependent inactivation mechanisms of complex I during I/R injury in both tissues.
Patients with coronavirus disease 19 (COVID-19) commonly show abnormalities of liver tests (LTs) of undetermined cause. Considering drugs as tentative culprits, the current systematic review searched for published COVID-19 cases with suspected drug-induced liver injury (DILI) and established diagnosis using the diagnostic algorithm of RUCAM (Roussel Uclaf Causality Assessment Method). Data worldwide on DILI cases assessed by RUCAM in COVID-19 patients were sparse. A total of 6/200 reports with initially suspected 996 DILI cases in COVID-19 patients and using all RUCAM-based DILI cases allowed for a clear description of clinical features of RUCAM-based DILI cases among COVID-19 patients: (1) The updated RUCAM published in 2016 was equally often used as the original RUCAM of 1993, with both identifying DILI and other liver diseases as confounders; (2) RUCAM also worked well in patients treated with up to 18 drugs and provided for most DILI cases a probable or highly probable causality level for drugs; (3) DILI was preferentially caused by antiviral drugs given empirically due to their known therapeutic efficacy in other virus infections; (4) hepatocellular injury was more often reported than cholestatic or mixed injury; (5) maximum LT values were found for alanine aminotransferase (ALT) 1.541 U/L and aspartate aminotransferase (AST) 1.076 U/L; (6) the ALT/AST ratio was variable and ranged from 0.4 to 1.4; (7) the mean or median age of the COVID-19 patients with DILI ranged from 54.3 to 56 years; (8) the ratio of males to females was 1.8–3.4:1; (9) outcome was favorable for most patients, likely due to careful selection of the drugs and quick cessation of drug treatment with emerging DILI, but it was fatal in 19 patients; (10) countries reporting RUCAM-based DILI cases in COVID-19 patients included China, India, Japan, Montenegro, and Spain; (11) robust estimation of the percentage contribution of RUCAM-based DILI for the increased LTs in COVID-19 patients is outside of the current scope. In conclusion, RUCAM-based DILI with its clinical characteristics in COVID-19 patients and its classification as a confounding variable is now well defined, requiring a new correct description of COVID-19 features by removing DILI characteristics as confounders.
Tight control over transcription factor activity is necessary for a sensible balance between cellular proliferation and differentiation in the embryo and during tissue homeostasis by adult stem cells, but mechanistic details have remained incomplete. The homeodomain transcription factor MEIS2 is an important regulator of neurogenesis in the ventricular–subventricular zone (V-SVZ) adult stem cell niche in mice. We here identify MEIS2 as direct target of the intracellular protease calpain-2 (composed of the catalytic subunit CAPN2 and the regulatory subunit CAPNS1). Phosphorylation at conserved serine and/or threonine residues, or dimerization with PBX1, reduced the sensitivity of MEIS2 towards cleavage by calpain-2. In the adult V-SVZ, calpain-2 activity is high in stem and progenitor cells, but rapidly declines during neuronal differentiation, which is accompanied by increased stability of MEIS2 full-length protein. In accordance with this, blocking calpain-2 activity in stem and progenitor cells, or overexpression of a cleavage-insensitive form of MEIS2, increased the production of neurons, whereas overexpression of a catalytically active CAPN2 reduced it. Collectively, our results support a key role for calpain-2 in controlling the output of adult V-SVZ neural stem and progenitor cells through cleavage of the neuronal fate determinant MEIS2.
Objective: Randomized trials have shown that concomitant methotrexate (MTX) augments the effectiveness of tumour necrosis factor (TNF) inhibitors in rheumatoid arthritis (RA), but its benefit in psoriatic arthritis (PsA) has not been demonstrated. The goal of this study was to examine whether the impact of concomitant MTX on therapeutic outcomes in patients with PsA was similar to its effects in RA.
Methods: We used data from highly comparable and concurrent observational studies of patients with PsA (N = 1424) or RA (N = 3148) who initiated adalimumab therapy during routine clinical care. The 28-joint Disease Activity Score (DAS28) and patient-reported pain scores were evaluated in patients who received 24 months of continuous treatment with adalimumab monotherapy or adalimumab + MTX and in patients who initiated or stopped concomitant MTX during ongoing adalimumab therapy.
Results: Twenty-four months of continuous treatment with adalimumab + MTX was superior to adalimumab monotherapy in RA patients, while no significant difference was observed in patients with PsA. RA patients who added MTX during the study showed significant individual improvements in DAS28 and pain scores at 6 months after the change in therapy, while those who removed MTX had slight increases in disease activity. In contrast, in patients with PsA, neither initiation nor removal of MTX during continuous adalimumab therapy had a significant effect on therapeutic outcomes.
Conclusion: Addition of MTX to adalimumab confers further therapeutic benefit in patients with RA, but not in those with PsA, suggesting differences in MTX effects in these two patient populations.
Clinicaltrials.gov NCT01078090, NCT01077258, NCT01111240
Hematopoietic mutations in epigenetic regulators like DNA methyltransferase 3 alpha (DNMT3A), play a pivotal role in driving clonal hematopoiesis of indeterminate potential (CHIP), and are associated with unfavorable outcomes in patients suffering from heart failure (HF). However, the precise interactions between CHIP-mutated cells and other cardiac cell types remain unknown. Here, we identify fibroblasts as potential partners in interactions with CHIP-mutated monocytes. We used combined transcriptomic data derived from peripheral blood mononuclear cells of HF patients, both with and without CHIP, and cardiac tissue. We demonstrate that inactivation of DNMT3A in macrophages intensifies interactions with cardiac fibroblasts and increases cardiac fibrosis. DNMT3A inactivation amplifies the release of heparin-binding epidermal growth factor-like growth factor, thereby facilitating activation of cardiac fibroblasts. These findings identify a potential pathway of DNMT3A CHIP-driver mutations to the initiation and progression of HF and may also provide a compelling basis for the development of innovative anti-fibrotic strategies.
Im Rahmen der vorliegenden Arbeit werden zum ersten Mal die Ökologie, Morphologie und Systematik von Pilzen untersucht, die assoziiert mit Haut- und Nagelläsionen von ambulanten Patienten sowie von Patienten dermatologischer Praxen in der Provinz Chiriquí im Westen Panamas nachgewiesen wurden. Die Pilze werden klassifiziert nach dem klinischen D-H-SSystem von Rieth und entsprechend ihrer Position im phylogenetischen System der Pilze. Die Morphologie der verschiedenen Arten wird dokumentiert auf der Grundlage von Kulturen und lichtmikroskopischer Untersuchungen durch Beschreibungen sowie Zeichnungen und Fotographien charakteristischer Strukturen. Die Pathogenität der einzelnen Pilzstämme wurde nicht nachgewiesen, sondern auf der Grundlage von Angaben aus der Literatur diskutiert. Außerdem lieferte die Literatur Daten zum Vorkommen der Pilze an Pflanzen und anderen Substraten in der Natur.
In Panama wurden zahlreiche klinische Proben untersucht, von denen ca. 100 Pilzstämme nach Deutschland geschickt wurden. Dort konnten 80 Stämme weiter kultiviert und detailliert untersucht werden. Mehr als 22 verschiedene Arten wurden beobachtet, die 17 verschiedenen Gattungen angehören. Sie entsprechen drei verschiedenen Arten von Dermatophyten, mindestens drei Arten von Hefen und 16 verschiedenen Schimmel- oder sonstigen Pilzarten.
Mit Ausnahme von Hormographiella verticillata wurden ausschließlich imperfekte Stadien beobachtet, und zwar überwiegend von verschiedenen Vertretern der Ascomycota: Dothideales: Scytalidium dimidiatum (6 Stämme), Eurotiales: Aspergillus spp. (4), Paecilomyces lilacinus (2), Penicillium sp. (2), Hypocreales: Fusarium lichenicola (3), F. solani (4), F. subglutinans (1), Microascales: Scopulariopsis brevicaulis (2), Onygenales: Trichophyton mentagrophytes (2), T. rubrum (9), T. tonsurans (7), Ophiostomatales: Sporothrix schenckii (1), Pleosporales: Curvularia geniculata (1), Polystigmatales: Colletotrichum gloeosporioides (1), Sordariales: Nigrospora sphaerica (1), Saccharomycetales: Candida spp. (12), Geotrichum candidum (8), incerte sedis: Pestalotiopsis cf. tecomicola (1), Tritirachium oryzae (1). Vertreter der Basidiomycota sind: Agaricales: Hormographiella verticillata bzw. Coprinellus domesticus (3), Polyporales: Unbekannter Basidiomycet (1), Trichosporonales: Trichosporon cutaneum (6).
Im Rahmen dieser Studie waren Schimmelpilze die am häufigsten bei Haut- und Nagelläsionen angetroffenen Pilze. Unter diesen waren Fusarium-Arten und Scytalidium dimidiatum besonders häufig vertreten. Candida-Arten wurden ebenfalls oft isoliert. Die wichtigste Art unter den Dermatophyten war Trichophyton rubrum. Die prozentualen Anteile der verschiedenen Gruppen entsprechen gut den von anderen Autoren aus anderen Regionen publizierten Ergebnissen. Dies erklärt sich aufgrund der ökologischen Tatsache, dass die Sporen der Schimmelpilze fast überall in der Natur vorhanden sind und diese Pilze viele verschiedene Substrate nutzen können. Candida-Arten gehören zur normalen Flora des Menschen, können aber bei immunodefizienten Patienten, Diabetikern u.a. schwere Haut- und Schleimhautinfektionen, sowie Organerkrankungen verursachen. Dermatophyten sind als Krankheitserreger oberflächlicher Hautmykosen bekannt.
Zum ersten Mal wird das Vorkommen von Hormographiella verticillata in Amerika nachgewiesen. Dieses imperfekte Stadium eines Basidiomyceten hat in Kultur Fruchtkörper gebildet, die als Coprinellus domesticus bestimmt wurden. Damit wurde zum ersten Mal die Anamorph-Teleomorph-Verbindung zwischen diesen beiden Arten festgestellt, die durch eine molekular-phylogenetische Analyse von LSU rDNA (große Untereinheit der ribosomalen DNA) unterstützt wird. Für diese Analyse wurden andere Stämme und Genbank-Daten zum Vergleich herangezogen.
In den Kulturen von H. verticillata entstehen vor der Entwicklung der Fruchtkörper asexuelle sterile Hyphen, die als Ozonium-Stadium bezeichnet werden können. Zum Vergleich wurden Herbarbelege von verschiedenen Arten dieser Gattung bearbeitet. Die Arten sind morphologisch nicht unterscheidbar, weshalb vorgeschlagen wird, nur den Gattungsnamen zur Bezeichnung des entsprechenden Entwicklungsstadiums zu benutzen.
Es war nicht möglich, aufgrund morphologischer Merkmale den Stamm des Unbekannten Basidiomyceten zu bestimmen. Erst eine molekular-phylogenetischer Analyse von LSU rDNA mit Vergleichssequenzen aus der Genbank zeigte, dass der Pilz nahe verwandt ist mit Vertretern der Polyporales.
In der präoperativen Diagnostik fokaler maligner Leberläsionen nimmt die MRT mit Resovist® einen zunehmend höheren Stellenwert ein.
T2 gewichtete TSE- und FS-Sequenzen der Resovist® unterstützten MRT sind dabei sensitiver als die in der präoperativen Diagnostik etablierte CTAP mit Ultravist® 370, einem nichtionischen jodhaltigen Kontrastmittel.
Die visuelle Differenzierung und Charakterisierung fokaler maligner Leberläsionen und deren Abgrenzung vom Leberparenchym in der Resovist® unterstützten MRT kann durch die Analyse der Signalintensitätskurven optimiert werden. Eine effektivere Charakterisierung der fokalen Leberläsionen wirkt sich damit positiv auf die Sensitivität und Spezifität der SPIO verstärkten MRT aus.
Es bleibt daher offen, die Signalintensitäten der primären und sekundären Leberläsionen der hier nicht untersuchten Gewebearten in weiteren Studien zu analysieren.
Desweiteren könnten nachfolgende Untersuchungen das Ziel haben, die Sensitivität und Spezifität der Resovist® unterstützten MRT durch eine Verminderung der Schichtdicke zu verbessern, womit sich die Detektion von Läsionen < 1 cm effektiver gestalten würde.
Die künstliche elektrische Stimulation bietet oftmals die einzige Möglichkeit, nicht vorhandene bzw. verloren gegangene motorische sowie sensorische Aktivitäten in gewissem Umfang wieder herzustellen. Im Falle von tauben Patienten wird zur Erlangung von Hörempfindungen die elektrische Stimulation des peripheren auditorischen Systems mit Hilfe von Cochlea- oder Hirnstammimplantaten standardmäßig eingesetzt. Es ist dabei notwendig, natürliche neuronale Entladungsmuster durch die elektrisch evozierten Entladungsmuster nachzubilden. Bei einkanaligen Systemen kann nur die Zeitstruktur des Signals dargeboten werden. Mehrkanalige Systeme bieten hier noch zusätzlich die Möglichkeit auch örtlich selektiv bestimmte Nervenfasergruppen zu stimulieren und damit die Ortsstruktur in den Entladungsmustern zu repräsentieren. So hat es sich gezeigt, dass die Sprachverständlichkeit durch Verwendung von Mehrkanal-Elektroden verbessert werden kann. Grundvoraussetzung hierfür ist die Optimierung der Kanalseparation durch Kleinst-Vielkanalelektroden und der Wahl einer optimalen Codierstrategie des Signals.
Die Codierstrategie ist abhängig von dem jeweiligen spezifischen Einsatzbereich. So gaben z.B. schon Clopton und Spelman (1995) zu bedenken, dass die als selektiv berechnete tripolare (S3) Konfiguration nur für einen bestimmten Stimulationsstrombereich gültig ist. Hinzu kommt es bei simultaner Verwendung benachbarter Kanäle zu schmerzhaften Lautheitssummationen. Ursache hierfür sind einerseits die Überlagerung der durch die Elektroden stimulierten neuronalen Bereiche und andererseits die Wechselwirkungen von Strömen benachbarter Elektrodenkanäle. Diese Effekte führen nicht nur zu einer Verringerung der räumlichen Stimulationsauflösung, sondern auch zu einer Einschränkung der exakten Abbildung der Zeitstruktur innerhalb der einzelnen Stimulationskanäle.
Die Techniken und Grundlagen der elektrischen Stimulation von neuronalem Gewebe mit Kleinst-Vielkanalelektroden sind bisher kaum untersucht worden. Ziel dieser Arbeit war es, ein mathematisches Modell zu implementieren und Qualitätsparameter zu definieren, mit deren Hilfe die Verteilung des elektrischen Feldes und die daraus resultierende neuronale Erregung beschrieben und optimiert werden kann. Zur Verifizierung des Modells sollten Methoden und Techniken entwickelt werden, die eine hochauflösende Abtastung der elektrischen Felder und Messung der neuronalen Daten innerhalb eines Messsystems ermöglichen.
Bei der neuronalen Stimulation mit Kleinst-Vielkanalelektroden ergibt sich eine Reihe von Problemen grundsätzlicher Art. So werden bei elektrodenferner Stimulation größere Stimulationsströme benötigt als bei elektrodennaher Stimulation, wobei für den Strombedarf die Stimulationskonfiguration eine entscheidende Rolle spielt: Der S1 Stimulationsmodus benötigt weniger Strom zur Erreichung großer Stimulationstiefen als der S2 Stimulationsmodus. Der größte Strom wird mit zunehmendem Elektrodenabstand gleichermaßen von dem S3 und S7 Stimulationsmodus benötigt. Gleichzeitig verfügen Kleinst-Vielkanalelektroden bauartbedingt aber nur über kleine Elektrodenkontaktoberflächen und lassen daher auf Grund der kritischen Feldstärke nur geringe Stimulationsströme zu.
Ein weiteres Problem besteht bei diesen Kleinst-Elektrodendimensionen in der konkreten Lage der Neurone an denen eine neuronale Erregung evoziert wird. Die Dimension der Kleinst-Vielkanalelektroden liegt bei einem Elektrodenkanalkontaktdurchmesser von 70 µm bereits in der Größenordnung der zu stimulierenden Neurone mit einem Durchmesser von 10 bis 15 µm. Dies macht sich bei den Messungen besonders dann deutlich bemerkbar, wenn nicht der Stimulationsstrom die Größe des überschwelligen Bereichs modelliert, sondern wenn der Elektrodenkanalabstand durch die Wahl der entsprechenden Elektrodenkanäle verändert wird. Hier weisen zwar die meisten neuronalen Antworten noch in die sich aus dem Modell ergebende Richtung, jedoch kommt es zu einer höheren Streuung der Ergebnisse als bei Messungen mit der Folienelektrode, die eine Kontaktfläche von 170 µm besitzt.
Es gibt also eine Reihe von begrenzenden Faktoren bei der optimalen Dimensionierung der Stimulationselektrode, die sowohl abhängig von der physiologischen Topologie ist als auch von den eingesetzten Stimulationskonfigurationen. Es ist also zur Stimulation die Wahl der optimalen Codierstrategie und die richtige Dimensionierung der Stimulationselektrode sowie der Elektrodenkanalabstände von entscheidender Bedeutung.
Die neuronalen Messungen wurden erstmalig für diese Fragestellung am Hirnschnitt durchgeführt, da sie, im Gegensatz zu in-vivo Versuchen, eine exakte Positionierung der Elektroden auf dem Hirnschnitt unter Sichtkontrolle durch das Mikroskop erlauben. Es wurden aus den neuronalen Messungen die Amplituden und Latenzen der exzitatorischen postsynaptischen Potenziale (EPSP) sowie der Feldpotenziale ausgewertet.
Der Versuchsaufbau macht es möglich, die Potenzialfelder mit genau den Konfigurationen abzutasten, mit denen auch die neuronalen Messungen des Hirnschnittes durchgeführt wurden. Das implementierte Programm zur Berechnung der Feldverteilung besitzt zum Messprogramm ein Interface, so dass es möglich ist, die Einstellungen des Experimentes, wie Stimulationskonfigurationen, Abtastraster des Feldes und die Koordinaten des Messraums, in der Modellrechnung zu verwenden. Somit ist ein direktes Vergleichen zwischen Messung und Berechnung möglich. In nachfolgenden Arbeiten können die vorliegenden Ergebnisse als Grundlage für in-vivo Versuche eingesetzt werden.
Zur Durchführung der Messungen wurden sehr kleine Elektroden aus eigener Herstellung verwendet und es wurden uns freundlicherweise neu entwickelte Folienelektroden des Fraunhofer Instituts St. Ingbert zur Verfügung gestellt. Die Größe der verwendeten Kleinst-Vielkanalelektroden aus eigener Herstellung lag um ca. eine Zehnerpotenz unter den aktuell eingesetzten Elektrodentypen und ist speziell für den direkten Kontakt zwischen Elektrode und Gewebe konzipiert. Dies entspricht dem typischen Einsatzbereich von Hirnstammimplantaten. Dies ist auch notwendig, um eine maximale räumliche Separation der erzeugten Felder zu ermöglichen. Außerdem erlaubte das Elektrodendesign auf Grund der hohen Anzahl der Elektrodenkanäle und durch variieren der Konfigurationen die Feldrichtung zu bestimmen, ohne die Elektrode neu auf den Hirnschnitt aufsetzen zu müssen.
Der in dieser Arbeit implementierte Algorithmus zur Berechnung der Feldverteilungen und die eingeführten Qualitätsparameter erlauben, die unterschiedlichen Stimulationskonfigurationen miteinander zu vergleichen und zu optimieren. Die Ergebnisse aus diesen Modellrechnungen wurden sowohl mit den Messungen der elektrischen Felder als auch mit den Ergebnissen aus den neuronalen Antworten verglichen.
Der im Rahmen dieser Arbeit erstellte Versuchsaufbau bestand aus einer über mehrere Mikromanipulatoren getriebene mikrometergenaue Positioniereinrichtung. Es konnten sowohl die Stimulationselektrode als auch die Elektrode zur Aufzeichnung der neuronalen Daten gesteuert werden. Die Steuerung des gesamten Setup, d.h. die Positionierung, die Aufzeichnung der neuronalen Daten und die Generierung der Stimulationsmuster wurde über den zentralen Messrechner durch ein hierfür entwickeltes Computerprogramm gesteuert. Die Versuche wurden über ein inverses Mikroskop durch eine CCD-Kamera aufgezeichnet.
Der entscheidende Vorteil des in dieser Arbeit gewählten Modellansatzes besteht in der grundsätzlichen Beschreibung der Feldverteilung bei vielkanaliger Stimulation, so dass diese auch auf andere Elektrodenformen bzw. Konfigurationen und Dimensionen übertragbar ist. Es lassen sich so den verschiedenen Konfigurationen nach bestimmten Qualitätskriterien bewerten und an die jeweilige Zielrichtung der Stimulation anpassen. Die berechneten Felder konnten erfolgreich in der Messeinrichtung generiert und nachgemessen werden. Außerdem ist es gelungen, differenzierte neuronale Aktivitäten auszuwerten, welche die Aussagen des Modells abstützen.
The mitochondrial matrix peptidase CLPP is crucial during cell stress. Its loss causes Perrault syndrome type 3 (PRLTS3) with infertility, neurodegeneration, and a growth deficit. Its target proteins are disaggregated by CLPX, which also regulates heme biosynthesis via unfolding ALAS enzymes, providing access for pyridoxal-5′-phosphate (PLP). Despite efforts in diverse organisms with multiple techniques, CLPXP substrates remain controversial. Here, avoiding recombinant overexpression, we employed complexomics in mitochondria from three mouse tissues to identify endogenous targets. A CLPP absence caused the accumulation and dispersion of CLPX-VWA8 as AAA+ unfoldases, and of PLPBP. Similar changes and CLPX-VWA8 co-migration were evident for mitoribosomal central protuberance clusters, translation factors like GFM1-HARS2, the RNA granule components LRPPRC-SLIRP, and enzymes OAT-ALDH18A1. Mitochondrially translated proteins in testes showed reductions to <30% for MTCO1-3, the mis-assembly of the complex IV supercomplex, and accumulated metal-binding assembly factors COX15-SFXN4. Indeed, heavy metal levels were increased for iron, molybdenum, cobalt, and manganese. RT-qPCR showed compensatory downregulation only for Clpx mRNA; most accumulated proteins appeared transcriptionally upregulated. Immunoblots validated VWA8, MRPL38, MRPL18, GFM1, and OAT accumulation. Co-immunoprecipitation confirmed CLPX binding to MRPL38, GFM1, and OAT, so excess CLPX and PLP may affect their activity. Our data mechanistically elucidate the mitochondrial translation fidelity deficits which underlie progressive hearing impairment in PRLTS3.
In recent years, the number and type of treatment options in advanced bladder cancer (BC) have been rapidly evolving. To select an effective therapy and spare unnecessary side effects, predictive biomarkers are urgently needed. As the host’s anti-cancer immune response is by far the most effective system to impede malignant tumor growth, immune system-based biomarkers are promising. We have recently described altered proteasomal epitope processing as an effective immune escape mechanism to impair cytotoxic T-cell activity. By altering the neoantigens’ characteristics through different proteasomal peptide cleavage induced by non-synonymous somatic mutations, the ability for T-cell activation was decreased (“processing escapes”). In the present study, we analyzed primary chemo-naïve tissue samples of 26 adjuvant platinum-treated urothelial BC patients using a targeted next-generation sequencing panel followed by the epitope determination of affected genes, a machine-learning based prediction of epitope processing and proteasomal cleavage and of HLA-affinity as well as immune activation. Immune infiltration (immunohistochemistries for CD8, granzyme B, CD45/LCA) was digitally quantified by a pathologist and clinico-pathological and survival data were collected. We detected 145 epitopes with characteristics of a processing escape associated with a higher number of CD8-positive but lower number of granzyme B-positive cells and no association with PD-L1-expression. In addition, a high prevalence of processing escapes was associated with unfavorable overall survival. Our data indicate the presence of processing escapes in advanced BC, potentially creating a tumor-promoting pro-inflammatory environment with lowered anti-cancerous activity and independence from PD-L1-expression. The data also need to be prospectively validated in BC treated with immune therapy.
The adult human body contains about 4 g of iron. About 1–2 mg of iron is absorbed every day, and in healthy individuals, the same amount is excreted. We describe a patient who presents with severe iron deficiency anemia with hemoglobin levels below 6 g/dL and ferritin levels below 30 ng/mL. Although red blood cell concentrates and intravenous iron have been substituted every month for years, body iron stores remain depleted. Diagnostics have included several esophago-gastro-duodenoscopies, colonoscopies, MRI of the liver, repetitive bone marrow biopsies, psychological analysis, application of radioactive iron to determine intact erythropoiesis, and measurement of iron excretion in urine and feces. Typically, gastrointestinal bleeding is a major cause of iron loss. Surprisingly, intestinal iron excretion in stool in the patient was repetitively increased, without gastrointestinal bleeding. Furthermore, whole exome sequencing was performed in the patient and additional family members to identify potential causative genetic variants that may cause intestinal iron loss. Under different inheritance models, several rare mutations were identified, two of which (in CISD1 and KRI1) are likely to be functionally relevant. Intestinal iron loss in the current form has not yet been described and is, with high probability, the cause of the severe iron deficiency anemia in this patient.
Background: To test the impact of urethral sphincter length (USL) and anatomic variants of prostatic apex (Lee-type classification) in preoperative multiparametric magnet resonance imaging (mpMRI) on mid-term continence in prostate cancer patients treated with radical prostatectomy (RP). Methods: We relied on an institutional tertiary-care database to identify patients who underwent RP between 03/2018 and 12/2019 with preoperative mpMRI and data available on mid-term (>6 months post-surgery) urinary continence, defined as usage 0/1 (-safety) pad/24 h. Univariable and multivariable logistic regression models were fitted to test for predictor status of USL and prostatic apex variants, defined in mpMRI measurements. Results: Of 68 eligible patients, rate of mid-term urinary continence was 81% (n = 55). Median coronal (15.1 vs. 12.5 mm) and sagittal (15.4 vs. 11.1 mm) USL were longer in patients reporting urinary continence in mid-term follow-up (both p < 0.01). No difference was recorded for prostatic apex variants distribution (Lee-type) between continent vs. incontinent patients (p = 0.4). In separate multivariable logistic regression models, coronal (odds ratio (OR): 1.35) and sagittal (OR: 1.67) USL, but not Lee-type, were independent predictors for mid-term continence. Conclusion: USL, but not apex anatomy, in preoperative mpMRI was associated with higher rates of urinary continence at mid-term follow-up.
The purpose of this study is to compare the efficacy and safety of microwave ablation (MWA) versus laser-induced thermotherapy (LITT) as a local treatment for hepatocellular carcinoma (HCC,) with regard to therapy response, survival rates, and complication rates as measurable outcomes. This retrospective study included 250 patients (52 females and 198 males; mean age: 66 ± 10 years) with 435 tumors that were treated by MWA and 53 patients (12 females and 41 males; mean age: 67.5 ± 8 years) with 75 tumors that were treated by LITT. Tumor response was evaluated using CEMRI (contrast-enhanced magnetic resonance imaging). Overall, 445 MWA sessions and 76 LITT sessions were performed. The rate of local tumor progression (LTP) and the rate of intrahepatic distant recurrence (IDR) were 6% (15/250) and 46% (115/250) in the MWA-group and 3.8% (2/53) and 64.2% (34/53) in the LITT-group, respectively. The 1-, 3-, and 5-year overall survival (OS) rates calculated from the date of diagnosis were 94.3%, 65.4%, and 49.1% in the MWA-group and 96.2%, 54.7%, and 30.2% in the LITT-group, respectively (p-value: 0.002). The 1-, 2-, and 3-year disease-free survival (DFS) rates were 45.9%, 30.6%, and 24.8% in the MWA-group and 54.7%, 30.2%, and 17% in the LITT-group, respectively (p-value: 0.719). Initial complete ablation rate was 97.7% (425/435) in the MWA-group and 98.7% (74/75) in the LITT-group (p-value > 0.99). The overall complication rate was 2.9% (13/445) in the MWA-group and 7.9% (6/76) in the LITT-group (p-value: 0.045). Based on the results, MWA and LITT thermal ablation techniques are well-tolerated, effective, and safe for the local treatment of HCC. However, MWA is recommended over LITT for the treatment of HCC, since the patients in the MWA-group had higher survival rates.
Brain metastases are a common finding upon initial diagnosis of otherwise locally limited non-small cell lung cancer. We present a retrospective case series describing three cases of patients with symptomatic, synchronous brain metastases and resectable lung tumors. The patients received local ablative treatment of the brain metastases followed by neoadjuvant immunochemotherapy with pemetrexed, cisplatin, and pembrolizumab. Afterwards, resection of the pulmonary lesion with curative intent was performed. One patient showed progressive disease 12 months after initial diagnosis, and passed away 31 months after initial diagnosis. Two of the patients are still alive and maintain a good quality of life with a progression-free survival and overall survival of 28 and 35 months, respectively, illustrating the potential of novel combinatorial treatment approaches.
Inguinal hernia repair (IHR) is a common procedure in childhood. Laparoscopic IHR has been evolving for the last three decades. Although clear advantages have been shown, adaptation in Germany has been slow. We aim to study the current status of pediatric laparoscopic IHR. A survey was sent to all 89 pediatric surgical departments in Germany on current practices and preferences of open versus laparoscopic IHR. Two nationwide databases of administrative claims data from 2019 were analyzed and correlated with responses from the survey. A total of 56% of the pediatric surgical departments supplied data through the quality reports. The recall of our survey was 58% of all pediatric surgery departments. According to the pooled data, laparoscopic IHR was performed in 8.2% of all inpatients treated. Laparoscopic IHR was considered a training procedure in 48% of the departments. Five different laparoscopic techniques were described (most commonly percutaneous closure of the hernia under laparoscopic vision). The choice between open and laparoscopic IHR was mainly determined by the child’s age. Currently, only a minority of German children undergo inguinal hernia repair by laparoscopy. More training opportunities in the form of hands-on and video workshops may lead to more widespread employment of the laparoscopic technique.
Reactive oxygen species (ROS) are important mediators of both physiological and pathophysiological signal transduction in the cardiovascular system. The effects of ROS on cellular processes depend on the concentration, localization, and duration of exposure. Cellular stress response mechanisms have evolved to mitigate the negative effects of acute oxidative stress. In this study, we investigate the short-term and long-term metabolic and transcriptomic response of human umbilical vein endothelial cells (HUVEC) to different types and concentrations of ROS. To generate intracellular H2O2, we utilized a lentiviral chemogenetic approach for overexpression of human D-amino acid oxidase (DAO). DAO converts D-amino acids into their corresponding imino acids and H2O2. HUVEC stably overexpressing DAO (DAO-HUVEC) were exposed to D-alanine (3 mM), exogenous H2O2 (10 µM or 300 µM), or menadione (5 µM) for various timepoints and subjected to global untargeted metabolomics (LC-MS/MS) and RNAseq by MACE (Massive analysis of cDNA ends). A total of 300 µM H2O2 led to pronounced changes on both the metabolic and transcriptomic level. In particular, metabolites linked to redox homeostasis, energy-generating pathways, and nucleotide metabolism were significantly altered. Furthermore, 300 µM H2O2 affected genes related to the p53 pathway and cell cycle. In comparison, the effects of menadione and DAO-derived H2O2 mainly occurred at gene expression level. Collectively, all types of ROS led to subtle changes in the expression of ribosomal genes. Our results show that different types and concentration of ROS lead to a different metabolic and transcriptomic response in endothelial cells.
Gram-negative Tripartite Resistance Nodulation and cell Division (RND) superfamily efflux pumps confer various functions, including multidrug and bile salt resistance, quorum-sensing, virulence and can influence the rate of mutations on the chromosome. Multidrug RND efflux systems are often characterized by a wide substrate specificity. Similarly to many other RND efflux pump systems, AcrAD-TolC confers resistance toward SDS, novobiocin and deoxycholate. In contrast to the other pumps, however, it in addition confers resistance against aminoglycosides and dianionic β-lactams, such as sulbenicillin, aztreonam and carbenicillin. Here, we could show that AcrD from Salmonella typhimurium confers resistance toward several hitherto unreported AcrD substrates such as temocillin, dicloxacillin, cefazolin and fusidic acid. In order to address the molecular determinants of the S. typhimurium AcrD substrate specificity, we conducted substitution analyses in the putative access and deep binding pockets and in the TM1/TM2 groove region. The variants were tested in E. coli ΔacrBΔacrD against β-lactams oxacillin, carbenicillin, aztreonam and temocillin. Deep binding pocket variants N136A, D276A and Y327A; access pocket variant R625A; and variants with substitutions in the groove region between TM1 and TM2 conferred a sensitive phenotype and might, therefore, be involved in anionic β-lactam export. In contrast, lower susceptibilities were observed for E. coli cells harbouring deep binding pocket variants T139A, D176A, S180A, F609A, T611A and F627A and the TM1/TM2 groove variant I337A. This study provides the first insights of side chains involved in drug binding and transport for AcrD from S. typhimurium.
In dieser Arbeit konnte gezeigt werden, daß die Proteinkinase Akt das
Zellzyklusprotein p21 in Endothelzellen an der Aminosäure Threonin 145
phosphoryliert und auf diese Weise p21 posttranskriptionell reguliert. So führt die Aktabhängige Phosphorylierung zur Aufhebung der PCNA-Bindungsfähigkeit und zu einer Abnahme der Komplexbildung von p21 mit Cdk2 und Cdk4. Dementsprechend reduziert die Akt-Phosphorylierung von p21 an Threonin 145 die Hemmung der Cdk2-Aktivierung durch p21, begünstigt damit die Phosphorylierung von Retinoblastoma-Protein und die Freisetzung des Transkriptionsfaktors E2F. Diese Daten weisen auf einen neuen Signaltransduktionsweg hin, über den Akt die Endothelzellproliferation reguliert.
Außerdem führt die Akt-vermittelte Phosphorylierung von p21 an T145 zur Stabilisierung von p21 gegenüber Caspase-abhängiger Degradation während der pro-apoptotischen Stimulation der Endothelzellen und schützt die Zellen gegenüber der Apoptose-Induktion durch TNFα. Die p21-Phosphorylierung durch Akt stellt dabei einen essentiellen Mechanismus der endothelzellprotektiven Wirkung von Akt dar, denn in Abwesenheit von p21 infolge Antisense-Transfektion vermag die Überexpression von Akt nicht mehr zu einer Senkung der endothelialen Apoptoserate nach TNFα -Stimulation führen.
Der retinoid-related orphan receptor α (RORα) ist ein nukleärer Rezeptor, der nach Bindung an sein Responselement die Transkription zahlreicher Gene reguliert. Pharmazeutisches Interesse erlangt der Rezeptor vor allem durch seine Verwicklung in pathophysiologische Prozesse wie Osteoporose und Arteriosklerose sowie durch seine antiinflammatorische Wirkung, die auf der negativen Interferenz mit dem NF-κB-Signalweg beruht. Bisher konnten vier RORα-Isoformen isoliert werden, die durch alternatives Spleißen sowie durch die Regulation über unterschiedliche Promotorregionen entstehen. In verschiedenen Studien konnte eine isoformspezifische Regulation als Antwort auf pathophysiologische Veränderungen der Zellen festgestellt werden, wie beispielsweise die Induktion der RORα4-Transkription in Leberzellen infolge einer Sauerstoffunterversorgung. Um Einblicke in die Mechanismen zu gewinnen, die der spezifischen Regulation der RORα4-Expression zugrunde liegen, wurde in der vorliegenden Arbeit der RORα4-Promotor als erster Promotor einer RORα-Isoform identifiziert und analysiert.
Sechs Fragmente mit einer Länge von bis zu 5,1 kbp der aus Datenbanken entnommenen, putativen Promotorsequenz wurden in einen Reportergenvektor kloniert. Transiente Transfektionsexperimente und Reportergenanalysen deckten die Promotoraktivität der gewählten Sequenz auf.
In dem durch einen hohen Gehalt an den Nukleotiden G und C auffallenden Promotor wurden drei einzelne GC-Boxen (A, B und C) sowie eine Viererkette (Box D) und eine Tandem-GCBox (Box E) als mögliche Bindungsmotive für Sp-Transkriptionsfaktoren gefunden. Mithilfe von Kotransfektionen konnte eine Induktion der Promotoraktivität durch die Transkriptionsfaktoren Sp1 und Sp4 nachgewiesen werden, während Sp3 die Promotoraktivität in diesen Experimenten nicht beeinflusste.
Durch die gezielte Mutation oder Deletion, bzw. die Inkubation mit verschiedenen Substanzen konnten diesen GC-Boxen unterschiedliche Funktionen zugeordnet werden. Durch transiente Transfektionen stark verkürzter Promotorfragmente wurde ein für die Promotoraktivität nötiger Sequenzbereich von 170 Basenpaaren eingegrenzt. In Mutationsanalysen wurde demonstriert, dass die beiden proximalen GC-Boxen A und B für die basale Promotoraktivität essentiell sind.
Die RORα4-Promotoraktivität ließ sich zelltypabhängig durch den Phorbolester TPA induzieren. In Deletionsanalysen ließ sich dieser Effekt teilweise auf die GC-Boxen C und D zurückführen. Der distalen GC-Box E konnte ebenfalls eine Funktion zugeordnet werden. In Reportergenanalysen konnte demonstriert werden, dass sie die Induktion der Promotoraktivität durch den HDAC-Inhibitor Trichostatin A vermittelt.
Durch die Untersuchungen an den TK-luc-Konstrukten mit RORα-Responselementen konnte gezeigt werden, dass der virale Promotor aufgrund der einklonierten RORα-Responselemente sehr stark auf die Kotransfektion der RORα-Isoformen reagiert. Die Reportergenanalyse mit diesen Konstrukten stellt daher eine effiziente Methode dar, um die RORα-vermittelte Transaktivierung zu bestimmen.
Obwohl der RORα4-Promotor zahlreiche RORα-Responselemente trägt, konnte in den Kotransfektionen mit Expressionsplasmiden für die einzelnen Isoformen in keiner der drei Zelllinien eine Autoregulation gefunden werden. Ebensowenig zeigte sich ein Einfluss des putativen RORα-Liganden Melatonin auf die Promotoraktivität.
Des Weiteren wurde gezeigt, dass die RORα4-Promotoraktivität in HeLa und MCF-7-Zellen durch das cAMP-Analogon DbcAMP induzierbar ist, während in HEK 293 keine Beeinflussung der Promotoraktivität erzielt wurde. Neben der Steigerung der Promotoraktivität durch TPA, konnte mit der DbcAMP-Induktion folglich ein zweiter, zelltypabhängiger Effekt auf die RORα4-Promotoraktivität identifiziert werden.
In recent decades, mass spectrometry has moved more than ever before into the front line of protein-centered research. After being established at the qualitative level, the more challenging question of quantification of proteins and peptides using mass spectrometry has become a focus for further development. In this chapter, we discuss and review actual strategies and problems of the methods for the quantitative analysis of peptides, proteins, and finally proteomes by mass spectrometry. The common themes, the differences, and the potential pitfalls of the main approaches are presented in order to provide a survey of the emerging field of quantitative, mass spectrometry-based proteomics.
Diadenosinpolyphosphate (ApnAs), wirken in einer Vielzahl unterschiedlicher Gewebe als extrazelluläre Signalmoleküle. Ihre asymmetrische Hydrolyse durch Enzyme auf der Zelloberfläche oder durch lösliche Enzyme im extrazellulären Milieu wurde in der Literatur bereits mehrfach beschrieben. Die molekulare Identität dieser Enzyme war jedoch nicht bekannt.
In der vorliegenden Arbeit wurden die Fähigkeit von NPP1, NPP2 und NPP3, den drei Mitgliedern der E-NPP-Familie, Diadenosinpolyphosphate zu hydrolysieren, untersucht. Dazu wurden humanes NPP1 und NPP3 aus der Ratte heterolog in CHO-Zellen exprimiert und die enzymatische Aktivität wurde anhand von Membranfraktionen analysiert. Für die Charakterisierung der katalytischen Eigenschaften von NPP2 wurde eine lösliche sekretierte Form des humanen NPP2 aus partiell aufgereinigtem Vaccinia-Viruslysat eingesetzt. Es konnte gezeigt werden, dass die heterolog exprimierten Enzyme NPP1, NPP2 und NPP3 die untersuchten Diadenosinpolyphosphate Diadenosin-5´,5´´´-P1,P3-triphosphat (Ap3A), Diadenosin-5´,5´´´-P1,P4-tetraphosphat (Ap4A) und Diadenosin-5´,5´´´-P1,P5-pentaphosphat (Ap5A), sowie das Diguanosinpolyphosphat Diguanosin-5´,5´´´-P1,P4-tetraphosphat (Gp4G) hydrolysieren.. Ein Vergleich der Hydrolyseraten zeigte, dass NPP1-2 Ap3A, Ap4A, Ap5A und Gp4G mit vergleichbarer Rate hydrolysieren. NPP3 zeigte ebenfalls keine Präferenz für eines der untersuchten Diadenosinpolyphosphate, hydrolysierte aber Gp4G im Vergleich zu Ap4A deutlich langsamer. Die Hydrolyse der Dinukleotide erfolgte asymmetrisch durch Spaltung der α,β-Pyrophosphatbindung. Als primäre Hydrolyseprodukte entstanden Nukleosid-5´-Monophosphat und der verbleibende Mononukleotidrest Npn-1. Für Ap3A als Substrat wurde für NPP1-3 ein alkalisches pH-Optimum mit maximaler Aktivität bei pH 8.5-9 (NPP1 und NPP3), bzw. pH 10 (NPP2) nachgewiesen. Die enzymatische Aktivität von NPP1-3 wurde durch EDTA inhibiert und die Km-Werte für Ap3A lagen mit 5,1 ± 3,6 µM (NPP1), 8,0 ± 0,5 µM (NPP2) und 49,5 ± 17,7 µM (NPP3) im niedrigen mikromolaren Bereich. Untersuchungen zur Hemmbarkeit der NPP-vermittelten Diadenosinpolyphosphathydrolyse (Ap4A) zeigten, dass die Enzyme NPP1, NPP2 und NPP3 nach Koapplikation verschiedener P2-Rezeptorantagonisten in unterschiedlichem Ausmaß inhibiert wurden. Cibacron Blue inhibierte kräftig alle drei Enzyme, während PPADS einen stärkeren inhibitorischen Einfluss auf die katalytsiche Aktivität von NPP1 und NPP3 als auf die katalytische Aktivität von NPP2 zeigte. Suramin inhibierte dagegen nur die NPP1 und NPP2 katalysierte Ap4AHydrolyse und hatte keinen Einfluss auf NPP3. Eine Inhibierung von NPP1-3 durch NaF wurde nicht beobachtet. Eine geringe inhibitorische Wirkung auf NPP1 wurde durch die extrazellulären Matrix-Komponenten Heparin und Heparansulfat, durch ATP und die Nukleotidanaloga, AMP-CP, AMP-CPP und ATP-γ-S beobachtet. NPP2 und NPP3 wurden durch AMP-CP nicht inhibiert. Den schwächsten inhibitorischen Einfluss zeigte ATP auf die durch NPP3-vermittelte Ap4AHydrolyse.
Die hier für NPP1-3 ermittelten katalytischen Eigenschaften zeigten Übereinstimmgen aber auch Unterschiede gegenüber früheren Daten, die für die Hydrolyse von Diadenosinpolyphosphaten auf der Oberfläche von Zellen ermittelt wurden. Insgesamt sprechen die Ergebnisse dafür, dass NPP1-3 die Hauptvertreter der Diadenosinpolyphosphat-hydrolysierenden Enzyme in Säugergewebe darstellen. Möglicherweise gibt es aber auch ApnA-hydrolysierende Enzyme, die nicht zu den bisher charakterisierten Mitgliedern der E-NPP-Familie zugehören.
In einem zweiten Teil der Arbeit wurde die Expression von NPP1-3 im Gehirn der Ratte mittels Western-Blot-Analyse (Entwicklungsstadien P1, P21 und adult) untersucht. Aufgrund der geringen Spezifität der gegen NPP1 und NPP2 zur Verfügung stehenden Antikörper, konnten jedoch keine eindeutigen Aussagen zur Expression von NPP1 und NPP2 im Gehirn getroffen werden. NPP3 konnte im Rattengehirn nachgewiesen werden. Die Expression war entwicklungsabhängig und nahm mit zunehmendem Alter der Tiere deutlich ab. Die Entwicklung spezifischer Antikörper erscheint ein lohnender Ansatz, um die zelluläre Verteilung von NPP1-3 im Nervengewebe zu bestimmen.
Biallelic pathogenic variants in CLPP, encoding mitochondrial matrix peptidase ClpP, cause a rare autosomal recessive condition, Perrault syndrome type 3 (PRLTS3). It is characterized by primary ovarian insufficiency and early sensorineural hearing loss, often associated with progressive neurological deficits. Mouse models showed that accumulations of (i) its main protein interactor, the substrate-selecting AAA+ ATPase ClpX, (ii) mitoribosomes, and (iii) mtDNA nucleoids are the main cellular consequences of ClpP absence. However, the sequence of these events and their validity in human remain unclear. Here, we studied global proteome profiles to define ClpP substrates among mitochondrial ClpX interactors, which accumulated consistently in ClpP-null mouse embryonal fibroblasts and brains. Validation work included novel ClpP-mutant patient fibroblast proteomics. ClpX co-accumulated in mitochondria with the nucleoid component POLDIP2, the mitochondrial poly(A) mRNA granule element LRPPRC, and tRNA processing factor GFM1 (in mouse, also GRSF1). Only in mouse did accumulated ClpX, GFM1, and GRSF1 appear in nuclear fractions. Mitoribosomal accumulation was minor. Consistent accumulations in murine and human fibroblasts also affected multimerizing factors not known as ClpX interactors, namely, OAT, ASS1, ACADVL, STOM, PRDX3, PC, MUT, ALDH2, PMPCB, UQCRC2, and ACADSB, but the impact on downstream metabolites was marginal. Our data demonstrate the primary impact of ClpXP on the assembly of proteins with nucleic acids and show nucleoid enlargement in human as a key consequence.
The accumulation of functionally impaired mitochondria is a key event in aging. Previous works with the fungal aging model Podospora anserina demonstrated pronounced age-dependent changes of mitochondrial morphology and ultrastructure, as well as alterations of transcript and protein levels, including individual proteins of the oxidative phosphorylation (OXPHOS). The identified protein changes do not reflect the level of the whole protein complexes as they function in-vivo. In the present study, we investigated in detail the age-dependent changes of assembled mitochondrial protein complexes, using complexome profiling. We observed pronounced age-depen-dent alterations of the OXPHOS complexes, including the loss of mitochondrial respiratory supercomplexes (mtRSCs) and a reduction in the abundance of complex I and complex IV. Additionally, we identified a switch from the standard complex IV-dependent respiration to an alternative respiration during the aging of the P. anserina wild type. Interestingly, we identified proteasome components, as well as endoplasmic reticulum (ER) proteins, for which the recruitment to mitochondria appeared to be increased in the mitochondria of older cultures. Overall, our data demonstrate pronounced age-dependent alterations of the protein complexes involved in energy transduction and suggest the induction of different non-mitochondrial salvage pathways, to counteract the age-dependent mitochondrial impairments which occur during aging.