Monitoring of oxidative and metabolic stress during cardiac surgery by means of breath biomarkers: an observational study

Research articl

The LifeCycle Project-EU Child Cohort Network : a federated analysis infrastructure and harmonized data of more than 250,000 children and parents

Early life is an important window of opportunity to improve health across the full lifecycle. An accumulating body of evidence suggests that exposure to adverse stressors during early life leads to developmental adaptations, which subsequently affect disease risk in later life. Also, geographical, socio-economic, and ethnic differences are related to health inequalities from early life onwards. To address these important public health challenges, many European pregnancy and childhood cohorts have been established over the last 30 years. The enormous wealth of data of these cohorts has led to important new biological insights and important impact for health from early life onwards. The impact of these cohorts and their data could be further increased by combining data from different cohorts. Combining data will lead to the possibility of identifying smaller effect estimates, and the opportunity to better identify risk groups and risk factors leading to disease across the lifecycle across countries. Also, it enables research on better causal understanding and modelling of life course health trajectories. The EU Child Cohort Network, established by the Horizon2020-funded LifeCycle Project, brings together nineteen pregnancy and childhood cohorts, together including more than 250,000 children and their parents. A large set of variables has been harmonised and standardized across these cohorts. The harmonized data are kept within each institution and can be accessed by external researchers through a shared federated data analysis platform using the R-based platform DataSHIELD, which takes relevant national and international data regulations into account. The EU Child Cohort Network has an open character. All protocols for data harmonization and setting up the data analysis platform are available online. The EU Child Cohort Network creates great opportunities for researchers to use data from different cohorts, during and beyond the LifeCycle Project duration. It also provides a novel model for collaborative research in large research infrastructures with individual-level data. The LifeCycle Project will translate results from research using the EU Child Cohort Network into recommendations for targeted prevention strategies to improve health trajectories for current and future generations by optimizing their earliest phases of life.Peer reviewe

Dysfunktion und Regulation

Die Kernpore ist ein dynamischer makromolekularer Komplex, dessen strukturelle und funktionelle Eigenschaften je nach physiologischem Zustand der Zelle reguliert werden. In Anbetracht der Tatsache, dass der Austausch apoptotischer Signal- und Effektormoleküle zwischen Kern und Zytoplasma essentiell für deren Funktion ist, könnte die Regulation dieses Austausches ein Schlüsselelement apoptotischer Signalwege darstellen.Ziel dieser Arbeit war es, zum Verständnis der Regulation struktureller und funktioneller Veränderungen der Kern-Zytoplasma-Barriere beizutragen. Hierzu wurden drei unabhängige experimentelle Ansätze verfolgt.Im ersten Ansatz wurde ein in vitro Kernpermeabilitätsassay (NPA) verwendet, um den Einfluss einzelner molekularer Faktoren auf die Kernpermeabilität zu untersuchen. Hierzu wurde der NPA optimiert und validiert um statistisch aussagekräftige Probengrößen untersuchen zu können.Inhibitorstudien mittels NPA zeigten, dass ein durch den Serinproteaseinhibitor Pefablock inhibierbares Enzym die Kernpermeabilität beeinflusst.Als potentiell an der Regulation der Kernpermeabilität beteiligte Serinproteasen wurden sowohl Chymotrypsin-ähnliche Proteasen als auch die mitochondriale Serinprotease HtrA2/OMI näher untersucht. Es konnte gezeigt werden, dass Chymotrypsin-ähnliche Serinproteasen in dem untersuchten apoptotischen System in geringen, aber messbaren Mengen aktiviert werden. Die Aktivität der mitochondrialen Serinprotease HtrA2/OMI konnte nicht durch Pefablock gehemmt werden. Es konnte jedoch gezeigt werden, dass HtrA2/OMI einen Einfluss auf die Kernpermeabilität und die proteolytische Prozessierung von Kernporenproteinen hat. Diese Ergebnisse legen nahe, dass, abhängig vom zellulären sowie apoptotischen Modelsystem, unterschiedliche Faktoren einen Einfluss auf die Kernpermeabilität haben.Im zweiten Ansatz wurde der Einfluss des membranständigen, anti-apoptotischen Proteins Bcl-2 auf die Kernpermeabilität untersucht. Es wurde in zwei verschiedenen, stabil mit murinem oder humanem Bcl-2 transfizierten Zellinien, sowie nach transienter Transfektion gezeigt, dass Überexpression von Bcl-2 eine basale Erhöhung der Kernpermeabilität zur Folge hat. Dieses Ergebnis wurde mit drei verschiedenen experimentellen Ansätzen verifiziert: NPA, Bead Loading von fluoreszentem 70 kDa Dextran sowie Transfektion des Permeabilitätsmarkers 4xCherry.Es wurde gezeigt, dass ausschließlich an den Mitochondrien lokalisiertes Bcl-2 keinen Einfluss auf die Kernpermeabilität hat, wohingegen sich die Lokalisation and der ER und/oder Kernmembran als essentiell für die Bcl-2-bedingte Erhöhung der Kernpermeabilität erwies.Durch Überexpression der Calcium ATPase SERCA in Bcl-2 überexprimierenden Zellen konnte die erhöhte Kernpermeabilität wieder erniedrigt werden. Dieses Ergebnis legt nahe, dass Bcl-2 durch Calcium als Signalmolekül eine Erhöhung der Kernpermeabilität vermitteln könnte.Der dritte Ansatz der vorliegenden Arbeit bestand aus der Entwicklung eines Systems zur Visualisierung und Charakterisierung funktioneller sowie struktureller Veränderungen der Kernpore während der Apoptose mittels konfokaler Lebendzellmikroskopie. Es wurde gezeigt, dass transient mit fluoreszent markierten Kernporenproteinen transfizierte HeLa Zellen ein hierfür geeignetes zelluläres System darstellen. Nup153-GFP, wurde als geeignetes Markerprotein zur Visualisierung der Caspase-abhängigen Proteolyse von Kernporenproteinen identifiziert und validiert. Es konnte gezeigt werden, dass Spaltung an der Caspase-3-Schnittstelle notwendig für den Apoptose-bedingten Verlust von Nup153-GFP von der Kernpore ist. Im Gegensatz dazu, hat eine putative Caspase-8 Schnittstelle keinen Einfluss auf die Verankerung von Nup153-GFP in der Kernpore. Um Apoptose-induzierte Veränderungen der Kernpermeabilität zu visualisieren, wurde ein tetrameres fluoreszentes Markerprotein (4xCherry) hergestellt und als Permeabilitätsmarker validiert.Mit Nup153-GFP und 4xCherry als fluoreszente Markerproteine wurde eine auf konfokaler Lebendzellmikroskopie basierende Methode entwickelt, welche die spezifischen Anforderungen zur Visualisierung von Veränderungen der Kern-Zytoplasma-Barriere während der Apoptose erfüllt. Zur graphischen Auswertung der Ergebnisse wurde eine entsprechende Bildanalyseprozedur entwickelt.Mit Hilfe der entwickelten experimentellen Methode konnte gezeigt werden, dass die Kernpermeabilität sich, je nach apoptotischem Modelsystem, zu verschiedenen Zeitpunkten ändert. In zwei untersuchten Modellsystemen erfolgt die caspase-abhängige Spaltung von Kernporenproteinen zu einem späten Zeitpunkt, zeitgleich mit Veränderungen der Chromatinstruktur

In vitro assay for the quantitation of apoptosis-induced alterations of nuclear envelope permeability

This protocol describes how to perform comparative measurements of the permeability of the nuclear envelope in adherent cells. The plasma membrane is permeabilized at low digitonin concentrations, leaving the nuclear membrane intact. These semi-permeabilized cells are incubated with cytosolic extracts prepared in advance and with a fluorescent reporter molecule whose molecular weight exceeds the size-exclusion limit of the nuclear envelope. Images are taken with a confocal microscope and subsequently analyzed using a custom-made software program that recognizes the nuclei automatically and calculates the mean nuclear fluorescence signal. Here, we measure the increase in nuclear permeability triggered by cytosolic extracts from cells dying by apoptosis. This method can be employed for the study of processes that affect the nucleocytoplasmic distribution of fluorescent molecules in cell populations. The large size of the samples means that subtle fluctuations in nuclear fluorescence can be detected with a high confidence level. Isolation of cell extracts takes 5–6 h, and the preparation and imaging of 15 or so specimens takes 4–5 h

Commuting (to) suicide : An update on nucleocytoplasmic transport in apoptosis

Commuting is the process of travelling between a place of residence and a place of work. In the context of biology, this expression evokes the continuous movement of macromolecules between different compartments of a eukaryotic cell. Transport in and out of the nucleus is a major example of intracellular commuting. This article discusses recent findings that substantiate the emerging link between nucleocytoplasmic transport and the signalling and execution of cell death

Progressive resistance training in cachectic head and neck cancer patients undergoing radiotherapy: a randomized controlled pilot feasibility trial

Abstract Background Cancer cachexia is a prevalent symptom of head and neck neoplasms. The reduction in skeletal muscle mass is one of the main characteristics which can lead to poor physical functioning. The purposes of this pilot randomized controlled trial were to determine the feasibility of progressive resistance training in cachectic head and neck cancer patients during radiotherapy and to explore possible risks and benefits. Methods Twenty cachectic participants with head and neck cancer receiving radiation were randomized to obtain either a machine supported progressive resistance training (n = 10) or usual care (n = 10). The training took place 3 times weekly for 30 min. Intervention included 3 exercises for major muscle groups with 8–12 repetition maximum for 3 sets each. Bioelectrical impedance analysis, hand-held dynamometry, Six-Minute Walk Test and standardized questionnaires for fatigue and quality of life were used for evaluating outcomes at baseline before radiotherapy (t1), after 7 weeks of radiotherapy (t2) and 8 weeks after the end of radiotherapy (t3). Results All participants (n = 20) completed the trial. No serious adverse events occurred. At the initial assessment the cachectic patients had already lost 7.1 ± 5.2% of their body weight. General fatigue (score 10.7 ± 3.3) and reduced quality of life (score 71.3 ± 20.6) were prevalent in cachectic head and neck cancer patients even before radiotherapy. An average improvement of weight loading for leg press (+ 19.0%), chest press (+ 29.8%) and latissimus pull-down (+ 22.8%) was possible in the intervention group. Participants had at least 13 training sessions. The outcome measures showed nonsignificant changes at t2 and t3, but a trend for a better course of general fatigue and quality of life at t2 in the intervention group. Conclusions Despite advanced tumor stage and burdensome treatment the intervention adherence is excellent. Progressive resistance training in cachectic head and neck cancer patients during radiotherapy seems to be safe and feasible and may have beneficial effects of general fatigue and quality of life. Trial registration ClinicalTrials.gov, NCT03524755. Registered 15 May 2018 - Retrospectively registered

Regulation of nuclear envelope permeability in cell death and survival

The nuclear pore complex (NPC) mediates macromolecular exchange between nucleus and cytoplasm. It is a regulated channel whose functional properties are modulated in response to the physiological status of the cell. Identifying the factors responsible for regulating NPC activity is crucial to understand how intracellular signaling cues are integrated at the level of this channel to control nucleocytoplasmic trafficking. For proteins lacking active translocation signals the NPC acts as a molecular sieve limiting passage across the nuclear envelope (NE) to proteins with a MW below ~40 kD. Here, we investigate how this permeability barrier is altered in paradigms of cell death and cell survival, i.e., apoptosis induction via staurosporine, and enhanced viability via overexpression of Bcl-2. We monitor dynamic changes of the NPC's size-exclusion limit for passive diffusion by confocal time-lapse microscopy of cells undergoing apoptosis, and use different diffusion markers to determine how Bcl-2 expression affects steady-state NE permeability. We show that staurosporine triggers an immediate and gradual leakiness of the NE preceding the appearance of apoptotic hallmarks. Bcl-2 expression leads to a constitutive increase in NE permeability, and its localization at the NE is sufficient for the effect, evincing a functional role for Bcl-2 at the nuclear membrane. In both settings, NPC leakiness correlates with reduced Ca²⁺ in internal stores, as demonstrated by fluorometric measurements of ER/NE Ca²⁺ levels. By comparing two cellular models with opposite outcome these data pinpoint ER/NE Ca²⁺ as a general and physiologically relevant regulator of the permeability barrier function of the NPC

Short-Term Effects of Dietary Protein Supplementation on Physical Recovery in Older Patients at Risk of Malnutrition during Inpatient Rehabilitation: A Pilot, Randomized, Controlled Trial

It is estimated that about 50% of geriatric rehabilitation patients suffer from sarcopenia. Thereby, malnutrition is frequently associated with sarcopenia, and dietary intake is the main modifiable risk factor. During hospitalization, older adults are recommended to consume more dietary protein than the current recommended dietary allowance of 0.8 g/kg body weight per day to optimize the recovery of muscular strength and physical function. This prospective pilot study examined the feasibility and preliminary efficacy of short-term protein supplementation with protein-enriched foods and drinks on the hand-grip strength, nutritional status, and physical function of older patients at risk of malnutrition during a three-week inpatient orthopedic rehabilitation stay. The Mini Nutritional Assessment (MNA) tool was used to assess malnutrition. Patients with an MNA score ≤ 23.5 points were randomly assigned to an intervention group (goal: to consume 1.2–1.5 g protein/kg body weight per day) or a control group (standard care). Both groups carried out the same rehabilitation program. Physical recovery parameters were determined at admission and discharge. A trend was recognized for participants in the intervention group to consume more protein than the control group (p = 0.058): 95.3 (SD 13.2) g/day as compared to 77.2 (SD 24.2) g/day, which corresponds to a mean protein intake of 1.6 (SD 0.3) g/kg/day vs. 1.3 (SD 0.5) g/kg/day. Dietary protein supplementation increased body weight by an average of 0.9 (SD 1.1) kg and fat mass by an average of 0.9 (SD 1.2) kg as compared to the baseline (p = 0.039 and p = 0.050, respectively). No significant change in hand-grip strength, body composition, or physical function was observed. In conclusion, short-term intervention with protein-enriched foods and drinks enabled older patients at risk of malnutrition to increase their protein intake to levels that are higher than their required intake. In these older individuals with appropriate protein intake, dietary protein supplementation did not result in a greater improvement in physical recovery outcomes during short-term inpatient rehabilitation. The intervention improved dietary protein intake, but further research (e.g., a full-scale, randomized, controlled trial with sufficient power) is required to determine the effects on physical function outcomes