Funktionsstudien mit dem in situ mono-ubiquitinierten peroxisomalen Importrezeptor PEX5

:Der Importrezeptor PEX5 ist für die peroxisomale Biogenese essentiell. Er versorgt die Peroxisomen mit den notwendigen Enzymen, um deren Funktion aufrecht zu erhalten. Die peroxisomalen Matrixproteine werden im Zytosol an den freien Ribosomen synthetisiert und von PEX5 durch ihre peroxisomale Erkennungssequenz (PTS1 oder PTS2) gebunden. Der PEX5-Matrixproteinkomplex wird zur peroxisomalen Membran transportiert und bindet dort an den membranverankerten Dockingkomplex (PEX13/PEX14). Anschließend erfolgt die Insertion von PEX5 in die peroxisomale Membran, wobei zeitgleich die Translokation durch die Membran und Freilassung des Matrixproteins in das Peroxisom erfolgt. Im nächsten Schritt wird PEX5 am N-terminalen konservierten Cystein monoubiquitiniert (mUb-PEX5) und ATP-abhängig durch die Exportmaschinerie (PEX26, PEX1/PEX6) aus der Membran gezogen. Nach der Deubiquitinierung kann ein neuer Zyklus von PEX5 stattfinden. In dieser vorliegenden Arbeit sollten folgende Aspekte untersucht werden: 1) Inwiefern kann mUb-PEX5 den PEX5-Zyklus aufrechterhalten? 2) Findet eine direkte Interaktion zwischen mUb-PEX5 und der Exportmaschinerie statt? Zur Beantwortung dieser Fragestellung war es zunächst notwendig, mUb-PEX5 in größeren Mengen zur Verfügung zu haben. Eine rekombinante Gewinnung von mUb-PEX5 wurde in der Literatur bisher nicht beschrieben. Diese Gewinnung wurde im ersten Schritt unter Verwendung der Klickchemie etabliert. Zunächst wurde durch eine Nonsense-Suppression in PEX5 die unnatürliche Aminosäure p-Azidophenylalanin (AzF) anstelle des konservierten N-terminalen Cysteins eingebaut (PEX5C11AzF). Im weiteren Schritt wurde Ubiquitin (Ub(ΔGG)) rekombinant exprimiert und durch Inteinspaltung zu einem Ub(ΔGG)-Alkin generiert. Aus beiden Komponenten konnte durch eine Kupferkatalytische Klickreaktion ein stabiles rPEX5-Ub(ΔGG) hergestellt werden. Es konnte in in vitro und in vivo Versuchen gezeigt werden, dass rPEX5-Ub(ΔGG) nicht an PTS1-Proteine binden, jedoch PTS2-Proteine in die Peroxisomen transportieren kann. rPEX5-Ub(ΔGG) ist in der Lage, an den Dockingkomplex zu binden und in die peroxisomale Membran zu inserieren. rPEX5-Ub(ΔGG) und die PTS2- Matrixproteine erlangen somit einen Protease-geschützten Status. Es ist stark anzunehmen, dass rPEX5-Ub(ΔGG) durch den Exportkomplex aus der Membran entfernt werden kann. In vitro Interaktionsstudien zeigen, dass rPEX5-Ub(ΔGG) gleichermaßen wie rPEX5L an PEX14 binden kann und dass diese Bindung durch die Anwesenheit des Exportkomplexes nicht beeinflusst wird. Aus den Versuchen geht auch hervor, dass rPEX5-Ub(ΔGG) stets besser an die Exportproteine binden kann als das normale rPEX5 (rPEX5L). Eine Interaktion zwischen PEX26 und rPEX5-Ub(ΔGG) konnte erstmals gezeigt werden. In weiteren Studien konnte eine Bindung zwischen PEX14 und PEX26 nachgewiesen werden, die in Anwesenheit von rPEX5-Ub(ΔGG) verstärkt wurde. Eine Analyse zur Faltung mittels einer limitierten Proteolyse der rekombinanten Proteine ergab, dass der N-Terminus im Vergleich zum C-Terminus von rPEX5L instabil und damit vermutlich weniger gefaltet ist. Im Unterschied dazu weist der N-Terminus von rPEX5-Ub(ΔGG) drei stabile Bereiche auf, die möglicherweise durch die Ubiquitinierung geschützt werden. Der C-Terminus in rPEX5-Ub(ΔGG) ist hingegen instabiler und wird zunehmend abgebaut. Die in dieser Arbeit beschriebene Herstellung von rPEX5-mUb(ΔGG) ermöglichte eine Vielzahl von Interaktionsstudien, durch die neue Aspekte der PEX5-Importmaschinerie gewonnen werden konnten. Dadurch wurde eine Ausgangsbasis für weitere Einblicke in die Peroxisom-Biogenese geschaffen. Weitere Studien könnten darauf aufbauend beispielsweise klären, inwiefern mUb-PEX5 mit dem PEX1/PEX6-Komplex interagiert und ob es sich hierbei um direkte oder indirekte Interaktionen handelt

Neonatal visual assessment in congenital heart disease: A pilot study

This study assessed neonatal visual maturity in infants with congenital heart disease (CHD) and its predictive value for neurodevelopmental outcomes. Neonates with CHD underwent a standardized visual assessment before and after cardiopulmonary bypass surgery. Visual maturity was rated as normal versus abnormal by means of normative reference data. Twelve-month neurodevelopment was assessed with the Bayley-III. Twenty-five healthy controls served as the reference group. Neonatal visual assessment was performed in five neonates with CHD preoperatively and in 24 postoperatively. Only postoperative assessments were considered for further analysis. Median [IQR] age at assessment was 27.0 [21.5, 42.0] days of life in postoperative neonates with CHD and 24.0 [15.0, 32.0] in controls. Visual performance was within reference values in 87.5% in postoperative CHD versus 90.5% in healthy controls (p = 1.0). Visual maturity was not predictive of neurodevelopment at 12 months. These results demonstrate the limited feasibility and predictive value of neonatal visual assessments in CHD

Sleep-disordered breathing on respiratory polygraphy in neonates with spina bifida

INTRODUCTION Studies have shown a high prevalence of sleep-disordered breathing (SDB) in children with spina bifida. International standards for regular testing for SDB in this population are lacking. While there are studies investigating the prevalence of SDB in children with spina bifida, there are close to no studies in neonates. AIM AND OBJECTIVE To evaluate if routine respiratory polygraphy (RPG) testing is indicated for neonates with spina bifida and if yes, with what therapeutic consequence. METHODS We conducted a retrospective cohort study of all neonates with spina bifida at the University (Children's) Hospital Zurich after fetal spina bifida repair born between 2017 and 2022, who had undergone at least 1 RPG evaluation during hospitalization on the neonatal ward. RPG were evaluated by a blinded group of experienced pediatric pulmonologists. Based on the neonatal RPG results and pediatric pulmonologist's recommendation for caffeine therapy the spina bifida cohort was divided into two groups. Neonatal baseline RPG and follow-up RPG at the age of the 3 months were evaluated. RESULTS 48 neonates with RPG were included. Compared to the standard values in healthy neonates, the RPG results of this spina bifida cohort showed findings of SDB with central apnea and hypopnea. 22 (45.8%) neonatal RPG evaluations detected central SDB, prompting caffeine therapy. Follow-up RPG conducted after 3 months showed significant improvement of SDB with (almost) no need for continuation of caffeine. CONCLUSION We recommend the implementation of routine RPG testing in neonates with spina bifida to detect SDB and facilitate early targeted treatment

Inhibition abilities and functional brain connectivity in school-aged term-born and preterm-born children

Background Inhibition abilities are known to have impact on self-regulation, behavior, and academic success, and they are frequently impaired in children born preterm. We investigated the possible contributions of resting-state functional brain connectivity to inhibition following preterm birth. Methods Forty-four preterm and 59 term-born participants aged 8–13 years were administered two inhibition tasks and resting-state functional MRI was performed. Functional connectivity (FC) networks were compared between groups using network-based statistics. Associations of FCNs and inhibition abilities were investigated through multivariate linear regression models accounting for the interaction between birth status and inhibition. Results NBS revealed weaker FC in children born preterm compared to term-born peers in connections between motor and supplementary motor regions, frontal lobe, precuneus, and insula. Irrespective of birth status, connections between the cerebellum, frontal, and occipital lobes and inter-lobar, subcortical, intra-hemispheric long-range connections were positively correlated with one of the two inhibition tasks. Conclusions Preterm birth results in long-term alterations of FC at network level but these FCN alterations do not specifically account for inhibition problems in children born very preterm. Impact Irrespective of birth status, significant associations were found between the subdomain of response inhibition and functional connectivity in some subnetworks. A group comparisons of functional brain connectivity measured by rsfMRI in school-aged children born very preterm and at term. The investigation of network-level functional connectivity at rest does not appear adequate to explain differences in inhibition abilities between children born very preterm and at term, hence other imaging techniques might be more suited to explore the underlying neural mechanisms of inhibition abilities in school-aged children born very preterm

Neuromonitoring, neuroimaging, and neurodevelopmental follow-up practices in neonatal congenital heart disease: a European survey

BACKGROUND Brain injury and neurodevelopmental impairment remain a concern in children with complex congenital heart disease (CHD). A practice guideline on neuromonitoring, neuroimaging, and neurodevelopmental follow-up in CHD patients undergoing cardiopulmonary bypass surgery is lacking. The aim of this survey was to systematically evaluate the current practice in centers across Europe. METHODS An online-based structured survey was sent to pediatric cardiac surgical centers across Europe between April 2019 and June 2020. Results were summarized by descriptive statistics. RESULTS Valid responses were received by 25 European centers, of which 23 completed the questionnaire to the last page. Near-infrared spectroscopy was the most commonly used neuromonitoring modality used in 64, 80, and 72% preoperatively, intraoperatively, and postoperatively, respectively. Neuroimaging was most commonly performed by means of cranial ultrasound in 96 and 84% preoperatively and postoperatively, respectively. Magnetic resonance imaging was obtained in 72 and 44% preoperatively and postoperatively, respectively, but was predominantly reserved for clinically symptomatic patients (preoperatively 67%, postoperatively 64%). Neurodevelopmental follow-up was implemented in 40% of centers and planned in 24%. CONCLUSIONS Heterogeneity in perioperative neuromonitoring and neuroimaging practice in CHD in centers across Europe is large. The need for neurodevelopmental follow-up has been recognized. A clear practice guideline is urgently needed. IMPACT There is large heterogeneity in neuromonitoring, neuroimaging, and neurodevelopmental follow-up practices among European centers caring for neonates with complex congenital heart disease. This study provides a systematic evaluation of the current neuromonitoring, neuroimaging, and neurodevelopmental follow-up practice in Europe. The results of this survey may serve as the basis for developing a clear practice guideline that could help to early detect and prevent neurological and neurodevelopmental sequelae in neonates with complex congenital heart disease

Neonatal transport in Switzerland: a retrospective single-centre analysis - quo vadis?

AIM: To assess neonatal transport activities by the neonatal transport teams of the University Children’s Hospital Zurich in order to identify opportunities for improvement in the organisation of these transports. METHODS: Retrospective analysis of prospectively collected data on neonatal transports by the neonatal transport teams of the University Children’s Hospital Zurich between January 2014 and December 2018. Data on transports affecting neonates with a corrected gestational age of up to 44 weeks and a weight less than 5 kg were extracted from registration forms, transport forms, transport reports and the neonates’ medical charts. Transport data were assessed separately for urgent, non-urgent and re-transfers. RESULTS: During the study period, 1110 transport runs, including 883 (79.5%) urgent, 105 (9.5%) non-urgent and 122 (11.0%) re-transfers were performed. Ground transport accounted for 90.7% of the cases. The majority (77.7%) of the transported neonates were born at term and 59.1% were transported within the first 24 hours of life. The most common reason for transport was respiratory distress (39.9%), followed by cardiac diseases (14.6%). Medical procedures performed by the neonatal transport teams during transport mostly addressed peripheral intravenous line placement (41.8%) and feeding tube placement (41.8%). The median preparation time for urgent transfers was 35 min (range 8–225) for ground and 50 min (range 20–260) for air transport. CONCLUSIONS: The high proportion of urgent transfers emphasises the need for an efficient neonatal transport system and dedicated neonatal transport teams staffed by members with training in neonatal transport and expertise in handling neonatal emergencies. To provide the best possible care to the vulnerable neonates, the heterogeneous nature of the cohort of transported neonates regarding the diagnoses transport demand was made for and the medical procedures performed during transport should be considered in simulation training of neonatal transport team staff. Additionally, processes to improve preparation time should be defined and implemented in order to reduce it to less than 30 min so as to guarantee efficient care. Further studies are needed to assess the quality and efficacy of neonatal transports in Switzerland. National guidelines on the standard of neonatal transport and quality metrics should be established in order to set benchmarks and to improve the quality of the transports

Altered brain metabolism contributes to executive function deficits in school-aged children born very preterm

BACKGROUND Executive function deficits in children born very preterm (VPT) have been linked to anatomical abnormalities in white matter and subcortical brain structures. This study aimed to investigate how altered brain metabolism contributes to these deficits in VPT children at school-age. METHODS Fifty-four VPT participants aged 8-13 years and 62 term-born peers were assessed with an executive function test battery. Brain metabolites were obtained in the frontal white matter and the basal ganglia/thalami, using proton magnetic resonance spectroscopy (MRS). N-acetylaspartate (NAA)/creatine (Cr), choline (Cho)/Cr, glutamate + glutamine (Glx)/Cr, and myo-Inositol (mI)/Cr were compared between groups and associations with executive functions were explored using linear regression. RESULTS In the frontal white matter, VPT showed lower Glx/Cr (mean difference: -5.91%, 95% CI [-10.50, -1.32]), higher Cho/Cr (7.39%, 95%-CI [2.68, 12.10]), and higher mI/Cr (5.41%, 95%-CI [0.18, 10.64]) while there were no differences in the basal ganglia/thalami. Lower executive functions were associated with lower frontal Glx/Cr ratios in both groups (β = 0.16, p = 0.05) and higher mI/Cr ratios in the VPT group only (interaction: β = -0.17, p = 0.02). CONCLUSION Long-term brain metabolite alterations in the frontal white matter may be related to executive function deficits in VPT children at school-age. IMPACT Very preterm birth is associated with long-term brain metabolite alterations in the frontal white matter.Such alterations may contribute to deficits in executive function abilities.Injury processes in the brain can persist for years after the initial insult.Our findings provide new insights beyond structural and functional imaging, which help to elucidate the processes involved in abnormal brain development following preterm birth.Ultimately, this may lead to earlier identification of children at risk for developing deficits and more effective interventions

Inhibition is associated with whole-brain structural brain connectivity on network level in school-aged children born very preterm and at term

Inhibition abilities are often impaired in children born very preterm. In typically-developing individuals, inhibition has been associated with structural brain connectivity (SC). As SC is frequently altered following preterm birth, this study investigated whether aberrant SC underlies inhibition deficits in school-aged children born very preterm. In a group of 67 very preterm participants aged 8–13 years and 69 term-born peers, inhibition abilities were assessed with two tasks. In a subgroup of 50 very preterm and 62 term-born participants, diffusion tensor imaging (DTI) data were collected. Using network-based statistics (NBS), mean fractional anisotropy (FAmean) was compared between groups. Associations of FAmean and inhibition abilities were explored through linear regression. The composite score of inhibition abilities was lower in the very preterm group (M ​= ​−0.4, SD ​= ​0.8) than in the term-born group (M ​= ​0.0, SD ​= ​0.8) but group differences were not significant when adjusting for age, sex and socio-economic status (β ​= ​−0.13, 95%-CI [-0.30, 0.04], p ​= ​0.13). In the very preterm group, FAmean was significantly lower in a network comprising thalamo-frontal, thalamo-temporal, frontal, cerebellar and intra-hemispheric connections than in the term-born group (t ​= ​5.21, lowest p-value ​= ​0.001). Irrespective of birth status, a network comprising parietal, cerebellar and subcortical connections was positively associated with inhibition abilities (t ​= ​4.23, lowest p-value ​= ​0.02). Very preterm birth results in long-term alterations of SC at network-level. As networks underlying inhibition abilities do not overlap with those differing between the groups, FAmean may not be adequate to explain inhibition problems in very preterm children. Future studies should combine complementary measures of brain connectivity to address neural correlates of inhibition abilities

Executive function deficits mediate the association between very preterm birth and behavioral problems at school-age

Background & aims Children and adolescents born very preterm are at increased risk to develop executive function deficits and to suffer from social, emotional and attentional problems. This study investigated whether executive function deficits contribute to behavioral problems in children and adolescents born very preterm at school-age. Study design Thirty-eight children and adolescents born very preterm and 41 age-matched term-born peers were assessed at a mean age of 12.9 (±1.8) years with a comprehensive battery of executive function tests, including working memory, planning, cognitive flexibility, and verbal fluency. A composite score was calculated to reflect overall executive function abilities. To assess behavioral problems, parents completed the Strengths and Difficulties Questionnaire (SDQ). Mediation analysis was applied to quantify the effect of preterm birth on behavioral problems with executive function abilities as a mediating variable. Results Executive function abilities were poorer in the very preterm compared to the term-born group (d = 0.62, p = .005) and the parents of very preterm children reported more behavioral problems on the SDQ Total Difficulties Score (d = 0.54, p = .01). The effect of birth status on behavioral problems was significantly mediated by executive function abilities while adjusting for age at assessment, sex, and socioeconomic status (F(2, 76) = 6.42, p = .002, R2 = 0.14). Conclusion Results from this study suggest that the increase in behavioral symptoms in very preterm children at school-age compared to term-born peers may partly be explained by their executive function deficits. These findings highlight the importance of continuously monitoring the development of children born very preterm to provide optimal care as they grow up

Cognitive outcome of early school-aged children born very preterm is not predicted by early short-term amplitude-integrated electroencephalography

AIM We investigated the association between early amplitude-integrated electroencephalography (aEEG) and cognitive outcome in very preterm infants at early school-age. METHODS This prospective cohort study, conducted in the Department of Neonatology, University Hospital Zurich, Switzerland, from 2009-2012, comprised infants born at less than 32 weeks of gestation, who underwent continuous aEEG recording during the first four days of life. Cognitive outcome was assessed with the Kaufman-Assessment Battery for Children at five years. Univariate and multivariate logistic regressions were calculated between aEEG parameters and normal cognitive outcome, defined as an intelligence quotient (IQ) of at least 85. RESULTS The 118 (52.5% male) infants were born at a mean gestational age of 29.9 weeks and a mean birth weight of 1,235 ±363 grams. We followed up 89 children at the age of five and they had a mean IQ of 97.8 ±12.7 with 21.3% under 85 - and 2.2% had cerebral palsy. Univariate analyses found associations between aEEG measures and normal cognitive outcome, but these were no longer significant after adjustment for confounders. Socioeconomic status and neonatal morbidity were independent predictors of cognitive outcome. CONCLUSION Early short-term aEEG did not predict later cognitive outcome in our cohort of very preterm infants. This article is protected by copyright. All rights reserved