Chimeric piggyBac transposases for genomic targeting in human cells.

Integrating vectors such as viruses and transposons insert transgenes semi-randomly and can potentially disrupt or deregulate genes. For these techniques to be of therapeutic value, a method for controlling the precise location of insertion is required. The piggyBac (PB) transposase is an efficient gene transfer vector active in a variety of cell types and proven to be amenable to modification. Here we present the design and validation of chimeric PB proteins fused to the Gal4 DNA binding domain with the ability to target transgenes to pre-determined sites. Upstream activating sequence (UAS) Gal4 recognition sites harbored on recipient plasmids were preferentially targeted by the chimeric Gal4-PB transposase in human cells. To analyze the ability of these PB fusion proteins to target chromosomal locations, UAS sites were randomly integrated throughout the genome using the Sleeping Beauty transposon. Both N- and C-terminal Gal4-PB fusion proteins but not native PB were capable of targeting transposition nearby these introduced sites. A genome-wide integration analysis revealed the ability of our fusion constructs to bias 24% of integrations near endogenous Gal4 recognition sequences. This work provides a powerful approach to enhance the properties of the PB system for applications such as genetic engineering and gene therapy

Influence of RNA structural stability on the RNA chaperone activity of the Escherichia coli protein StpA

Proteins with RNA chaperone activity are able to promote folding of RNA molecules by loosening their structure. This RNA unfolding activity is beneficial when resolving misfolded RNA conformations, but could be detrimental to RNAs with low thermodynamic stability. In order to test this idea, we constructed various RNAs with different structural stabilities derived from the thymidylate synthase (td) group I intron and measured the effect of StpA, an Escherichia coli protein with RNA chaperone activity, on their splicing activity in vivo and in vitro. While StpA promotes splicing of the wild-type td intron and of mutants with wild-type-like stability, splicing of mutants with a lower structural stability is reduced in the presence of StpA. In contrast, splicing of an intron mutant, which is not destabilized but which displays a reduced population of correctly folded RNAs, is promoted by StpA. The sensitivity of an RNA towards StpA correlates with its structural stability. By lowering the temperature to 25°C, a temperature at which the structure of these mutants becomes more stable, StpA is again able to stimulate splicing. These observations clearly suggest that the structural stability of an RNA determines whether the RNA chaperone activity of StpA is beneficial to folding

Young age at school entry and attention-deficit hyperactivity disorder-related symptoms during primary school : results of a prospective cohort study conducted at German Rudolf Steiner schools

Objectives Young age at school entry (ASE) for students has been related to their impaired mental health in higher grades. To avoid the negative health consequences of young ASE, preschool examinations and individual school entry deferral for young children are routinely performed by some school authorities. We aimed to investigate whether ASE was associated with attention-deficit hyperactivity disorder (ADHD)-related symptoms in pupils attending schools using a selective school enrolment procedure. Design Prospective open cohort study with baseline assessments at school entry and two follow-ups in the second and fourth grades. Setting Up to 128 Rudolf Steiner Schools (Waldorf Schools) located within Germany. Participants Of the 3079 children from whom data were gathered in the second or fourth grade, 2671 children born between 1 July 2001 and 31 October 2002 (age at baseline: mean 6.7, min 5.91, max 7.24 years, 50% girls) were selected for analysis to avoid bias introduced by individuals at the edges of the ASE distribution. Main outcome measures ADHD-related symptoms were assessed at school entry and second and fourth grades by parent-reported and teacher reported versions of the Strengths and Difficulties Questionnaire (Hyperactivity-Inattention Subscale). Results The agreement between parent-reported and teacher-reported symptoms was poor (intra-class correlation: 0.41 and 0.44 in second and fourth grade assessments, respectively). Regarding teacher reports, ASE was negatively associated with ADHD-related symptoms in the second grade (regression coefficient β=−0.66 per year, P=0.0006) and fourth grade (β=−0.56, P=0.0014). Associations remained after adjusting for potential confounders and pre-existing symptoms at baseline. Regarding parent reports, associations were markedly weaker in both grades (second grade: β=−0.22, P=0.12; fourth grade: β=−0.09, P=0.48).Conclusions Using a prospective study design and comprehensive adjustment for confounding and baseline symptoms, we confirmed prior evidence of the association between young ASE and teacher-reported ADHD symptoms in primary school

The COPE-Trial-Communicating prognosis to parents in the neonatal ICU: Optimistic vs. PEssimistic: study protocol for a randomized controlled crossover trial using two different scripted video vignettes to explore communication preferences of parents of preterm infants.

BACKGROUND One of the numerous challenges preterm birth poses for parents and physicians is prognostic disclosure. Prognoses are based on scientific evidence and medical experience. They are subject to individual assessment and will generally remain uncertain with regard to the individual. This can result in differences in prognostic framing and thus affect the recipients' perception. In neonatology, data on the effects of prognostic framing are scarce. In particular, it is unclear whether parents prefer a more optimistic or a more pessimistic prognostic framing. OBJECTIVE To explore parents' preferences concerning prognostic framing and its effects on parent-reported outcomes and experiences. To identify predictors (demographic, psychological) of parents' communication preferences. DESIGN, SETTING, PARTICIPANTS Unblinded, randomized controlled crossover trial (RCT) at the Division of Neonatology of the University Medical Center Mainz, Germany, including German-speaking parents or guardians of infants born preterm between 2010 and 2019 with a birth weight < 1500 g. Inclusion of up to 204 families is planned, with possible revision according to a blinded sample size reassessment. INTERVENTION Embedded in an online survey and in pre-specified order, participants will watch two video vignettes depicting a more optimistic vs. a more pessimistic framing in prognostic disclosure to parents of a preterm infant. Apart from prognostic framing, all other aspects of physician-parent communication are standardized in both videos. MAIN OUTCOMES AND MEASURES At baseline and after each video, participants complete a two-part online questionnaire (baseline and post-intervention). Primary outcome is the preference for either a more optimistic or a more pessimistic prognostic framing. Secondary outcomes include changes in state-anxiety (STAI-SKD), satisfaction with prognostic framing, evaluation of prognosis, future optimism and hope, preparedness for shared decision-making (each assessed using customized questions), and general impression (customized question), professionalism (adapted from GMC Patient Questionnaire) and compassion (Physician Compassion Questionnaire) of the consulting physician. DISCUSSION This RCT will explore parents' preferences concerning prognostic framing and its effects on physician-parent communication. Results may contribute to a better understanding of parental needs in prognostic disclosure and will be instrumental for a broad audience of clinicians, scientists, and ethicists. TRIAL REGISTRATION German Clinical Trials Register DRKS00024466 . Registered on April 16, 2021

Chronic health conditions and school performance in first graders : a prospective cohort study

Objective Children with chronic health conditions may perform poorer at school. Associations may be confounded by numerous social factors. We aimed to estimate the effects of a chronic health condition on overall school performance in first graders with an emphasis on rigorous adjustment for potential confounders. Methods A population-based cohort study was performed in the area of Mainz-Bingen (Germany). In 2015 all preschoolers were approached and the presence of a chronic health condition was assessed by parental questionnaires and preschool health examination data. The identification of a chronic health condition was based on special health care needs and presence of a doctor’s diagnosis out of 24 school-relevant diseases. At the end of the first school year, overall school performance was assessed by teachers and rated on a 5-item scale ranging from -10 to 10. Results Of 3683 children approached, 2003 were enrolled. Overall school performance was available for 1462 children (51% boys). Of these, 52% suffered from a chronic health condition. Compared to children without a chronic health condition, children with special health care needs (15%) performed worse at school (adjusted mean difference: -0.95, 95% CI: [-1.55; -0.35], P = 0.002). Children with a doctor’s diagnosis but without special health care needs (37%) did not perform worse at school. The effect was further analysed considering the extent of special health care needed. Conclusions Chronic health conditions affect overall school performance early in primary school. To identify academically at-risk children, a chronic health condition identification based on special health care needs may be used

Ultra-Long Pharmacokinetic Properties of Insulin Degludec are Comparable in Elderly Subjects and Younger Adults with Type 1 Diabetes Mellitus

BACKGROUND: Management of diabetes in elderly subjects is complex and careful management of glucose levels is of particular importance in this population because of an increased risk of diabetes-related complications and hypoglycaemia. OBJECTIVE: The aim of this study was to evaluate the pharmacokinetic and pharmacodynamic properties of insulin degludec (IDeg), a basal insulin with an ultra-long duration of action, in elderly subjects with type 1 diabetes compared with younger adults. METHODS: This trial was a randomised, double-blind, two-period, crossover trial conducted in a single centre and included both inpatient and outpatient periods. Subjects were men and women aged 18–35 years inclusive (younger adult group) or ≥65 years (elderly group) with type 1 diabetes who received IDeg (0.4 U/kg) via subcutaneous injection in the thigh once-daily for six days. Following 6-day dosing, a 26-hour euglycaemic glucose clamp procedure was conducted to evaluate the steady-state pharmacodynamic effects of IDeg. Blood samples were taken for pharmacokinetic analysis up to 120 h post-dose. Pharmacokinetic endpoints included the total exposure of IDeg, ie the area under the IDeg serum concentration curve during one dosing interval at steady state (AUC(IDeg,τ,SS)) (τ = 0–24 h, equal to one dosing interval) and the maximum IDeg serum concentration at steady state (C(max,IDeg,SS)). Pharmacodynamic endpoints included the total glucose-lowering effect of IDeg, ie the area under the glucose infusion rate (GIR) curve at steady state (AUC(GIR,τ,SS)), and the maximum GIR at steady state (GIR(max,IDeg,SS)). RESULTS: Total exposure (AUC(IDeg,τ,SS)) and maximum concentration (C(max,IDeg,SS)) of IDeg were comparable between elderly subjects and younger adults. Estimated mean age group ratios (elderly/younger adult) for AUC(IDeg,τ,SS) and C(max,IDeg,SS) and corresponding two-sided 95 % confidence intervals (CIs) were 1.04 (95 % CI 0.73–1.47) and 1.02 (95 % CI 0.74–1.39), respectively. Mean AUC(IDeg,0–12h,SS)/AUC(IDeg,τ,SS) was 53 % in both younger adult and elderly subjects, showing that in both age groups IDeg exposure was evenly distributed across the first and second 12 h of the 24-hour dosing interval. No statistically significant differences were observed between younger adult and elderly subjects with regard to AUC(GIR,τ,SS) (the primary endpoint of this study) and GIR(max,IDeg,SS). Estimated mean age group ratios (elderly/younger adult) for AUC(GIR,τ,SS) and GIR(max,IDeg,SS) and corresponding two-sided 95 % CIs were 0.78 (95 % CI 0.47–1.31) and 0.80 (95 % CI 0.54–1.17), respectively. Duration of action was beyond the clamp duration of 26 h in all subjects. CONCLUSIONS: The exposure of IDeg at steady state during once-daily dosing was similar in younger adult and elderly subjects. The glucose-lowering effect of IDeg was numerically lower in elderly subjects compared with younger adults, but no significant differences were observed between age groups. The ultra-long pharmacokinetic and pharmacodynamic properties of IDeg observed in younger adults were preserved in elderly subjects with type 1 diabetes. Clinical trials.gov number: NCT0096441

Effects of closed-loop automatic control of the inspiratory fraction of oxygen (FiO(2)-C) on outcome of extremely preterm infants - study protocol of a randomized controlled parallel group multicenter trial for safety and efficacy

Background: Most extremely low gestational age neonates (ELGANS, postmenstrual age at birth (PMA) < 28 completed weeks) require supplemental oxygen and experience frequent intermittent hypoxemic and hyperoxemic episodes. Hypoxemic episodes and exposure to inadequately high concentrations of oxygen are associated with an increased risk of retinopathy of prematurity (ROP), chronic lung disease of prematurity (BPD), necrotizing enterocolitis (NEC), neurodevelopmental impairment (NDI), and death beyond 36 weeks PMA. Closed-loop automated control of the inspiratory fraction of oxygen (FiO2-C) reduces time outside the hemoglobin oxygen saturation (SpO2) target range, number and duration of hypo- and hyperoxemic episodes and caregivers’ workload. Effects on clinically important outcomes in ELGANs such as ROP, BPD, NEC, NDI and mortality have not yet been studied. Methods: An outcome-assessor-blinded, randomized controlled, parallel-group trial was designed and powered to study the effect of FiO2-C (in addition to routine manual control (RMC) of FiO2), compared to RMC only, on death and severe complications related to hypoxemia and/or hyperoxemia. 2340 ELGANS with a GA of 23 + 0/7 to 27 + 6/ 7 weeks will be recruited in approximately 75 European tertiary care neonatal centers. Study participants are randomly assigned to RMC (control-group) or FiO2-C in addition to RMC (intervention-group). Central randomization is stratified for center, gender and PMA at birth (< 26 weeks and ≥ 26 weeks). FiO2-C is provided by commercially available and CE-marked ventilators with an FiO2-C algorithm intended for use in newborn infants. The primary outcome variable (composite of death, severe ROP, BPD or NEC) is assessed at 36 weeks PMA (or, in case of ROP, until complete vascularization of the retina, respectively). The co-primary outcome variable (composite outcome of death, language/cognitive delay, motor impairment, severe visual impairment or hearing impairment) is assessed at 24 months corrected age. Discussion: Short-term studies on FiO2-C showed improved time ELGANs spent within their assigned SpO2 target range, but effects of FiO2-C on clinical outcomes are yet unknown and will be addressed in the FiO2-C trial. This will ensure an appropriate assessment of safety and efficacy before FiO2-C may be implemented as standard therap

Measuring social participation in children with chronic health conditions: validation and reference values of the child and adolescent scale of participation (CASP) in the German context

Background: While ICF-CY-based models of care are promising avenues for improving participation of children with chronic health conditions, feasible and valid instruments to assess participation as an outcome in routine are still needed. We aimed to validate a German parent-report version of the Child and Adolescent Scale of Participation (CASP) in children with chronic health conditions of different severity. Methods: Cross-sectional data were collected in 327 children (mean age 7.8 years, 55% boys) from two paediatric centres (n = 112) and one population-based sample (n = 215). Cronbach’s alpha, factor analyses, face validity assessments, correlation analyses, receiver operating characteristics (ROC) curves, and parent-reported health-related quality of life (HRQoL: KINDL) were used to examine internal consistency, test-retest reliability, and capacity to differentiate between disease severity groups. Disease severity was operationalized according to ICD-diagnosis groups and/or parent-reports on health problems, medical and educational support, and medication. A newly developed item “overall perceived participation” was added to the CASP and evaluated. Results: We found good to excellent content validity, excellent internal consistency, and good-to-excellent test-retest reliability of the instrument. While children with mild disease had a significantly greater extent of participation (higher CASP scores) than children with severe disease, they did not differ from healthy children. Children with mild compared to severe disease much more differed in participation as measured by the CASP compared to the KINDL (area under the ROC curve: 0.92 vs. 0.75). In addition, the item “overall perceived participation” was highly correlated (r = 0.86) with the CASP total score, indicating the potential value of this specific single item. Finally, we provided preliminary reference values for the CASP obtained in a population-based sample of children without chronic health conditions. Conclusions: The German version of the CASP and the new item are efficient, valid and reliable measures of social participation in childhood. The CASP-measured participation focuses more on attendance than on involvement into social circumstances of everyday life. To detect children with a high burden of disease on everyday life, the CASP may be more accurate than HRQoL instruments such as the KINDL. As outcome measurement, the CASP may facilitate the implementation of patient-centred paediatric health care

Sleep-disordered breathing and cognitive functioning in preschool children with and without Down syndrome

BACKGROUND: Sleep affects children's cognitive development, preparedness for school and future academic outcomes. People with Down syndrome (DS) are particularly at risk for sleep‐disordered breathing (SDB). To our knowledge, the association between SDB and cognition in preschoolers with DS is unknown. METHODS: We assessed sleep by using cardiorespiratory polygraphy in 22 typically developing (TD) preschoolers and 22 with DS. Cognition was assessed by using the Mullen Scales of Early Learning and behaviour by using the Strengths and Difficulties Questionnaire (SDQ). The MacArthur Communicative Development Inventory (MCDI) measured language level. We predicted that sleep problems would be associated with lower cognitive and behavioural functioning. RESULTS: In TD children, longer sleep duration was associated with higher scores on MCDI expressive language and fewer emotional symptoms such as fear and unhappiness on the SDQ, whilst SDB was associated with increased conduct problems and less prosocial behaviour on the SDQ. Conversely, for children with DS, SDB was associated with increased language understanding and use of actions and gestures on the MCDI. CONCLUSIONS: The findings in the TD group support our hypotheses. We recommend that sleep problems are screened for and treated as even mild SDB may prompt poorer cognition and behaviour. For children with DS, we expect that multiple factors in this complex syndrome mask or mediate the association between sleep and cognitive development and tighter controls are necessary to uncover effects of sleep. We propose longitudinal studies as a necessary tool to assess the precise impact of sleep on cognitive development in accounting for individual differences in DS