Profiling of chemonaive osteosarcoma and paired-normal cells identifies EBF2 as a mediator of osteoprotegerin inhibition to tumor necrosis factor–related apoptosis-inducing ligand–induced apoptosis

Osteosarcoma is the most prevalent bone tumor in children and adolescents. At present, the mechanisms of initiation, maintenance, and metastasis are poorly understood. The purpose of this study was to identify relevant molecular targets in the pathogenesis of osteosarcoma. EXPERIMENTAL DESIGN: Tumor chemonaive osteoblastic populations and paired control normal osteoblasts were isolated and characterized phenotypically from seven osteosarcoma patients. Global transcriptomic profiling was analyzed by robust microarray analysis. Candidate genes were confirmed by real-time PCR and organized in molecular pathways. EBF2 and osteoprotegerin (OPG) levels were determined by real-time PCR and OPG protein levels were assessed by ELISA. Immunohistochemical analysis was done in a panel of 46 osteosarcoma samples. Silencing of EBF2 was achieved by lentiviral transduction of short hairpin RNA. Apoptosis was determined by caspase-3/7 activity. RESULTS: A robust clustered transcriptomic signature was obtained in osteosarcoma. Transcription factor EBF2, a known functional bone regulator, was among the most significantly overexpressed genes. Immunohistochemical analysis showed that osteosarcoma is expressed in approximately 70% of tumors analyzed. Because EBF2 was shown previously to act as a transcriptional activator of OPG, elevated levels of EBF2 were associated with high OPG protein levels in osteosarcoma samples compared with normal osteoblastic cells. Knockdown of EBF2 led to stunted abrogation of OPG levels and increased sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. CONCLUSIONS: These findings suggest that EBF2 represents a novel marker of osteosarcoma. EBF2 up-regulation may be one of the mechanisms involved in the high levels of OPG in osteosarcoma, contributing to decrease TRAIL-induced apoptosis and leading to TRAIL resistance

BRCA1 and CtIP promote alternative non-homologous end-joining at uncapped telomeres

Loss of telomere protection occurs during physiological cell senescence and ageing, due to attrition of telomeric repeats and insufficient retention of the telomere-binding factor TRF2. Subsequently formed telomere fusions trigger rampant genomic instability leading to cell death or tumorigenesis. Mechanistically, telomere fusions require either the classical non-homologous end-joining (C-NHEJ) pathway dependent on Ku70/80 and LIG4, or the alternative non-homologous end-joining (A-NHEJ), which relies on PARP1 and LIG3. Here, we show that the tumour suppressor BRCA1, together with its interacting partner CtIP, both acting in end resection, also promotes end-joining of uncapped telomeres. BRCA1 and CtIP do not function in the ATM-dependent telomere damage signalling, nor in telomere overhang removal, which are critical for telomere fusions by C-NHEJ. Instead, BRCA1 and CtIP act in the same pathway as LIG3 to promote joining of de-protected telomeres by A-NHEJ. Our work therefore ascribes novel roles for BRCA1 and CtIP in end-processing and fusion reactions at uncapped telomeres, underlining the complexity of DNA repair pathways that act at chromosome ends lacking protective structures. Moreover, A-NHEJ provides a mechanism of previously unanticipated significance in telomere dysfunction-induced genome instability.FWN – Publicaties zonder aanstelling Universiteit Leide

Cortactin (CTTN) overexpression in osteosarcoma correlates with advanced stage and reduced survival

The cortactin (CTTN) gene has been found, by transcriptomic profiling, to be overexpressed in pediatric osteosarcoma. The location of CTTN at 11q13 and the role of cortactin in cytoskeleton restructuring make CTTN of interest as a potential biomarker for osteosarcoma. MATERIALS AND METHODS: Osteoblasts were isolated from 20 high-grade osteosarcomas before chemotherapy, and paired with cell samples from normal tissue, prior to RNA expression analysis on HG-U133A chips (Affymetrix). Semiquantitative CTTN mRNA expression was analyzed by real-time PCR. An osteosarcoma tissue microarray (TMA) containing 233 tissue spots from 48 patients was used for an immunohistochemical (IHC) study of cortactin. RESULTS: Transcriptomic profiling and real-time PCR analysis indicated increased CTTN expression in osteosarcomas (p = 0.001, Student's T test). TMA IHC showed cortactin to be present more frequently and in greater abundance in osteosarcomas than non-tumoral osteoblastic samples (p< 0.006, Mann-Withney test). Analysis of clinical outcomes indicated that overall survival for patients with primary tumors positive for cortactin was significantly lower than that for patients with cortactin negative (or only weakly staining) tumors (p = 0.0278, Log-rank test). CONCLUSIONS: Our preliminary data support the hypothesis that over-expression of cortactin, contained in the 11q13 amplicon, is involved in osteosarcoma carcinogenesis. The potential of cortactin overexpression as a biomarker for osteosarcoma is consolidated

Fine Motor Control Underlies the Association Between Response Inhibition and Drawing Skill in Early Development

Previous research shows that the development of response inhibition and drawing skill are linked. The current research investigated whether this association reflects a more fundamental link between response inhibition and motor control. In Experiment 1, 3- and 4-year-olds (n = 100) were tested on measures of inhibition, fine motor control, and drawing skill. Data revealed an association between inhibition and fine motor control, which was responsible for most of the association observed with drawing skill. Experiment 2 (n = 100) provided evidence that, unlike fine motor control, gross motor control and inhibition were not associated (after controlling for IQ). Alternative explanations for the link between inhibition and fine motor control are outlined, including a consideration of how these cognitive processes may interact during development

A systematic review of randomized and case‐controlled trials investigating the effectiveness of school‐based motor skill interventions in 3‐ to 12‐year‐old children

Background Research suggests that children identified with impaired motor skills can respond well to intensive therapeutic interventions delivered via occupational and physical therapy services. There is, however, a need to explore alternative approaches to delivering interventions outside traditional referral‐based clinic settings because limited resources mean such health services often struggle to meet demand. This review sets out to systematically assess the evidence for and against school‐based interventions targeted at improving the motor skills of children aged between 3‐12 years old. Method Five electronic databases were searched systematically (AMED, CINAHL, Cochrane, Medline & PsycINFO) for peer‐reviewed articles published between January 2012 and July 2018. Studies were eligible if they implemented a school‐based motor skill intervention with a randomised or case‐controlled trial design that objectively measured motor skills as an outcome, which were not specific to an athletic or sporting skill. Participants had to be aged between 3‐12 years old and free from neurological disorders known to affect muscle function. Risk of bias was assessed using the Cochrane risk of bias tool. Results Twenty‐three studies met the inclusion criteria. These studies encompassed interventions targeted at training: fundamental movement skills; handwriting; fine; and global motor skills. The majority of these studies reported beneficial impact on motor function specifically, but some interventions also assessed subsequent impacts on activity and participation (but not wellbeing). A number of the studies had methodological shortcomings that means these results need to be interpreted with caution. Conclusions Schools appear to be an effective setting for motor skill interventions, but the extent of benefit likely depends on the type of intervention. Moreover, confirmation is needed as to whether benefits extend beyond motor function into everyday activities, participation and wellbeing. Future research should include follow‐up measures to assess the longer‐term efficacy of school‐based interventions

The effect of incorrect scanning distance on boundary detection errors and macular thickness measurements by spectral domain optical coherence tomography: a cross sectional study

BACKGROUND: To investigate the influence of scan distance on retinal boundary detection errors (RBDEs) and retinal thickness measurements by spectral domain optical coherence tomography (SD-OCT). METHODS: 10 eyes of healthy subjects, 10 eyes with diabetic macular edema (DME) and 10 eyes with neovascular age-related macular degeneration (AMD) were examined with RTVue SD-OCT. The MM5 protocol was used in two consecutive sessions to scan the macula. For the first session, the device was set 3.5 cm from the eye in order to obtain detectable signal with low fundus image quality (suboptimal setting) while in the second session a distance of 2.5 cm was set with a good quality fundus image. The signal strength (SSI) value was recorded. The score for retinal boundary detection errors (RBDE) was calculated for ten scans of each examination. RBDE scores were recorded for the whole scan and also for the peripheral 1.0 mm region. RBDE scores, regional retinal thickness values and SSI values between the two sessions were compared. The correlation between SSI and the number of RBDEs was also examined. RESULTS: The SSI was significantly lower with suboptimal settings compared to optimal settings (63.9+/-12.0 vs. 68.3+/-12.2, respectively, p = 0.001) and the number of RBDEs was significantly higher with suboptimal settings in the "all-eyes" group along with the group of healthy subjects and eyes with DME (9.1+/-6.5 vs. 6.8+/-6.3, p = 0.007; 4.4+/-2.6 vs. 2.5+/-1.6, p = 0.035 and 9.7+/-3.3 vs. 5.1+/-3.7, p = 0.008, respectively). For these groups, significant negative correlation was found between the SSI and the number of RBDEs. In the AMD group, the number of RBDEs was markedly higher compared to the other groups and there was no difference in RBDEs between optimal and suboptimal settings with the errors being independent of the SSI. There were significantly less peripheral RBDEs with optimal settings in the "all-eyes" group and the DME subgroup (2.7+/-2.6 vs. 4.2+/-2.8, p = 0.001 and 1.4+/-1.7 vs. 4.1+/-2.2, p = 0.007, respectively). Retinal thickness in the two settings was significantly different only in the outer-superior region in DME. CONCLUSIONS: Optimal distance settings improve SD-OCT SSI with a decrease in RBDEs while retinal thickness measurements are independent of scanning distance

Chlorambucil targets BRCA1/2-deficient tumours and counteracts PARP inhibitor resistance.

Due to compromised homologous recombination (HR) repair, BRCA1- and BRCA2-mutated tumours accumulate DNA damage and genomic rearrangements conducive of tumour progression. To identify drugs that target specifically BRCA2-deficient cells, we screened a chemical library containing compounds in clinical use. The top hit was chlorambucil, a bifunctional alkylating agent used for the treatment of chronic lymphocytic leukaemia (CLL). We establish that chlorambucil is specifically toxic to BRCA1/2-deficient cells, including olaparib-resistant and cisplatin-resistant ones, suggesting the potential clinical use of chlorambucil against disease which has become resistant to these drugs. Additionally, chlorambucil eradicates BRCA2-deficient xenografts and inhibits growth of olaparib-resistant patient-derived tumour xenografts (PDTXs). We demonstrate that chlorambucil inflicts replication-associated DNA double-strand breaks (DSBs), similarly to cisplatin, and we identify ATR, FANCD2 and the SNM1A nuclease as determinants of sensitivity to both drugs. Importantly, chlorambucil is substantially less toxic to normal cells and tissues in vitro and in vivo relative to cisplatin. Because chlorambucil and cisplatin are equally effective inhibitors of BRCA2-compromised tumours, our results indicate that chlorambucil has a higher therapeutic index than cisplatin in targeting BRCA-deficient tumours.This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska‐Curie grant agreement No. 722729. Research in M.T. laboratory is supported by Cancer Research UK, Medical Research Council and University of Oxford

Study protocol: an early intervention program to improve motor outcome in preterm infants: a randomized controlled trial and a qualitative study of physiotherapy performance and parental experiences

Background Knowledge about early physiotherapy to preterm infants is sparse, given the risk of delayed motor development and cerebral palsy. Methods/Design A pragmatic randomized controlled study has been designed to assess the effect of a preventative physiotherapy program carried out in the neonatal intensive care unit. Moreover, a qualitative study is carried out to assess the physiotherapy performance and parents' experiences with the intervention. The aim of the physiotherapy program is to improve motor development i.e. postural control and selective movements in these infants. 150 infants will be included and randomized to either intervention or standard follow-up. The infants in the intervention group will be given specific stimulation to facilitate movements based on the individual infant's development, behavior and needs. The physiotherapist teaches the parents how to do the intervention and the parents receive a booklet with photos and descriptions of the intervention. Intervention is carried out twice a day for three weeks (week 34, 35, 36 postmenstrual age). Standardized tests are carried out at baseline, term age and at three, six, 12 and 24 months corrected age. In addition eight triads (infant, parent and physiotherapist) are observed and videotaped in four clinical encounters each to assess the process of physiotherapy performance. The parents are also interviewed on their experiences with the intervention and how it influences on the parent-child relationship. Eight parents from the follow up group are interviewed about their experience. The interviews are performed according to the same schedule as the standardized measurements. Primary outcome is at two years corrected age. Discussion The paper presents the protocol for a randomized controlled trial designed to study the effect of physiotherapy to preterm infants at neonatal intensive care units. It also studies physiotherapy performance and the parent's experiences with the intervention

Assessment of motor functioning in the preschool period

The assessment of motor functioning in young children has become increasingly important in recent years with the acknowledgement that motor impairment is linked with cognitive, language, social and emotional difficulties. However, there is no one gold standard assessment tool to investigate motor ability in children. The aim of the current paper was to discuss the issues related to the assessment of motor ability in young pre-school children and to provide guidelines on the best approach for motor assessment. The paper discusses the maturational changes in brain development at the preschool level in relation to motor ability. Other issues include sex differences in motor ability at this young age, and evidence for this in relation to sociological versus biological influences. From the previous literature it is unclear what needs to be assessed in relation to motor functioning. Should the focus be underlying motor processes or movement skill assessment? Several key assessment tools are discussed that produce a general measure of motor performance followed by a description of tools that assess specific skills, such as fine and gross motor, ball and graphomotor skills. The paper concludes with recommendations on the best approach in assessing motor function in pre-school children