Absence of the dystrophin protein in Duchenne muscular dystrophy: effects on transcriptome, proteome and microRNA secretome

Duchenne muscular dystrophy (DMD) is a classical monogenic disorder characterised by severe muscle weakness. Progressive muscle loss ultimately culminates in fatality due to failure of the heart and diaphragm muscles, typically in the 2nd-4th decade of life. The genetic cause of DMD is disruption of the DMD gene encoding for the dystrophin protein. Dystrophin is important for cytoskeletal structure and normal muscle function, and it plays a vital role in maintaining membrane stability and signalling functions. However, the downstream molecular events that follow dystrophin loss are incompletely understood. Moreover, pharmacodynamic biomarkers that can inform on disease progression are severely lacking. This thesis aimed to identify the effects of the dystrophin protein absence on the cardiac transcriptome, skeletal muscle proteome and extracellular microRNA (miRNA) secretome with pathological progression. To improve the understanding of the dystrophic cardiomyopathic advancement in the various anatomical regions of the heart, transcriptomic arrays were performed on RNA taken from the distinct chambers to identify novel pathways involved in pathology. Analysis identified early molecular changes in the right atrium and ventricle, mostly related to cell migration and cardiac remodelling. Furthermore, Sfrp2 (secreted Frizzled-related protein 2), a major regulator of the Wnt signalling pathway, was found to be highly differentially expressed between control and dystrophic hearts. It was observed that expression of the non-canonical c-Jun N-terminal kinase (JNK)-arm of the Wnt cascade was reduced. Interestingly, cellular fractionation demonstrated decreased levels in the cytoplasmic/extracellular region and increased expression surrounding the nucleus, which suggests a potential mislocalisation of the sFRP2 protein in dystrophic hearts. Protein expression in skeletal muscle was characterised by high-resolution mass spectrometry-based proteomics in two dystrophic mouse models over time. Analysis of the two models at different time points allowed for protein identification irrespective of mutation or age. The results highlighted the involvement of many proteins in DMD, such as Rho GTPases, SLC25A24, RICTOR, CAV3 and MVP. Expression of major vault protein (MVP) increased during differentiation of myoblasts in vitro and interestingly, Mvp knockdown was observed to potentially influence Dmd expression levels, suggesting a novel role for MVP in DMD pathology. The miRNA secretome was examined to investigate the relationship between skeletal muscle dystrophin expression and extracellular miRNA abundance. Interestingly, while muscle-related miRNAs (myomiRs, indicative of muscle turnover) were not restored to wild-type levels in mdx-XistΔhs mice, which express low levels of dystrophin from birth (~8-44%), other miRNAs such as miR-193b-3p, miR-370-3p and miR-483-3p did exhibit a decrease in extracellular abundance. In contrast, exon skipping treatment achieving similar dystrophin re-expression levels was able to completely restore serum miRNA levels. The disparity between the mdx-XistΔhs and mdx mice treated with exon skipping was attributed to uneven dystrophin distribution at the sarcolemma in the former. The observed patchy, within-fibre mosaicism was unable to restore extracellular myomiR abundance, and by extension muscle turnover. These results are directly relevant to the development of therapeutic strategies for dystrophin re-expression. In conclusion, this work has identified novel molecular pathways involved in cardiac and skeletal muscle dystrophic pathology. Furthermore, the results have demonstrated the importance of uniform dystrophin distribution at the sarcolemma for pathological correction. Together, these findings provide new insights into the molecular pathology of DMD with implications for therapy

Implementing Growth Mindset Principles for Girls in STEM Elementary Classrooms Through the Creation of a Children\u27s Book

With an emphasis on STEM education in schools, young girls begin to have an idea that math and science skills are based on one’s natural ability. A fixed mindset is the belief that one possesses an ability that comes naturally. Many girls, starting at the elementary level tend to interpret a lack of skill for being dumb, and therefore, give up on difficult subjects like math and science. On the other hand, a fluid theory of intelligence, or growth mindset is when a student values effort and understands that these “abilities” come from hard work and taking on new challenges. Students in a growth mindset see intelligence as something that can be developed overtime, while every learning opportunity, challenge, and failure is seen as an important step to becoming more knowledgeable. As a teacher, promoting a growth mindset in the classroom is key for student success; praising and encouraging students through the process of learning is more valuable then giving a grade for the final product. This thesis not only researched the differences between a growth versus fixed mindset, but also the value of fluid theories of intelligence, and the effects on elementary aged girls. This thesis includes a children’s book that promotes the idea of a growth mindset with a protagonist who learns to see the importance of persevering, working hard, and attaining success. With the picturebook intended for elementary aged students, its hope is to bring awareness to students and teachers that having a growth mindset mentality is important

Making Distribution State Estimation Practical: Challenges and Opportunities

In increasingly digitalized and metered distribution networks, state estimation is generally recognized as a key enabler of advanced network management functionalities. However, despite decades of research, the real-life adoption of state estimation in distribution systems remains sporadic. This systematization of knowledge paper discusses the cause for this while comparing industrial and academic experiences and reviewing well- and less-established research directions. We argue that to make distribution system state estimation more practical and applicable in the field, new perspectives are needed. In particular, research should move away from conventional approaches and embrace generalized problem specifications and more comprehensive workflows. These, in turn, require algorithm advancements and more general mathematical formulations. We discuss lines of work to enable the delivery of tangible research.Comment: 10 page

Two-year neurodevelopmental outcome in children born extremely preterm:the EPI-DAF study

OBJECTIVE: In 2010, the Dutch practice regarding initiation of active treatment in extremely preterm infants was lowered from 25 completed weeks' to 24 completed weeks' gestation. The nationwide Extremely Preterm Infants - Dutch Analysis on Follow-up Study was set up to provide up-to-date data on neurodevelopmental outcome at 2 years' corrected age (CA) after this guideline change. Design: National cohort study. PATIENTS: All live born infants between 240/7 weeks' and 266/7 weeks' gestational age who were 2 years' CA in 2018-2020. MAIN OUTCOME MEASURE: Impairment at 2 years' CA, based on cognitive score (Bayley-III-NL), neurological examination and neurosensory function. RESULTS: 651 of 991 live born infants (66%) survived to 2 years' CA, with data available for 554 (85%). Overall, 62% had no impairment, 29% mild impairment and 9% moderate-to-severe impairment (further defined as neurodevelopmental impairment, NDI). The percentage of survivors with NDI was comparable for infants born at 24 weeks', 25 weeks' and 26 weeks' gestation. After multivariable analysis, severe brain injury and low maternal education were associated with higher odds on NDI. NDI-free survival was 48%, 67% and 75% in neonatal intensive care unit (NICU)-admitted infants at 24, 25 and 26 weeks' gestation, respectively. CONCLUSIONS: Lowering the threshold has not been accompanied by a large increase in moderate-to-severely impaired infants. Among live-born and NICU-admitted infants, an increase in NDI-free survival was observed from 24 weeks' to 26 weeks' gestation. This description of a national cohort with high follow-up rates gives an accurate description of the range of outcomes that may occur after extremely preterm birth

Increasing trends in a low 5-min Apgar score among (near) term singletons:a Dutch nationwide cohort study

Objective: To investigate trends in low Apgar scores in (near) term singletons using the Dutch Perinatal Registry. Methods: In a cohort of 1,583,188 singletons liveborn ≥35 weeks of gestation in the period 2010–2019, we studied trends in low 5-min Apgar scores (&lt;7 and &lt;4) using Cochrane Armitage trend tests. Results: The proportion of infants with low Apgar scores &lt;7 and &lt;4 increased significantly between 2010–2019 (1.04–1.42% (p &lt; 0.001), 0.17–0.19% (p = 0.009), respectively). Neonatal mortality remained unchanged. Induction of labour, epidural analgesia and planned caesarean section showed an increasing trend. Instrumental vaginal delivery and emergency caesarean section were performed less frequently over time, but these intervention subgroups showed the highest relative increase in infants with low Apgar scores. Conclusions: In the Netherlands, the risk of a low 5-min Apgar score increased over the last decade. The highest relative increase was observed in subgroups of instrumental vaginal delivery and emergency caesarean section.</p

Neurodevelopmental outcome at 5.5 years in Dutch preterm infants born at 24-26 weeks' gestational age: the EPI-DAF study

Objective After lowering the Dutch threshold for active treatment from 25 to 24 completed weeks' gestation, survival to discharge increased by 10% in extremely preterm live born infants. Now that this guideline has been implemented, an accurate description of neurodevelopmental outcome at school age is needed.Design Population-based cohort study.Setting All neonatal intensive care units in the Netherlands.Patients All infants born between 24(0/7) and 26(6/7 )weeks' gestation who were 5.5 years' corrected age (CA) in 2018-2020 were included.Main outcome measures Main outcome measure was neurodevelopmental outcome at 5.5 years. Neurodevelopmental outcome was a composite outcome defined as none, mild or moderate-to-severe impairment (further defined as neurodevelopmental impairment (NDI)), using corrected cognitive score (Wechsler Preschool and Primary Scale of Intelligence Scale-III-NL), neurological examination and neurosensory function. Additionally, motor score (Movement Assessment Battery for Children-2-NL) was assessed. All assessments were done as part of the nationwide, standardised follow-up programme.Results In the 3-year period, a total of 632 infants survived to 5.5 years' CA. Data were available for 484 infants (77%). At 5.5 years' CA, most cognitive and motor (sub)scales were significantly lower compared with the normative mean. Overall, 46% had no impairment, 36% had mild impairment and 18% had NDI. NDI-free survival was 30%, 49% and 67% in live born children at 24, 25 and 26 weeks' gestation, respectively (pDevelopmen

Neurodevelopmental outcome at 5.5 years in Dutch preterm infants born at 24–26 weeks’ gestational age: the EPI-DAF study

Objective After lowering the Dutch threshold for active treatment from 25 to 24 completed weeks’ gestation, survival to discharge increased by 10% in extremely preterm live born infants. Now that this guideline has been implemented, an accurate description of neurodevelopmental outcome at school age is needed. Design Population-based cohort study. Setting All neonatal intensive care units in the Netherlands. Patients All infants born between 240/7 and 266/7 weeks’ gestation who were 5.5 years’ corrected age (CA) in 2018–2020 were included. Main outcome measures Main outcome measure was neurodevelopmental outcome at 5.5 years. Neurodevelopmental outcome was a composite outcome defined as none, mild or moderate-to-severe impairment (further defined as neurodevelopmental impairment (NDI)), using corrected cognitive score (Wechsler Preschool and Primary Scale of Intelligence Scale-III-NL), neurological examination and neurosensory function. Additionally, motor score (Movement Assessment Battery for Children-2-NL) was assessed. All assessments were done as part of the nationwide, standardised follow-up programme. Results In the 3-year period, a total of 632 infants survived to 5.5 years’ CA. Data were available for 484 infants (77%). At 5.5 years’ CA, most cognitive and motor (sub)scales were significantly lower compared with the normative mean. Overall, 46% had no impairment, 36% had mild impairment and 18% had NDI. NDI-free survival was 30%, 49% and 67% in live born children at 24, 25 and 26 weeks’ gestation, respectively (p<0.001). Conclusions After lowering the threshold for supporting active treatment from 25 to 24 completed weeks’ gestation, a considerable proportion of the surviving extremely preterm children did not have any impairment at 5.5 years’ CA

Dystrophin involvement in peripheral circadian SRF signalling

Absence of dystrophin, an essential sarcolemmal protein required for muscle contraction, leads to the devastating muscle-wasting disease Duchenne muscular dystrophy. Dystrophin has an actin-binding domain, which binds and stabilises filamentous-(F)-actin, an integral component of the RhoA-actin-serum-response-factor-(SRF) pathway. This pathway plays a crucial role in circadian signalling, whereby the suprachiasmatic nucleus (SCN) transmits cues to peripheral tissues, activating SRF and transcription of clock-target genes. Given dystrophin binds F-actin and disturbed SRF-signalling disrupts clock entrainment, we hypothesised dystrophin loss causes circadian deficits. We show for the first time alterations in the RhoA-actin-SRF-signalling pathway, in dystrophin-deficient myotubes and dystrophic mouse models. Specifically, we demonstrate reduced F/G-actin ratios, altered MRTF levels, dysregulated core-clock and downstream target-genes, and down-regulation of key circadian genes in muscle biopsies from Duchenne patients harbouring an array of mutations. Furthermore, we show dystrophin is absent in the SCN of dystrophic mice which display disrupted circadian locomotor behaviour, indicative of disrupted SCN signalling. Therefore, dystrophin is an important component of the RhoA-actin-SRF pathway and novel mediator of circadian signalling in peripheral tissues, loss of which leads to circadian dysregulation