This conceptual analysis examines the relationship between play-based learning and the science of reading, addressing a growing tension in early literacy policy and practice. While contemporary mandates increasingly emphasize structured, skills-based instruction grounded in cognitive science, play-based pedagogies are often positioned as incompatible with these approaches. This paper challenges that dichotomy by positioning play-based learning as a powerful and complementary context for the development of foundational literacy skills. Drawing on interdisciplinary research from education, developmental psychology, and cognitive science, the paper examines how explicit, evidence-based instruction can be integrated within play-based classroom activities. It highlights how key components of reading development, such as alphabetics, phonological awareness, and vocabulary, can be supported through intentional, teacher-guided play. Building on existing conceptualizations of guided play, the paper proposes active child participation, rather than strict child-directedness, as an alternative defining characteristic of guided play. This reframing provides greater flexibility for integrating explicit and systematic literacy instruction within play-based contexts while preserving children's engagement in the learning process. Ultimately, the paper reframes play-based learning and instruction not as competing priorities, but as complementary components of effective literacy education. By bridging divides between policy mandates and developmentally appropriate practice, it offers a nuanced perspective that supports flexible, intentional, and evidence-informed approaches to early reading instruction.

Pathogenic variants in cytochromecoxidase assembly factor 5 (COA5), a proposed complex IV (CIV) assembly factor, have been shown to cause clinical mitochondrial disease with two siblings affected by neonatal hypertrophic cardiomyopathy manifesting a rare, homozygousCOA5missense variant (NM_001008215.3: c.157G>C, p.Ala53Pro). The most striking observation in the affected individuals was an isolated impairment in the early stage of mitochondrial CIV assembly. In this study, we report an unrelated family in whom we have identified the sameCOA5variant with patient-derived fibroblasts and skeletal muscle biopsies replicating an isolated CIV deficiency. A CRISPR/Cas9-edited homozygousCOA5knockout U2OS cell line with a similar biochemical profile was generated to interrogate the functional role of the human COA5 protein. Mitochondrial complexome profiling pinpointed a role of COA5 in early CIV assembly, more specifically, its involvement in the stage between MTCO1 maturation and the incorporation of MTCO2. We therefore propose that the COA5 protein plays an essential role in the biogenesis of MTCO2 and its integration into the early CIV assembly intermediate for downstream assembly of the functional holocomplex.

BACKGROUND:Children born to women who carry pathogenic variants in mitochondrial DNA (mtDNA) are at risk for a range of clinical syndromes collectively known as mtDNA disease. Mitochondrial donation by pronuclear transfer involves transplantation of nuclear genome from a fertilized egg from the affected woman to an enucleated fertilized egg donated by an unaffected woman. Thus, pronuclear transfer offers affected women the potential to have a genetically related child with a reduced risk of mtDNA disease. METHODS:We offered mitochondrial donation (by pronuclear transfer) or preimplantation genetic testing (PGT) to a series of women with pathogenic mtDNA variants who sought to reduce the transmission of these variants to their children. Patients with heteroplasmy (variants present in a proportion of copies of mtDNA) were offered PGT, and patients with homoplasmy (variants present in all copies of mtDNA) or elevated heteroplasmy were offered pronuclear transfer. RESULTS:Clinical pregnancies were confirmed in 8 of 22 patients (36%) and 16 of 39 patients (41%) who underwent an intracytoplasmic sperm injection procedure for pronuclear transfer or for PGT, respectively. Pronuclear transfer resulted in 8 live births and 1 ongoing pregnancy. PGT resulted in 18 live births. Heteroplasmy levels in the blood of the 8 infants whose mothers underwent pronuclear transfer ranged from undetectable to 16%. Levels of the maternal pathogenic mtDNA variant were 95 to 100% lower in 6 newborns and 77 to 88% lower in 2 newborns than in the corresponding enucleated zygotes. Heteroplasmy levels were known for 10 of the 18 infants whose mothers underwent PGT and ranged from undetectable to 7%. CONCLUSIONS:We found that mitochondrial donation through pronuclear transfer was compatible with human embryo viability. An integrated program involving pronuclear transfer and PGT was effective in reducing the transmission of homoplasmic and heteroplasmic pathogenic mtDNA variants. (Funded by NHS England and others.).

Early math learning is a fundamental goal of early years education, which is commonly supported through play. In practice, however, educators' differing views of play lead to different implementations of play that either dichotomize or integrate the constructs of play and academic learning. Despite this, little work has addressed the connections between teachers' perspectives of play and mathematics and their approaches to math instruction in kindergarten. The present study responded to this need through qualitative analysis of educators' perspectives of play and math learning. Results identified how the majority of participating educators supported more indirect, or child-led without adult intervention, approaches to math learning, while only a minority of educators described more direct, or teacher-facilitated, approaches to math. These findings point to a larger conversation about the need to move away from more child-led approaches and towards more teacher-facilitated approaches to play and early math learning in kindergarten.

Heteroplasmic mitochondrial DNA (mtDNA) variants accumulate as humans age, particularly in the stem-cell compartments, and are an important contributor to age-related disease. Mitochondrial dysfunction has been observed in osteoporosis and somatic mtDNA pathogenic variants have been observed in animal models of osteoporosis. However, this has never been assessed in the relevant human tissue. Mesenchymal stem cells (MSCs) are the progenitors to many cells of the musculoskeletal system and are critical to skeletal tissues and bone vitality. Investigating mtDNA in MSCs could provide novel insights into the role of mitochondrial dysfunction in osteoporosis. To determine if this is possible, we investigated the landscape of somatic mtDNA variation in MSCs through a combination of fluorescence-activated cell sorting and single-cell next-generation sequencing. Our data show that somatic heteroplasmic variants are present in individual patient-derived MSCs, can reach high heteroplasmic fractions and have the potential to be pathogenic. The identification of somatic heteroplasmic variants in MSCs of patients highlights the potential for mitochondrial dysfunction to contribute to the pathogenesis of osteoporosis.

As efforts to use learning through play (LtP) expand globally, it is important to explore how children's perspectives impact the efficacy and experience of this pedagogical approach. LtP has been conceptualized as a spectrum from free play to guided play to teacher-directed play. This spectrum describes different ways in which play happens-with varying levels of adult support-and acknowledges that children's agency is characterized by choice and the ability to direct, participate in, and/or initiate play. Previous research has primarily focused on adult perceptions of LtP for preschool children in high-resource contexts. We present the perspectives on LtP of children (3-12 years) in Bangladesh, Colombia and Uganda. We photographed learning activities in community centres and schools that incorporated play-based practices. In group discussions, we used these photographs to elicit children's perspectives on the difference between play and learning in the classroom, the factors that influence their construction of play and learning, and the role that teachers play in these activities. Conceptualizations across the three research sites and ages were similar: Children associated learning with play if the activity was fun and social; distinctions between play and learning were defined by content, modality, materials and location; and teachers were seen as involved in play under limited conditions. We discuss the implications of our findings for the implementation of LtP in majority-world contexts. By moving teachers from a primarily teacher-directed approach to more guided-play approaches, we are not only asking teachers to give up some control but are also asking children to think about adults in fundamentally different ways. This research highlights that we may need to couple professional development for teachers with approaches that support children to change their perceptions of agency and choice in the classroom.

The early years of education are a crucial time to develop a strong foundation of critical mathematical skills. A growing body of research continues to demonstrate that this foundational knowledge can be successfully built through teacher-facilitated, or guided, approaches to play. Despite its benefits, the implementation of guided play is relatively uncommon, as educators have expressed uncertainty regarding how to support guided play within the realities of classroom practice. The current study addressed this central challenge through qualitative inquiry, and identified three overarching approaches, each with specific strategies for implementation, that educators used to guide children’s play to support early math learning. Results also identified how guided play is situated within an ongoing and iterative process of play, whereby educators play a crucial role in supporting children’s learning before, during, and after periods of play. Illustrative examples of these approaches and strategies for guiding play will be shared and discussed relative to their potential for supporting educators within their practice of integrating teacher-facilitated play and early mathematics learning.

In this study, we explore teacher perspectives on the utility of a formative assessment tool, Teacher RePlay, to support their implementation of learning through play (LTP) activities in classrooms for students age 3 to 12 years in Bangladesh, Colombia, and Uganda. Research suggests that formative assessment tools may be useful to bridge support from professional development trainings to the classroom. The Teacher RePlay tool was designed to provide continued support to teachers subsequent to their participation in training sessions on implementing LTP. Through interviews, we explored the benefits and challenges teachers experienced while using the tool. Teachers reported the tool improved their planning and delivery of LTP activities and cited positive outcomes, including enhanced student engagement, attendance, and overall learning. Challenges in implementing LTP included class size limitations, time constraints, and technology access. The study underscores the importance of considering cultural contexts in implementing LTP and suggests future considerations, including alternative resource delivery formats and the creation of a support community for teachers. We highlight, as an alternative to the traditional professional development model, the promise of formative assessment tools as a means of providing continual and productive support to teachers.

Children identified with developmental disabilities or delays (DD) are increasingly being educated in inclusive mainstream classroom environments. At the kindergarten level, there have been pedagogical shifts towards the promotion of play-based learning, with few resources addressing how teachers can support inclusion in play. Forty-two Ontario kindergarten teachers completed an online survey with open-ended questions regarding the promotion of play and inclusion for children with DD. While participants shared differing conceptualizations of inclusion, the most common definition underscored the need to provide all children with appropriate academic support. In line with this definition, the majority of participants (74%) discussed the need for inclusive pedagogical strategies to promote greater academic inclusion in classroom learning activities. Several teachers (43%) also highlighted the need for strategies to promote a respectful classroom community. While teachers' views on the role of play complemented themes of academic and community inclusion, few teachers (24%) shared strategies to help support academic or community inclusion within children's play. These results provide insight into how kindergarten teachers conceptualise and promote inclusion primarily outside of the context of play and underscore a critical gap in research and practice.

Teacher-facilitation of play is proposed as an effective method for supporting early literacy learning, however, educators remain uncertain how to balance child-autonomy in play while also directing play toward explicit academic objectives. In response, this study sought to understand how kindergarten teachers can successfully facilitate play to support early literacy development. Classroom observations and semistructured interviews were gathered and qualitatively analyzed to identify key perspectives and classroom practices that lead to the educators' successful facilitation of play to support literacy. Results of this study showed how different core literacy skills can be supported through different types of play, with each offering unique and critical opportunities for learning. Results also demonstrated how a multitude of core literacy skills can be supported through guided approaches to play, and begin to illustrate how teachers are facilitating a continuum of guided play to support literacy learning.

Mitochondrial function is critical to continued cellular vitality and is an important contributor to a growing number of human diseases. Mitochondrial dysfunction is typically heterogeneous, mediated through the clonal expansion of mitochondrial DNA (mtDNA) variants in a subset of cells in a given tissue. To date, our understanding of the dynamics of clonal expansion of mtDNA variants has been technically limited to the single cell-level. Here, we report the use of nanobiopsy for subcellular sampling from human tissues, combined with next-generation sequencing to assess subcellular mtDNA mutation load in human tissue from mitochondrial disease patients. The ability to map mitochondrial mutation loads within individual cells of diseased tissue samples will further our understanding of mitochondrial genetic diseases.

Metachromatic leukodystrophy (MLD) is a rare - typically paediatric - sphingolipid storage disorder resulting from bi-allelic pathogenic variants in the ARSA gene, encoding the lysosomal arylsulphatase A (ASA). Heterozygous variants in ARSA are associated with risk of Lewy body diseases (LBD), a group of age-associated neurodegenerative disorders characterised by the accumulation of the protein alpha-synuclein; however, no study has yet determined whether alpha-synuclein with putative pathological features is observed in MLD brain tissue. We examined post-mortem brain tissue from MLD cases (N=5, age 2-33) compared to matched control cases using histological approaches and alpha-synuclein seeding amplification assay (SAA). Juvenile-onset MLD cases exhibited granular alpha-synuclein deposits in neurons of regions prone to neuronal pathology in MLD, and seed-competent conformers that generated atypical short, twisted fibrils on SAA. In contrast, infantile-onset MLD cases gave only variably positive reactions on SAA. In summary, this study suggests MLD cases manifest alpha-synuclein pathology reminiscent of that observed in LBD, even in juvenile populations, further expanding the spectrum of sphingolipid storage disorders associated with the aggregation of alpha-synuclein. These findings have important implications for understanding the disease process of both LBD and MLD, potentially highlighting novel pathways for therapeutic interventions in both conditions. ### Competing Interest Statement The authors have declared no competing interest.

The response to the COVID-19 pandemic impacted educational systems throughout the world. School closures, virtual schooling, and strict safety protocols for in-person learning changed instructional approaches to teaching. These changes were particularly disruptive in early childhood education which relies on play-based learning to support students' socioemotional and academic development. This study examines the impact of school disruptions on play-based learning during the pandemic and the lasting effect on classroom practices from the perspective of kindergarten educators in Ontario, Canada. An online survey was administered to 100 kindergarten educators with open-ended questions regarding how the pandemic shaped their implementation of play, challenges they faced during this time, and how their practices have changed in the years following the pandemic. Results indicated significant impacts on play-based learning due to changes to the physical space and materials available, decreases in student choice, limits to social interaction, and masking protocols. Post-pandemic, educators indicated some lasting effects on the implementation of play in their classrooms and noted concerns regarding students' social-emotional development and gaps in their academic skills. These findings provide insight into the continued influence of the pandemic on education and how educators are responding to these lasting impacts.

Neurodegeneration in Parkinson’s disease (PD) precedes diagnosis by years. Early neurodegeneration may be reflected in RNA levels and measurable as a biomarker. Here, we present the largest quantification of whole blood linear and circular RNAs (circRNA) in early-stage idiopathic PD, using RNA sequencing data from two cohorts (PPMI = 259 PD, 161 Controls; ICICLE-PD = 48 PD, 48 Controls). We identified a replicable increase in TMEM252 and LMNB1 gene expression in PD. We identified novel differences in the expression of circRNAs from ESYT2, BMS1P1 and CCDC9, and replicated trends of previously reported circRNAs. Overall, using circRNA as a diagnostic biomarker in PD did not show any clear improvement over linear RNA, minimising its potential clinical utility. More interestingly, we observed a general reduction in circRNA expression in both PD cohorts, accompanied by an increase in RNASEL expression. This imbalance implicates the activation of an innate antiviral immune response and suggests a previously unknown aspect of circRNA regulation in PD.

Abstract Pathogenic variants in either the mitochondrial or nuclear genomes are associated with a diverse group of human disorders characterized by impaired mitochondrial function. Within this group, an increasing number of families have been identified, where Mendelian genetic disorders implicate defective mitochondrial RNA biology. The PDE12 gene encodes the poly(A)-specific exoribonuclease, involved in the quality control of mitochondrial non-coding RNAs. Here, we report that disease-causing PDE12 variants in three unrelated families are associated with mitochondrial respiratory chain deficiencies and wide-ranging clinical presentations in utero and within the neonatal period, with muscle and brain involvement leading to marked cytochrome c oxidase (COX) deficiency in muscle and severe lactic acidosis. Whole exome sequencing of affected probands revealed novel, segregating bi-allelic missense PDE12 variants affecting conserved residues. Patient-derived primary fibroblasts demonstrate diminished steady-state levels of PDE12 protein, whilst mitochondrial poly(A)-tail RNA sequencing (MPAT-Seq) revealed an accumulation of spuriously polyadenylated mitochondrial RNA, consistent with perturbed function of PDE12 protein. Our data suggest that PDE12 regulates mitochondrial RNA processing and its loss results in neurological and muscular phenotypes.

BackgroundEvidence from the science of learning suggests that playful learning pedagogical approaches exist along a spectrum and can support student learning. Leveraging active engagement, iterative, socially interactive, meaningful, and joyful interactions with content also supports student learning. Translating these concepts into guidance and support for teachers is lacking.MethodWe introduce a tool designed to support teachers in implementing across the facilitation spectrum and leverage the characteristics that help children learn. Across three international contexts, we engaged with 1207 teachers and the tool was used 4,911 times.ResultsStudent age, the intended learning goal, and context influenced teachers’ use of the tool, suggesting that contextualization is critical, even when basing programs on evidence-based, universal principles given by the science of learning.ConclusionScience of learning research must be effectively translated but we must use evidence from teachers and real-life classrooms to inform those studying the science of learning.

Playful learning has seen a resurgence of interest in the past decade, particularly in contexts where play is not traditionally part of a teacher’s repertoire. Teachers interested in exploring the integration of play in their classrooms need formative tools and resources that help them to reflect and assess their own practice and their ability to create a playful learning experience for their students. This study presents the results of two rounds of pilot testing in three countries for Teacher RePlay, a new open-source toolkit designed to support teachers interested in reflecting on and deepening their learning through play practice. The toolkit includes the main Teacher RePlay observation protocol for teachers, as well as Children ReAct, a complementary protocol for a photo-elicited focus group discussion with children, intended to directly assess children’s experiences and reflections on learning through play. Upon observation, teachers receive customized coaching suggestions and tips designed to strengthen their learning through play practice. Initial results from the piloting indicate that the toolkit holds strong potential for teachers interested in better understanding and deepening their playful learning practice. This paper discusses the development, validation, successes, and challenges of the Teacher RePlay toolkit, and identifies future directions for its use.

Mitochondrial disorders exhibit clinical and genetic diversity. Nearly 400 distinct genes, located in both the mitochondrial and nuclear genomes, harbor pathogenic variants that can produce a broad spectrum of mitochondrial diseases. This work aims to explore the genetic etiology of a cohort of Egyptian pediatric patients who were clinically suspected of having a mitochondrial disorder. A total of 49 patients from 44 unrelated families were studied. Selection criteria included age below 18 years and meeting Morava criteria (a score ≥ 3). The mitochondrial disease criteria (MDC) have been developed to quantify the clinical picture and evaluate the probability of an underlying mitochondrial disorder Exome sequencing, including mitochondrial genome sequencing, was carried out for each participant. Causative variants likely responsible for the phenotypes were identified in 68% of the study population. The mitochondrial subgroup constituted 41% of the studied population with a median age of 4 years. No primary pathogenic variants in mitochondrial DNA were detected. Pathogenic or likely pathogenic variants in eight mitochondrial genes were identified in 78% of the mitochondrial cohort. Additionally, seven novel variants were identified. Nonmitochondrial diagnoses accounted for 27% of the study population. In 32% of cases, disease-causing variants were not identified. The current study underscores the diverse phenotypic and genetic landscape of mitochondrial disorders among Egyptian patients.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder typically resulting from biallelic loss-of-function variants in the ARSA gene which encodes the lysosomal enzyme, arylsulphatase A, leading to the accumulation of its substrate, sulphatide, and widespread demyelination. Although gene therapy is available for MLD, it is limited by high cost and a narrow window for intervention, which means the development of therapies for MLD remains a key goal. The aim of the present study was to explore disease mechanisms in MLD with a view to identifying novel targets for therapeutic intervention for patients who cannot avail of gene therapy. Postmortem globus pallidus and dentate nucleus tissue was obtained from MLD cases (N=5; age 2-33 years old) and compared to age-, sex and ethnicity matched controls (N=5) and studied using discovery proteomics which demonstrated a marked inflammatory response, activation of the mTOR pathway, oxidative stress and metabolic remodelling in MLD cases. Histological analysis of inflammatory markers, including the terminal fragment of complement pathway activation, C3d, and the secreted glycoprotein YKL-40, a commonly used biomarker for inflammation, demonstrated their enrichment in MLD cases. Given that the mTOR pathway plays a key role in supressing autophagy, we next investigated autophagy and identified the accumulation of autophagosomes in MLD cases, consistent with deficient autophagy. Taken together, these findings suggest inflammation and autophagy dysfunction are key processes involved in MLD and that the mTOR pathway could be a novel therapeutic target for MLD.

Play occupies a critical role in the kindergarten classroom, and the expansion of formal play-based learning programmes have brought connections between play and learning to the forefront. With respect to social and emotional learning (SEL), child-directed play has been viewed as critical, while teacher direction has been framed as a potential disruption to this learning. However, teachers facilitate different types of play in kindergarten with varying degrees of assistance, yet few studies have looked at the ways teachers target children's SEL across different play configurations. The current study gathered observational data in 20 play-based kindergarten classrooms. Video data were coded according to the type of play (child-directed or teacher-facilitated), SEL competency, and type of teacher promotion. Across both types of play, teachers focused primarily on supporting children's relationship skills and self-management skills in an incidental way, while other competencies not practiced spontaneously in play largely went overlooked. Skills were targeted most often during teacher-facilitated play, underscoring the importance of this context for children's SEL. Overall, these findings illustrate positive examples of teacher promotion of SEL in play, while underscoring the need for more intentional targeting of broader competencies to promote all areas of children's SEL in a developmentally appropriate manner.