The Healthy Lifestyles Programme (HeLP), a novel school-based intervention to prevent obesity in school children: study protocol for a randomised controlled trial

Background: Over the last three decades there has been a substantial increase in the proportion of children who are overweight or obese. The Healthy Lifestyles Programme (HeLP) is a novel school-based intervention, using highly interactive and creative delivery methods to prevent obesity in children. Methods/Design: We describe a cluster randomised controlled trial to evaluate the effectiveness and cost effectiveness of HeLP. The intervention has been developed using intervention mapping (involving extensive stakeholder involvement) and has been guided by the Information, Motivation, Behavioural Skills model. HeLP includes creating a receptive environment, drama activities, goal setting and reinforcement activities and runs over three school terms. Piloting showed that 9 to 10 year olds were the most receptive and participative. This study aims to recruit 1,300 children from 32 schools (over half of which will have ≥19% of pupils eligible for free school meals) from the southwest of England. Participating schools will be randomised to intervention or control groups with baseline measures taken prior to randomisation. The primary outcome is change in body mass index standard deviation score (BMI SDS) at 24 months post baseline. Secondary outcomes include, waist circumference and percent body fat SDS and proportion of children classified as overweight or obese at 18 and 24 months and objectively measured physical activity and food intake at 18 months. Between-group comparisons will be made using random effects regression analysis taking into account the hierarchical nature of the study design. An economic evaluation will estimate the incremental cost-effectiveness of HeLP, compared to control, from the perspective of the National Health Service (NHS)/third party payer. An in-depth process evaluation will provide insight into how HeLP works, and whether there is any differential uptake or engagement with the programme. Discussion: The results of the trial will provide evidence on the effectiveness and cost effectiveness of the Healthy Lifestyles Programme in affecting the weight status of children

Imaging nanoscale nuclear structures with expansion microscopy

Commonly applied super-resolution light microscopies have provided insight into subcellular processes at the nanoscale. However, imaging depth, speed, throughput and cost remain significant challenges, limiting the numbers of three-dimensional (3D) nanoscale processes that can be investigated and the number of laboratories able to undertake such analysis. Expansion microscopy (ExM) solves many of these limitations, but its application to imaging nuclear processes has been constrained by concerns of unequal nuclear expansion. Here, we demonstrate the conditions for isotropic expansion of the nucleus at a resolution equal to or better than 120–130 nm (pre-expansion). Using the DNA damage response proteins BRCA1, 53BP1 (also known as TP53BP1) and RAD51 as exemplars, we quantitatively describe the 3D nanoscale organisation of over 50,000 DNA damage response structures. We demonstrate the ability to assess chromatin-regulated events and show the simultaneous assessment of four elements. This study thus demonstrates how ExM can contribute to the investigation of nanoscale nuclear processes

Imaging nanoscale nuclear structures with expansion microscopy

Commonly applied super-resolution light microscopies have provided insight into subcellular processes at the nanoscale. However, imaging depth, speed, throughput and cost remain significant challenges, limiting the numbers of three-dimensional (3D) nanoscale processes that can be investigated and the number of laboratories able to undertake such analysis. Expansion microscopy (ExM) solves many of these limitations, but its application to imaging nuclear processes has been constrained by concerns of unequal nuclear expansion. Here, we demonstrate the conditions for isotropic expansion of the nucleus at a resolution equal to or better than 120–130 nm (pre-expansion). Using the DNA damage response proteins BRCA1, 53BP1 (also known as TP53BP1) and RAD51 as exemplars, we quantitatively describe the 3D nanoscale organisation of over 50,000 DNA damage response structures. We demonstrate the ability to assess chromatin-regulated events and show the simultaneous assessment of four elements. This study thus demonstrates how ExM can contribute to the investigation of nanoscale nuclear processes

Disruption Of PML Nuclear Bodies Cooperates In The Pathogenesis Of Acute Promyelocytic Leukemia

Abstract Acute promyelocytic leukemia (APL) is characterised by the t(15;17)(q22;q21) leading to fusion of PML to the gene encoding the myeloid transcription factor Retinoic Acid Receptor α (RARα). Chromosomal translocations such as the t(15;17) are considered to be initiating events in leukemogenesis; however, sequencing of APL genomes has provided further evidence that the PML-RARα fusion is insufficient to induce leukemia, which depends upon the acquisition of cooperating mutations. The PML-RARα oncoprotein exerts a profound effect on nuclear architecture, disrupting multiprotein structures known as PML nuclear bodies (NBs). The function of these structures remains an enigma; however, their disruption in PML-RARα+ APL and acute lymphoblastic leukemia with the t(9;15)(p13;q24)/PAX5-PML fusion is associated with delocalisation of a number of component proteins including PML, which have been implicated in growth control and neoplastic transformation. It is now established that the PML moiety contributes to APL pathogenesis by conferring via the translocation a novel dimerisation capacity to RARα, but it has been unclear whether deregulation of PML and other NB components cooperates in leukemic transformation or impacts the response to differentiating agents. To address these questions, we generated a knock-in mouse model with targeted NB disruption achieved through mutation of key zinc-binding cysteine residues in the amino-terminal RING domain of Pml. Homozygous Pml RING mutant mice are viable, with no overt developmental defect; however, analysis of the bone marrow revealed significant expansion of the Lin(-)Sca-1(+)c-Kit(+) (LSK) population compared to wild type (WT) controls (p<0.01), accompanied by increased LSK cell proliferation (p<0.0001) as evaluated by in vivo labelling through incorporation of 5-ethynyl-2'-deoxyuridine (EdU). In addition, hematopoietic cells derived from homozygous Pml RING mutant mice exhibited markedly elevated levels of DNA damage compared to WT cells from age-matched controls, as evidenced by increased numbers of γH2AX foci (p=0.009). This was associated with significantly delayed DNA damage repair responses in Pml RING mutant cells following γ-irradiation (p=0.005). Accordingly, expression of PML-RARα in human hematopoietic cells, which led to disruption of NBs, also induced a significant increase in γH2AX foci (p=0.0023). While no leukemias arose in homozygous Pml RING mutant mice, they developed an excess of T- and B-cell lymphomas (p=0.03), consistent with the proposed tumour suppressor function of PML and the NBs. Since a key property conferred by the PML moiety required for leukemogenicity of the PML-RARα oncoprotein is the capacity to dimerise, we evaluated whether Pml NB disruption could cooperate with forced RARα homodimerisation (mediated artificially by linking RARα to the p50 dimerisation motif of NFκB). While Pml NB disruption or p50-RARA expressed under the control of the MRP8 promoter in murine hematopoietic stem/progenitor cells conferred limited replating capacity, in combination they exhibited marked cooperativity, with a significant increase in third round colonies (p=0.03). Moreover, NB disruption was found to cooperate with forced RARα homodimerisation in vivo with a doubling in the rate of leukemia development in p50-RARα mice with mutated Pml (p<0.0001), leading to a penetrance comparable to that observed in previously published PML-RARα transgenic models. Moreover, the latency to onset of leukemia was significantly shorter in p50-RARα mice with the Pml RING mutation, occurring from 213 days of age vs 310 days with WT Pml (p=0.008). While Pml NB disruption did not affect engraftment of p50-RARα leukemias in serial transplantation, the in vitro differentiation response of p50-RARα leukemias to All transretinoic acid (ATRA) as determined by nitroblue tetrazolium assay was significantly impaired in the context of NB disruption (p<0.05). Moreover, prolongation of survival following ATRA treatment in mice transplanted with p50-RARα leukemic blasts was dependent upon Pml NB integrity (p=0.03). Overall, these data suggest that the NB disruption mediated by the PML-RARα oncoprotein plays a key role in APL pathogenesis contributing to expansion of the LSK population and defective DNA repair predisposing to the acquisition of cooperating mutations, but also implicate NBs in the response to differentiating agents. Disclosures: No relevant conflicts of interest to declare

Disruption Of PML Nuclear Bodies Cooperates In The Pathogenesis Of Acute Promyelocytic Leukemia

Acute promyelocytic leukemia (APL) is characterised by the t(15;17)(q22;q21) leading to fusion of PML to the gene encoding the myeloid transcription factor Retinoic Acid Receptor α (RARα). Chromosomal translocations such as the t(15;17) are considered to be initiating events in leukemogenesis; however, sequencing of APL genomes has provided further evidence that the PML-RARα fusion is insufficient to induce leukemia, which depends upon the acquisition of cooperating mutations. The PML-RARα oncoprotein exerts a profound effect on nuclear architecture, disrupting multiprotein structures known as PML nuclear bodies (NBs). The function of these structures remains an enigma; however, their disruption in PML-RARα+ APL and acute lymphoblastic leukemia with the t(9;15)(p13;q24)/PAX5-PML fusion is associated with delocalisation of a number of component proteins including PML, which have been implicated in growth control and neoplastic transformation. It is now established that the PML moiety contributes to APL pathogenesis by conferring via the translocation a novel dimerisation capacity to RARα, but it has been unclear whether deregulation of PML and other NB components cooperates in leukemic transformation or impacts the response to differentiating agents. To address these questions, we generated a knock-in mouse model with targeted NB disruption achieved through mutation of key zinc-binding cysteine residues in the amino-terminal RING domain of Pml. Homozygous Pml RING mutant mice are viable, with no overt developmental defect; however, analysis of the bone marrow revealed significant expansion of the Lin(-)Sca-1(+)c-Kit(+) (LSK) population compared to wild type (WT) controls (p<0.01), accompanied by increased LSK cell proliferation (p<0.0001) as evaluated by in vivo labelling through incorporation of 5-ethynyl-2'-deoxyuridine (EdU). In addition, hematopoietic cells derived from homozygous Pml RING mutant mice exhibited markedly elevated levels of DNA damage compared to WT cells from age-matched controls, as evidenced by increased numbers of γH2AX foci (p=0.009). This was associated with significantly delayed DNA damage repair responses in Pml RING mutant cells following γ-irradiation (p=0.005). Accordingly, expression of PML-RARα in human hematopoietic cells, which led to disruption of NBs, also induced a significant increase in γH2AX foci (p=0.0023). While no leukemias arose in homozygous Pml RING mutant mice, they developed an excess of T- and B-cell lymphomas (p=0.03), consistent with the proposed tumour suppressor function of PML and the NBs. Since a key property conferred by the PML moiety required for leukemogenicity of the PML-RARα oncoprotein is the capacity to dimerise, we evaluated whether Pml NB disruption could cooperate with forced RARα homodimerisation (mediated artificially by linking RARα to the p50 dimerisation motif of NFκB). While Pml NB disruption or p50-RARA expressed under the control of the MRP8 promoter in murine hematopoietic stem/progenitor cells conferred limited replating capacity, in combination they exhibited marked cooperativity, with a significant increase in third round colonies (p=0.03). Moreover, NB disruption was found to cooperate with forced RARα homodimerisation in vivo with a doubling in the rate of leukemia development in p50-RARα mice with mutated Pml (p<0.0001), leading to a penetrance comparable to that observed in previously published PML-RARα transgenic models. Moreover, the latency to onset of leukemia was significantly shorter in p50-RARα mice with the Pml RING mutation, occurring from 213 days of age vs 310 days with WT Pml (p=0.008). While Pml NB disruption did not affect engraftment of p50-RARα leukemias in serial transplantation, the in vitro differentiation response of p50-RARα leukemias to All transretinoic acid (ATRA) as determined by nitroblue tetrazolium assay was significantly impaired in the context of NB disruption (p<0.05). Moreover, prolongation of survival following ATRA treatment in mice transplanted with p50-RARα leukemic blasts was dependent upon Pml NB integrity (p=0.03). Overall, these data suggest that the NB disruption mediated by the PML-RARα oncoprotein plays a key role in APL pathogenesis contributing to expansion of the LSK population and defective DNA repair predisposing to the acquisition of cooperating mutations, but also implicate NBs in the response to differentiating agents