Furthermore, targeting the RNA4.9 promoter with CRISPR-Cas9 or genetic relocalization of oriLyt leads to reduced levels of the viral single-stranded DNA-binding protein (ssDBP), suggesting that the levels of ssDBP are coupled to the oriLyt activity. We further identified a similar, oriLyt-embedded, G+C-rich lncRNA in murine cytomegalovirus (MCMV). These results indicate that HCMV RNA4.9 plays an important role in regulating viral DNA replication, that the levels of ssDBP are coupled to the oriLyt activity, and that these regulatory features may be conserved among betaherpesviruses.Viruses are known for their extremely compact genomes composed almost entirely of protein-coding genes. Nonetheless, four long noncoding RNAs (lncRNAs) are encoded by human cytomegalovirus (HCMV). Although these RNAs accumulate to high levels during lytic infection, their functions remain largely unknown. Here, we show that HCMV-encoded lncRNA4.9 localizes to the viral nuclear replication compartment, and that its depletion restricts viral DNA replication and viral growth. RNA4.9 is transcribed from the HCMV origin of replication . In this study, we show that one of human cytomegalovirus (HCMV) encoded lncRNAs, named RNA4.9, is important for viral DNA replication and viral propagation. RNA4.9 is embedded in the viral origin of replication and its transcription causes the formation of a RNA-DNA hybrid, a structure which is likely important for the viral origin of replication unwinding and initiation of viral DNA replication. Furthermore, interfering with viral origin of replication or with RNA4.9 promoter activities leads to reduced levels of the viral single-stranded DNA-binding protein (ssDBP), suggesting that the ssDBP levels are coupled to the origin activity. Finally, we discovered a new lncRNA encoded by the murine cytomegalovirus, which seems to have similar features and function as the HCMV encoded RNA4.9. These results suggest a novel mechanism, conserved among betaherpesviruses, by which a viral lncRNA, embedded in the viral origin of replication, regulates viral DNA replication and may play a role in coupling origin activity with the level of ssDBP. I have sent your manuscript to three experts in the Herpesvirus field who all have experience with Viral DNA replication. All three reviewers viewed your work as very important and appreciated the use of highly innovative tools to answer critical questions about HCMV replication. Their views were wide ranging on the acceptance of the manuscript and two reviewers  had significant issues with the interpretation and presentation of the data which undermined their support of the manuscript. I however find the manuscript of high value and am inclined to allow you to read the comments and see if you think they are addressable in a reasonable time frame. As such, I rendered a decision of Major Revision. If you are able to address the concerns of Reviewer #s 2 & 3, I would be inclined to send it back to them for review. While this is not the decision you were hoping for, do understand that if you are able to accommodate those two reviewers, I will be highly supportive of the work.Cheers,Eain MurphyReviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #1: Upon characterizing the subcellular distribution of the four major lncRNAs encoded by HCMV, Tai-Schmiedel et al commence this study by showing that RNA1.2, RNA2.7 and RNA5.0 accumulate in the cytoplasm of infected cells while RNA 4.9 largely accumulates within nuclear viral replication compartments. Using Crispr-cas9 and CRISPRi methods, the authors create the first hypomorphic functional allele for RNA4.9 that maintains the critical integrity of oriLyt, elegantly overcoming a significant impediment that has prevented the investigation of RNA4.9 function during HCMV reproduction. They conclusively show that interfering with RNA4.9 accumulation directly inhibits viral DNA synthesis and reduces virus productive growth. Furthermore, effects of RNA4.9 on virus gene expression were shown to be secondary to effects on DNA synthesis. Using DNA-RNA hybrid immunoprecipitation, the authors provide evidence that GC rich regions specifically from the RNA4.9 RNA are enriched in these structures. Finally, they present evidence that RNA4.9 post-transcriptionally controls levels of the HCMV UL57 ssDBP and that ectopic expression of ssDBP restores replication of recombinant viruses containing RNA4.9 hypomorphic alleles. A similar RNA4.9 counterpart is identified in MCMV and shown to regulate viral DNA replication.The work is significant as it is the first to establish biological function for the HCMV lncRNA4.9 in viral DNA synthesis and characterize the phenotype of RNA4.9 hypomorphic alleles in the productive growth cycle. It uses creative cutting edge genome editing methods to overcome a key obstacle limiting genetic analysis in large viral genomes that encode overlapping cis and trans-acting functions within discrete, compact regions and creates the first hypomorphic allele of lncRNA4.9 that doesn't incidentally damage orilyt. Besides this creative engineering strategy, the experiments are well executed, the data are rigorous and compelling and the manuscript is well written. The work is of broad interest to many investigators studying the many roles of non coding RNAs in infection biology and seeking to engineer mutations in overlapping genes without damaging critical cis-acting regulatory elements. I have no substantial, essential comments that would significantly improve this already strong manuscript.Reviewer #2: The functions of the four long noncoding RNAs encoded by human cytomegalovirus are not well-characterized. This manuscript investigates the function of lncRNA4.9. The authors observe localization in the viral nuclear replication compartment during HCMV infection of fibroblasts. They apply a loss-of-function approach using gene editing in the lncRNA4.9 region. By this approach, they uncover an impact on viral DNA replication and the level of the viral ssDNA binding protein. A parallel approach in the MCMV system led to similar outcomes. The authors provide a model whereby the lncRNA4.9 binds the oriLyt to form an RNA-DNA hybrid that impacts the initiation of DNA replication and reduces ssDNA binding protein levels. However, the experimental approach used to edit the lncRNA4.9 will lead to dsDNA breaks that make the impact on oriLyt function and neighboring transcription very difficult to parse.Reviewer #3: The manuscript by Tai-Schmiedel at al. “Human cytomegalovirus long noncoding RNA4.9 regulated viral DNA replication” is an impressive paper. Many of their experiments answer some of the basic questions regarding the expression and localization of the viral lncRNAs. This is an important area of research given the abundance of the viral lncRNA and how little is known about the functions during viral replication. The identification of a RNA4.9 homologue in MCMV is an interesting discovery and extends the work to a possible mechanism used by other beta herpesviruses. There are concerns regarding the approach to KD RNA4.9 and the interpretation of the role of RNA4.9 in DNA replication given its overlap with oriLyt, and how UL57 (ssDBP) is regulated by RNA4.9. These concerns damp some of the enthusiasm for the manuscript. Specific questions are listed below.**********Part II – Major Issues: Key Experiments Required for Acceptanceabsolutely required to validate study conclusions.Please use this section to detail the key new experiments or modifications of existing experiments that should be Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".Reviewer #1: none.Reviewer #2: Major concern1. Their approach to knock-down the gene of interest using WT Cas9 will lead to complex and varied mutations and possibly indirect host DNA damage responses.The authors have used gene-editing with wt Cas9 to generate dsDNA breaks in the CMV genomes. This is not an appropriate way to transiently knock-down a specific gene of interest in the context of infection (why not use siRNA?). This type of approach is especially troublesome in the area of the genome that houses both the origin of lytic replication and the regulatory region of the DNA replication proteins that is focused on. A break in this area can cause non-specific host DNA damage responses, loss-of-function of the origin and disruption of the normal architecture of the genome that mediates transcription of genes in that area. Given the complexity of this region and the lack of characterization of the ensuing genome edits, it is impossible to interpret the downstream effects. The use of multiple gRNAs and seeing disparate effects merely indicates the efficiency of edits and damaged genomes differs. Their control gRNA will likely not cut at all- they might want to target an innocuous region (this is obviously hard to do). They do not sequence the edited genomes. Cas9 systems lead to a spectrum of mutations. Conclusions about the direct effect of loss of the lncRNA4.9 can not be made using this approach.If the authors wish to use this method to edit the virus, they might purify and characterize the mutants prior to use in infection experiments to better ensure that only lncRNA4.9 is directly impacted. This will be very difficult to do. More refined mutations such as disruption of initiation or insertion of a transcriptional stop would seem much cleaner.The use of a mutant Cas9 that impairs transcription initiation is a more reasonable approach and there is one supplemental figure demonstrating an impairment in lncRNA expression and replication. How does the alteration of transcription in this region impact the ‘firing’ of the lytic origins? Rescue by ectopic lncRNA4.9 expression should be demonstrated.2. Experimental reproducibility and rigor throughout the paper.In general, the MOI varies within each figure and across nearly all figures in the paper. This makes integration of RNA, DNA, Protein, and IFA data very difficult. Specific concerns with each figure are described below.Figure 1.1A appears to be previously published data. What was the cell type and MOI? How do these levels relate to coding transcripts typical of IE, E, L.1B. What is the nuclear to cytoplasmic ratio? Why not show dual color as in 1C. Is there a software-based enumeration of this data so we can learn from hundreds of cells, not single cell staining?1B-1C. Where is the mock control? MOI5.0 vs MOI1.0 in 1B. There is no direct demonstration of viral DNA replication compartmentsFigure 2.2A. There seem to be disparities in the impact on RNA, DNA, virus production between sgRNA3 and 5. The impact on gene expression seems to be greater with sgRNA3 at 24 hpi and with sgRNA5 at 48 hpi. Why was sgRNA 3 used moving forward?Figure 3.3A. This was based on one experiment. Why was a low MOI used for this? Why weren’t more genes impacted.3B. The normalization of this data is really difficult to understand. There is no data regarding the oriLyt.Figure 4.A and b. 24 hpi is shown without PFA and the 48 hr timepoint is with PFA. This is a strange comparison. PFA abolishes viral replication compartments, does this change the nuclear localization of RNA4.9?C. Isn’t it expected that PFA or anything that abrogates lytic replication will reduce viral gene expression?D. no error barsE. This should be provided with the exact coordinates of the sgRNAs used earlier in the manuscript.Figure 5.How many experimental replicates does this represent?It is difficult to compare transcripts to protein. Such correlations are not very valuable and no viral DNA replication data is provided for reference. How is the quantitation of transcripts and proteins from an overexpressed gene informative? If PFA is used, what happens to ssDNADBP?Figure 6.Different sgRNAs in the lncRNA4.9 region have differential editing of the genome that leads to dsDNA breaks. This does not show a direct effect of one transcript to another.Figure 7.7D. This is an unusual mutant. Does the previous publication describe a replication defect at this MOI?7F. Does ssDNA overexpression increase DNA copy number?F and G. What was the statistical test used? The impact of ssDBP is very slight.B-F, if you overexpress full-length lncRNA4.9, do you rescue ssDBP, replication and infectious virus production back to WT levels?Figure 8.A Where do the sgRNA bind and edit in MCMV? What are those edits?3. The model is not supported by the data.There are no experiments using proteasome inhibitors to support premise of ssDNABP instability. No data for direct interaction of lncRNA4.9 with the oriLyt. No data for impact on nascent DNA replication or unwinding at oriLyt.Reviewer #3: The timing of the onset of DNA replication and the expression of RNA4.9 needs to be clarified. DNA replication begins around 24 hrs post infection. The expression kinetics of RNA4.9 needs to be confirmed at earlier time points before DNA replication begins in order to determine the role of RNA4.9 in DNA synthesis. The question is, what is the level of RNA4.9 between 24 and 36 hrs? The relationship between the levels of RNA4.9 and amount of newly synthesized DNA resulting in an increase in template which could be used to produce more RNA4.9. This brings into question if the results are really due to a decrease in RNA4.9 or if it is disrupting the cis-elements within oriLyt which would result in a decrease in DNA replication and then a decrease in RNA4.9. The other issue with the kinetics of RNA4.9 expression is that some of the experiments were performed at 24 hrs  when they have not established that RNA4.9 is expressed at significant levels by 24 hpi. Fig. 1A extrapolates the levels of RNA4.9 from 24 to 72 hpi and it’s not clear if RNA4.9 is an early transcript or expressed after the onset of DNA synthesis. The knockdown of RNA4.9  is at 48 hpi, but that is already after the onset of DNA replication at 24-36 hpi.In regards to the CRISPR experiments , what was the percentage of HCMV genomes that carried the sgRNA induced RNA4.9 promoter mutation? The authors attempt to answer this question with the relative induced mutations shown in Figure S6 but it doesn’t give an absolute value to how many genomes have the mutation since it is a relative comparison between the specific gRNA to the control gRNA.This brings up another issue, the control elements within the origin and the RNA4.9 promoter. Promoters are often associated with activity of lytic origins, and the act of transcription is thought to help open the DNA for initiation of DNA synthesis or allow for assemble of the core enzymatic machinery. The SV40 promoter can functionally replace the HCMV oriLyt promoter, demonstrating that the act of transcription is an important process for oriLyt-dependent DNA replication. The RNA4.9 promoter may have additional functions beyond the expression of the RNA4.9 transcript, it may in fact be a control element within the origin for initiating. The authors have not ruled out the possibility that they are disrupting the function of the cis-elements in the origin. This is a very difficult issue to resolve but there is no evidence that the phenotype described is due to the disruption of the RNA4.9 promoter which causes a decrease in the expression of RNA4.9 or if the disruption of the RNA4.9 promoter is directly affecting the ability of the origin to function normally.The R-loop within the origin has previously been described. In addition to the RNA4.9 R-loop there are other RNAs that have been identified from the origin. This was first reported in Prichard et al. J Virol. 1998 Sep;72(9):6997-7004 “Identification of persistent RNA-DNA hybrid structures within the origin of replication of human cytomegalovirus”. Interestingly, the 5’ region of RNA4.9 is within the transient plasmid-based replication assay and maybe functional in that system as well. The viral protein UL84 was also shown to interact with the stem-loop structure at the 5’ region of the RNA-DNA hybrid in Colletti et al. J Virol. 2007 Jul;81(13):7077-85 “Human Cytomegalovirus UL84 Interacts with an RNA stem-loop sequence found within the RNA/DNA hybrid region of oriLyt”.Fig. 4 panel c and d. Based on the western blot it seems like there might also be a reduction in the amount of UL84 and UL44 protein in cells treated with the sgRNA3 and not a specific reduction in the ssDBP levels. The graph in panel d shows the ssDBP relative to GAPDH, what are the levels of UL84 and UL44 in the same graph. Additionally, with panel f what are the relative levels of early transcripts UL84 and UL44 compared to UL57?Fig. 6 and 7. In figure 6 there is a decrease in RNA4.9 from the specific sgRNA and a decrease in UL57 RNA, while in Figure 7 there decrease in RNA4.9 in the OriShift mutant but the expression of UL57 is not significantly different in the WT and OriShift mutant (panel e). This difference in the amount of UL57 transcript detected  is not adequately discussion as to why there is an inconsistency in the method used to reduce RNA4.9 and the levels of UL57 that are reported.Figure S2. The other compounding factor is if the CRISPRi protein, which binds to DNA to block transcription without the nuclease activity, could block the ability of the viral DNA initiation protein in a similar manner, and if that’s true then the results could be due to the blocking IE2 or UL84 at the origin in addition to blocking transcription.**********Part III – Minor Issues: Editorial and Data Presentation ModificationsPlease use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.Reviewer #1: 1) It would be helpful to include a second guide RNA (ie:sgRNA5) in at least panel F of figure 42) Does PFA treatment limit accumulation of ectopically expressed ssDBP flag in HCMV infected cells? This would be predicted to occur and is simple to test.Editorial comments to address in the text:Line 199-200 revise to state,” We next tested the possibility that RNA4.9 KD affects ssDBP stability perhaps as an outcome of faulty unwinding of oriLyt. “Line 205, replace “started” with “first observed”Line 207-209: As written, this is somewhat confusing, whereas it is very clear in the figure 9 legend. I think the authors are combining too much in a single sentence and as a result may make it harder for the generalist reader to separate experimental findings from more interpretative speculation.Figure 5 and S5 legends: the authors measure DNA accumulation, which of course reflects DNA replication. The term “initiation”, however, reflects a discrete phase of the replication process that is inferred by the authors, and while likely correct, has not been demonstrated experimentally. Perhaps the term “initiation” could be removed from the legend and retained in the text in the speculative manner in which it is already used by the authors.Reviewer #2: (No Response)Reviewer #3: Minor points:Page 4 line 66-68: In regards to the reports of viral lncRNAs, Noriega et al. J Virol. 2014 Aug;88(16):9391-405 “Human cytomegalovirus modulates monocyte-mediated innate immune responses during short-term experimental latency in vitro” has also identified HCMV lncRNAs during latency.**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article : 30 Mar 2020Dear Dr. Stern-Ginossar,We are delighted to inform you that your manuscript, "Human cytomegalovirus long noncoding RNA4.9 regulates viral DNA replication," has been formally accepted for publication in PLOS Pathogens.We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication.The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles  are generated on a different schedule and may not be made available as quickly.Soon after your final files are uploaded, the early version of your manuscript, if you opted to have an early version of your article, will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens. Best regards,Kasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064


Implantable cardioverter-defibrillator (ICD) lead perforation is a rare but serious complication of cardiac device implantation. Subacute (24 h to one month) and delayed (>1 month) presentations of rupture are also rare. Here we report a case of right ventricular perforation by a ventricular ICD lead in a 61-year-old man that was detected four months’ postimplantation. The lead was present out from his chest wall and was palpable beneath the skin. However, subacute (24 h to 1 month) and delayed (> 1 month) presentations also occur. Given its thin wall relative to the rest of the right ventricle, the apex is typically the perforated location.2 Diagnosis is made via transthoracic echocardiogram and chest computed tomography (CT).3 Lead extraction is performed by either transvenous manual traction or a hybrid surgical approach, both of which are safe and effective.4 Pericar-diocentesis or a pericardial window is necessary for cardiac tamponade.4 Here, we report a case of right ventricular perforation by a ventricular ICD lead in a 61-year-old man that was detected four months after implantation.Implantable cardioverter-defibrillator (ICD) lead perforation is a rare but serious complication of cardiac device implantation with a mean incidence of 0.82%.. A CT chest scan was performed and indicated lead perforation with the tip extending through the right ventricular apex into the right anterior abdominal wall. The lead tip was observed in the subcutaneous tissues anterior to the costochondral junction and appeared to be less than 1 cm from the skin surface. There was no pericardial effusion or fluid collection surrounding the distal aspect of the lead and no evidence of pneumomediastinum or pneumothorax .A 61-year-old man with a past medical history of non-ischemic cardiomyopathy, a left ventricular ejection fraction of 35% to 40%, permanent atrial fibrillation while on anticoagulation, and a single-chamber ICD implanted four months earlier in a different state as a secondary prevention device presented to an outside hospital with syncope. A chest X-ray suggested misplacement of the ICD lead The patient was transferred to our facility for further management. Upon arrival, he was in no distress, with a heart rate of 86 bpm and a blood pressure rate of 132/ 100 mmHg. Physical examination was significant for a firm, pointed object along the fifth intercostal space at the midclavicular line. A transthoracic echocardiogram revealed the ICD lead in the right ventricle, with trace pericardial effusion.Interrogation of the present Inogen™ EL ICD  showed an impedance of 419 ohm and a pacing threshold of 20V@ 10 ms, as well as numerous device-labeled episodes of ventricular tachycardia and fibrillation, which were determined to be noise. The records from the facility where the device was implanted were not available for comparison. The patient was subsequently scheduled for device extraction in the operating room.. Following skin marking, the lead tip was cut, and the lead was manually pulled from the pocket under fluoroscopy. An intraoperative transesophageal echo-cardiogram demonstrated no pericardial effusion. A new ICD lead  was then implanted into the apical right ventricular septum. The patient had an uneventful postoperative course and was discharged two days after surgery.During extraction, the device generator was disconnected from the leads and explanted, the leads were dissected down to the tie-down sleeve, and fluoroscopy was performed to locate the protruding ICD lead on the chest wall. The lead location was then marked on the skin. Notably, the lead itself was actually moving in and out of the chest wall with each heartbeat


The Dose Adjustment For Normal Eating (DAFNE) intervention is a group education course designed to help adults with T1D develop and sustain the complex self-management skills needed to adjust insulin in everyday life. It leads to improved glucose levels in the short term , reduced rates of hypoglycaemia and sustained improvements in quality of life but overall glucose levels remain well above national targets. The DAFNEplus. Primary clinical outcome is the change in HbA1c and the primary endpoint is HbA1c at 12 months, in those entering the trial with HbA1c >7.5% (58 mmol/mol), and HbA1c at 6 months is the secondary endpoint. Sample size is 662 participants (approximately 47 per centre); 92% power to detect a 0.5% difference in the primary outcome of HbA1c between treatment groups. The trial also measures rates of hypoglycaemia, psychological outcomes, an economic evaluation and process evaluation.A pragmatic cluster randomised controlled trial in adults with T1D, delivered in diabetes centres in National Health Service secondary care hospitals in the UK. Centres will be randomised on a 1:1 basis to standard DAFNE or DAFNEEthics approval was granted by South West-Exeter Research Ethics Committee (REC ref: 18/SW/0100) on 14 May 2018. The results of the trial will be published in a National Institute for Health Research monograph and relevant high-impact journals.ISRCTN42908016. Comparison of group therapy against another group therapy will standardise the treatment comparison.Cluster randomisation to avoid contamination of the intervention material.Number of sites in both England and Scotland representing a wide range of National Health Service Trusts.Use of a covariate constrained methodology to randomise means that sites are matched which can create issues if sites drop out.Blinding not possible in trial due to the intervention and design.Type 1 diabetes (T1D) is characterised by absolute insulin deficiency, requiring insulin to be injected subcutaneously several times a day. Successful management requires those affected (>3 00 000 adults in the UK)Achieving the blood glucose control to help prevent complications depends on an individual’s ability to self-manage their condition, calculating precise insulin doses based on accurate estimations of food intake before every meal, frequent blood glucose measurements and account for fluctuations in physical activity, illness and hormones. If people with T1D are unable or unwilling to calculate and administer their insulin doses correctly, their blood glucose either runs high, increasing the risks of complications or else falls too low leading to hypoglycaemia. Hypoglycaemia, if severe, can result in acute cognitive impairment, confusion, collapse and injury, coma or even death.‘Dose Adjustment For Normal Eating’ (DAFNE) is a structured education programme run within the National Health Service (NHS), designed to enable adults with T1D to learn or enhance their self-management skills in flexible intensive insulin therapy to improve both glucose control and quality of life. It is a five-day training course, delivered in small groups. DAFNE has been delivered to over 51 000 adults in the UK.plus intervention has been developed through modifying the existing DAFNE programme by incorporating techniques for initiating and sustaining behaviour change, and supplementing this with structured follow-up support and enhanced information technology. The aim of this trial is to investigate whether the DAFNEplus programme will produce improved and sustainable diabetes self-management behaviour and better glucose outcomes than currently achieved with standard DAFNE, without compromising quality of life in the longer term.The DAFNEplus intervention to the existing DAFNE programme to answer the following question:The primary aim of this study is to conduct a cluster RCT comparing the new DAFNEIn adults with T1D, will modifying the existing DAFNE programme and developing structured professional input, using learning from our recent research, behavioural change theory and new forms of technological support, produce improved and sustained diabetes self-management behaviours, leading to better glucose control than currently achieved, using the existing DAFNE intervention, without compromising quality of life?The primary objective is to assess the effects of the intervention on glycaemic control, as measured by HbA1c at 12 months.The secondary objectives of this trial are:plus) to standard DAFNE on diabetes-specific quality of life.To compare the effects of the intervention (DAFNEplus) to standard DANFE on glycaemic control as measured by HbA1c using data at 6 months.To compare the medium term effect of the intervention (DAFNEplus) to standard DAFNE on other biomedical outcomes.To compare the effects of the intervention (DAFNEplus) to standard DAFNE on psychological outcomes.To compare the effects of the intervention .Diagnosis of T1D for at least 6 months, or posthoneymoon. The honeymoon period refers to the time when, postdiagnosis, people start taking insulin injections, and their insulin producing cells sometimes recover temporarily  .Serious diabetic complications . .Other serious comorbidities, for example, psychosis, diagnosed eating disorder. .Previous participation in standard DAFNE course less than five years before proposed study enrolment date.Unable to speak/hear/understand/read or write in English.Unable to give written informed consent.Patient participants will be identified from current caseloads of adults with T1D from each participating centre. They will be sent an invitation letter and information sheet before the course. A member of the clinical team in participating centres will then telephone potential participants to discuss whether or not they are interested in principle in taking part. If interested, they will be asked to consent to participate at their baseline visit. In both trial arms, if they do not want to take part in the research they will be offered attendance at a standard DAFNE course that is not part of this trial, if that is their wish. Reasons for non-participation in the trial will be recorded.In order to maximise recruitment to the courses, a reserve list of eligible patients will be held at participating centres. Eligible patients may also be invited to take part by their HCP during routine face-to-face appointments or via telephone. Trial information meetings may also be held during the recruitment period at various locations in centres.Written informed consent will be obtained from all participants. Members of the local study teams will be responsible for taking informed consent from potentially eligible study participants at the DAFNE centres. The process for obtaining participant informed consent will be in accordance with the REC guidance, and Good Clinical Practice (GCP) and any other regulatory requirements that might be introduced.Written informed consent to contribute to the process evaluation will also be taken from HCPs in participating sites by the central study team.DAFNE is a skill-based structured education programme for adults with T1D delivered in the NHS. Two evidence-based models of delivering standard DAFNE are in operation, whereby the five sessions of the course are delivered weekly or daily, as described elsewhere.The aim of the course is to train adults with T1D in the skills to manage their condition effectively. It covers numerous topics in a progressive modular based structure. In addition to the five days of the course, participants are asked to attend a baseline appointment before the DAFNE course, and they are also typically invited to attend an optional group follow-up session 6–8 weeks after the course. They may also attend routine appointments every 6–12 months and seek ad hoc support from local diabetes clinicians post-course.plus. All participants in the control arm will be given access to a stand-alone bolus calculator to assist them with calculating insulin doses. There will be no structured follow-up appointments beyond those provided in usual care. To qualify as adherent for statistical purposes, participants need to have attended the equivalent of 4 days of the course including days one and two which are mandatory; it will be acceptable to include half days in the total.For the purposes of this study, the control arm will be the 5-week model of standard DAFNE to match the frequency of sessions offered in DAFNEplus will be delivered by trained DAFNE educators in the NHS. In DAFNEplus, those delivering the intervention are referred to as ‘facilitators’, as opposed to ‘educators’ in standard DAFNE. These will be HCPs including diabetes specialist nurses, dietitians and physicians, all of whom will be using DAFNE principles as an integral part of the management of T1D in adults. DAFNEplus is a complex intervention, defined by the Medical Research CouncilDAFNEplus programme was informed by the Behaviour Change Wheel framework.plus programme , as well as a bolus calculator.The group-based course component of the DAFNEplus course include technology assisted individual review, emotional aspects of living with diabetes and its management, harnessing social support and behavioural change—including additional support for action planning and relapse prevention to help participants achieve their self-management goals. The curriculum was revised to be consistent with modern approaches to the recommended language used in diabetes care.New sessions included in the DAFNEplus principles to improve self-management and achieve/sustain glycaemic targets. As part of the trial, up to five one-to-one consultations  with a DAFNE facilitator will be offered, delivered at progressively wider spaced intervals during the 12 months after the course. Appointments are supported by paperwork to ‘activate’ both the participant and the facilitator prior to meeting.The model of structured follow-up support builds on the clinical and behavioural skills introduced during the course to enable participants to maximise the efficacy of key DAFNEplus website, revise course material, address any additional clinical needs and signpost participants to any relevant sources of support. In addition, ad hoc support by telephone, email or web-based video calling will be available, as necessary. To qualify as adherent for statistical purposes, participants will need to have attended a minimum of three follow-up sessions.The purpose of these individual sessions is to review participants’ progress with managing their diabetes, including progress with their action plans, review blood glucose data on the DAFNEplus programme incorporates two forms of digital technology via the DAFNEplus website and box. Participants will be given access and training at the pre-course appointment, so that they can use the technology before and throughout the 12-month programme. The DAFNEplus box (Withcare+) transmits, stores and displays blood glucose (and other) data on a secure-server via the DAFNEplus website in formats to help people with T1D and their HCPs recognise and interpret blood glucose patterns. The website also includes an e-learning section to help maintain knowledge of the DAFNEplus approach.The DAFNEplus facilitator training and supervision. The training programme is delivered over a maximum of five days and will build on the existing skill-set of DAFNE facilitators but also draw on additional behavioural science to deliver the revised curriculum.A clinical psychologist who specialises in diabetes and is experienced in training diabetes professionals in behavioural change skills will lead the development and delivery of DAFNEplus facilitator. Supervision will comprise weekly teleconferences before and during the first DAFNEplus course, weekly email supervision (for subsequent courses) and ad hoc remote support to allow issues that arise to be addressed in a timely manner during the trial.Throughout the trial, facilitators in each centre will be offered supervision by the clinical psychologist and a DAFNEThe decision regarding participation in the study is entirely voluntary, and consent regarding study participation may be withdrawn at any time without affecting the quality or quantity of future medical care. No study-specific interventions will be undertaken before informed consent has been obtained.A participant will be classed as complete if they have continued in the study until the last protocol defined intervention , although there may be missing data for individual participants.On recruitment of centres and following ethical approval, the participating centres will be randomised on a 1:1 basis to control or the intervention arm of the trial by the trial statistician. As there are numerous stratification variables that have been identified as clinically important and the small number of randomising centres, a covariate constrained methodologyDue to the nature of the intervention, it is not possible for members of the study team working directly with participants or the intervention to be blinded. Additionally, the blinding of the statistician is problematic due to the cluster level randomisation. Statisticians are usually involved within Trial Management Group (TMG) discussions and have access to status reports where the potential for unintentional unblinding is a high possibility. It is considered important for the statistician to be included in these aspects of the trial management and so after discussion with senior statisticians at the Clinical Trials Research Unit (CTRU) and the independent statistician on the Trial Steering Committee (TSC), it has been deemed acceptable that the statisticians are not blind within this study.10.1136/bmjopen-2020-040438.supp4Supplementary dataThe primary biomedical outcome is an integrated measure of glucose levels over the previous 4–6 weeks, defined by HbA1c . The primary endpoint is HbA1c at 12 months, in those entering the trial with HbA1c >7.5% (58 mmol/mol), and HbA1c at 6 months is the secondary endpoint.Our primary aim is to compare HbA1c between the two arms and we have therefore confined our primary analysis to those with raised A1c values at baseline. We, therefore, excluded those with an HbA1c below 7.5% (58 mmol/mol) when calculating the primary endpoint as these people have less need to reduce their HbA1c.However, we have included participants with lower A1c values to ensure we can calculate important secondary outcomes part rates of hypoglycaemia, and other biomedical and psychological outcomes. We have estimated the expected proportion of participants with A1c values above 7.5% at 75% of those currently undertaking DAFNE courses based on a national research database.Other secondary outcomes are the number of participants achieving either an HbA1c <7.5% (58 mmol/mol) or a decrease in HbA1c of ≥0.5% (≥5.5 mmol/mol) which will be calculated at both 6 and 12 months post course. These cut-off points are recognised throughout the diabetes research community as being clinically relevant.Other secondary biomedical outcomes will include: Severe hypoglycaemia, as defined by the American Diabetes Association,Psychological outcomes and process measures will be collected via self-completed postal or online questionnaires at baseline, course completion, 3, 6, 9 and 12 months see .The primary psychological outcome is the impact of diabetes on quality of life assessed at 12 months using a 15-domain version of the Audit-Dependent Diabetes Quality of Life Questionnaire.plus intervention development and possible treatment mechanisms.Additional psychological constructs are assessed with validated questionnaires and study-specific individual items, based on: existing knowledge about their association with the trial’s primary biomedical outcome (HbA1c) and primary psychological outcome , including the findings of the YourSAY surveyplus in routine care.Interviews will be undertaken with a subset of participants randomised to the intervention at baseline, course completion, 3 months and 12 months  to exploplus manuals is delivered as intended: self-report checklists completed by educators/facilitators, and objectively analysed delivery from session audio recordings. Fidelity of delivery will be assessed in standard DAFNE as well as DAFNEplus in order to assess any loss of treatment differentiation and potential contamination between the two arms.We will explore fidelity of delivery using two methods to assess the extent to which the intervention content specified in the DAFNE/DAFNESelf-report checklists: Facilitators will complete checklists after each session. Each checklist lists the components intended to be delivered in each session . These components correspond to different BCTs. Each component will be rated as fully, partially or not delivered, with space for additional comments. The proportion of intended components rated as partially/fully delivered by educators/facilitators will be calculated, with <50% of intended content delivered classified as low fidelity; 51%–79% as moderate fidelity, and 80%–100%’ as high fidelity.Objectively analysed delivery: A subsample of group course sessions in both arms will be audio-recorded and transcribed verbatim. Transcripts will be coded into component BCTs using an established taxonomy.Detailed plans for the process evaluation are in 10.1136/bmjopen-2020-040438.supp2Supplementary dataplus website will be used to cost the intervention. The analysis population for the health economic analyses will include all trial participants, as it is important that the analysis of health economic data includes all participants who would be eligible to receive DAFNEplus (if it were to be implemented). In line with the statistical analysis, we will conduct subgroup analyses in participants with an HbA1c ≤7.5% and >7.5% (58 mmol/mol).plus versus DAFNE to standard National Institute for Health and Care Excellence thresholds to determine cost-effectiveness.Two health economic analyses will be conducted, a primary long-term analysis using the Sheffield T1D Policy Model and a secondary analysis of the data collected in the trial. All health economic analysis will compare the incremental cost-effectiveness ratio of DAFNE10.1136/bmjopen-2020-040438.supp3Supplementary dataStudy centres are only required to report as adverse events episodes of diabetic ketoacidosis and severe hypoglycaemia which while not requiring admission to hospital have been noted by either the participant or their relative/partner etc. These will be recorded on the data collection form and database.plus or standard DAFNE but will report any which are deemed related to the study intervention and or are unexpected to the Sponsor and the REC in line with best practice.We do not anticipate many Serious Adverse Events related specifically to DAFNEIt is expected that there will be 882 patients referred for DAFNE courses within the 15-month recruitment window and of these it is expected 75% (662 patients) will be recruited, equivalent to 47 participants at each of the 14 centres. From current DAFNE data, a further 25% are expected not to meet the primary analysis population criteria of baseline HbA1c greater than 7.5% (58 mmol/mol), leaving 497 participants. Finally, we anticipate 15% of participants to be lost to follow-up by the 12-month stage, therefore giving a primary analysis population of 422 participants. The sample size takes into account the design effect associated with the cluster design of the study. With an intracluster correlation coefficient (ICC) of 1.5% (from previous DAFNE data) and 30 participants per cluster (422 participants over 14 centres) the design effect is 1.435 leaving the effective total sample size of n=294 participants (n=147 per arm).Using a two sample comparison of mean HbA1c at the 12-month follow-up with two-sided alpha of 5%, a correlation of 0.5 between baseline and final values and a SD of 1.45 (from previous DAFNE data), the trial sample gives 92% power to detect a 0.5% difference in HbA1c  between the two treatment groups in the study.The primary analysis population will be participants that had an HbA1c greater than 7.5% (58 mmol/mol) at baseline and the analysis will be completed on an intention-to-treat (ITT) basis. This primary analysis is to assess the difference between the two treatment groups on the mean HbA1c at 12 months which will be completed using a multiple linear regression model with coefficients estimated using generalised estimating equations to account for the clustering design. A 95% confidence interval for the difference between the two treatment groups will be presented. Appropriate covariates will be included in the model, along with the participant’s baseline HbA1c, to adjust the treatment effect accordingly.The secondary analysis population is all consenting participants in the trial and analysis will again be completed on an ITT basis. This population will also be used to assess the difference in psychological outcomes between the two treatment groups using the same model as for the primary analysis.A full statistical analysis plan has been written and was circulated to the TMG and TSC before being signed-off. This is available in 10.1136/bmjopen-2020-040438.supp5Supplementary dataCase report forms will be completed by DAFNE facilitators/educators at each study visit. Follow-up questionnaires will be self-completed by participants at each follow-up point. Participants will be allocated a unique identification number to identify them throughout the trial.Plans to promote retention and follow-up of all trial participants include research appointments being scheduled and followed up by their clinical teams at 6 months and 12 months. Overdue questionnaires are followed-up with an email reminder and then telephone call from CTRU. All participants received email newsletters to update them on trial progress.plus database on CTRU’s secure online system, hosted on University of Sheffield servers. Access is restricted such that users can enter and view only information required to perform their role.Data will be entered onto the DAFNEplus website teams. Consent will be obtained from the participant for this to occur. Data will be stored securely on access-restricted network drive folders in accordance with CTRU standard operating procedures (SOPs).Identifiable data will be shared with CTRU and the supporting study team and DAFNEAll consent forms and questionnaires will be kept in a locked filing cabinet in a secured area and will be retained for a minimum of 5 years after study completion, in accordance with the sponsor’s archiving requirements. Sheffield CTRU may request consent forms to be sent from the research site to the CTRU via post or email as part of remote monitoring procedures.The nature, frequency and intensity of trial monitoring will be outlined in the site monitoring plan, which will be devised in accordance with CTRU SOPs.In addition to the patient representation on the trial oversight committees, this trial is supported by a patient advisory group who have and will continue to meet regularly during the conduct of the trial (and the wider programme grant). Patient input has been sought throughout on the trial and intervention design, the informational material to support trial conduct and patient burden.Two oversight committees have been established to oversee the conduct of this trial— the TSC and TMG, the composition of each is listed at the end of this paper. A Data Monitoring and Ethics Committee has not been convened, on the grounds that the study is low risk, in line with CTRU SOP GOV003. This has been approved by the Sponsor and TSC.The RCT was not initiated until the protocol, informed consent forms and participant information sheets received approval from the Research Ethics Committee, the Health Research Authority and local Capacity and Capability is confirmed by the respective NHS Research and Development departments. MHRA  approval was not required for this study.The RCT is being conducted in accordance with the ethical principles that have their origin in the Declaration of HelsinkiOutputs from the trial will be generated in accordance with the communication and dissemination strategy. A number of academic outputs will be produced as the data are analysed from the trial. Journals will be selected based on the highest possible impact. Other stakeholder-specific outputs in relevant formats will also be produced for commissioners, third sector and user advocacy organisations.


Pregnancy during adolescence is prevalent in low- and middle-income countries (LMICs), which is associated with various adverse outcomes that can be prevented with home visiting programs. However, testing these interventions in LMICs can be challenging due to limited resources. The use of electronic data collection via smartphones can be an alternative and ideal low-cost method to measure outcomes in an environment with adverse conditions.Our study had two objectives: to test the efficacy of a nurse home visiting intervention on maternal parenting and well-being measured by an electronic daily diary (eDiary), and to investigate the compliance rate of the eDiary measurement method.We conducted a randomized controlled trial to test the efficacy of Primeiros Laços, a nurse home visiting program, for adolescent mothers living in an urban deprived area of São Paulo, Brazil. A total of 169 pregnant adolescents were assessed for eligibility criteria, 80 of whom were included and randomized to the intervention (n=40) and control group . Primeiros Laços is a home visiting intervention delivered by trained nurses tailored to first-time pregnant adolescents and their children, starting during the first 16 weeks of pregnancy until the child reaches 24 months of age. Participants were assessed by blind interviewers at 8-16 weeks of pregnancy (baseline), 30 weeks of pregnancy, and when the child was 3, 6, and 12 months of age. At 18 months, participants were assessed regarding maternal parenting and parental well-being using a 7-consecutive-day eDiary. The smartphone app was programmed to notify participants every day at 9:00 PM over a period of seven days.P=.02) and the maternal parenting behavior of the mother telling a story or singing to the child .Our analyses showed a significant effect of the intervention on parental well-being  and the maternal parenting behavior of the mother telling a story or singing to the child .We were able to contact 57/80 (71%) participants (29 from the intervention group and 28 from the control group) when the child was 18 months of age. Forty-eight of the 57 participants (84%) completed at least one day of the eDiary protocol. The daily compliance rate ranged from 49% to 70%. Our analyses showed a significant effect of the intervention on parental well-being (B=0.32, 95% CI , The Primeiros Laços intervention improved maternal parenting and parental well-being, demonstrating its promise for low-income adolescent mothers. The compliance rate of the eDiary assessment showed that it was generally accepted by adolescent mothers with limited resources. Future studies can implement ambulatory assessment in LMICs via smartphones to measure mother and child behaviors.ClinicalTrials.gov NCT02807818; https://clinicaltrials.gov/ct2/show/NCT02807818 Every year, 16 million 15 to 19-year-old adolescents give birth, predominantly in low and middle-income countries (LMICs) , increasStudies focusing on maternal parenting usually rely on direct or recorded observations of dyads by trained and certified experts  or self-Ambulatory assessment encompasses methods designed to study people in their natural environment. Owing to the ubiquitous presence of smartphones, ambulatory assessment methods are now most commonly delivered via mobile technology. Subjects can be notified to answer questions during specific times of the day throughout days, weeks, or months. Given the numerous advantages of ambulatory assessment, its use has recently grown in many fields -11. NeveTherefore, we implemented an electronic daily diary (eDiary), a specific type of ambulatory assessment through which participants report a set of behaviors that occurred during the day to measure maternal parenting and parental well-being, in the context of a randomized controlled trial. In particular, we assessed the efficacy of Primeiros Laços, a nurse home visiting program for adolescent mothers living in an urban deprived area of São Paulo, Brazil. The intervention aimed to foster the mother-child relationship and improve child development. The present study had two objectives: (1) to test the efficacy of a nurse home visiting intervention on child maternal parenting and parental well-being measured by an eDiary, and (2) to investigate the compliance rate of the eDiary measurement method. The first hypothesis was that mothers who receive the home visiting intervention will present a higher frequency of maternal parenting behaviors and higher scores of well-being. The second hypothesis was that the eDiary method will present a compliance rate similar to that reported in previous studies, despite the fact that our sample is composed of low-income adolescent mothers living in adverse conditions.The present study is part of a parallel group randomized controlled trial that was originally designed to test the efficacy of Primeiros Laços, a nurse home visiting program for adolescent mothers living in an urban deprived area of São Paulo, Brazil. Previous findings from this trial can be found elsewhere -26. FromPrimeiros Laços is a home visiting intervention delivered by trained nurses that is tailored to first-time adolescent pregnant women and their children, which starts during the first 16 weeks of pregnancy until the child reaches 24 months of age. The frequency of visits was (a) biweekly during gestation and from 2 to 20 months of the child’s age; (b) weekly during the first and last month of pregnancy, and during puerperium; and (c) monthly from 21 to 24 months of the child’s age. The Primeiros Laços program is based on three theoretical frameworks: attachment theory , self-efThe content is directed to five domains. The first domain is health and social care, in which the nurse provides education regarding maternal and child health such as nutrition, hygiene, common pathologies in childhood, domestic care, vaccination, prevention of accidents, and child development. The second domain is environmental health, in which the nurse provides support and help to identify resources to guarantee adequate living conditions, safe housing, daycare and school, and access to health services. The third domain is life course, which involves life course planning to help participants achieve life goals such as finishing high school, finding a part-time job, starting college, and postponing the birth of a second child. The needs and goals of the participants were discussed individually to respect their personal goals and wishes. Nurses also help mothers gain access to primary care services and government-sponsored social programs. The fourth domain includes parenting skills, which involves education on parenting skills and child behavior considering each development stage, aiming to develop a sensitive and responsive pattern of care. The last domain is family and social support, as nurses frequently highlighted the role of family members and friends to support parental needs. Nurses were supervised weekly by the developers of the program (senior nurses and a child psychologist) to discuss cases, plan visits, and to guarantee fidelity and quality of visits. More details about the intervention can be found elsewhere ,36.Participants allocated to the control group received care from Unified Health System (Sistema Único de Saúde), Brazil’s public health system , accordiParticipants were assessed by interviewers blinded to group allocation at 8-16 weeks of pregnancy (baseline), 30 weeks of pregnancy, and when the child was 3, 6, 12, 18, and 24 months of age. Interviews were conducted by trained psychologists who underwent a 1-month training program provided by senior psychiatrists, psychologists, and pediatricians. Before study interviews commenced, interviewers trained by assessing volunteers living in the western region of São Paulo. Ambulatory assessment was administered when the child was 18 months old. At baseline (8-16 weeks of pregnancy), we measured depression symptoms using the Beck Depression Inventory ,42 and aWe used the LifeData system , a mobilThe present study focused on the following questions of the eDiary protocol: (1) How do you feel about the day today?  (2) Did you take care of your child or spend some time with him/her today? (3) What was it like taking care of your child today?  (4) Did you read or show a book to your child today? (5) Did you tell stories or sing to your child today? (6) Did you go out or go for a stroll with your child today? (7) Did you play with your child today? (8) Did you talk to your child today? (9) Did you eat or have a meal with your child today? (10) Did you kiss, hug, tickle  your child today? (11) How many hours did you spend with your child today? Maternal parenting behavior was operationalized as parenting practices related to mother-child interaction and positive activities known to stimulate child development. Questions 2, and 4 to 11 were related to maternal parenting. Screenshots of the eDiary protocol can be found elsewhere (imgur.com/a/eTMAp84).t tests were used to examine differences between groups (intervention vs control) at each time point. To examine intervention effects on eDiary outcomes, we used generalized estimating equation models [P values <.05 were considered statistically significant. Parameter estimates are reported with the 95% CI. Analyses were conducted using STATA 15.1 software.The sample size was calculated based on the difference in the electroencephalogram alpha wave frequency between the groups (30%) with a probability of type I error of 5% and statistical power of 80%. Frequencies and distribution of eDiary outcomes are provided for each of the 7 days of the intervention. The Fisher exact test and n models . Continun models . Fitted n models . All ranOur study was approved by the Ethics Committee of the University of São Paulo Medical School, University Hospital of the University of São Paulo, and São Paulo Municipal Health Department. All participants and their primary caregiver signed written informed consent forms. The study was registered at clinicaltrials.gov (NCT02807818).We were able to contact 57/80 (71%) participants (29 in the intervention group and 28 in the control group) when the child was 18 months old. P=.02), read to child at day 6 (P=.04), and telling a story or singing at day 2 (P=.04) and at day 6 (P=.047). P=.01) and increased behavior of the mother telling a story or singing to the child   .This is the first ECD randomized controlled trial testing an intervention using a smartphone-based ambulatory assessment to measure outcomes. The results showed that our nurse home visiting program had a positive effect on parental well-being and maternal parenting as measured by a smartphone eDiary. Previous studies have shown that maternal parenting behaviors in adolescent mothers  and low-It is worth mentioning that Brazil is a country currently facing many educational challenges. The last report from the Programme for International Student Assessment (PISA) showed that reading performance among students has not improved in the last 18 years . In the We also found an intervention effect on daily reports of parental well-being. Parenting a child is known to have positive effects on adult parents such as higher levels of life satisfaction, happiness, general feeling of positive emotion, and more meaning in life . HoweverThe compliance rate of the eDiary assessment was 84% (participants who responded at least one day of assessment), demonstrating that it was generally accepted by adolescent mothers living in an urban deprived area. This compliance rate was similar to that reported in other studies conducted with participants in the same age range. For example, a study conducted in Canada using a compliance criterion of participants responding to questions less than 7 times reported a rate of 90.4% . A recenOur findings should be viewed in light of some limitations. The sample size calculation of our clinical trial was based on different outcomes than those reported herein. The eDiary outcomes that were analyzed are secondary outcomes of the clinical trial, which were conceptualized and implemented after the clinical trial started. Therefore, our study may not have had sufficient power to find differences in these secondary outcomes, resulting in potential false negatives when compared to primary outcomes analyses. Additionally, our sample size limited the scope of our analyses. For instance, it would be interesting to use a structural equation modeling approach such as growth curves to validate the constructs assessed; however, the small sample sizes did not allow for convergence of these models. Moreover, the lack of variability and a ceiling effect in some variables may have also influenced these potential analyses. Therefore, even though we presented findings suggesting that we adequately measured the constructs of interest, we were not able to validate these measures quantitatively. However, we found that maternal parenting behaviors were negatively associated with maternal depression measured at the same time point , which iOur findings demonstrate the efficacy of Primeiros Laços, a nurse home visiting program for pregnant youth living in an urban deprived area, on improving maternal parenting and well-being assessed by an eDiary. Assessments with the eDiary were successfully conducted in this specific population. The implications are two-fold. First, Primeiros Laços is a promising intervention to promote maternal parenting and well-being among low-income adolescent mothers. As a structured and manualized intervention, this could be implemented in the primary care system, potentially benefiting millions of Brazilian mothers and children. Other LMICs similar to Brazil may also benefit from the program given appropriate adaptations for language, culture, and context. Second, our findings demonstrate the potential for future ECD intervention studies to implement ambulatory assessment in LMICs via smartphones for measuring mother and child behaviors. More frequent assessments of maternal parenting behaviors and well-being should be implemented to further enhance temporal and ecological validity, and to also expand the scope of measured behaviors.


ATP-dependent chromatin remodelling enzymes are molecular machines that act to reconfigure the structure of nucleosomes. Until recently, little was known about the structure of these enzymes. Recent progress has revealed that their interaction with chromatin is dominated by ATPase domains that contact DNA at favoured locations on the nucleosome surface. Contacts with histones are limited but play important roles in modulating activity. The ATPase domains do not act in isolation but are flanked by diverse accessory domains and subunits. New structures indicate how these subunits are arranged in multi-subunit complexes providing a framework from which to understand how a common motor is applied to distinct functions. Conversion between chromatin states involves changes to chromatin at different levels, including post-translational modifications to histones and DNA, recruitment of chromatin-binding factors and direct changes to the structure of nucleosomes as a result of the action of chromatin remodelling ATPases.In addition to packaging DNA within nuclei, chromatin provides a means of segmenting the genome into distinct chromatin states that ensure that transcription is regulated correctly in time and spaceATP-dependent chromatin remodelling enzymes are molecular machines that act to reconfigure nucleosomes so as to enable gene regulation in response to developmental and environmental signals. Interest in this fundamental process has heightened with the finding that many subunits of these complexes are mutated at high frequencies in cancers and neurological disorders.2. Many of the genes identified in these screens were found to associate as multi-subunit SWI/SNF complexes that had the ability to reconfigure chromatin3. Subsequently, budding yeast were found to encode a related complex, remodels the structure of chromatin (RSC), in which many  subunits are paralogs of those found in SWI/SNF4. Multicellular organisms also encode related complexes. InDrosophila, the Brahma complex falls within the trithorax group of developmental regulators7. In mammalian cells, three forms of SWI/SNF-related complexes have been identified8  and sucrose fermentation (SNF) in budding yeastified8 . Homozyg11. Consistent with this, the complexes are linked to the maintenance of accessible chromatin structure at promoters in yeast and at enhancers in mammalian cells18. The complexes contain a catalytic subunit that is related to ancient ATP-dependent DNA translocases and that acts to drive DNA across the surface of nucleosomes. These specialised ATPase domains are found in an extended family of some 20 yeast and 40 human proteins that regulate DNA–protein interactions19. In the context of SWI/SNF complexes, ATPase subunits act in the repositioning, destabilisation and dissociation of histones from DNA20. Until recently, a structural framework on which the mechanism of action can be built has been lacking.Biochemical characterisation of the complexes indicated that they can disrupt nucleosome structureHere, we summarise recent insights into the structure of ATP-dependent remodelling enzymes. These show that remodelling enzymes share an ATPase module that acts on DNA within nucleosomes and that this motor domain is tuned to different purposes within distinct multi-subunit complexes.23. More recently, cryogenic electron microscopy (cryo-EM) has been used to obtain structures of yeast Snf2, human (SNF2H) and yeast ISWI (imitation SWI) proteins and yeast Chd1 proteins in complex with nucleosomes29  6 and histone H2A/H2B dimer surface and generates an unwrapping of one turn of nucleosomal DNA15. In the case of Arp5/Ies6, the interaction is internal to the nucleosome. INO80 and SWR1 also contain a second grouping of actin fold proteins: the Arp8/Arp4/actin module. This module has been studied in isolation and is known to interact with a helicase-SANT-associated (HSA) domain present N-terminal to the ATPase domains of many remodelling ATPases where it acts as a linker DNA-binding module that regulates activity19. Density for the Arp8/Ar4/actin module is not observed in one structure32, but weak density is observed in the other33 Adjacent to the Rsc8 dimerisation helices, the armadillo repeats of the Rsc9/Swi1/ARID1A protein are visible. The ARID1A and ARID2 proteins are distinguishing features of the BAF and PBAF forms of mammalian complex positioned some distance from the ATPase domains and likely provide as-yet-uncharacterised complex-specific functionality.ii)The tandem bromodomain-containing Rsc2 and Rsc4 proteins are located close to the C-terminus of Rsc8. In mammalian PBAF complex, the polybromo protein PBRM1 is likely to interact at a similar location.iii)en route to the motor domains which engage with the nucleosome in an orientation similar to that observed with the isolated Snf2 protein. The HSA domain interacts with the actin fold subunits Arp7 and Arp9 and, together with Rtt102, has been crystallised independently36. This HSA-ARP module links the ATPase domains to the central core based around the Rsc8 dimer. This module is shared in SWI/SNF, RSC, INO80 and SWR1 complexes19.In between these regions, the HSA domain of Sth1/Snf2/SMARCA4 protrudesiv)The nucleosomal linker DNA extends back towards the Rsc8 core and may make contact with a DNA-binding domain which, at present, is poorly resolved but likely to be composed of the Rsc3 and Rsc30 subunits.v)46. This contact is conserved in human forms of SWI/SNF complex47 and mutations in this region observed in patients with Coffin–Siris syndrome also affect the ability of complexes to reconfigure nucleosomes52. It is possible that contact with this region confers specificity for histone variants53.The Sfh1/Snf5/SMARCB1 protein C-terminus extends back forming a distinct nucleosome-binding module located such that it is placed to interact with the acidic patch region on the lateral surface of the nucleosome. This contact is shared in SWI/SNF and RSC complexes that also have the ability to both reposition and evict nucleosomes but is not present in SNF2H and Chd1 complexes which only reposition nucleosomes. Furthermore, mutation of the C-terminal region of Sfh1 that interacts with the acidic patch specifically decreases the ability of RSC complexes to evict nucleosomes with little effect on ATPase activityvi)The structures of nucleosome-bound complexes are in all cases relatively low and combined with higher-resolution structures of nucleosome-free complexes. This means that it is not possible to determine whether detail observed in the higher-resolution structures of the Snf2 ATPase fragment bound to a nucleosome, including the location of the histone H4 tail, hold true in the context of nucleosome-bound complexes.Overall, the structure can be considered a central hub from which modules representing sites of association for ARID-containing proteins, multi-bromodomain–containing subunits, the ATPase domains, a linker DNA-binding domain and a module that interacts with the lateral surface of the nucleosome are projected in different directions . The neeSMARCC1 andSMARCC2. These can be present as either hetero- or homo-dimers within SWI/SNF complexes, but the published structure contains only SMARCC2.The major features of the RSC nucleosome structure are conserved in the yeast SWI/SNF complex and many of the subunits have equivalents in human forms of SWI/SNF complex . A major28. This is consistent with the idea that individual modules are largely independent. The exception is the core of the complex which, through acting as a scaffold for association of many components, plays a more significant role in complex integrity. Consistent with this, perturbing SMARCC1 and SMACC2 levels results in substantial degradation of SWI/SNF complexes30. Similarly, loss of all SMARCD isoforms  results in disruption of core complexes and recovery of a residual core module7. Subunits that are not assembled correctly are subject to ubiquitinylation and proteasome-mediated degradation30. Differences in the potency of this surveillance system may explain some differences in the effects of deleting subunits in specific cell types. For example, the peripheral association of the Sfh1/Snf5/SMARCAB1 subunits would suggest that these are not required for complex integrity. Consistent with this, SWI/SNF complexes remain largely intact following deletion of the Snf5 subunit with only Swp82 and Taf14 dissociating27. Deletion of the human homologSMARCB1 does not severely compromise complex integrity in HEK293T cells7 but does in rhabdoid cell lines17. Thus, subtle effects on subunit associations may have different effects on complex integrity in different tissue types. The partial or complete dissociation of complexes following loss of subunits is, of course, relevant to diseases in which these subunits are lost. However, it does not necessarily inform on the pathway by which complexes are assembled which may be distinct. To characterise how complexes assemble requires study of the order in which nascently translated peptides associate, as observed for assembly of SAGA complexes59.Many features of the modular organisation of the complex are consistent with previous observations. For example, SWI/SNF-related complexes remain substantially intact following loss of most subunits9. However, the ARID1A, PBRM1 and SMARCA4 subunits are mutated at highest frequencies and these are mutated at much higher rates in tumours of some tissues in comparison with others (58 are mutated at substantially lower rates (31. Similarly, the PBRM1 component contains six bromodomains and is likely related to the Rsc2, Rsc4 and Rsc58 subunits which occupy a location at one extremity of the core region. Deletion of PBRM1 does not affect association of other subunits28. Mutations to SMARCB1 which is related to the yeastSFH1 andSNF5 genes are relatively rare but debilitating as they drive malignant rhabdoid tumours60. The relatively low frequency of SMARCB1 mutations may be related to the fact it has no obvious partially redundant paralogs and is present in two forms of human SWI/SNF-related complex. SMARCA4 is mutated at relatively high frequency, but unlike other subunits, these mutations are predominantly missense mutations clustering to the ATPase domains and appear to act in a dominant fashion16. In contrast, the high frequency of mutations in ATPase domains, the HSA and N-terminal region of SMARCA4 is mutated at relatively low frequency (It is estimated that 20% of all human cancers contain a mutation to at least one subunit of one form of SWI/SNF complex others . It is nr rates . It is pr rates . The ARIequency . As a reThis first wave of structures provides a first insight into the layout of different classes of chromatin remodelling enzyme. Though spectacular, substantial proportions of many subunits are not defined, meaning that it is not possible to assign functions to the bromodomain and armadillo repeat-containing subunits that define different forms of complex. In addition, the current structures represent snapshots of moving machines. To build a complete picture of their function will require views of different stages of the reactions they drive. It will also be critical to determine where and how their activity is regulated by contacts with interaction partners. Nonetheless, insight into the complexes is proceeding at a dramatic pace and provides a structural framework encompassing many of the subunits and domains present.HSA, helicase-SANT-associated; Ies, inositol eighty subunit; INO80, INOsitol requiring; RSC, remodels the structure of chromatin; SMARCC, SWI/SNF-related matrix-associated actin-dependent regulator of chromatin; SWI/SNF, mating type SWItching/sucrose non-fermenting; SWR1, SWI-related 1; cryo-EM, cryogenic electron microscopy.


Background: Child cardiac arrest is rare, but more frequent among infants, requiring immediate cardiopulmonary resuscitation (CPR). Many studies have reported that simulation-based training (SBT) increased CPR performance of healthcare providers. However, the CPR performance of laypeople using basic life support remains poorly known. The aim of this study was to assess nursery assistants'  CPR performance and knowledge, before and after SBT.Methods: The study was carried out from January to June 2018 in the city of Poitiers, France. Two teaching sessions (T1 and T2) and two evaluation sessions (E1 and E2) were performed. Performance in infant CPR on a manikin at E1 and E2 were videotaped and assessed automatically with Resusci Baby QCPR® and a SimPad PLUS SkillReporter  and by an observer using an original CPR performance checklist . Nursery assistant's CPR knowledge was assessed by a questionnaire at the beginning and the end of the session T1, E1, and E2.Results: Twenty-Seven nursery assistants over 30 contacted were included. There was an improvement between E1 and E2 in QCPR Global-Score , MCPR Global-Score  and theoretical knowledge with score (over 45) of 16.9+5.4 before T1 and 35.2+2.7 after E2, respectively (p < 0.001). The improvement mainly concerned QCPR and MCPR compression steps scores. MCPR Global-Score was strongly correlated to QCPR Global-Score  and predictive to CPR quality determined by QCPR Global-Score  with a high sensitivity and negative predictive values. Moreover, these improvements were maintained 2 months after training with no difference between scores obtained by the three groups 15, 30, or 60 days after simulation-based training session T2.Conclusion: SBT could significantly improve knowledge and skills in infant CPR management by nursery assistants especially for chest compression. CPR performance checklist appeared as an interesting tool to assess CPR performance quality. Pediatric prehospital cardiac arrest are rare, with an overall incidence of 8 to 10 per 100,000 persons  but occuTo improve quality CPR of rescuers, the use of simulation-based training (SBT) increased these last years and has proven its efficiency to improve CPR skills and maintain them over time –13. MoreBecause infant OHCA are more frequent than in children , occur mWe hypothesized that SBT with mastery training would increase infant CPR performance of nursery assistants.The aim of this study was to assess infant CPR performance among a population of nursery assistants working alone at home, before and after mastery training including didactics and SBT. The primary objective was to improve nursery assistants' infant CPR performance quality. Secondary objectives were:  To assesThis monocentric prospective pilot study with assessment before and after intervention took place in Poitiers, France, from January to June 2018 and involved nursery assistant (NA), managed by the “Early Childhood department” of the City hall of Poitiers, working alone at home. After the agreement of the “Early childhood department,” the 30 nursery assistants were contacted for the study. Inclusion criteria were: being a certified NA (involving previous participation in certified CPR training), having participated in CPR training within the last 2 years and giving a written informed consent to participate. Participants who did not completed all the study procedure were excluded. The study received the approval of the simulation center and the local ethics committee of the Faculty of Medicine of Poitiers.The study design is represented in Except for T2, knowledge test was performed at the beginning and the end of each session. Content of the scenario was explained out to participants during briefing just before simulation and of the simulation room. The same scenario  was usedBasic life support (BLS) competencies were measured using Resusci Baby QCPR®  and a SimPad PLUS SkillReporter  connected defining a QCPR Score, a QCPR Breathing Score  and a QCPR Compression Score . Scores were expressed by the device as percentage of success. According to manufacturer instructions, the CPR performance quality was determined by the QCPR scores which reflect a basic (<50%), intermediate (≥50 and <75%), or advanced (≥75%) CPR performance quality. A CPR performance checklist created for the study based on the European Resuscitation Council recommendations , was alsThe same QCPR Global-Scores thresholds were used for MCPR scores to determine CPR quality.A written theoretical knowledge test based on questionnaire created for the study was used to measure infant CPR knowledge and included 36 items  for a ma25-IQR75]. Comparisons of continuous variables were made with paired t-test or ANOVA for paired and parametric values, Wilcoxon or Friedman tests for non-parametric paired values and Mann–Whitney or ANOVA test for non-parametric and unpaired values. Categorial variables were analyzed with Chi square test or Fisher's test. Pearson test was used to assessed correlation for parametric values or Spearman test for non-parametric values. Bland and Altman test was used to assess intra-observers reproducibility. A p-value < 0.05 was considered significant.Statistical analysis was performed with SPSS 26.0 . Results are presented as mean + standard deviation (SD) or median with interquartile range [IQROut of the thirty contacted nursery assistants, two refused to participate, and one was excluded because she did not complete the study protocol . CharactWhile the QCPR Global-Score was significantly improved between E1 and E2, the increase in the number of participants with a QCPR Global-Score between 50 and 75% or over 75% was not significant .Considering MCPR scores, intra-observer agreement and intra-class correlation over time were high and lack of intra-observer agreement over time was low at E1, E2 or for all observations together . MCPR GlThere was a significant improvement in participants positioning the manikin in neutral position, opening airways, inspecting airways and performing CPR during the call to emergency medical system. There was no significant change for stimulating the manikin, putting it on hard surface and calling emergency medical system item performed by most of participant at E1.There was no significant difference of QCPR B-Score between E1 and E2, however the number of participants starting and performing 5 rescue breathes before chest compressions and with chest expansion observed during rescue breathes. The time of the first rescue breath after the discovery of the manikin was decreased at E2 compared to E1 but the difference was not significant. The percentage of ventilation administering the correct volume remained very low at E2, with most of the volumes administered exceeding the maximum expected volume.QCPR C-Score and number of participants with a QCPR C-score over 75% increased significantly between E1 and E2. Moreover, the percentage of compressions performed at a correct rate, the number of participants with an average compression rate of 100–120 per minute, and the use of the two fingers technique for chest compression and performing 2 rescue breathes each 30 chest compressions increased significantly between E1 and E2. The time to first compression and the percentage of no flow duration were decreased between E1 and E2 but the difference was not significant. However, the no flow duration after the first compression decrease significantly between the session. Chest compression depth and recoil, or correct hand position were not different between E1 and E2.Score obtained before any intervention at the beginning of T1 (QT1A) increased significantly after didactic intervention (QT1B) with an improvement of knowledge in infant cardiac arrest management, order of CPR steps of management, chest compression, ventilation, and emergency call  and afteBLS competencies assessed with Skill Reporter or with BLS checklist and QE2A questionnaire score were not significantly different between the three groups performing E2 15, 30, or 60 days after SBT training session T2 .This simulation-based study demonstrated an improvement in infant CPR performance, skills and knowledge—especially concerning Chest compression steps—with a satisfactory retention at 15, 30, or 60 days in a population of nursery assistants. Scores assessed with checklist were strongly correlated to automatically assessment and predictive of the of CPR performance quality determined by skill recorder.In this study, most of participants had CPR performance scores and Compression scores above 50% at baseline while they were previously trained to CPR, suggesting that first aid management of cardiac arrest training was insufficient and that scores of untrained laypeople would be similar or worse . Most siAs previously demonstrated, mastery training, led to an improvement in participants' skills and knowledge in different steps of infant CPR management , 16, 17.Surprisingly QCPR B-Score was not increased after SBT. This lack of result could be explained by difficulties for participants to administrate a correct volume during rescue breathes. Previous studies highlighted that rescue breathes in CPR did not improve survival or morbidity –35 excepAs described previously, an optimal CPR could improve survival after CA , 6, 7, hThis study showed that CPR performance was correlated to laypeople's CPR theoretical knowledge after didactic reminder but not after practice training. However, after the didactic reminder, performance scores remained low. This result highlighted that improved theoretical knowledge in CPR was not enough to obtain a satisfactory CPR quality and the interest of practical training , 40. MorThis study had some limitations. First, while the inclusion rate was high number of participants included was small leading to little group to assess retention over time. However, the results obtained by the three groups were very similar and it seems unlikely that the lack of difference is related to a lack of statistical power.Secondary, the equipment used for simulation was only presented during briefing before CPR performance while most of participant had never used QCPR devices or manikin Resusci Baby QCPR before the study. The lack of awareness of the possibilities of these elements by the participants may had influenced results of E1 and artificially increased those of E2. However, the influence of equipment on score may be low because all participants were previously trained to CPR on different types of manikin. The use of the same scenario for E1 and E2 may have contributed to artificially increasing the results on E2. However, before E2 and until the briefing, participants were not informed that scenario used for E2 would be the same as for E1 to prevent external training. Moreover, using the same scenario was necessary to compare performance.Another limitation of the study was the absence of evaluation before any intervention to know CPR performance at baseline and to correlate it to theoretical knowledge. However, score and skills at E1 were low and were probably not or only slightly modified by the didactic intervention. Moreover, previous studies have demonstrated the low quality of CPR performance of laypeople at baseline , 9. The The used of non-standardized checklist was a limitation of the study however the checklist was developed from ERC recommendations and allowed to assess skills unrecorded by the QCPR device. Moreover, MCPR score determined with the checklist was strongly correlated to the CPR quality assess automatically with the QCPR device. The knowledge questionnaire was also non-standardized however no standardized questionnaire was previously available. In addition, each answer can be deduced from the didactic reminder performed at T1 and ensured that the participant had sufficient knowledge to perform a CPR. In addition,To our knowledge, this is the first simulation-based study about infant CPR performance of nursery assistants working alone at home proposing an easy and accessible method to assess infant CPR quality. The chosen population included motivated and very engaged nursery assistant women previously trained once every 2 years in infant CPR. In addition to the small number of participants, it would be difficult to generalize these results to all of the nursery assistant, other caregivers or laypeople and further study with a larger number of participants would be necessary.Mastery training including didactics and SBT could improve performance, skills, knowledge with satisfactory retention over time in infant CPR management performed by nursery assistants especially for chest compression step. CPR performance quality could be assessed with detailed CPR checklist. This training represents an interesting method to train laypeople in charge of infants in management of different emergencies requiring immediate support while waiting for help. However, further study would be useful in determining the number of SBT training sessions required for each participant to achieve advanced quality. The repetition of simulation sessions would be mandatory for long-term memory retention as infant cardiac arrest represents a low volume-high stakes situation. Further studies should include larger area-based populations and measure critical events management in infants taken care of by nursery assistants.The datasets generated for this study are available on request to the corresponding author.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study.Conception and design of the study, or acquisition of data, or analysis and interpretation of data: FB, BB, VG-M, and DO. Drafting the article or revising it critically for important intellectual content: FB, AG, and DO. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.