Streptococcus pneumoniae (the pneumococcus) is commonly carried as a commensal bacterium in the nasopharynx but can cause life-threatening disease. Transmission occurs by human respiratory droplets and interruption of this process provides herd immunity. A 2017 WHO Consultation on Optimisation of pneumococcal conjugate vaccines (PCV) Impact highlighted a substantial research gap in investigating why the impact of PCV vaccines in low-income countries has been lower than expected. Malawi introduced the 13-valent PCV (PCV13) into the national Expanded Programme of Immunisations in 2011, using a 3+0 (3 primary +0 booster doses) schedule. With evidence of greater impact of a 2+1 (2 primary +1 booster dose) schedule in other settings, including South Africa, Malawi’s National Immunisations Technical Advisory Group is seeking evidence of adequate superiority of a 2+1 schedule to inform vaccine policy.A pragmatic health centre-based evaluation comparing impact of a PCV13 schedule change from 3+0 to 2+1 in Blantyre district, Malawi. Twenty government health centres will be randomly selected, with ten implementing a 2+1 and 10 to continue with the 3+0 schedule. Health centres implementing 3+0 will serve as the direct comparator in evaluating 2+1 providing superior direct and indirect protection against pneumococcal carriage. Pneumococcal carriage surveys will evaluate carriage prevalence among children 15–24 months, randomised at household level, and schoolgoers 5–10 years of age, randomly selected from school registers. Carriage surveys will be conducted 18 and 33 months following 2+1 implementation.The primary endpoint is powered to detect an effect size of 50% reduction in vaccine serotype (VT) carriage among vaccinated children 15–24 months old, expecting a 14% and 7% VT carriage prevalence in the 3+0 and 2+1 arms, respectively.The study has been approved by the Malawi College of Medicine Research Ethics Committee , the University College London Research Ethics Committee (Ref: 8603.002) and the University of Liverpool Research Ethics Committee (Ref: 5439). The results from this study will be actively disseminated through manuscript publications and conference presentations.NCT04078997. The study is supported by more than 20 years of routine pneumococcal surveillance.The study design is both pragmatic and robust, offering a methodology that is relevant to diverse settings in the region.By engaging community, institutional and government partners, we have optimised its relevance and increased the likelihood of community uptake.ie, children receiving a 2+1 vaccine schedule who reside in or relocate to a cluster implementing the 3+0 vaccine schedule or vice-versa).Limitations to this study include a risk of contamination between clusters  carriage as a prerequisite for the development of disease but also a key process for developing natural immunity.via human respiratory air droplets. With serotype-specific differences, carriage duration decreases with age, lasting from 2 weeks in adults up to 4 months among children.6–14In HICs, routine administration of pneumococcal conjugate vaccines (PCV) through the infant immunisation schedule has contributed to a rapid decline of vaccine serotype invasive pneumococcal disease (VT-IPD) in both vaccinated and unvaccinated populations.29Studies undertaken prior to PCV introduction in The Gambia, Kenya, Mozambique, Malawi and South Africa reported VT carriage prevalence ranging from 28% to nearly 50% among children <5 years old.38Currently, WHO recommends the implementation of the PCV vaccine using either a 3+0 schedule  or a 2+1 schedule (two primary infancy doses at 6 and 14 weeks of age and one booster at 9 months of age or after the first year of life). The WHO further recommends that the decision on which schedule to use be based on the epidemiology of disease in the local setting.40A 2017 WHO Technical Expert Consultation on Optimisation of PCV Impact highlighted a substantial research gap in investigating why the impact of PCV vaccines in low-income countries has been less than expected, underlining the need to define an optimal PCV vaccination schedule that will maximise their benefit in such settings.et alMalawi introduced the 13-valent PCV (PCV13) into the national Expanded Programme of Immunisations (EPI) in November 2011, using a 3+0 schedule , with a three-dose catch-up vaccination campaign among all infants <1 year of age. This introduction has been highly successful, with field studies showing an EPI vaccine coverage exceeding 90%.In this context, the Malawi Ministry of Health (MoH) and the National Immunisations Technical Advisory Group are now seeking evidence of adequate superiority of a 2+1 vaccine schedule to inform a change to Malawi’s current EPI schedule. To this aim, a pragmatic health centre-based evaluation comparing the current 3+0 schedule to a 2+1 schedule will be implemented in Blantyre District, southern Malawi. Two pneumococcal carriage surveys, conducted 18 and 33 months following the implementation of the 2+1 schedule, will have the objective of comparing the effect of the two schedules against carriage reduction among otherwise-healthy children and evaluate their potential to enhance herd immunity.The study is a pragmatic health centre-based randomised evaluation of the direct effect of a 2+1 PCV13 vaccine schedule on pneumococcal carriage in vaccinated infants and the indirect effect on non-vaccine eligible children and high-risk adults.2. Healthcare is delivered through a network of private and government hospitals and 28 government primary health centres, where EPI vaccinations are administered. Queen Elizabeth Central Hospital (QECH) is the government referral hospital providing free medical care to the 1.3 million urban, periurban and rural residents of Blantyre District. Children <5 years account for 16% of the total population.The study will be conducted in Blantyre district, southern Malawi. The District is 240 kmWe will investigate whether a 2+1 PCV13 vaccination schedule (two primary doses at 6 and 14 weeks of age +1 booster dose at 9 months of age), compared with the current 3+0 schedule , is superior in reducing VT carriage prevalence among vaccinated children and, consequently, creating a superior herd effect among older children and HIV-infected adults. To assess the effect of a district-wide change from 3+0 to 2+1, this study will address two specific research questions: First, will a 2+1 schedule provide enduring vaccine-induced protection against pneumococcal VT carriage into the second year of life? Second, will the 2+1 vaccine schedule generate stronger herd protection and result in decreased VT carriage in older children and unvaccinated high-risk adults in the general population?The primary objective of the study is to evaluate the direct effect of a 2+1 PCV13 vaccination schedule on VT pneumococcal carriage among children aged 15–24 months, 3 years after introducing the 2+1 schedule. This objective will answer the question of whether the 2+1 schedule induces a more enduring direct protection against pneumococcal VT carriage into the second year of life, compared with that observed with a 3+0 schedule.The secondary objectives of the study are to evaluate, 3 years after introducing the 2+1 schedule, (1) the indirect effect of a 2+1 schedule on VT pneumococcal carriage among children aged 5–10 years (PCV age-ineligible at time of implementing the 2+1 schedule); (2) the indirect effect of a 2+1 vaccination schedule on VT pneumococcal carriage among HIV-infected adults 18–40 years old and on antiretroviral therapy (ART).To address these questions, the Malawi MoH and Blantyre District Health Office (DHO) will randomly select 10 health centres  in which the routine PCV13 schedule will be switched to a 2+1 schedule. An additional 10 health centres will be randomly selected to continue with the current 3+0 schedule but will serve as the direct comparator in evaluating the effectiveness of the 2+1 schedule.This switch to a 2+1 schedule is an initiative led by the MoH and will be implemented within the scope of the routine EPI programme, subject to EPI standard procedures for delivery, monitoring and performance assessment. Implementation of all vaccination activities will be implemented, as per routine practices, by MoH EPI vaccinators through the routine EPI programme. The MoH will monitor completeness of dosing following standard reporting practices within the scope of the EPI. Routine study activities throughout the duration of the 3-year study period will include research nurses providing support and guidance to the EPI vaccination teams through weekly site visits. In addition, research enumerators will monitor patient-retained health passports of a representative sample population of vaccinees to confirm they are receiving the proper vaccine schedule (2+1 or 3+0) assigned to the catchment population of their respective health centre.The pragmatic design of this study has been assessed through the tool The PRagmatic-Explanatory Continuum Indicator Summary 2 (PRECIS-2).To ensure the 2+1 schedule is implemented efficiently, and to limit the risk of confusion during the transition to the 2+1 schedule (including not deferring PCV13 vaccination to a later visit), a 3+1 schedule will be implemented during the first several months at health centres implementing the 2+1 schedule. At these health centres, children who have received either their 10 weeks or 14 weeks PCV13 dose before 2+1 implementation will receive a dose at their 14 weeks (third dose PCV13) and their 40 weeks (fourth dose PCV13). Approximately 6 months after 2+1 implementation, all first-contact vaccine visits (post 2+1 implementation) will be for the scheduled visit at 6 weeks of age and the 3+1 schedule will no longer be required.The study will include two cross-sectional carriage surveys, implemented 18 and 33 months after the switch to 2+1. Carriage surveys will be conducted using a well-established methodology implemented extensively in the setting,Sampling will include:Children 15–24 months of age, PCV13-vaccinated  with either the 2+1 or the 3+0 schedule. Evaluation within this age group will allow us to evaluate for our primary objective, evaluating the direct effect of vaccination on carriage. Within each of the catchment areas of the 20 participating health centres, we will conduct a random walk method to systematically identify households with eligible children. A ‘egg and yolk’ strategy will be applied, defining two geographic sampling perimeters around each health centre. Sampling will prioritise those living within the geographic perimeter closest to the health centre. In the event that recruitment targets are not met, sampling will move into the second geographic perimeter. This strategy will maximise the likelihood of recruiting children who have received their vaccines at the selected health centre, minimising recruitment from buffer zonal borders and therefore minimising risk of contamination .Children 5–10 years of age, PCV13-vaccinated with the 3+0 schedule. These children, having received PCV13 at infancy, will be recruited from government schools. Evaluation within this age group will allow us to evaluate for a secondary objective, evaluating the indirect effect of a 2+1 schedule on VT pneumococcal carriage among children aged 5–10 years, the majority of whom received PCV13 infancy EPI vaccination using a 3+0 schedule. Six schools will be selected, three located centrally in each of the 3+0 clusters and the 2+1 clusters. Children will be chosen at random from school registers. This component will allow the evaluation of indirect effects of the 2+1 compared with 3+0 schedule among children with waning vaccine-induced immunity.Adults 18–40 years of age, HIV-infected on ART and PCV13-unvaccinated. Evaluation within group will allow us to achieve the secondary objective of evaluating the indirect effect of a 2+1 schedule on VT pneumococcal carriage among HIV-infected adults (18–40 years) on ART, a population at high risk of pneumococcal disease. Participants will be recruited from the QECH, Lighthouse ART Clinic in Blantyre.To assess the differential effects of 3 vs 2 primary vaccine doses in the first year of life, an additional carriage survey will be implemented at health centres approximately 9 months after 2+1 implementation. A convenience sample of children aged 9 months will be recruited at health centres providing either the 2+1 or 3+0 schedule. This will allow an evaluation of carriage prevalence among children receiving 3 (according to the 3+0 schedule) vs 2 (according to the 2+1 schedule and presenting for their booster dose) primary doses. Additionally, this additional carriage survey will contribute to evaluating the coverage of the 2+1 vaccination schedule and the proportion of children receiving all three PCV13 doses at health centres providing the 2+1 schedule.Inclusion criteria for all individuals recruited includes permanent resident in Blantyre District. Among children 15–24 months of age (assigned to either 2+1 or 3+0 schedule), inclusion criteria additionally include: aged between 15 and 24 months, parent/legal guardian providing written informed consent, evidence  of having received a full schedule of PCV13. Among children 5–10 years of age, inclusion criteria include: aged between 5–10 years, parent/legal guardian providing written informed consent, providing written informed assent (if the child is aged ≥8), and either verbal or documented evidence of having received PCV13. Among adults, inclusion criteria include: aged 18–40 years, providing written informed consent, and being HIV-infected and receiving ART.Among children 9 months of age, recruited as part of the safety component, inclusion criteria include: aged 9 months, parent/legal guardian providing written informed consent, and evidence  of having received either a full 3+0 PCV13 vaccine schedule  or both primary doses of PCV13 at approximately 6 and 14 weeks of age .Exclusion criteria for all screened individuals include receiving TB treatment at time of screening, hospitalisation for pneumonia within 14 days prior to study screening and terminal illness. Exclusion criteria for all children include parental/legal guardian not providing consent, not providing assent (for children aged ≥8 years), having received antibiotic treatment within 14 days prior to study screening. Exclusion criteria for adults include not providing written informed consent or prior vaccination with a pneumococcal vaccine.S. pneumoniae. VT pneumococcal carriage will be serotyped by latex agglutination. Samples from participants with confirmed pneumococcal carriage will be sent to the UK for assessment of multiple serotype carriage (genomic microarray) and for whole genome sequencing.The intervention will consist of two carriage surveys conducted 18 and 33 months after the 10 randomised health centres switch from a 3+0 to a 2+1 PCV13 schedule. In both surveys, a single NP swab will be collected from each participant. Following previously described WHO-recommended procedures,The primary endpoint of this study will be the difference in VT pneumococcal carriage prevalence among children aged 15–24 months, comparing those vaccinated with PCV13 in either a 2+1 or 3+0 schedule, 3 years after implementing the switch to a 2+1 schedule. Additionally, the study will evaluate four secondary outcomes: (1) difference in VT carriage prevalence among children 5–10 years old, 18 months and 33 months after 2+1 implementation, (2) VT carriage prevalence among HIV-infected adults aged 18–40 years and receiving ART at the time of sampling, (3) VT carriage prevalence among infants aged 9 months, who will have received three primary doses  or two primary doses  prior to the booster dose, 9 months after the implementation of the 2+1 schedule and (4) prevalence of multiple serotype carriage, 18 and 33 months after 2+1 implementationPrior to development of the protocol, key stakeholders were informed of the study, including the study sites  and their surrounding communities , the DHO, MoH and the Ministry of Education. We actively sought and incorporated input from these stakeholders into the study objectives and overall design. Community sensitisation will be further strengthened through a community advisory board.The primary endpoint is powered to detect an effect size of 50% reduction in VT carriage, expecting a 14% and 7% VT carriage prevalence among vaccinated children 15–24 months old in the 3+0 and 2+1 arms, respectively. Sample sizes were calculated based on a power of 80% and a statistical significance of 0.05. The calculations accounted for household similarities using an intraclass correlation (ICC) of 0.005 (based on previous experience in this setting) and adjusted for a design effect (dependent on both ICC and cluster size.) of 1.21. Minimum sample sizes needed to achieve the necessary power under these assumptions are shown in To assess the primary and secondary endpoints (1) One child 15–24 months of age (2+1 or 3+0) will be recruited from each of 40 households randomly selected within each of the 20 clusters. This is a total 800 vaccinated children per survey . (2) A total of 125 children will be recruited from each of the six schools per survey. This is a total 750 children per survey . (3) A total of 1200 HIV-infected adults will be recruited from the QECH ART Clinic over the course of the 3-year study period. (4) A total of 800 children 9 months of age will be recruited from the vaccination centres 9 months after schedule change .Demographic data and relevant medical history will be collected using password-protected electronic data capturing. Each participant will be assigned a unique participant identification number (PID) at recruitment. This PID will be used in all datasheets and files, and will be linked to the laboratory data, hence, only anonymised data will be used for the analysis. Fully anonymised data will be uploaded daily to a secured on-site server, which is backed up daily to both local and off-site facilities. A logbook containing identifiable information (including name) will be kept separate in a secured location by an authorised member of the study team and will only be accessed by authorised study members. This will allow the study team to recover any missing epidemiological information at a later date  and to facilitate any participants who choose to withdraw consent at any time.Continuous variables will be summarised by means and SD, or medians and IQRs if the distribution exhibits skew. Categorical variables will be summarised by frequency distributions. Direct effects of the PCV13 schedule change on VT carriage will be ascertained by comparing carriage prevalence in children aged 15–24 months residing in the recruitment clusters of health centres randomised to the 2+1 schedule and those in recruitment clusters of health centres randomised to the 3+0 schedule. Indirect effects will be evidenced by comparing children in the recruitment clusters of health centres randomised to the 3+0 schedule among older children (5–10 years) and HIV +adults. Additionally, data obtained in this study will be compared with those obtained from previous carriage surveys to ascertain any changes in VT carriage prevalence before and after the PVC13 schedule change. Statistical tests will be selected depending on the distribution patterns of the data. Potential confounders and sources of interaction  will be identified by testing the association between variables and VT carriage and included in the multivariable models when p<0.1. Sensitivity analyses will include assessing impact of (1) receiving only one, only two or all three doses PCV; (2) having document-confirmed PCV vaccination or (3) schedule adherence to within 2 weeks of each scheduled dose on VT prevalence and on the VT distribution.The study has been approved by the Malawi College of Medicine Research Ethics Committee , the University College London Research Ethics Committee (Ref: 8603.002) and the University of Liverpool Research Ethics Committee (Ref: 5439).The implementation of the study protocol will be reviewed and monitored by an External Advisory Group, providing oversight of the study activities and advise the study team at the time of the interim and final analyses. The group will include experts from the University of Malawi, College of Medicine, the London School of Hygiene & Tropical Medicine and the Medical Research Council.On completion of the first carriage survey, an interim analysis will be implemented using data obtained on the VT carriage prevalence among vaccinated children 15–24 months of age. These results will be used to assess possible adaptation of the second community carriage survey. Three possible scenarios are considered: (1) If convincing evidence of major change in carriage prevalence is demonstrated, immediate action will be discussed with the MoH to move to change the schedule and adapt year-3 sampling; (2) If no major change in carriage prevalence is identified, the study will continue as planned; (3) If carriage prevalence has fallen by more than 30% but does not meet the primary threshold of 50%, the second carriage survey will be brought forward by 6 months. Decisions will be reached in consensus between investigators, the Expert Advisory group, and the MoH.The study will only recruit children whose parents/legal guardians have the capacity to provide informed consent or, in the case of adults, are capable of giving consent. Children who are minors but ≥8 years of age will be required to provide informed assent, in addition to parents/legal guardians providing informed consent. Participants will receive both verbal and written information about the study and will be given the opportunity to ask questions and express their doubts and concerns before accepting to take part. They will also be given time to reflect before they come to a decision. An informed consent and/or assent form will be signed and dated by the participant and a member of the research team. The participant will keep a copy of the document, and a second one will be kept in the study file with the Principal Investigator based in Blantyre, Malawi. Participants will be informed of their right to withdraw consent at any point until study ends without the need to provide a reason and without penalty.Study results will be shared with local stakeholders and published in peer-reviewed journals. Partial results and interim analyses will be shared with the Malawi MoH and other relevant policymakers and decision-making stakeholders. Partial and final findings will be presented at relevant international conferences and meetings. Copies of all published materials and reports will be shared with the research ethics committees and collaborators. We will return to the community partners and work with the community advisory board to report and further disseminate our results into those communities where we worked.


Bioinformatics (2020) doi: 10.1093/bioinformatics/btz595In the above article, the image currently appearing in the "Figure 2" position belongs instead in the "Figure 1" position; the image currently appearing in the "Figure 3" position belongs instead in the "Figure 2" position; the image currently appearing in the "Figure 1" position belongs instead in the "Figure 3" position. The publisher apologises for the error.


Following the publication of this article , concernIn the left panel of Fig 1C, the left side of the bands do not appear to align correctly with the right sides of the bands.The Day 5 anti-β-actin panel of Fig 1Db appears similar to the Day 5 β-actin panel of The Sox9 panel and the GFP panel of The Day 2 β-actin panel of The corresponding author has provided the original images underlying Fig 1C and indicated that black/white inversion was performed to improve the presentation of the results. The uncropped underlying figures provided in the The similarity between the Sox9 and GFP panel in The similarity between the western blot panels in Fig 1D and The PLOS ONE Editors issue this Expression of Concern to notify readers of the above concerns and relay the supporting data and updated figures provided by the corresponding author.S1 File(TIF)Click here for additional data file.S2 File(TIF)Click here for additional data file.S3 File(XLS)Click here for additional data file.S4 File(XLSX)Click here for additional data file.S5 File(TIF)Click here for additional data file.S6 File(TIF)Click here for additional data file.S7 File(TIF)Click here for additional data file.S8 File(XLSX)Click here for additional data file.


Exercise improves cognition in the aging brain and is a key regulator of neuronal plasticity genes such as BDNF. However, the mechanism by which exercise modifies gene expression continues to be explored. The repressive histone modification H3K9me3 has been shown to impair cognition, reduce synaptic density and decrease BDNF in aged but not young mice. Treatment with ETP69, a selective inhibitor of H3K9me3’s catalyzing enzyme (SUV39H1), restores synapses, BDNF and cognitive performance. GABA receptor expression, which modulates BDNF secretion, is also modulated by exercise and H3K9me3. In this study, we examined if exercise and ETP69 regulated neuronal plasticity genes by reducing H3K9me3 at their promoter regions. We further determined the effect of age on H3K9me3 promoter binding and neuronal plasticity gene expression. Exercise and ETP69 decreased H3K9me3 at BDNF promoter VI in aged mice, corresponding with an increase in BDNF VI expression with ETP69. Exercise increased GABRA2 in aged mice while increasing BDNF 1 in young mice, and both exercise and ETP69 reduced GABRA2 in young mice. Overall, H3K9me3 repression at BDNF and GABA receptor promoters decreased with age. Our findings suggest that exercise and SUV39H1 inhibition differentially modulate BDNF and GABRA2 expression in an age dependent manner. Exercise has been shown to improve cognition in the aged brain across a range of clinical and animal studies . UnderlyOur lab has focused on elucidating the role of H3K9me3 in cognitive function. We first determined that H3K9me3 is elevated in the hippocampi of aged mice. To investigate if H3K9me3 is involved in cognitive decline, we inhibited the histone methyltransferase (SUV39H1) that catalyzes H3K9 trimethylation. We used a selective SUV39H1 inhibitor called ETP69, a chaetocin A analog developed by Dr. Larry Overman . TreatmeStudies have begun to examine how exercise affects the regulation of neuronal plasticity genes such as BDNF, which enhances learning and memory . BDNF isIn this study, we examined how exercise, SUV39H1 inhibition and age affect H3K9me3 promoter repression and expression of BDNF and GABA receptors. We first separated aged and young mice into sedentary, exercised and ETP69 treatment groups. We then measured levels of H3K9me3 at the promoters of BDNF transcripts I, IV, and VI, and at the promoters of GABBR1 and GABRA2. Lastly, we quantified transcript mRNA to determine the correlation between promoter H3K9me3 levels and total gene expression. Exercise and ETP69 reduced H3K9me3 at BDNF promoter VI in aged mice, corresponding with an increase in BDNF VI expression with ETP69. In young mice, exercise increased BDNF I expression, suggesting an age-dependent change in BDNF regulation. Exercise increased GABRA2 expression in aged mice while both exercise and ETP69 reduced GABRA2 expression in young mice. Overall, H3K9me3 repression at BDNF and GABA receptor promoters decreased with age. Our findings suggest that exercise and SUV39H1 inhibition differentially modulate BDNF and GABRA2 expression in an age dependent manner.n = 21 for each age; Jackson Laboratory) were individually housed with food and water ad libitum, and allowed 1 week acclimation to the vivarium prior to experiments. Lights were maintained on a 12 h light–dark cycle. Mice were sacrificed by carbon dioxide gas and cervical dislocation, and brains were rapidly frozen on isopentane cooled in liquid nitrogen. 1 mm punches of the dorsal hippocampus were collected from 500 μm slices.All experiments were conducted in accordance with the National Institutes of Health guidelines for animal care and use, and were approved by the Institutional Animal Care and Use Committee of the University of California, Irvine. Male 18 month old and 3 month old C57BL/6J mice (All mice were single housed in either standard cages (sedentary and ETP69 injected mice) or cages with a running wheel (exercised mice). Mice were single housed due to the design of the running wheel apparatus, which allows for only a single animal per cage. Single housing also gave each animal free and equal access to a running wheel . ExercisChIP was performed as described previously based on the protocol from the Millipore Magna ChIP kit . Tissue qRT-PCR was performed as previously described . RNA waspost hoc analysis using Prism software. In all cases, p ≤ 0.05 was considered significant. Excel was used for percent input analysis of ChIP data and Pfaffl analysis of qRT-qPCR data.Results were analyzed using one way ANOVA, Welch’s ANOVA, or two way ANOVA, followed by p = 0.0036; Dunnett’s post hoc test, *p = 0.011 ETP69 vs. sedentary). This suggests that the neuronal activity caused by an OLM task changes the regulation of BDNF promoter regions.We first investigated if ETP69 and exercise affected H3K9 trimethylation levels at BDNF promoter regions. We examined BDNF I, IV, and VI as these regulatory exons are highly expressed in the hippocampus . Our labp = 0.0021 exercise vs. sedentary), displaying a specific and parallel effect of exercise and ETP69 on BDNF promoters. Surprisingly, neither ETP69 nor exercise changed H3K9me3 at BDNF promoter transcripts in young mice . There was no change in BDNF I or IV expression with ETP69 or exercise in aged mice . Exercise significantly elevated BDNF IV levels in comparison to ETP69 treatment, but failed to reach significance relative to sedentary controls . Exercise and ETP69 did not increase BDNF VI levels in young mice (p = 0.24). This is in accordance with previous findings that exercise induces BDNF I and IV expression but not BDNF VI expression in young mice after 3 weeks of exercise . These results suggest that GABA receptor promoters are resistant to epigenetic changes by exercise and global reductions in H3K9me3.We next investigated how the repression and expression of GABA receptors correlate with BDNF, as GABA receptor activation increases BDNF signaling . We prevp = 0.024; Tukey’s post hoc test, ex vs. sed *p = 0.052, ex vs. etp *p = 0.044). In contrast, exercise and ETP69 reduced GABRA2 expression in young mice . ETP69’s effect on GABRA2 is surprising considering it does not reduce H3K9me3 at its promoter, suggesting that a global reduction in H3K9me3 activates a pathway that decreases inhibitory signaling. The exercise-induced increase in GABRA2 in aged mice is supported by our research in aged postmortem brains, suggesting that the need for inhibitory signaling increases in the aged brain . Therefore, exercise and ETP69 regulate GABRA2 expression independently of promoter H3K9me3 in an age-dependent manner.We next asked if exercise and ETP69 changed GABA receptor expression levels and found that the direction of change was highly dependent on mouse age. Exercise significantly increased GABRA2 expression in aged mice (ed brain . There wOur lab previously found that H3K9me3 is increased with age in the hippocampi of aged mice, leading us to ask if promoter bound H3K9me3 was also elevated with age in our study . We compOur findings demonstrate that exercise and SUV39H1 inhibition affect the expression of BDNF exons and GABRA2 in an age dependent manner. Exercise and ETP69 significantly decreased H3K9me3 at BDNF promoter VI in aged mice, corresponding to an increase in BDNF VI expression with ETP69 treatment. Apart from this important exception, exercise and ETP69 did not significantly alter H3K9me3 levels at the promoters of neuronal plasticity genes, although they did regulate mRNA levels. These findings suggest that H3K9me3 promoter repression is not a primary driver of neuronal plasticity gene expression. Age had a strong effect on H3K9me3 promoter binding, as H3K9me3 repression of BDNF and GABA receptors decreased significantly in old mice. Exercise and ETP69 altered neuronal gene expression in an age-dependent manner and reduced H3K9me3 recruitment at the BDNF VI promoter region.Of the promoters we examined, exercise and ETP69 only reduced H3K9me3 at the BDNF VI promoter in aged mice, suggesting that promoter bound H3K9me3 is a largely stable epigenetic modification. This finding differs from previous research showing that ETP69 reduces H3K9me3 at BDNF I following an OLM task . OLM incIn addition to increasing BDNF VI levels in aged mice, exercise also elevated BDNF I expression in young mice. This finding is supported by previous studies in our lab showing that 3 weeks of wheel running increase BDNF I levels in the hippocampi of young rats and mice . There wIntriguingly, exercise and SUV39H1 inhibition significantly affected GABRA2 expression, yet the direction of change was dependent on mouse age. While exercise increased GABRA2 expression in aged mice, ETP69 and exercise both reduced GABRA2 levels in young mice. GABA signaling is reduced in the hippocampus with age, contributing to overexcitation and memory impairment . A studyExercise induced changes in BDNF and GABRA2 expression in our models, yet GABRA2’s role in BDNF regulation remains unclear. In aged and exercised mice, the increase in GABRA2 correlated with an increase in BDNF VI expression, suggesting that GABRA2 may upregulate BDNF in aged models of exercise. In contrast, exercise induced a significant decrease in GABRA2 and an increase in BDNF I expression in young mice. GABRA2 may only be involved in BDNF upregulation in systems where BDNF and GABA signaling is impaired, such as models of aging. Future studies should examine the signaling pathway between GABRA2 and BDNF in models of aging and exercise to determine if GABRA2 differentially upregulates BDNF expression.Although promoter H3K9me3 was largely unaffected by our manipulations, its stability and binding affinity appear to decline with age. With the exception of GABBR1, H3K9me3 binding was greater in young mice than aged mice at the promoters of neuronal plasticity genes. Our findings demonstrate that H3K9me3 at promoter regions decreases with age even as overall H3K9me3 increases, which may indicate a change in H3K9me3’s localization and function . AlthougThis study opens up avenues for future investigation into the regulation of neuronal plasticity genes. The role of GABRA2 in BDNF regulation has yet to be explored, and studies involving GABRA2 inhibition or overexpression could elucidate its effect on BDNF expression. Studies on H3K9me3 localization throughout the lifespan will further suggest how its function changes with age. Our SUV39H1 inhibitor, ETP69, globally reduces H3K9me3 but cannot modify this marker at specific genes. ChIP-sequencing could be used to identify how H3K9me3 binding changes with age and exercise, and RNA-sequencing can determine if these changes are correlated with gene expression. A comprehensive examination of H3K9me3 will clarify its effect on neuronal gene expression and specific sites of pharmacological manipulation.We have demonstrated that H3K9me3 regulates the expression of BDNF VI in aged mice. Reducing H3K9me3 with ETP69 increases BDNF VI expression, thus increasing synaptic plasticity at distal dendrites. Increased age reduces H3K9me3 promoter binding and changes the regulation of BDNF and GABRA2 by exercise and ETP69. Overall, exercise and SUV39H1 inhibition can effectively modify the expression of genes involved in cognition.The original contributions presented in the study are included in the article/The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of the University of California, Irvine.AI-T and NB conceived of the presented ideas. CB and AI-T performed the experiments. DM and MW assisted with experimental design and provided PCR equipment. AI-T analyzed the data and wrote the manuscript. CC reviewed the manuscript and supervised the project. 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Wolbachia can manipulate arthropod reproduction and invade host populations by inducing cytoplasmic incompatibility (CI). Some host species are coinfected with multiple Wolbachia strains which may have sequentially invaded host populations by expressing different types of modular CI factor (cif) genes. The tephritid fruit fly Rhagoletis cerasi is a model for CI and Wolbachia population dynamics. It is associated with at least four Wolbachia strains in various combinations, with demonstrated , predicted (wCer1) or unknown (wCer5) CI phenotypes.The endosymbiont Wolbachia strains wCer1, wCer4 and wCer5, and compared these with the previously sequenced genome of wCer2 which currently invades R. cerasi populations. We found complete cif gene pairs in all strains: four pairs in wCer2 , two pairs in wCer1 (both Type I) and wCer4 , and one pair in wCer5 (Type IV). Wolbachia genome variant analyses across geographically and genetically distant host populations revealed the largest diversity of single nucleotide polymorphisms (SNPs) in wCer5, followed by wCer1 and then wCer2, indicative of their different lengths of host associations. Furthermore, mitogenome analyses of the Wolbachia genome-sequenced individuals in combination with SNP data from six European countries revealed polymorphic mitogenome sites that displayed reduced diversity in individuals infected with wCer2 compared to those without.We sequenced and assembled the draft genomes of the Wolbachia are common in arthropods and affect options for Wolbachia-based management strategies of pest and vector species already infected by Wolbachia. Our analyses of Wolbachia genomes of a host naturally coinfected by several strains unravelled signatures of the evolutionary dynamics in both Wolbachia and host mitochondrial genomes as a consequence of repeated invasions. Invasion of already infected populations by new Wolbachia strains requires new sets of functionally different cif genes and thereby may select for a cumulative modularity of cif gene diversity in invading strains. Furthermore, we demonstrated at the mitogenomic scale that repeated CI-driven Wolbachia invasions of hosts result in reduced mitochondrial diversity and hitchhiking effects. Already resident Wolbachia strains may experience similar cytoplasmic hitchhiking effects caused by the invading Wolbachia strain.Coinfections with The online version contains supplementary material available at 10.1186/s12864-021-07906-6. Wolbachia bacteria (Alphaproteobacteria) of arthropods can affect host reproduction and fitness, including host immunity, in a multitude of ways . Therefore, wCer2 had four complete sets of cif genes and the largest number of cif modules in this host species.Orthology to verified r5 Table . OriginaifB gene , but reacifA maximum likelihood tree comprised 41 cifA orthologues, representative of all five types, and was built on an alignment of 1884 nucleotide sites, of which 1267 were parsimony-informative  and 99.6% (cifB) amino acid similarity with CI inducing cifwPip[T4].Both contiguous nes Fig. . wCer4 a96_03720 , which acifwCer4T genes whe et al. . wCer5 iwCer2 gene (E3V96_03405) with an identical amino acid sequence to wmk of wMel (WD0626) which had previously been found to cause MK when highly expressed in transgenic D. melanogaster. No orthologues for this gene were found in wCer4 and wCer5. However, for a wmk homologue in wMel, WD0508, for which transgenic expression did not alter sex ratios in D. melanogaster, orthologues were found in wCer2 (similarity of 89%), wCer4 (94%) and wCer5 (93.6%). No orthologues of wmk or its homologues were found in wCer1.Furthermore, we found a R. cerasi individuals were representatives of three  populations with different Wolbachia infection types  and these individuals did not have wCer2, and the mitogenome of wCer2-infected RcerAS was HT2  between the three mitogenomes , 8 of the 12 sites were polymorphic ; for HT2 individuals (with wCer2) only 10 sites were represented by ddRADseq, and none was polymorphic . The variance in the Euclidean distance within the HT1 group of mitochondrial haplotypes (var = 0.07) was higher than group HT2 (var = 0.001). Furthermore, the PERMANOVA showed strong influence of the wCer1/wCer2 grouping on the distance measures between haplotypes (p = 0.001), but presence of wCer4 (p = 0.736) or wCer5 (p = 0.206) had no effect , wCer5  and wCer2  was almost 60% higher than the number of SNPs between the two wCer1 variants in the same two samples . The consensus sequences for each strain for each individual were calculated by majority consensus with no lower coverage threshold, however the additional variant analysis also showed strain variation within host individuals. The wCer1 of RcerHB showed at least two possible nucleotides (with minimum coverage of 35% of a given variant with minimum coverage threshold of 5 reads) at 16 sites; at 12 of these sites both nucleotides were present in the wCer1 of RcerIZ, and at 10 sites both nucleotides were present in the wCer1 of RcerAS. The overlap of variant sites in RcerIZ and RcerAS was at 11 sites . However, our data indicated at most a single SNP in a transposase gene that was single copy in wCer1 but multicopy in wAu and wMel may be a false positive. For wCer5, the single SNP in the phage gene patatin and nine of the 10 SNPs in the major tail sheath protein gene in RcerAS are true differences (> 58% frequency) from the reference sequence, while one heterozygous site may be a false positive. Notwithstanding these possible misassemblies, the number of changes still suggests wCer5 has a greater number of SNPs than wCer1 between RcerIZ and RcerAS.The variation across the ces Fig. , 8 and 9ces Fig.  and 12 wDsimRC50 ) showed wCer2, eight SNPs between the Wolbachia genome of Ccap88.6 and its donor RcerAS  from their donor, but three SNPs were common to the two recipients, and an additional two were common to the Ccap88.6 and D. simulans lines , wCer1 had ~20x coverage, wCer4 had ~2x coverage and wCer5 had ~5x coverage, thereby confirming wCer1 and wCer2 as high titre infections, and wCer4 and wCer5 as low titre infections in this species.We also searched the ddRadseq reads mapped to the wCer1 variants and between the HT1 mitogenomes of RcerIZ and RcerHB (11 SNPs and 17 SNPs respectively), equated to 117x relatively more mutations in the mitogenome (Wolbachia genome is approximately 76x larger than the mitogenome). The wCer1 genome comparison of RcerAS and RcerHB revealed 14 SNPs, the mitogenomes of those samples had 24 SNPs, which equated to 130x relatively more mutations in the mitogenome. The wCer1 genome comparison of RcerAS and RcerIZ revealed 19 SNPs, the mitogenomes of those samples differed by 29 SNPs, i.e. 116x relatively more mutations in the mitogenome.The number of mutations between the Wolbachia strain genomes, wCer1, wCer4 and wCer5, of the European cherry fruit fly, R. cerasi, and analysed these in conjunction with the previously sequenced genome of wCer2  genes that recapitulate CI  gene pair [cifwCer2[T5] gene pair for which only cifB had previously been annotated  subclade, however the wMel-like Type I cifA gene could also play a role. Such a prediction is supported by the demonstrated CifA rescue of CI induced by a similar but non-cognate CifA/B pair  may induce CI that is not rescued by Type I cif due to their dissimilarity. While there is no experimental evidence for CI induction by cifwCer2[T5], Type V cif genes have characteristics of other cif types: (i) the cifA and cifB genes are adjacent, transcribed in the same direction and located in prophage regions; (ii) the domain structure is similar to cifwPip[T4]  but only had 36 and 45% similarity with cifwCer2[T5]. Both cif gene pairs are potentially responsible for CI in wCer4 and one or both are presumably incompatible with the wCer2 cif gene pairs. The substantial divergence between the Type V cif genes in wCer2 and wCer4 suggests that they are likely incompatible. The repertoire of cif genes in wCer4 also indicates this strain may be incompatible with wCer1, due to the absence of Type V cif genes in wCer1.Furthermore, capitata . We founwCer5 can induce CI, the genome sequencing of wCer5 showed a high similarity of cifwCer5[T4] to CI-inducing cifwPip[T4] (over 99.6% amino acid identity), and is therefore likely to induce CI. Furthermore, wCer5 is the only wCer genome with Type IV cif genes in this host species, and unlike the other wCer genomes has no Type I cif genes. Therefore, wCer5 is likely bidirectionally incompatible with wCer1, wCer2 and wCer4, and this could cause issues, e.g. slowing down of a wCer2 invasion between populations that are polymorphic in infection status. One would expect that for invasion to be successful either wCer2 and wCer5 spread should be linked, or wCer5 should already exist at a high enough prevalence in populations that are being invaded.While it has not been demonstrated in crossing experiments that wCer genomes contain orthologues of the wmk gene sequence which can simulate a MK phenotype in transgenic D. melanogaster  [cifBwPip[T4]  cannot be rescued by either cognate cifA or non-cognate cifwMel[T1], so lethality is not strictly through CI   and cifBwPip[T4]  when therough CI , and thisimulans .wCer5 variants is larger than the divergence between the respective wCer1 variants (by 60%). This indicates that wCer5 has been associated with R. cerasi for a relatively longer period than wCer1. Furthermore, the variant analysis showed that strain variants comprised a polymorphic population. For wCer5 there were few variable site overlaps, and therefore more accrued  differences between the two sequenced individuals; in contrast, there were many variable site overlaps for wCer1 variants of the same two individuals. The linkage of wCer1 with HT1 is a clear indication of a more recent invasion of wCer1 than wCer5. No such link for wCer5 with a mitochondrial haplotype was detected, and this could indicate that a previously existing linkage may have broken down as a consequence of the wCer1 invasion in R. cerasi. wCer5 maintains high prevalence in some R. cerasi populations where it routinely co-occurs with wCer1, wCer2 and wCer4  or multiple strains in a single host, and resolve the fitness costs (toxicity or incomplete CI self-rescue) that have been demonstrated for single infections of wCer2 and wCer4 in novel hosts. Furthermore, mitogenomes and Wolbachia genomes from WGS projects can guide and increase resolution of SNP analyses from reduced representation genomic datasets, such as ddRadSeq. This enabled us to link Wolbachia strain infection with mitogenome haplotypes in individuals and clearly demonstrated haplotype variation associated with Wolbachia infections and the more recent acquisition of wCer2.Our analyses of the four R. cerasi: RcerAS from Stillfried, Austria (approximately 40 km NE of Vienna), collected in 2001; RcerIZ from Zafferana in eastern Sicily, Italy, collected in 2001; and RcerHB from Bajna, Hungary (approximately 40 km NW of Budapest), collected in 2000  by microinjection, using a donor population of R. cerasi from Sicily , wNo [T3], wPip [T4] and wStri [T5]. The nomenclature of cif gene pairs has recently been proposed to follow the format of cifwStrain[T1] as an example for a Type I pair [cifA alignment excluded orthologues if they were truncated and did not contain the unannotated N-terminal region, or the catalase-rel or DUF domains, because mutations in any of these essential regions can diminish CI and rescue [cifB gene alignment included orthologues only if they contained the unannotated N-terminal region and the two PDDEXK domains common to Type I and Type IV cifB genes which have both been determined experimentally to induce CI. Maximum likelihood trees were estimated from the gene alignments as described above, using models TPM3 + F + G4 for cifA and TPM3 + F + I + G4 for cifB.Orthologues of e I pair . Proteine I pair , furin ce I pair  and the e I pair . The gene I pair  and the e I pair . The cifd rescue . The cifwmk orthologues were identified in the wCer genomes from the orthogroup containing wMel WD0626 (wmk). No orthologues were found in wCer1, but each orthologue from wMel (seven genes), wRec (one gene), wCer2 (eight genes), wCer4 (five genes) and wCer5 (three genes) were codon-aligned in MEGA v7 using Muscle, and the amino acid pairwise distances were calculated.wCer1 variants of three populations , and between wCer5 variants of two populations , were identified by read mapping using CLC Genomics Workbench to the final draft genomes at a similarity of 97% over 97% of the read length, and only properly paired reads were kept. These parameters differed from the parameters used to verify the draft genome sequences because here we wanted to capture strain variation within an individual. For the RcerAS library , reads were competitively mapped to the wCer2 (GenBank Accession No: SOZK01000000) as well as the wCer1 and wCer5 genomes simultaneously to restrict errors, primarily due to wCer2 reads mapping to the wCer1 genome. This problem was likely to occur because wCer1 and wCer2 are both supergroup A strains and wCer2 reads were more abundant than wCer1 reads and would therefore inflate the outcome of variant detection. The RcerIZ library was competitively mapped to wCer1 and wCer5, and RcerHB was only mapped to wCer1. These stringency parameters allowed for polymorphisms to be detected, while minimising off target reads.Polymorphisms between wCer1 variant SNP calling, the RcerAS and RcerHB libraries were subsampled (26 million and 10 million reads respectively) to normalize the number of wCer1 reads against the full RcerIZ library that had 258,194 properly paired reads mapped to wCer1 (average 20x coverage). For wCer5 variant SNP calling, the RcerAS library was not subsampled to the level of RcerIZ, because the low number of reads from RcerIZ  gave very low and sparse coverage (average 4x). The full RcerAS library was used and provided ~18x coverage. In order to aid analysis, variants of wCer2 from two D. simulans lines and one C. capitata line carrying single infections of wCer2 [wCer2 from RcerAS subsampled to 87 million reads. To determine the consensus sequences, no minimum read number threshold was applied and the majority (> 50% reads) nucleotides were extracted for each Wolbachia strain derived from each library to determine a library specific genome. Alignment of these consensus sequences  generate a library-specific consensus sequence for each strain and (ii) detect variation within individuals for each strain. For of wCer2  were alsg Popart . For varR. cerasi de novo assembly via BLASTn match to the C. capitata complete mitochondrial genome (GenBank Acc: AJ242872). The sequencing reads were mapped at high stringency (97% similarity and 90% length) and the circular genomes closed and verified by mapping at 99% similarity and 95% length. Protein coding genes (PCGs), tRNAs and rRNAs were annotated using Mitos2 [The mitochondrial genome contigs were extracted from each R. cerasi mitochondrial genomes were identified by mapping a subsample of each library to the RcerHB mitochondrial genome at 97% similarity and 97% length. Each library was subsampled to achieve approximately 500-fold coverage of the mitogenome, hence RcerHB , RcerIZ  and RcerAS  were sampled and mapped to the RcerHB mitogenome. Variant SNPs were called with a low frequency cut-off of 1%, and differences between populations were identified when found at > 99% frequency.SNPs across the three R. cerasi individuals from six countries  published as part of a population study [Wolbachia genomes  and the mitochondrial genome of RcerHB at 85% of read length and 98% similarity, and only reads specific to a single genome were retained. This low length stringency was chosen so the overhanging barcode of 8 to 10 nucleotides met the parameters. The barcodes were used to identify the samples that mapped to regions on the mitochondrial genome that showed variability. These samples were scored for Wolbachia strain presence by examining mapping coverage over the four Wolbachia genomes. The threshold selected was at least one perfect read over at least five mapped regions of the genome. This threshold meant that low titre strains were reliably detected (even at one-fold coverage over many regions) but eliminated misallocation of reads to a different Wolbachia strain where the genome was incomplete; this could have occurred if wCer5 was present but the conservative approach to its genome assembly resulted in some of its reads mapping to another genome.A ddRADseq dataset representing 192 on study  was downwCer2, if they had wCer4 or wCer5, and by country of origin (with Sicily divided into Sicily West and Sicily East). Adonis of the R package vegan [Mitochondrial SNPs for each of 46 samples were identified from the ddRadseq mapped reads, tabulated and converted to a genind object and a Euclidean distance matrix using adegenet , 89 in Rge vegan  was implAdditional file 1. UpSet graph showing shared Wolbachia orthogroups.Additional file 2. PHASTER identification of prophage regions in wCer1, wCer4 and wCer5.Additional file 3. cifA and cifB locus names from Wolbachia strains present in phylogeny.Additional file 4. Mitochondrial genomes and the polymorphic sites between RcerHB (HT1a), RcerIZ (HT1b) and RcerAS (HT2).Additional file 5. Polymorphisms in mitogenomes of three complete mitogenomes of RcerHB (HT1a), RcerIZ (HT1b) and RcerAS (HT2) and an additional 46 samples with SNP representation at 12 of these poymorphic sites from ddRadSeq data. Sample names and location are from Bakovic et al [ic et al , togetheAdditional file 6. PERMANOVA of mitochondrial genetic distances between samples grouped by presence of Wolbachia strains.Additional file 7. FASTA aligned wCer1 consensus sequences from RcerHB, RcerIZ and RcerAS.Additional file 8. FASTA aligned wCer5 consensus sequences from RcerIZ and RcerAS.Additional file 9. FASTA aligned wCer2 consensus sequences from RcerAS, DsimRC45, DsimRC50 and Ccap88.6.Additional file 10.wCer1 variant calling. Reference position refers to the wCer1 genome position after the 16 contigs were joined in order. Reads from three libraries: RcerHB (sampled to 10 million reads), RcerIZ  and RcerAS (sampled to 26 milion reads), were mapped at 97% length and 97% similarity, and RcerIZ and RcerAS were mapped competitively to the genomes of wCer1 and wCer5; and wCer1, wCer2 and wCer5, respectively. Variants were called with a minimum cut-off of 35% frequency, so a frequency of 65% for a variant is considered homozygous. Location of SNPs within genes is based on PROKKA annotation, and determination of copy number (single or multiple copy) was based on Orthofinder assessment of orthogroups including wCer1, wAu and wMel genomes.Additional file 11. wCer5 variant calling. Reference position refers to the wCer5 genome position after the 57 contigs were joined in order. Reads from two libraries: RcerIZ  and RcerAS , were mapped at 97% length and 97% similarity, and RcerIZ and RcerAS were mapped competitively to the genomes of wCer1 and wCer5, and wCer1, wCer2 and wCer5, respectively. Variants were called with a minimum cut-off of 35% so a frequency of 65% for a variant is considered homozygous. Location of SNPs within genes is based on PROKKA annotation, and determination of copy number (single or multiple copy) was based on Orthofinder assessment of orthogroups including wCer5, wMeg and wPip genomes.Additional file 12. wCer2 variant calling. Reference position refers to the wCer2 genome position after the 11 contigs were joined in order. RcerAS (sampled to 87 million reads) was mapped competitively to the genomes of wCer1, wCer2 and wCer5, respectively at 97% length and 97% similarity. Variants were called with a minimum cut-off of 35% frequency.


All the synthesised compounds inhibit HDAC1 and HDAC2 selectively over other isoforms and many inhibit DLD1 and HCT116 cells more effectively than a parent compound. Compounds 8 and 16 inhibit HCT116 cells by activation of the apoptosis pathway.Five pathways involving different ring structures led to generation of fourteen thienylbenzamides (


Following the publication of this article , the aut◦ The ◦ The ◦ The In In In Fig 5D, lane 1 of the β-actin of MRC5 panel for the ATII-MRC5 experiment is identical to lane 2 of the β-actin of MRC5 panel for the ATII-MRC5 experiment in Contradictory to the figure legend, the blots presented in Quantitative data files in the Supporting Information tables  Tables iThe authors commented that the issues in In relation to the issues highlighted by the authors in Figs 5D and The authors submitted image data to support the western blots presented in Figs The available quantitative data underlying the results presented in this article are provided in the Furthermore, the authors notified PLOS of the below errors in the Supporting Information tables. Updated Supporting Information tables in which these issues have been addressed are provided with this notice.In In In In In In In PLOS ONE editorial team, and it was concluded that the errors did not impact the results presented in the corresponding graphs. Additionally, as The updated tables were reviewed by the PLOS ONE Editors issue this Expression of Concern to notify readers of the above concerns and relay the supporting data and updated figures provided by the authors.The S2 File(A) The blots in (TIF)Click here for additional data file.S3 File(PDF)Click here for additional data file.S4 File(PDF)Click here for additional data file.S5 File(PDF)Click here for additional data file.S6 FileNormalized data for Figs (ZIP)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table(DOC)Click here for additional data file.S5 Table(DOC)Click here for additional data file.S6 Table(DOC)Click here for additional data file.S7 TableUpdated (DOC)Click here for additional data file.S8 TableUpdated (DOC)Click here for additional data file.S9 Table(DOC)Click here for additional data file.S10 Table(DOC)Click here for additional data file.


NF1 deletions often exhibit more severe clinical manifestations than patients with intragenic NF1 gene mutations, including facial dysmorphic features, overgrowth, severe global developmental delay, severe autistic symptoms and considerably reduced cognitive abilities, all of which are detectable from a very young age. Type 1 NF1 deletions encompass 1.4 Mb and are associated with the loss of 14 protein-coding genes, including NF1 and SUZ12. Atypical NF1 deletions, which do not encompass all 14 protein-coding genes located within the type 1 NF1 deletion region, have the potential to contribute to the delineation of the genotype/phenotype relationship in patients with NF1 microdeletions. Here, we review all atypical NF1 deletions reported to date as well as the clinical phenotype observed in the patients concerned. We compare these findings with those of a newly identified atypical NF1 deletion of 698 kb which, in addition to the NF1 gene, includes five genes located centromeric to NF1. The atypical NF1 deletion in this patient does not include the SUZ12 gene but does encompass CRLF3. Comparative analysis of such atypical NF1 deletions suggests that SUZ12 hemizygosity is likely to contribute significantly to the reduced cognitive abilities, severe global developmental delay and facial dysmorphisms observed in patients with type 1 NF1 deletions.Patients with neurofibromatosis type 1 (NF1) and type 1  NF1 gene and its flanking regions at 17q11.2  17q11.2(28997893-29695563)x1). The centromeric deletion breakpoint is located within NF1-REPa, and the telomeric deletion breakpoint within intron 57 of the NF1 gene, as confirmed by MLPA analysis. The deletion identified in patient 310221 encompasses the NF1 gene, its three embedded genes and the five genes located centromeric to NF1, namely CRLF3, ATAD5, TEFM, ADAP2 and RNF135  are loss-of-function intolerant, as determined by the metric ‘probability of being loss-of-function intolerant (pLI)’ ’ . The pLINeither array analysis nor MLPA analysis of blood and saliva-derived genomic DNA of the patient indicated the presence of somatic mosaicism with normal cells. The deletion of patient 310221 was not identified in the blood of his parents or his healthy sister and is hence considered to have occurred de novo.NF1 deletions have been reported [NF1 deletions can be classified into two groups: The first encompasses very large deletions extending beyond one or both of the boundaries of the 1.4 Mb type 1 NF1 microdeletion region which is flanked by NF1-REPa and NF1-REPc. In total, 31 such large atypical NF1 deletions have been identified, which together constitute group #1 . Mutatifeatures . Since irelation . SUZ12 mutations exhibit an overgrowth phenotype [NF1 deletions. In contrast to the short stature observed in most patients with intragenic NF1 mutations, tall stature in adults and overgrowth during childhood have been reported in patients with NF1 microdeletions [RNF135 gene located centromeric to NF1 within the type 1 NF1 deletion region has been suggested to be associated with the overgrowth seen in patients with NF1 microdeletions [NF1 deletions that do not encompass the RNF135 gene but are associated with the loss of SUZ12 [SUZ12 resulting in SUZ12 haploinsufficiency is likely to contribute to the overgrowth phenotype observed in patients with type 1 NF1 microdeletions. This conclusion is supported by the observation that SUZ12 is not included within the deletion interval in patient 310221 who is small for age. Overgrowth or tall stature was also not reported for other patients with atypical NF1 deletions of group #2  of 21 children with type 1 NF1 deletions, which was significantly more frequent than in the general NF1 population [In addition to ADHD, a high incidence of autism has been observed in patients with NF1 ,99,100. pulation . AutistiNF1 microdeletion-associated phenotype and in particular autism has recently been demonstrated for the cytokine receptor-like factor 3 (CRLF3) gene located within the NF1 microdeletion region. CRLF3 is deleted in patient 310221 reported here  present in addition to pathogenic variants in NF1. Taken together, these findings suggest an essential role for CRLF3 in both human brain development and autism [CRLF3 gene in the patient described here may have contributed to his autistic symptoms. Further analyses of patients with different types of NF1 microdeletion will confirm or refute the possible contribution of the CRLF3 gene to the deletion-associated phenotype.A specific role in the development of the ted here . InducedNF1 deletions of group #2, such as the 698 kb deletion observed in patient 310221, are potentially very informative in terms of genotype/phenotype correlations. However, the possibility that atypical NF1 deletions can be associated with mosaicism with normal cells not harbouring the deletion must be considered. Vogt et al. [NF1 deletions. In 6 of these 10 patients, mosaicism was analysed by FISH which allowed the determination of the proportion of cells harbouring the deletion. In the blood samples of these six patients, the proportion of cells with the deletions was 70%, 75%, 80%, 93%, 96% and 98%, respectively. In three (50%) of the six patients, mosaicism was also detected by MLPA, which is considered to have an intrinsic detection limit of ~10%  in those patients whose NF1 microdeletions encompass this gene. However, further studies are necessary to investigate this in greater detail. To date, genotype/phenotype correlations for group #2 atypical NF1 deletions are limited by the scarcity of clinical details available for many of the reported patients and by limited breakpoint definition due to the use of MLPA instead of microarray analysis. Moreover, information about possible mosaicism with cells not harbouring the deletion is not available for many patients with atypical NF1 deletions. The future analysis of genetically and clinically well-characterized patients with atypical NF1 deletions will serve to further clarify the role of the loss of genes such as SUZ12 and CRLF3 for the NF1 microdeletion-associated phenotype.Group #2 atypical


After publication of this article , concernThe authors provided underlying image data for all panels shown in Figs 3 and The raw image data revealed that the pAkt and pERK blot images were spliced to remove data between lanes 4 and 5 on the published Figure 3C see  Files.In regard to the control panels included in Figs 3 and The raw data underlying other results in this article are available from the authors.The authors apologize for the errors in the published article.S1 FileOriginal blot data supporting the results in Figure 3A.(PDF)Click here for additional data file.S2 FileOriginal blot data supporting the results in Figure 3C.(PDF)Click here for additional data file.S3 FileOriginal blot data supporting the results in (PDF)Click here for additional data file.S4 FileOriginal blot data supporting the results in the corrected (PDF)Click here for additional data file.S5 FileAdditional original blot data supporting the results in Figs 3 and (PDF)Click here for additional data file.


In-stent restenosis and stent thrombosis are the major concerns while choosing a coronary stent. This single-centre, retrospective study evaluated the one and three-year clinical outcomes following implantation of Yukon Choice Flex (YCF) sirolimus-eluting stent. A total of 168 consecutive patients with 217 lesions underwent stenting with YCF stent. The presentation was with acute coronary syndrome in 158 (94%) patients. At 3 years, 9 (5.3%) patients died due to cardiac cause. Myocardial infarction, and definite stent thrombosis occurred in 10 (6%) and 4 (2.4%) patients respectively. Redo stenting and coronary artery bypass surgery was performed in 3 (1.8%) and 1 (0.6%) patient respectively. The use of YCF sirolimus eluting stent was associated with a favourable safety and efficacy profile at one and three-years of follow-up in a high-risk population. Patients who died before PCI or had significant renal dysfunction or sepsis, severe thrombocytopenia or coagulation abnormalities, those who had a delayed presentation with refractory left ventricular failure (LVF) or refractory cardiogenic shock in whom PCI could not be performed, were not enrolled. The study aimed at studying the efficacy and outcomes of patients who underwent PCI with YCF stent at 1 and 3 years.The study protocol conformed to the ethical guidelines of the declaration of Helsinki and was reviewed and cleared by the institute's ethical committee.Clinical diagnosis included 88 (52.4%) patients with ST-segment elevation myocardial infarction (STEMI), 70 (41.6%) patients with non–ST-segment elevation ACS [including non-ST elevation myocardial infarction (NSTEMI) in 42 (25%) patients and unstable angina in 28 (16.6%) patients], and 10 (6%) patients with chronic coronary syndromes . The culStatistical analysis was performed with Statistical Package for the Social Sciences version 26 .3A total 217 YCF stents were deployed in 168 patients. The mean age of the participants were 56.4 ± 12.68 years. The study population were predominantly male (85.9%). The presentation was with ACS in 158 (94%) of study subjects. The baseline characteristics of patients were summarized in The baseline angiographic characteristics were tabulated in Clinical follow up was performed for all patients for three years. At the end of 3 years, a total of 12 (7.1%) deaths occurred, out of which 9 (5.3%) patients died due to presumed cardiac cause. Other outcomes assessed at the end of 3 years was myocardial infraction in 10 (6%) patients, definite stent thrombosis in 4 (2.4%) patients and target lesion revascularization was needed in 2 (1.2%) patients. Individual outcomes at the end of 1 year and from 1 to 3 years are summarized in Redo PCI was done in 3 patients. Coronary artery bypass graft surgery was done in 1 patient during the study period.4,,PCI is the revascularisation modality of choice in most patients with ACS and suitable coronary anatomy. The odds of the development of stent failure in the form of in-stent restenosis and stent thrombosis are important considerations while selecting a stent.Even though the debate over the efficacy and safety of DES versus BMS is continuing; contemporary data suggest that the benefits of DES outweigh the risks compared with BMS mirroring the current increase in DES usage.,Our study population consisted predominantly of ACS patients. More than half of the patients with ACS had STEMI which was similar to other studies from India and west including the INTERHEART study.In an observational study of YCF stent by Xhepa et al, the rates of death, myocardial infarction, definite stent thrombosis and ischemia-driven target lesion revascularization were 2.4%, 1.9%, 0.3% and 11.3% respectively at 1 year.,15We would like to reiterate the fact that the study aimed to study the efficacy of YCF stent and the intermediate and long-term outcomes of patient undergoing PCI with YCF. It was not designed to look at outcomes of ACS patients as such. Still the lower 1 year mortality at 4.2% in our study can be explained by the relatively younger patient powith mean patient age of 56.4 ± 12.68 years. Much lesser patients had diabetes compared to other larger studies and the mean ejection fraction was higher at 46.3 ± 9.21% compared to prior studies.A study similar to ours by Liu et al which enrolled patients of STEMI and NSTEMI undergoing PCI with DES had an overall mortality rate of 5.6% at 5 years and was comparable to our data of 7.1% mortality at 3 years.The major limitations of our study was the absence of routine angiographic follow up and a scarce use of intravascular imaging for PCI optimization.The mean left ventricle ejection fraction in our study was 46.3 ± 9.21%, which was higher for a cohort predominantly comprising of an ACS population. The sickest of patients who either died before PCI could be performed or had significant renal dysfunction or sepsis, severe thrombocytopenia or coagulation abnormalities or had a delayed presentation with refractory LVF or refractory cardiogenic shock in whom PCI could not be performed, were not enrolled. One would expect these patients to be having a lower LVEF compared to patients undergoing PCI in our study. As a result, selection bias cannot be ruled out.This study provides 3-year clinical outcome data of Sirolimus-eluting Yukon choice flex stent in a real-world setting and highlights the fact that improved stent design technology will lead to comparable clinical outcomes.In conclusion, the use of YCF sirolimus eluting stent was associated with a favourable safety and efficacy profile at one and three-years of follow-up in a high-risk population.There is no conflict of interest amongst the authors in regards to the present study.


Better markers of early response to neoadjuvant chemotherapy (NACT) in patients with breast cancer are required to enable the timely identification of non-responders and reduce unnecessary treatment side-effects. Early functional imaging may better predict response to treatment than conventional measures of tumour size. The purpose of this study was to test the hypothesis that the change in tumour blood flow after one cycle of NACT would predict pathological response.In this prospective cohort study, dynamic contrast-enhanced MRI was performed in 35 females with breast cancer before and after one cycle of epirubicin and cyclophosphamide-based NACT (EC90). Estimates of tumour blood flow and tumour volume were compared with pathological response obtained at surgery following completion of NACT.Tumour blood flow at baseline  reduced slightly after one cycle of NACT (0.28 ± 0.18 ml/min/ml). Following treatment 15 patients were identified as pathological responders and 20 as non-responders. There were no relationships found between tumour blood flow and pathological response. Conversely, tumour volume was found to be a good predictor of pathological response  at both baseline (area under the receiver operating characteristic curve 0.80) and after one cycle of NACT (area under the receiver operating characteristic curve 0.81).The change in breast tumour blood flow following one cycle of EC90 did not predict pathological response. Tumour volume may be a better early marker of response with such agents. Typically, a change in tumour size is demonstrated after two or more cycles of NACT2 but several studies have examined functional imaging techniques to assess response after only one cycle.3–5 Amongst the functional imaging techniques 18F-FDG PET has been used to assess changes in tumour glucose metabolism following NACT6 and a number of PET studies have looked specifically at breast tumour blood flow with either [15-O] H2O PET7 or dynamic 18F-FDG PET.8 However, repeated PET imaging is impractical for routine clinical use9 and so MRI techniques that measure parameters related to flow, via dynamic contrast-enhanced (DCE) MRI, have also been studied.10 A difference between these MRI approaches and PET imaging of blood flow is the lack of absolute quantification by MRI. Moreover, there has been a lack of consistency in study methodology and the reporting of pathological response in MRI studies over the years.10–12Neoadjuvant chemotherapy (NACT) offers an increased rate of breast conserving surgery, can downsize advanced breast tumours to allow surgery and with the addition of human epidermal growth factor receptor 2 (HER2)-targeted therapy (to NACT or adjuvant chemotherapy) can improve survival in HER2-positive breast cancer.13 to measure tumour blood flow at baseline and after one cycle of NACT in order to test the hypothesis that the change in blood flow after one cycle of NACT would predict pathological response as defined according to international recommendations.14 Analysis of the HTR data to estimate blood flow also allowed us to assess related haemodynamic parameters such as blood volume, capillary permeability surface–area product (PS) and interstitial volume and, by using the HSR data, we were able to assess tumour volume.In this study, we used a recently developed interleaved high spatial resolution (HSR)/high temporal resolution (HTR) MRI technique2) and cyclophosphamide (600 mg/m2)] followed by three 3-weekly cycles of docetaxel (100 mg/m2). In patients with HER2 positive tumours, docetaxel was accompanied by trastuzumab, and in some (more recent) cases pertuzumab. Blood samples, taken as standard of care during clinic visits, were used to estimate haematocrit at each MRI visit.In this prospective study consecutive patients, with newly diagnosed primary invasive carcinoma of the breast due to undergo NACT with curative intent, were approached and asked if they were willing to take part. All those agreeing to join the local Research Ethics Committee approved study provided written informed consent after the nature of the procedures had been fully explained. Each underwent a block sequential regimen of NACT: three 3-weekly cycles of EC90  when they were not (assessed using the Shapiro–Wilk test). To determine whether differences existed between the measured parameters, estimates from pR and pNR were compared using the non-parametric Mann–Whitney Between August 2015 and April 2018, 40 female patients  gave written informed consent to enter the study. MRI data were obtained in all 40 patients at baseline (7 [4 - 12] days before the first administration of NACT). However, one patient was unable to tolerate the complete imaging protocol and technical problems in one other (back coil was switched on and off intermittently) precluded the measurement of tumour blood flow. Following a single cycle of EC90, 37 patients were scanned (three patients declined further MRI scans). Technical problems (as previously) affected one further scan, so blood flow estimates were obtained in 36 patients. Paired baseline and cycle one data were available for 35 patients and the results presented below reflect these 35 patients only .The immunohistochemical subtype (IHS) of the 35 tumours were: 2 HER2 enriched (oestrogen receptor and progesterone receptor negative and HER2 positive), 9 triple negative and 24 luminal B (oestrogen receptor and/or progesterone receptor positive and HER2 positive or HER2 negative with high levels of Ki-67). Following the completion of NACT and surgery, pathological analysis revealed 6 patients (17%) had a pathological complete response (RCB-0), 9 patients had minimal residual disease (RCB-I), 12 patients moderate (RCB-II) and 8 patients extensive residual disease (RCB-III). Thus, there were deemed to be 15 pR (43%) and 20 pNR (57%).3 and patients going on to a pR had tumours that were significantly smaller than those that went on to pNR (p = 0.003). There was a small but significant decrease in tumour volume following one cycle of NACT  and tumour volume at baseline and after cycle one were good predictors of pR (area under the ROC curve 0.80 (95% CI 0.66 to 0.95) and 0.81 (95% CI 0.66 to 0.95), respectively). Using a volume threshold at cycle one to predict response failure, 12 patients with tumours of 9 cm3 or larger went on to pNR  and all 15 patients who went on to a pR had tumours smaller than 9 cm3 (100% specificity). This test had a positive predictive value of 100%.Pathological complete response to NACT varied as a function of IHS – neither of the patients with HER2 enriched tumours, 3/9 (33%) patients with triple negative tumours and 3/24 (12.5%) patients with luminal B tumours had a complete pathological response. Less variation was seen in pR where 1/2 (50%) patients with HER2 enriched tumours showed pR compared to 3/9 (33%) with triple negative tumours and 11/24 (46%) with luminal B tumours. Luminal B tumours that were HER2 positive responded better to NACT: 9/12 (75%) HER2 positive tumours had a pR compared to only 2/12 (17%) HER2 negative tumours. Median enhancing tumour volume at baseline was 4.8 [2.3 to 22.5] cmT1 at baseline (1287 ± 87 ms) showed no change following one cycle of NACT (1285 ± 92 ms) and showed no significant variation as a function of IHS (Tumour blood flow at baseline (0.32 ± 0.17 ml/min/ml) and after cycle one (0.28 ± 0.18 ml/min/ml) showed little variation as a function of IHS  or pathon of IHS .p = 0.007) after one cycle of NACT and this was mirrored in an increase of blood T1 from a baseline value of 1660 ± 64 ms to 1766 ± 109 ms (p = 0.00002) after one cycle of NACT.15Patient haematocrit decreased from a baseline value of 0.41 ± 0.03 to 0.39 ± 0.03  of NACT.7 We did not see this effect in our study following just one cycle of NACT. There was, on average, a decrease in blood flow of 12% but this was subject to considerable variation, with both increases and decreases seen in both pR and pNR (8 who saw a large variability in blood flow response in HER2 negative tumours after one cycle of EC chemotherapy (HER2 positive patients in that study experienced a large decrease in blood flow but they were selectively treated with docetaxel and trastuzumab). The primary hypothesis of this study - that change in blood flow after one cycle would predict pathological response - was not supported by our resultsAt least one previous [15-O] H and pNR . Our res10 was predictive of response. This mirrors results seen in the ACRIN 6657 trial that highlighted the importance of enhancing tumour volume, measured at cycle one, to recurrence-free survival.19 At cycle one of our study, 12 of the 35 tumours were 9 cm3 or larger . These patients could potentially have been spared further NACT since all failed to respond (pNR). Of the remaining 23 patients with tumours smaller than 9 cm3, 15 went on to a pR leaving only 8 non-responding patients. Further work is required to identify a specific imaging phenotype to identify those tumours at the early stages of NACT.Conversely, tumour volume, which was identified as a more reliable marker of response than tumour diameter in a previous review,20 Previous quantitative MRI studies have typically reported parameters such as Ktrans (volume transfer constant) without measuring patient-specific arterial input functions21–23 or by measuring the input function inadequately24 . It is well recognised that measurement of the arterial input function is very challenging.11 As in the current study, some reports have highlighted the importance of tumour size after a single cycle of NACT as a predictor of response while tracer kinetic parameters have been less useful21,25; others found neither size nor tracer kinetic parameters useful at this timepoint.22 Although the current study lacks direct validation of the blood flow estimates, the values reported very closely reflect previously published estimates obtained using the ‘gold-standard’ of [O-15] H2O PET in a similar group of patients.26 Moreover, the lack of variation in tumour blood flow as a function of IHS seen in the PET study26 is similarly seen in our data . Despite the well-established variation in response to NACT of the different IHS of breast cancer, there appears to be very little difference in their haemodynamic characteristics at baseline and no clear patterns of change following one cycle of NACT. The decrease of blood flow seen in responders later in the course of NACT by others7 is not yet apparent after one cycle. Measurements of blood flow may prove to be more revealing when assessing NACT regimens with expected antiangiogenic effects.8This is the first published study to report absolute estimates of breast tumour blood flow and related parameters measured using MRI in response to treatment.T1 measurements plus a few additional DCE acquisitions) and this could be reduced if a faster B1-insensitive T1 measurement sequence were employed. The interleaving of HTR data with the clinical HSR data did not have a significant impact on clinical reporting.13 Perhaps more significant limitations are the small number of patients studied and the post-processing overhead both for tumour volume estimation and tracer kinetic analysis, which currently rely on in-house software. Further work is required to develop user-friendly tailored software for these applications.The additional time required to acquire data for blood flow estimation amounts to less than 10 min  show little variation with tumour IHS at baseline or in response to one cycle of NACT and, contradicting our primary hypothesis, blood flow change following one cycle of NACT did not predict pathological response. In contrast and reflecting the findings of a recent multicentre trial, enhancing tumour volume measured after one cycle of NACT appears to show promise for prediction of pathological response.


Background and Objectives: The aim of this study was to evaluate longitudinal changes using both upper limb muscle Magnetic Resonance Imaging (MRI) at shoulder, arm and forearm levels and Performance of upper limb (PUL) in ambulant and non-ambulant Duchenne Muscular Dystrophy (DMD) patients. We also wished to define whether baseline muscle MRI could help to predict functional changes after one year. Materials and Methods: Twenty-seven patients had both baseline and 12month muscle MRI and PUL assessments one year later. Results: Ten were ambulant (age range 5–16 years), and 17 non ambulant (age range 10–30 years). Increased abnormalities equal or more than 1.5 point on muscle MRI at follow up were found on all domains: at shoulder level 12/27 patients (44%), at arm level 4/27 (15%) and at forearm level 6/27 (22%). Lower follow up PUL score were found in 8/27 patients (30%) at shoulder level, in 9/27 patients (33%) at mid-level whereas no functional changes were found at distal level. There was no constant association between baseline MRI scores and follow up PUL scores at arm and forearm levels but at shoulder level patients with moderate impairment on the baseline MRI scores between 16 and 34 had the highest risk of decreased function on PUL over a year. Conclusions: Our results confirmed that the integrated use of functional scales and imaging can help to monitor functional and MRI changes over time. In the last few years, the development of new outcome measures has allowed a better definition of the natural history of Duchenne Muscular Dystrophy (DMD), a progressive, X-linked neuromuscular disorder caused by mutation in the dystrophin gene, affecting one in 3600–5000 live male births. The absence of functional dystrophin protein leads to a progressive muscle degeneration, weakness and to a predictable pattern of loss of specific functional milestones ,8,9. While in the past the severity of muscle involvement was mainly assessed by muscle biopsy, over the last two decades there has been increasing attention to the use of non-invasive techniques, such as Magnetic Resonance Imaging (MRI) and spectroscopy (MRS) as markers of muscle pathology and disease progression ,18,19,20The aim of the present study was to evaluate longitudinal changes using both upper limb muscle MRI at shoulder, arm and forearm levels and PUL in ambulant and non-ambulant DMD patients. Furthermore, following evidence from lower limb MRI studies showing that subtle muscle MR changes may precede functional changes in boys with DMD ,29 we alThe study is part of a project aimed at establishing muscle MRI changes in DMD. The study was approved by the Ethics committee of our Institution . Written informed consent was obtained for all the patients who agreed to participate. For the minors, consent was obtained by their parents. As part of this study, we enrolled DMD patients attending their routine follow up clinics between June 2017 and December 2018. The exclusion criteria were related to the impossibility to perform MRI without sedation, therefore excluding very young children or those with severe cognitive or behavioral problems. We also excluded patients with severe joint contractures, pacemakers, respiratory or cardiac problems that would interfere with positioning or performing MRI. The investigations were carried out following the rules of the revised Declaration of Helsinki.Unilateral upper-limb MRI was performed at 1.5T  using a flexible body coil (body SENSE 32 Channel Flex Coil). Subjects lay in the scanner in the head-first supine position, with the dominant upper limb to be imaged lying in a comfortable position on the scanner bed alongside the torso. The upper limb was stabilized using a fabricated thermoplastic splint and sandbags were placed over the forearm and hand to minimize motion. Boys were encouraged to watch a movie or cartoon during scan acquisition and usually a parent and/or a staff member was present in the scan room during scan acquisition. The patients did not receive any sedation and the total examination time was approximately 20 to 30 min.Non contrast-enhanced images were obtained from the dominant upper limb.TSE T1-weighted spin-echo was acquired on axial plane selected in respect to the long axis of the humerus for the shoulder and arm, and in respect of the long axis of the radius for the forearm. The slices were set up to cover the entire extension of the upper limb. Each muscle was evaluated throughout its length.Scan parameters were as follows: TSE T1 .Descriptive analysis was used to identify the muscles that were more frequently affected in the different segments. At shoulder level the muscles examined were: deltoid, supraspinatus, infraspinatus, subscapularis, coraco-brachialis, pectoralis major and minor, teres minor, latissimus dorsi, serratus anterior. At arm level the muscles examined were: biceps brachii, brachialis and triceps brachii. At the forearm level the muscles examined were: supinator, pronator teres, flexor carpi radialis, palmar, flexor digitorum superficialis, flexor carpi ulnaris, flexor digitorum profundus, anconeus, flexor pollicis longus, extensor carpi ulnaris, extensor digiti minimi, extensor digitorum, extensor carpi radialis, brachioradialis, extensor pollicis longus.For each segment, we identified the muscles that were more often spared or affected. Although in the present paper we did not aim to quantify the level of involvement, as this will be separately reported, we used a previously reported classification to have a rough estimate of the level of involvement. All muscles MRI scans were assessed for normal or abnormal signal intensity within the different muscles groups and scored using Mercuri classification ,17, as fThree examiners  scored the scans separately with consensus on the scores of over 90%, In the discordant cases the scans were reviewed by all examiners and an agreement was found.The PUL 2.0  includesIn the PUL 2.0 there is a maximum score of 12 for the shoulder level, 17 for the middle level, and 13 for the distal level. A total score can be achieved by adding the three level scores . In order to predict functional changes we reported MRI scores at baseline and PUL scores at follow-up.p < 0.05.Descriptive statistics  were used. T student was used to compare differences in total scores and subtotal scores of each level  between baseline and follow up for both MRI and PUL. The correlation between MRI scores at baseline and PUL scores at follow up for each level was explored with Pearson correlation test. The level of significance was set at Thirty-one patients who had muscle MRI at baseline were enrolled in the study. Twenty-seven of the 31 patients completed the study one year later. Three of the remaining 4 were enrolled in clinical trials and the other one refused to repeat the scan. Ten of the 27 patients were ambulant (age range 5–16 years), and 17 non ambulant (age range 10–30 years). All the patients were on steroids.p = 0.01) was found between MRI shoulder scores at baseline (between 16 and 34), and PUL shoulder scores at follow up.No significant differences were found at baseline and at follow up for both MRI and PUL in total scores and subtotal scores at each level . No significant correlation was found between baseline MRI total scores, baseline MRI subtotal scores of each level and PUL scores at follow up but for the shoulder level where a moderate correlation ; the total score at follow up ranged between 12.5 and 110 ; MRI changes ranged between 0 and + 14  ; the total score at follow up (1 year later) ranged between 6 and 42 ; PUL changes ranged between −9 and +2  (For details At shoulder level 11/27 (41%) patients had stable scores between baseline and 1 year (changes = 0/0.5), 4/27(15%) + 1 point and 12/27 (44%) showed changes equal or more than 1.5.  of patients. Ten of 27 patients (37%) showed a worsening (a higher MRI score than baseline) in at least 2 or more than 2 muscles. The changes were most frequent in the deltoid muscle (10/27) (37%), followed by latissimus dorsi (8/27) (30%). Pectoralis major and minor and coracobrachialis were the muscles that showed less often changes at follow up. Of the 10 patients with baseline MRI shoulder scores lower than 16, five (50%) had higher scores (>1.5) on follow up MRI.Of the 11 patients with baseline MRI scores between 16 and 34, 6 (54%) patients had higher shoulder scores (>1.5) on follow up MRI.Of the 6 patients with baseline MRI scores >34, 1 (17%) had higher scores (>1.5) on follow up MRI.At shoulder level 19/27 (70%) patients had stable scores between baseline and 1 year (changes = 0/± 1); 5/27 (18%) showed changes between −2 and −3, and 3/27 (11%) showed changes between −4 and −5 (For details Ten patients had baseline shoulder MRI scores lower than 16: 9 of the 10 (90%) showed stable and 1 (10%) lower PUL scores.Eleven patients had baseline MRI scores between 16 and 34: 4 of the 11 (36%) had stable and 7 (64%) lower PUL scores.Six patients had baseline MRI scores >34: all 6 had a baseline PUL score of 0 with no possibility to show further loss (For details At arm level 17/27 (63%) patients had stable scores between baseline and 1 year (changes = 0), 6/27 (22%) +1 point, 4/27 (15%) showed changes equal or >1.5.  with a similar proportion of patients with a score of 2 or higher with the brachialis showing the highest number of scores >3.Of the 11 patients with baseline MRI scores equal or lower than 4, 9 (82%) had stable scores on follow up MRI, 2 (18%) had a score of +1 point.Of the 10 patients with baseline MRI scores between 6 and 7.5, 4 of the 10 (40%) had higher scores (>1.5) on follow up MRI.Of the 6 patients with baseline MRI scores >7.5, 5 (83%) had stable scores on follow up MRI, 1 (17%) had a score of +1 point.At Mid-level 17/27 (63%) patients had stable scores between baseline and 1 year (changes = 0/±1), 8/27 (30%) showed changes between −2 and −3, and 1/27 (4%) with changes below 4 (For details Eleven patients had baseline arm MRI scores lower than 4: 10 of the 11 (91%) had stable and 1 (9%) lower PUL scores.Ten patients had baseline MRI scores between 6 and 7.5: 4 of the 10 (40%) had stable and 6 (54%) lower PUL scores.Six patients had baseline MRI scores >7.5: 3 of the 6 (50%) showed stable, 1 (17%) had + 2 points, and 2 (33%) lower PUL scores  patients had stable scores between baseline and 1 year (changes = 0), 1/27 (4%) + 1 point, 6/27 (22%) showed a changes score above 1.5.  and brachioradialis (52%).Of the 11 patients with baseline MRI scores lower than 6 all (100%) had stable scores on follow up MRI.Of the 11 patients with baseline MRI scores between 6.5 and 36, 4 (36%) had higher scores on follow up MRI.Of the 5 patients with baseline MRI scores more than 36, 2 (40%) had higher scores on follow up MRI (2/5 in supinator and extensors muscles), 1 (2%) had a score of +1.At distal level 26 of the 27 (96%) patients had stable scores at follow up (changes = 0/± 1), and the remaining one (4%) had changes above 2.  ,22,23,24n = 6), or three (n = 3) domains. MRI changes were more often found at shoulder domain, probably related to the higher number of relatively young ambulant patients in our cohort. Although the numbers were relatively small to allow a meaningful statistical analysis taking into account so many variables, it is of interest that in each domain the changes indicating a more severe impairment mainly occurred in muscles which had mild to moderate impairment (between 19 and 34) at baseline and more rarely occurred in the muscles who were more severely impaired or were completely spared at baseline.The possibility to explore all the domains allowed to define which muscles and domains are at higher risk of becoming progressively involved with increased age. MRI changes of at least two points were found in 15/27 (55%) patients on at least one domain explored, with patients showing changes in two (n = 55%) and an overall mean reduction of 2 points/year, in line with previous studies reporting 12 month changes in larger cohorts [p = 0.01). Smaller functional changes were found at the two ends of the MRI spectrum. In patients with low MRI scores at baseline the low risk of fucntional redution was probably related to the relatively preserved muscles. At the other end of the spectrum, in patients with very high MRI scores indicating severe muscle impairment, the PUL scores were already extremely low at baseline and could therefore not show much further deterioration.The MRI scores at baseline could not always predict the risk of showing functional changes over one year. The PUL total scores showed a decrease in 15/27 (At arm and forearm level MRI did not appear to be equally sensitive to detect PUL changes. These results should however be interpreted with caution because of the limited overall number of patients in our cohort and the relatively low number of non-ambulant patients that may have reduced the possibility to observe more changes in the mid or distal domain. Another limitation of the study is that we only performed a visual analysis and better correlation may have was related to the been obtained by using a quantitative MR approach.Another limitation was that related to the exclusion criteria as MRI cannot be easily perfomed in patients with severe behavioural or cognitive problems or severe contractures.Even with these limitations our results confirm that muscle MRI could represent a reliable biomarker to monitor upper limb muscle involvement also in young DMD patients ,29. Our This information can be useful at the time of designing clinical trials or intervention studies to exclude or select patients or to select the most appropriate imaging protocol. The follow up of these patients and the results of the quantitative analysis, both in progress, are needed to provide more accurate information on longitudinal changes at all levels.