Musculoskeletal injuries account for 10 million work-limited days per year and often lead to both acute and/or chronic pain, and increased chances of re-injury or permanent disability. Conservative treatment options include various modalities, nonsteroidal anti-inflammatory drugs, and physical rehabilitation programs. Sustained Acoustic Medicine is an emerging prescription home-use mechanotransductive device to stimulate cellular proliferation, increase microstreaming and cavitation in situ, and to increase tissue profusion and permeability. This research aims to summarize the clinical evidence on Sustained Acoustic Medicine and measurable outcomes in the literature.A systematic literature review was conducted using PubMed, EBSCOhost, Academic Search Complete, Google Scholar and ClinicalTrials.gov to identify studies evaluating the effects of Sustained Acoustic Medicine on the musculoskeletal system of humans. Articles identified were selected based on inclusion criteria and scored on the Downs and Black checklist. Study design, clinical outcomes and primary findings were extracted from included studies for synthesis and meta-analysis statistics.A total of three hundred and seventy-two participants (372) were included in the thirteen clinical research studies reviewed including five (5) level I, four (4) level II and four (4) level IV studies. Sixty-seven (67) participants with neck and back myofascial pain and injury, one hundred and fifty-six (156) participants with moderate to severe knee pain and radiographically confirmed knee osteoarthritis (Kellgren–Lawrence grade II/III), and one hundred forty-nine (149) participants with generalized soft-tissue injury of the elbow, shoulder, back and ankle with limited function. Primary outcomes included daily change in pain intensity, change in Western Ontario McMaster Osteoarthritis Questionnaire, change in Global Rate of Change, and functional outcome measures including dynamometry, grip strength, range-of-motion, and diathermic heating (temperature measurement).Sustained Acoustic Medicine treatment provides tissue heating and tissue recovery, improved patient function and reduction of pain. When patients failed to respond to physical therapy, Sustained Acoustic Medicine proved to be a useful adjunct to facilitate healing and return to work. As a non-invasive and non-narcotic treatment option with an excellent safety profile, Sustained Acoustic Medicine may be considered a good therapeutic option for practitioners. The significant and clinically meaningful reduction in musculoskeletal pain  and improvement in joint function (20–87%) were reported in n = 9 and n = 6 studies, respectively on Sustained Acoustic Medicine.In addition to measurable clinical outcomes reported in the literature, n = 2 studies reported on vigorous therapeutic heat (Δ4 °C to Δ12 °C) and n = 1 study on biological clearance of lactic acid from Sustained Acoustic Medicine treatment in human participants.Clinical evidence, health economic cost effectiveness and health provider positive opinions on Sustained Acoustic Medicine support treatment utilization in musculoskeletal conditions such as Osteoarthritis, tendinopathy, and myofascial pain.Musculoskeletal pain is a common issue experienced by most of the general population at some point over the lifetime . ChronicMusculoskeletal pain can be caused by a variety of conditions including maximal or submaximal concentric contractions, joint contractures, and direct trauma, leading to the abnormal release of acetylcholine resulting in increased tension, blood flow restriction, inflammation, and tissue damage –13. A coRecently, noninvasive nonpharmacological treatments such as transcutaneous nerve stimulation therapy (TENS), laser, and ultrasound therapy have been added to treatment regimens as standalone or adjunctive therapies –47. TENS2 intensity delivering 18,720 J over 4 h of the treatment [The Food and Drug Administration (FDA) in March 2020 approved Sustained Acoustic Medicine  #K191568, Class II, Medical Device) for prescription home use to treat pain, increase local circulation and improve joint function . SAM utireatment –52. The reatment , 53, 54.reatment . The ultreatment . The intreatment –61. CollThis systematic review and meta-analysis aim to summarize the clinical effects of SAM treatment on musculoskeletal injuries including diathermy (tissue heating), functional outcomes (strength and range of motion), quality of life, pain reduction, and safety profile of the intervention.This systematic review and meta-analysis were performed and reported in accordance with the guidelines described by The PRISMA 2020 statement .Studies were included if they applied SAM treatment to human participants (aged 18 and over) with institutional review board approval or exemption; and if they were published in English, original research or peer-reviewed, related to the musculoskeletal treatment ; level IV (case cohort) or higher evidence based on Levels of Evidence, Oxford Centre for Evidence-Based Medicine, 2009; used validated outcome measurement methods ; included study designs of comparative, case cohort or qualitative studies.Relevant literature was searched to identify studies of level IV or higher (Oxford Centre) measuring clinical benefit of the SAM device in clinical research applications up to 09/10/2021. Measurable clinical outcomes included: pain, function, tissue-heating (diathermy), strength, recovery, and return to work. The PRISMA flow diagram for identifying relevant research is shown in Fig. All references were exported, and duplicates removed. Two investigators  screened titles and abstracts as per the inclusion criteria and retrieved full text for further analysis. Disagreements were resolved by a third reviewer (AU).The two investigators (SW and TB) independently extracted data from the selected studies and complied them into tables. The data collected included study characteristics , study musculoskeletal focus area , and variables associated with measurable outcomes . Extractable variables were pooled and stratified to similar conditions on reported outcomes in the literature for synthesis.A total of 362 records were identified and a total of n = 13 clinical studies including five (5) level I, four (4) level II and four (4) level IV studies were selected for assessment. The quality of each selected study was scored by two investigators (SW and TB) using the Downs and Black checklist  and inve2 statistic was used to assess study heterogeneity within the meta-analysis. Analysis was conducted on studies grouped by body location and condition being treated, and according to outcomes measure . Given the limited evidence uncovered (13 studies) and variability in design amongst the studies , data groupings were made according to time points analyzed and reported and standardized for comparison controls where available. In cases where it was not possible to undertake meta-analysis such as limited evidence for a specified comparison (lack of a control group) and/or only one study available on an outcome, data was extracted into tables and main findings reported.Two authors (SW and TB) completed the analysis using both Microsoft Excel  and Review Manager Version 5.4 . A fixed effects meta-analysis with standardized mean difference (SMD) statistics was used to analyze the results where two or more controlled studies could be analyzed. The IThe PRISMA flow diagram is shown in Fig. The study characteristics and participants for upper neck, back and shoulder conditions are reported in Table 2 for 1–4 h, respectively [2 for 4 h [The characteristics and methodology of SAM treatment for upper neck back and shoulder conditions are reported in Table ectively , 65. One for 4 h . SAM tre for 4 h , 64, andThe level of evidence and quality assessment of the studies is shown in Table p < 0.05) compared to placebo, and a 60% GROC improvement over the placebo group after 10 SAM treatment sessions (p < 0.05). In the pilot study by Lewis et al. [p < 0.05). In a 33 subject RCT on upper neck and shoulder pain conducted by Petterson et al. [p < 0.001) and a 1.03 points decrease over placebo (p = 0.003) after 4 weeks of intervention. Petterson et al. [p < 0.001).The primary outcomes and main findings from the included studies are shown in Table s et al.  5 patienn et al.  pain wasn et al.  also rep2 = 0%; n = 63) and Global Health Improvement . There were significant between-group differences found in pain (p = 0.005) and health improvement (p < 0.0001) with low heterogeneity between studies . The two studies in these outcomes were graded as good to excellent [The meta-analysis and forest plot of SAM treatment outcomes on pain and global health improvement compared to placebo treatment are shown in Fig. xcellent , 64.Fig.The study characteristics and participants for knee joint conditions treated by SAM are reported in Table 2 for 4 h daily [The characteristics and methodology of SAM treatment for knee joint pain related to Osteoarthritis is shown in Table  h daily , 66, 67. h daily , 52, 66. h daily , 52, 67 The level of evidence and quality assessment of the studies is shown in Table p < 0.03). A 90-subject double-blinded, placebo-controlled study by Draper et al. 2018 reported a 1.96-point decrease in NRS pain relative to placebo treatment (p < 0.01) [p < 0.01). In a 32 patient multicenter study on knee Osteoarthritis, Madzia et al. [p < 0.001) and 2.96-point 75% pain decrease in responders (p < 0.001). The WOMAC score improved by 351 points in the entire population (p < 0.001) and 510 points (p < 0.001) in the rapid responder cohort. A high usability rate over 95% patient satisfaction and no adverse events were also reported.The primary outcomes and main findings from the included knee joint studies are shown in Table  < 0.01) . WOMAC fa et al.  reported2 = 93%; n = 63). There were significant between-group differences found in pain (p < 0.00001). The included studies had high heterogeneity  which was not meaningfully reduced by exclusion of a data set. The three studies included in these outcomes were graded as poor to excellent quality. Other measures such as WOMAC were not sufficiently available to conduct analysis on.The meta-analysis and forest plot of SAM treatment outcomes on pain reduction compared to placebo treatment are shown in Fig. The study characteristics and participants for patient-self-treatment and soft tissue injuries treated by SAM are reported in Table 2 for 4 h daily [The characteristics and methodology of SAM treatment for patient self-treatment and soft tissue injuries is shown in Table  h daily , 68–71.  h daily . The oth h daily , 68–70. The level of evidence and quality assessment of the studies is shown in Table The primary outcomes and main findings from the included studies on self-treatment and soft tissue injury are shown in Table A placebo-controlled study by Rigby et al. 2015  measured the diathermic effects of one and two SAM transducer setups at 1.5 cm and 3 cm intramuscular depth over 3 h . The 3–4p = 0.002) and a 2.38 kg improvement in grip strength (p = 0.04). An overall reduction in pain was observed within the 4-h treatment sessions (p < 0.001) as well.Best et al. 2015 reported the efficacy of SAM therapy in controlled case studies including Achilles and elbow tendinopathy (n = 25 subjects) . Patient−1 compared to the sham treatment 318.5 ± 86.0 mmol min L−1 (p = 0.002), reflecting a 20% average decrease in total blood lactate levels after 1 h of recovery with SAM. There were also improvements in muscle performance with active versus placebo SAM treatment, including increased peak torque at 90° sec−1 into extension (p = 0.031), increased total work at 90° sec−1 into extension (p = 0.027) and average power output at 90° sec−1 into extension (p = 0.024). Similarly, Draper et al. 2020 conducted a set of case studies (n = 18) using SAM as an adjunct therapy in athletic injuries from sports [p < 0.05), 87% improvement in quality of life, and 55% of the athletes successfully returned to active sports.A randomized placebo-controlled cross-over study design by Langer et al. 2017 reported on the efficacy of SAM treatment to improve healing and recovery after muscle injury from high-intensity resistant exercise (n = 16 subjects) . Subjectm sports . The the2 = 97%; n = 114). There were significant between-group differences found in tissue heating (p < 0.00001). The included studies had high heterogeneity  related to location  diathermy measurement on the body. The two studies included in this outcome were graded as fair to good quality. Other measures such as pain, lactic acid, and functional measures were not sufficiently available to conduct meta-analysis on.The meta-analysis and forest plot of SAM treatment outcomes on diathermy compared to placebo/no-treatment are shown in Fig. Musculoskeletal pain and soft-tissue injuries are highly prevalent with a significant impact on quality of life and the economy . Acute p2 and delivers 18,720 J of energy over 4 h of treatment [As a recently approved FDA home-use treatment in 2020 , this syreatment , 65, 75.reatment , 65.The studies conducted by Lewis et al. 2013 and Petterson et al. 2020 show SAM’s ability to manage pain in fibrous and skeletal tissue in the upper shoulder and neck, alleviate pain, and increase shoulder mobility , 64. In Case series by Best et al. 2015 and Draper et al. 2020 reported the effects of SAM treatment on fibrous and skeletal tissue , 70. TheAcross the thirteen studies (n = 372 subjects) measurable outcomes on device usability, safety profile, diathermy, pain relief, health improvement, and functional assessment using dynamometry, range of motion and grip strength were measured. Table SAM has shown excellent results in rehabilitation and pain management, but there are various other potential applications for low-intensity continuous ultrasound Fig. A. This mAlthough the systematic review focused on Sustained Acoustic Medicine (SAM) for the treatment of musculoskeletal injuries, it is possible that other relevant studies using similar treatment parameters (3 MHz ultrasound at 1.3 W) are available in the scientific literature to further aggregate and synthesize the clinical literature. This limitation was beyond the scope of this research but could be considered in a future analysis paying close attention to time, duration, dose delivered and regularity of ultrasound treatment. The literature search strategy we employed found 13 relevant articles specific to SAM that are more than other past reviews on Sustained Acoustic Medicine ever, it is possible that some relevant studies were missed that were not available in English language or those in the grey literature which are emerging on this new therapeutic treatment , 92, 93.This systematic review and meta-analysis reported the current evidence for Sustained Acoustic Medicine on musculoskeletal injuries and chronic pain. SAM, a novel mechanobiological treatment, is clinically effective at reducing pain, improving overall health quality, generating deep therapeutic heat, and increasing mobility leading to a better-quality life and return to daily activities. The prescription home use treatment has excellent safety, usability and satisfaction characteristics for patients, and may be considered a good non-pharmacological and non-invasive treatment option in musculoskeletal injuries.


Estimation of influenza disease burden is necessary to monitor the impact of intervention programmes. This study aims to estimate the attributable fraction of respiratory and circulatory disease due to influenza among Australian adults 50–64 and ≥65 years of age.A semi‐parametric generalised‐additive model was used to estimate annual and average rate of influenza‐attributable hospitalisation and death per 100,000 population under the principal diagnosis of influenza/pneumonia, respiratory, circulatory and myocardial infarction (MI) from 2001 through 2017.Over the study period, seasonal influenza accounted for an estimated annual average respiratory hospitalisation rate of 78.9  and 287.5  per 100,000 population in adults aged 50–64 and ≥65 years, respectively. The corresponding respiratory mortality rates were 0.9  and 18.2  per 100,000 population. The 2017 season had the highest influenza‐attributable respiratory hospitalisations in both age groups, and respiratory complications were estimated approximately 2.5 times higher than the average annual estimate in adults aged ≥65 years in 2017. For mortality, on average, influenza attributed 1,080 circulatory and 361 MI deaths in adults aged ≥65 years per year. Influenza accounted for 1% and 2.8% of total MI deaths in adults aged 50–64 and ≥65 years, respectively.Rates of cardiorespiratory morbidity and mortality were high in older adults, whilst the younger age group contributed a lower disease burden. Extension of influenza vaccination programme beyond the targeted population could be an alternative strategy to reduce the burden of influenza. In Australia, annual influenza vaccination is recommended for people from 6 months of age and is available under the National Immunisation Program (NIP) for adults aged 65 years and over and at‐risk groups, including those with cardiovascular or respiratory diseases.Current evidence suggests that influenza infection is a precipitant for ischaemic events and that vaccination and prevention of infections may have an important role in reducing the ischaemic burden.Vaccination is the most effective public health intervention for prevention of influenza infection.2We used descriptive epidemiology and statistical modelling method to estimate the age‐specific rate of influenza‐attributable hospitalisations and deaths due to influenza/pneumonia, respiratory, circulatory and myocardial infarction (MI) in two age groups: 50–64 and ≥65 years, from July 2001 through December 2017. Our study aimed to highlight the gap in the vaccination programme between the two age groups to reduce influenza burden in Australia. We extracted surveillance data on weekly counts of laboratory‐confirmed influenza notifications in Australia between 2001 and 2018 from the National Notifiable Diseases Surveillance System (NNDSS), the Australian Government, Department of Health.2.1A semi‐parametric generalised‐additive model (GAM) was used to model the burden of influenza in 50–64 and ≥65 years.Y represents the weekly hospitalisation rate per 100,000 population for a specific age group and disease category. β0 is the model intercept and β1 (year), β2, β3, β4, …, β11 represent the value of parameter estimates of the respective independent or indicator variable. β12 (time) is a linear term for week number to control for any long‐term  linear time trend in the hospitalisation rate, and spline (time) is a smoothing spline of the week number to control for unmeasured time varying factors.The model equation was as follows:Similarly, to estimate influenza‐attributable mortality for the period, 2001–2017, the modified version of the model was used separately for both age groups and four diagnostic categories. It was assumed that death may occur sometimes after exposure to the infection and the interval between the onset of symptoms and its associated death may occur from 1–40 days after the infection.Y represents weekly mortality rate per 100,000 population for a specific age group and disease category. β0 is the model intercept, β1 (year) is the parameter estimate of the respective independent variable, β2 (time) is a linear term for week number, and spline (time) represents smoothing spline of the week number in the model.As mentioned previously, In addition, we split the years as pre‐ (July 2001 to December 2008) and post‐pandemic (January 2009 to June 2018) models for circulatory and MI due to variation in notification counts nationally since the pandemic year 2009.From the model, weekly age‐specific rate of hospitalisation or death per 100,000 population was estimated for each disease category, and annual total rates were calculated from the weekly estimates. For each disease category and age group, average seasonal estimate was calculated excluding the 2009 pandemic year in the analysis. A 95% conference interval (CI) was used to test the statistical significance in the study, and the analysis was conducted using SAS Enterprise Guide (SASEG) version 8.1 .3Over the study period, a total of approximately over 11 million  hospitalisations and nearly 1.2 million deaths  were extracted under four principal diagnosis category of influenza/pneumonia, respiratory, circulatory and MI among adults aged ≥50 years. Average annual rate of hospitalisation and death for both age groups by disease category are presented in Table Laboratory‐confirmed influenza notifications were extracted from the NNDSS for the study. The number of influenza notifications varied nationally from year to year over the study period Figure . During 3.1The estimated hospitalisation rates varied across the seasons Table . CompareEstimated yearly rate of influenza‐attributable hospitalisation by age group and principal diagnoses of circulatory and MI are shown in the Figure Table 3.2For elderly adults, statistically significant association was found between influenza and all four diseases; however, less mortality burden was seen in adults aged 50–64 years. For people aged ≥65 years, the estimated average annual mortality rates attributable to influenza were 37.7  for circulatory, 18.2  for respiratory, 12.8  for MI and 7.6  for influenza/pneumonia per 100,000 population  per 100,000 Table  was foun4The burden of influenza‐attributable complications was highest among adults ≥65 years old, but a considerable disease burden was also found in younger adults aged 50–64 years. Influenza was responsible for respiratory complications in both age groups, whereas less burden was seen in young adults 50–64 years old for cardiovascular complications. The estimated hospitalisation rates attributable to influenza varied by season and by age group. Across the years, 2017 season had the highest rate of influenza‐attributable respiratory complications in both age groups in the study. Australia experienced a severe A/H3N2 influenza season in 2017 with higher number of influenza notifications as well as increased reporting of influenza‐related hospitalisations and deaths recorded in the elderly during the season.Compared to pre‐pandemic period, reporting of influenza notifications increased nationally after 2009.Our study estimated an average annual respiratory hospitalisation rate of 288 per 100,000 population in adults aged ≥65 years. This is comparable to previous Australian study by Newall et al.,In addition to respiratory complications, we found that on average, influenza accounted for 2.8% and 1% of total MI deaths annually in adults aged ≥65 years and 50–64 years, respectively. For circulatory and MI, 2002 and 2005 showed higher rates of influenza‐attributable hospitalisations and deaths among elderly adults. These seasons have been reported with moderate seasonal activity in Australia; however, both influenza A and B outbreaks were also reported during the two seasons.Influenza triggers cardiovascular‐related complications, and many studies documented influenza‐attributable cardiovascular events.It is evident that influenza vaccine offers protective effect on influenza‐related cardiac events.This study has some limitations. First, in the study, weekly counts of total influenza were applied in the model; thus, type‐specific disease burden cannot be estimated in these age groups. Second, the seasonality of influenza infection may coincide with seasonality of respiratory syncytial viruses (RSV), and our study could not be able to adjust presence of RSV in the model; thus, study findings may have been confounded by RSV. Finally, in the study, we used national data on weekly counts of all‐age influenza infection for individual year in the model, to account for variation in testing or reporting practices by general practitioners as well as variation across states and territories in Australia over time. Strengths of the study include the use of weekly counts of total influenza infection as a proxy measure or covariate in the model, thus reflecting the incidence of infection in the population to predict better disease estimates across the studied years. Also, 17 years of influenza notifications data in the study could provide a stronger statistical power in model outputs compared to a short‐term data analysis.In conclusion, our study demonstrates that influenza attributed to increased respiratory morbidity in both age groups: 50–64 and ≥65 years and high circulatory mortality among the older population. Influenza and its associated complications can be prevented by seasonal vaccines. Improved vaccine uptake in the recommended age group, as well as in adults 50–64 years of age including the high‐risk population, might be the best available intervention to reduce cardiorespiratory disease burden associated with influenza.Aye Moa: Data curation; formal analysis; methodology. Robert Menzies: Formal analysis; methodology. J. Kevin Yin: Conceptualization; formal analysis; methodology. C. Raina MacIntyre: Conceptualization; formal analysis; methodology.Table S1. Estimated annual influenza‐attributable hospitalisation rate by principal diagnosis and year, 50–64 and 65 years and over, Australia, 2001–2017.Table S2. Estimated annual influenza‐attributable mortality rate by principal diagnosis and year, 50–64 and 65 years and over, Australia, 2001–2017.Table S3. Comparison of estimated influenza‐attributable hospitalisation and mortality between studies.Figure S1: Observed rate of hospitalisation with principal diagnosis of respiratory disease.Figure S2: Observed rate of hospitalisation with principal diagnosis of influenza/pneumonia.Figure S3: Observed rate of hospitalisation with principal diagnosis of circulatory disease.Figure S4: Observed rate of hospitalisation with principal diagnosis of myocardial infractionFigure S5. Laboratory‐confirmed influenza notifications by type, subtype and year, Australia, 2001–2017.Figure S6: Estimated, observed and baseline annual hospitalisation rate* of influenza‐attributable circulatory and myocardial infraction, by year and age group, Australia, 2001–2017Figure S7: Estimated, observed and baseline annual rate* of influenza‐attributable influenza/pneumonia and respiratory death, by year and age group, Australia, 2001–2017.Click here for additional data file.


For recurrent glioblastoma (GB) patients, several therapy options have been established over the last years such as more aggressive surgery, re-irradiation or chemotherapy. Age and the Karnofsky Performance Status Scale (KPSS) are used to make decisions for these patients as these are established as prognostic factors in the initial diagnosis of GB. This study’s aim was to evaluate preoperative patient comorbidities by using the age-adjusted Charlson Comorbidity Index (ACCI) as a prognostic factor for recurrent GB patients.In this retrospective analysis we could include 123 patients with surgery for primary recurrence of GB from January 2007 until December 2016 ). Preoperative age, sex, ACCI, KPSS and adjuvant treatment regimes were recorded for each patient. Extent of resection (EOR) was recorded as a complete/incomplete resection of the contrast-enhancing tumor part.P < 0.001) and EOR (P = 0.013) were associated with significantly improved survival in univariate analysis. Including these factors in multivariate analysis, preoperative KPSS < 80  and EOR are the only significant prognostic factor . ACCI was not shown as a prognostic factor in univariate and multivariate analyses.Median overall survival (OS) was 9.0 months (95% CI 7.1–10.9 months) after first re-resection. Preoperative KPSS > 80% (For patients with surgery for recurrent glioblastoma, the ACCI does not add further information about patient’s prognosis besides the well-established KPSS and extent of resection. Glioblastoma (GB) multiforme is the most aggressive primary brain tumor in adults with a median age of 64 years at the time of diagnosis , 2. AlthThis retrospective, non-interventional bicentric study was approved by the medical ethics committee of the Technical University Munich (5625–12) and is in accordance with the ethical standards of the 1964 Declaration of Helsinki and its later amendments .From 189 possible eligible patients, 33 were excluded due to progression from a lower grade of glioma and another 23 patients were excluded because of second or third recurrence, 10 were excluded due to biopsy. 123 consecutive patients with recurrent GB (WHO IV) between January 2007 and December 2016 met our inclusion criteria: surgery for primary recurrence of GB with available ACCI, preoperative age, sex, KPSS, and pre−/postoperative MRI. We retrospectively reviewed all the clinical data of these 123 patients and analyzed the comorbidities by using the age-adjusted ACCI . Non-normally distributed data are shown as median and interquartile range (IR), and normally distributed variables are shown as mean and standard deviation. OS was assessed with the Kaplan Meier method in univariate analysis (log-rank). Multivariate survival analysis was performed using a cox proportional hazard regression model. A The patient population comprises 123 patients  with surgery for recurrent GB (Table Median OS was 9.0 months (95% CI 7.1–10.9) after surgery for first recurrence of GB. Median preoperative ACCI was 4.0 (95% CI 3–6).P = 0.002). Preoperative ACCI was included in the further analysis as a continuous variable and could not reach statistical significance for survival analysis after the operation for first recurrence of GB. Patients with complete re-resection showed significantly improved survival after surgery (P = 0.013). Age, with a cutoff at 65 years, was not significant in relation to OS from operation for first recurrence of GB until death (P = 0.445)  and EOR  were significant prognostic factors. ACCI  and age < 65 years  did not show significant results. In a further analysis, preoperative KPSS was removed for multivariate analysis, and only the factors age, EOR, and preoperative ACCI were assessed , preoperative KPSS (</≥80), preoperative ACCI, and EOR . For survival after the surgery for first recurrence of GB, preoperative KPSS < 80 (HR 2.002 [95% CI: 1.246–3.216], 3. Each characteristic was assigned one point. They could show that patients with a score of three points expected a 1-month median survival, those with 1–2 points expected an intermediate survival with 4.5 months, and those with zero points presented the best survival with a median of 10.8 months [ACCI might be an additional prognostic factor for patients with recurrent GB; beneath the well-established KPSS, the ACCI, however, did not show statistical significance. The present study assessed 133 patients with surgery or biopsy for recurrent GB. Although there is a standardized treatment for newly diagnosed GB according to a complete surgical resection if possible, followed by concomitant radiochemotherapy as reported in the EORTC Trial , all tum8 months . The KPS8 months . Equally8 months . Therefo8 months  and its 8 months  are part8 months . The ext8 months –39. Like8 months , 41. Of 8 months , an ACCI8 months , 44. As The main limitation of this study is the retrospective design that might introduce an unavoidable bias. The validation of prognostic factors for the outcomes and for supporting therapy selection in recurrent GB would ideally have to be randomized and prospective. Therefore, prospective studies should be performed to assess the value of the ACCI in preoperative patient assessment.For patients with surgery for recurrent glioblastoma, the ACCI does not add further information about patient’s prognosis besides the well-established KPSS and extent of resection.


Although blood lead levels (BLLs) in children are gradually decreasing, low-concentration lead exposure can still exert adverse effects. We studied the factors that affect BLLs in children in Shenyang, China.We conducted a cross-sectional study by administering structured questionnaires on family demographics and food intake. The concentrations of lead in venous blood were determined by graphite furnace atomic absorption spectrometry.p = 0.004). Consumption of puffed grains and eggs had an adjusted OR (95%CI) for BLLs ≥20 μg/L of 1.714 (1.012–2.901) (p = 0.045) and 1.787 (1.000–3.192) (p = 0.050), respectively.A total of 273 children aged 1–6 years were enrolled. The geometric mean (geometric standard deviation) of BLLs was 24.94 (12.70) μg/L in boys and 23.75 (11.34) μg/L in girls. The prevalence of BLLs of ≥35 μg/L was 22.7% and was mainly observed in children aged under 3 years. Often hand washing before meals was protective against BLLs ≥20 μg/L (adjusted OR: 0.427, 95%CI: 0.238–0.767, BLLs of children in Shenyang are still higher than in developed countries. Consumption of puffed grains and eggs is associated with higher BLLs. Often hand washing before meals may be protective against high BLLs. Lead is considered one of the most toxic substances by the US Agency for Toxic Substances and Disease Registry . The ChiThis was a cross-sectional study conducted in children aged 1–6 years at the physical examination center of the Fourth Affiliated Hospital of China Medical University. The criteria for inclusion were children who were found to be healthy by medical examination and whose BLLs were measured. Children were excluded if they had received zinc, calcium, iron, or multivitamin supplementation during the preceding 3 months. The study period was December 2017 to December 2019. A total of 305 children were approached while 32 had the exclusion criteria; 273 children were eventually enrolled in the study. Ethics approval was granted by the Ethics Committee of the Fourth Affiliated Hospital of China Medical University and written informed consent was obtained from the parents/caretakers of the participants.A total sample size of 273 children was determined with a first error level of 5%, a standard deviation of 15 and accuracy of 12% of standard deviation error.2) was calculated by the hospital staff from height and weight measurements. Age- and sex-specific body mass index cutoffs developed for Chinese children were used to evaluated participant weight status  [The parents/caretakers of the participants completed a questionnaire, including information on the child’s age, sex, one-child family (yes/no), passive exposure to cigarette smoke (one or more family members had regularly smoked at home), socioeconomic status (low/above median income), maternal education (≤ high school/college or higher), parents’ occupations , whether the home had been remodeled within the past year, and frequency of hand washing before meals . Body mass index (kg/md obese) .A food frequency questionnaire was used in the present study. The original version of the food frequency questionnaire which is both reproducible and valid included 86 food items that were commonly consumed by the Chinese population , 11, whiVenous blood (4 mL) was collected by a phlebotomy nurse to determine the concentration of blood lead. Blood was collected into lithium heparin-coated trace metal-free tubes  and transported on ice to Shenyang Harmony Health Medical Laboratory. The testing credentials of the laboratory have been previously described . BLLs wet-test after logarithmic transformation. The chi-squared test was used to compare qualitative data and descriptive statistics are shown as n (%). Gilbert and Weiss have proposed 20 μg/L as a benchmark for successful prevention, therefore we took dichotomous BLLs as the dependent variable (< 20 μg/L and ≥ 20 μg/L) [p ≤ 0.2). Maternal education, hand washing frequency, and consumption of fruit, bean products, puffed grains, and eggs were included in the multivariate logistic regression model. Statistical analyses were performed in SPSS version 22.0 . p ≤ 0.05 was considered significant.BLLs were not normally distributed Fig. , and the20 μg/L) . Food in2 = 3.262, p = 0.660).A total of 273 children participated in this study, including 126 boys (46.2%) and 147 girls (53.8%). The participants ranged in age from 1 to 6 years (mean: 4.00 ± 1.13 years); 108 children (39.6%) were aged 1–3 years and 165 children (60.4%) were aged 4–6 years. There was no statistical difference in the distribution of boys and girls between the two age groups . However, no significant difference in BLLs was observed between the two age groups  (Table p = 0.019)  (Table p = 0.050) : 0.382, 95% confidence intervals (CI): 0.218–0.671, Lead is a ubiquitous metal and its blood concentrations are considered the best indicator of human exposure. No BLLs are considered safe in children, who are particularly susceptible to lead poisoning , 15 becaIn the present study, we found that often hand washing before meals was inversely associated with BLLs, perhaps indicating that dust was a source of lead and that washing it off hands prevented exposure by inhalation or ingestion . TherefoFood consumption is currently considered a major source of lead , but datWe also found an association between consumption of eggs and BLLs exceeding 20 μg/L, perhaps because environmental pollution increases lead content in eggs and lead absorption through the intestines increases with the number of eggs ingested. However, we did not find literature to support this view. Eggs contain a rich content of protein, and protein intake was reported to be a positive modulator of BLLs in humans . MoreovePrevious studies have shown positive associations between consumption of grains and vegetables and BLLs, supporting the hypothesis that these foods are sources of lead –28. WholOur study had several limitations. First, this was a single-center study and some selection bias may be present. As children with lead poisoning is rare in China, blood lead testing is not routine inspection item. In our study, only healthy children whose parents asked for blood lead determination were investigated by questionnaire. Therefore, our selected population may have better health care awareness and have limitations in representing the general population in Shenyang. In addition, most data were collected by questionnaire and recall bias may be possible. Second, weighed food records provide quantitative information on individual diets and are considered a “gold standard” for dietary assessment. However, this method is time consuming and generally suitable only for individuals or small groups of cooperative volunteers . Food frIn conclusion, we found that BLLs of children in Shenyang are still higher than in developed countries and that consumption of puffed grains and eggs is associated with higher BLLs in this cohort. Often hand washing before meals may be protective against high BLLs. Further prospective study is needed to verify these findings.


Polygonatum odoratum (Mill.) Druce  is a widely cultivated medicinal herb in China. However, this useful herb is understudied despite being known as a medicinal resource with top grade medical and edible properties since long. In this study, P. odoratum and four cultivars were investigated. The variations in morphological characteristics and vegetative phases of each cultivar were observed. For genetic aspect, the plastid genome of P. odoratum varies in length from 154,569 bp to 155,491 bp, containing a large single‐copy region of 83,486–84,459 bp, a small single‐copy region of 18,292–18,471 bp, and two inverted repeats of 26,302–26,370 bp. A total of 131 genes were predicted, including 85 protein‐coding, 38 tRNA, and eight rRNA genes. Genome comparisons revealed a slight variation in the sequence across the five accessions, but two highly variable regions (trnC‐petN and rpl32‐trnL) were detected when comparing the four different cultivars. For the RAD‐seq markers, a total of 33.64 Gb of clean data, with an average value of 1.08 Gb per sample, were analyzed for the presence of single nucleotide polymorphisms (SNPs). Well‐resolved phylogenies of the P. odoratum cultivars are constructed; the nonmonophyletic relationship in the plastome‐based phylogenetic trees, yet monophyletic form in the RAD‐based linkage map suggested possibility of hybrid cultivar for P. odoratum “Dazhu” (GDDZ), which was further supported by morphological observations. Quality assessment based on the standards of the Chinese Pharmacopoeia on Polygonati Odorati Rhizoma (POR) on the four cultivars used in this study recorded that PORs from P. odoratum “Zhongzhu” (GDZZ) met the minimum criteria for the acceptance as raw material for medicinal drug production. This study has provided insights on the morphological variations, genetic background, and medicinal qualities of P. odoratum cultivars that could be explored for future genetic improvement as well as breeding programs of P. odoratum for POR production. Polygonatum odoratum cultivars can be differentiated morphologically. The nonmonophyletic relationship revealed from the phylogenetic analyses based on the plastome sequences and a monophyletic form in the RAD‐based linkage map suggested the possible presence of hybrids in certain cultivars. Polygonati Odorati Rhizoma from the cultivar Zhongzhu (GDZZ) met the criteria in the Chinese Pharmacopoeia as raw material for medicinal drug production. In order to meet this surge of demand, herbs are now mass‐cultivated in open fields and chambers instead of relying on wild resources  Druce, commonly called the Solomon's seal, is a perennial herb from the family of Asparagaceae. It is mainly natural distributed in the East Asia region, including China, Japan, Korea, and Mongolia, and has also been documented in the northern Europe continent and eastern region of Russia. The P. odoratum produces fibrous rhizome, which is an essential ingredient in traditional medicine. The dried P. odoratum's rhizome, also known as Polygonati Odorati Rhizoma (POR), or Yuzhu (jade bamboo in Chinese), is classified as a medicinal resource with top grade medical and edible properties in ancient medicinal plant records  and asexually (via rhizome cuttings), the supply for planting material is often sufficient. With the aid of crop breeding techniques, new P. odoratum cultivars are introduced by medicinal plant breeders through selection breeding, with the aim to yield high‐quality PORs. Cultivars often use rhizome cuttings to propagate where identical clones can be replicated through asexual means. The morphological and genetic characteristics of these clones should correspond to the main body, in which the desired quality is thought to be conserved. There are two ways to distinguish types of P. odoratum that the cultivars exhibit in markets. One is from its morphological characteristics, such as rhizome shape, size, and taste, and the other is by its place of origin. According to the former technique, there have been claims that there are more than 10 different cultivars recorded in literatures that used as POR resources, mainly from the provinces of Jilin, Guangdong, and Hunan , Haimenyuzhu (Jiangsu origin), Xiyuzhu (Guangdong origin), Dongyuzhu (Zhejiang origin), Guanyuzhu (Northeast China origin), and Jiangbeiyuzhu (Jiangsu and Anhui origins), which all exhibit different phenotypic characteristics. Initially, the major cultivated POR‐producing provinces were confined to Jilin, Guangdong, and Hunan though the high demand in POR has led to the expansion in P. odoratum planting sites in neighboring provinces, such as Jiangsu, Zhejiang, Henan, and Liaoning. While there are slight morphological differences between P. odoratum cultivars  markers  technology warrants an opportunity to generate genome‐scale information to provide better resolution in genetic diversity and genome evolution analyses at a much lower cost. The plastid genome (plastome) is reported to be highly conserved in its gene contents and displays a maternal inheritance among angiosperms, thus being useful in phylogenetic studies, population genetics, phylogeography, and species characterization. Mutation hotspots identified in the plastome due to highly variable regions when comparing across several plastomes of closely related species could be developed for phylogeny and species identification at intraspecies or lower taxa levels. On the other hand, the assembly of the complete nuclear genome is a tedious work; thus, researchers have introduced a substitute method via restriction site‐associated DNA (RAD) sequencing technique to provide genome‐scale information of the nuclear genome. The RAD markers are short DNA fragments adjacent to the selected restricted enzyme recognition site orders , Zhongzhu  from Guangdong province, Xiangyuzhu  from Hunan province, and Guanyuzhu  from Jilin province, were collected for this study  following the manufacturer's protocol. In order to extract sufficient DNA concentration, each accession was extracted with at least two replicates which were pooled together after the DNA extraction process for quantification and sequencing. The quality and quantity of the DNA product were determined using NanoDrop™ , and DNA samples were kept in −20°C freezer storage prior to sequencing of the plastome and nuclear RAD data.2.3.2Polygonatum odoratum cultivars, one accession was selected as standard for each cultivar. A genomic library with an insert size of ~350 bp was prepared using a TruSeq DNA Sample Prep Kit  and sequenced on an Illumina NovaSeq platform  for pair‐ends of 150 bp at Guangzhou Jierui Biotechnology Company Ltd . Adapter sequences were then removed using NGS Toolkit (Patel and Jain rbcL gene sequence of P. odoratum (GenBank accession: KC704957) acting as the seed sequence. The assembled plastome was initially annotated using the online annotation tool GeSeq . The plastome sequences of the five Polygonatum odoratum accessions were aligned using MAFFT  was visualized under mVISTA program  at 37°C for 5 hr. The restriction–ligation reactions were heat‐inactivated at 65°C for 20 min with T4 DNA ligase , ATP , MluCI adapter, and SphI adapter. Subsequently, the mixture was incubated at 12°C for 12 hr. A 40‐µl preamplification reaction was conducted using 5 µl of the diluted restriction–ligation mixture using a pair of single‐selective‐nucleotide primers (EcoRI and MseI adapters). PCR amplification was conducted on a T100™ thermal cycler  with thermal settings programmed as follows: 20 cycles of 94°C for 30 s, 56°C for 60 s, and 72°C for 60 s. Prior to the PCR purification step, PCR products were pooled and incubated at 37°C with MluCI, SphI, T4 DNA ligase, ATP, and EcoRI+MseI adapters . The pooled samples were further purified using a E.Z.N.A.® Cycle Pure Kit  and separated on a 2% agarose gel via electrophoresis. Paired‐end sequencing was performed on purified fragments extracted from the agarose gel ranging from 350 to 500 bp (with indexes and adaptors) in size via Illumina NovaSeq platform .For RAD sequencing, a double‐digestion method was applied. Six accessions were selected as representatives for each cultivar, while three accessions—each one for the 2.3.5Prior to phylogenetic tree analyses, quality filtering and loci assembly were performed using Stacks v1.40 , and seven other Polygonatum species  that were downloaded from the GenBank. Plastome sequence alignment was carried out using MAFFT , Disporopsis fuscopicta (MW248136), and Heteropolygonatum ginfushanicum (MW363694) were included as outgroup. For RAD‐seq dataset, the vcf file was converted to fasta format using TASSEL v5.0  to 155,491 bp (GDDZ)  Figure . All fiv3.3Polygonatum odoratum accessions were 37.7% for GDDZ and JLGY, and 37.8% for GDWA, GDZZ, and HNXY. The alignment of the complete plastome sequences of the five P. odoratum accessions resulted in a 155,987‐bp long sequence, containing 337 singleton variable sites, in which 335 were with two variants and two were three variants. The singletons were abundant in IRA (n = 213), followed by the SSC region (n = 69), LSC region (n = 34), and IRB (n = 21). A total of 106 parsimony informative sites were detected, in which 79 were detected in the LSC region and 29 were located in the SSC region. Both the IR regions were detected in every three sites. The smallest pairwise distance was detected between GDWA and GDZZ (0.000), while the largest pairwise distance was detected between GDDZ and JLGY (0.0022)  in which three GDWA were clustered with all six GDDZ and two GDZZ (GDZZ_1 and GDZZ_2); four GDZZ  were clustered with all six JLGY; HNXY formed an independent clade . The GDZZ clade that consisted of four GDZZ samples  were well resolved and separated from the JLGY clade. Polygonatum humile was found to be sister to JLGY in both the ML and BI trees.A 9,024‐bp long aligned SNP dataset was obtained for genetic subdivision and phylogeny reconstruction. The SNP‐based ML tree displayed three distinct clades for the five 3.6For fresh POR samples, JLGZ was recorded with the highest percentage in water content, followed by GDDZ, while both the GDZZ and HNXY recorded the lowest water content percentage Table . Accordi44.1Polygonatum odoratum cultivars had similar morphological features but can still be differentiated when carefully compared. We found out that the distinct morphological characteristics that could differentiate these cultivars were their stem sizes and leaf size. Although the size of the rhizome differs between cultivars, we anticipated that the size of the rhizome could be influenced by the plant spacing  sequences were proven to have the strength in distinguishing closely related species and identifying the genetic source of hybrids and cultivars for economic crops . Therefore, the proposal to obtain a draft genome for P. odoratum could certainly aid in the gene identification of desired traits. The estimated genome size for P. odoratum was recorded to be 9,613.73 Mbp ; Data curation ; Formal analysis (lead); Investigation (lead); Methodology (lead); Writing‐original draft (lead); Writing‐review & editing . Zhihui Chen: Data curation ; Formal analysis (supporting); Investigation (supporting); Methodology (supporting); Writing‐original draft (supporting). Zhiming Chen: Data curation ; Investigation (supporting); Resources (supporting). Jingrui Chen: Data curation ; Formal analysis (supporting); Investigation (supporting); Methodology (supporting). Xinjian Zhang: Data curation ; Formal analysis (supporting); Investigation (supporting); Methodology (supporting); Writing‐original draft (supporting). Jiawen Pan: Data curation ; Investigation (supporting); Project administration (lead). Qiang Fan: Conceptualization ; Funding acquisition ; Resources (supporting); Supervision (supporting); Writing‐review & editing . Wenbo Liao: Conceptualization ; Funding acquisition ; Resources (lead); Supervision (lead); Writing‐review & editing .


The incidences of MACE were 7.4, 22.2, 25.7 and 56.5%, respectively, in nsHRV + nfQRS group, nsHRV + fQRS group, sHRV + nfQRS group and sHRV + fQRS group (p < 0.05). Multivariable Cox regression analysis showed that patients in the sHRV + fQRS group had a sixfold higher risk of MACE compared to patients in the nsHRV + nfQRS group . The predicting sensitivity and specificity on MACE were 81.4 and 58.2% by sHRV, 69.8 and 69.1% by fQRS in these AMI patients. The specificity (81.8%) was the highest with the combination of sHRV and fQRS. Adding sHRV and fQRS to clinical data offered incremental prognostic value. Present results indicate that fQRS is closely related to sHRV, suggesting significant impairment of sympathetic nerve function in AMI patients with fQRS. Combined assessment with fQRS and sHRV enhances the predicting efficacy on outcome in AMI patients.The association between fragmented QRS (fQRS) and autonomic nervous dysfunction, and major adverse cardiovascular events (MACE) is not fully clear in patients with acute myocardial infarction (AMI). This study aimed to observe whether combined assessment with fQRS and cardiac autonomic nervous function could enhance the predicting efficacy on outcome in AMI patients. A total of 153 consecutive hospitalized AMI patients were included in this retrospective study. Patients were divided into non-fQRS (nfQRS) group and fQRS group according to 12-lead electrocardiogram, into sHRV [severely depressed heart rate variability (HRV): standard deviation of NN intervals (SDNN) < 100 ms and very low frequency (VLF) < 26.7 ms] group and nsHRV (non-severely depressed HRV) group according to 24 h Holter monitoring, and into non-MACE (nMACE) group and MACE group according to 12 months’ follow-up results. The incidence of sHRV was significantly higher in the fQRS group than in the nfQRS group (71.9 vs. 39.3%, The online version contains supplementary material available at 10.1007/s00380-021-01930-y. Acute myocardial infarction (AMI) is the main pathogeny of sudden cardiac death in patients with ischemic cardiovascular diseases. Clinical adverse cardiovascular events are not rare despite successful percutaneous coronary intervention (PCI) in AMI patients . Therefon = 89) and fQRS group (n = 64) according to the 12-lead ECG at admission, into sHRV [severely depressed HRV: standard deviation of NN intervals (SDNN) < 100 ms [n = 81] group, and nsHRV  group according to 24 h Holter monitoring. Patients were also divided into non-MACE (nMACE) group (n = 110) and MACE group (n = 43) according to the 12 months’ follow-up results. Patients with cardiomyopathy, congenital heart disease, organic heart valvular disease, electrolyte disturbance, bundle branch block, Wolf–Parkinson–White syndrome, atrial fibrillation, pacemaker implantation, malignant tumor and digitalis medication were excluded. This study protocol was approved by the ethical committees of Wuhan Fourth Hospital, Puai Hospital affiliated to Tongji Medical College, Huazhong University of Science and Technology and conducted in compliance with the ethical principles of the Declaration of Helsinki. 24 h Holter monitoring results were obtained in the clinical medical records in this study and the requirement to obtain informed consent was waived by the ethical committees of Wuhan Fourth Hospital, Puai Hospital affiliated to Tongji Medical College, Huazhong University of Science and Technology.A total of 156 consecutive hospitalized AMI patients, who were hospitalized in our department from January 2017 to December 2018, were included in this retrospective study. Three patients were lost to follow-up and data from 153 patients were analyzed in this study. Patients were divided into non-fQRS group  within one week after infarction. 24 h mean heart rate and HRV parameters were analyzed according to the guidelines of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology . The maiAll patients were followed up for 12 months by clinic visit or phone call. The main endpoint was MACE, which included all-cause mortality, non-fatal reinfarction, non-fatal stroke, heart failure, and urgent revascularization for unstable angina .t test. Non-normal distribution data were expressed as median (inner-quartile distance) and assessed by two-tailed Mann–Whitney U test. Categorical variables were expressed as count and percentages, and compared using Chi-square test or Fischer’s exact test, as appropriate. Spearman correlation analysis was performed between fQRS and HRV parameters in the AMI patients. The cutoff value of VLF was derived from receiver operating characteristic curves (ROC) analysis by maximizing the sum of the sensitivity and specificity. The rates of MACE were compared by log-rank test of Kaplan–Meier curve. The hazard ratios (HRs) with 95% confidence intervals (CIs) of MACE were assessed by univariable and multivariable Cox proportional hazards regression analysis. Multivariable Cox regression models adjusting for clinical confounders with backward stepwise method (likelihood ratio) were used. Clinical confounders were identified as basic parameters (including age and sex) and parameters significantly associated all with fQRS, sHRV and MACE . NT-proBNP was normalized prior to regression analysis using natural logarithm values (Ln). Incremental model performance was assessed by changes in the Chi-square value for the regression models with enter method. Statistical analyses were performed using IBM SPSS, version 22.0 for Windows , with p < 0.05 (two-tailed test) as statistical significance.Kolmogorov–Smirnov test was used for normal distribution of all continuous variables. Continuous data with normal distribution were expressed as mean ± standard deviation and assessed by Student’s p < 0.05). LVEF, SDNN, SDANN, VLF, LF and LF/HF values were significantly lower in the fQRS group than in the nfQRS group (p < 0.05). The percentage of sHRV was significantly higher in the fQRS group than in the nfQRS group .Table p < 0.05). Percentages of male, LVEF, SDNN, SDANN, rMSSD, pNN50, VLF, LF, HF and LF/HF values were significantly lower in the sHRV group than in the nsHRV group . The percentage of fQRS was significantly higher in the sHRV group compared to the nsHRV group   Table .r = 0.322, p < 0.001) , re-hospitalization due to heart failure (n = 5), non-fatal reinfarction (n = 2), urgent revascularization for unstable angina (n = 1) and stroke (n = 3). There were 30 MACEs in the fQRS group: all-cause mortality , rehospitalization due to heart failure (n = 8), non-fatal reinfarction (n = 5), urgent revascularization for unstable angina (n = 4) and stroke (n = 8).Three patients were lost to follow-up, 1 in the nfQRS group and 2 in the fQRS group. There were 13 MACE in the nfQRS group: all-cause mortality , rehospitalization due to non-fatal reinfarction (n = 2), urgent revascularization for unstable angina (n = 1) and stroke (n = 4). There were 35 MACEs in the sHRV group: all-cause mortality , rehospitalization due to heart failure (n = 13), non-fatal reinfarction (n = 5), urgent revascularization for unstable angina (n = 4) and stroke (n = 7).According to the HRV classification, 1 patient in the nsHRV group and 2 patients in the sHRV group were lost to follow-up. There were 8 MACEs in the nsHRV group: all-cause mortality .p < 0.05). Percentages of STEMI and LVEF were significantly lower in the MACE group than in the nMACE group (p < 0.05). The SDNN, SDANN, LF, VLF, LF/HF values were significantly lower in the MACE group compared to the nMACE group (p < 0.05). The percentages of sHRV (81.4 vs. 41.8%) and fQRS (69.8 vs. 30.9%) were significantly higher in the MACE group compared to the nMACE group (both p < 0.001)    method (Table p < 0.001). The sHRV and fQRS were combined and the patients were further divided into nsHRV + nfQRS group (n = 54), nsHRV + fQRS group (n = 18), sHRV + nfQRS group (n = 35) and sHRV + fQRS group (n = 46). The incidences of MACE were 7.4, 22.2, 25.7 and 56.5%, respectively (p < 0.05). After adjusted for age, sex, Ln NT-proBNP, Scr and LVEF, sHRV + fQRS was an independent predictor (p = 0.021), using nsHRV + nfQRS as reference group, the risk of MACE was significantly increased in the other three groups, and patients in the sHRV + fQRS group had a sixfold higher risk of MACE . As the whole cohort was divided into subgroups of LVEF  ≥ 50% and LVEF  < 50%, Cox regression analysis showed that sHRV + fQRS was also an independent predictor on MACE in LVEF  ≥ 50% subgroup (p = 0.008). Using nsHRV + nfQRS as reference group, patients in the sHRV + fQRS group had a ninefold higher risk of MACE  . The risk of MACE was similar between the nsHRV + fQRS group and the nsHRV + nfQRS group , and the risk of MACE was significantly higher in the sHRV + fQRS group compared to the sHRV + nfQRS group , and the sHRV + fQRS group was associated with the highest increased risk of MACE compared to other groups (p < 0.001) . In model II, adding sHRV to the model I enhanced the explanatory power . Adding fQRS (model III) further improved the prognostic performance of the model II   including age, sex, NT-proBNP, Scr and LVEF were entered in the first step of a multivariable Cox model to predict MACE  Fig. .Fig. 3InThe major findings of the present study were as follows: (1) fQRS is closely related to sHRV, suggesting the presence of significant autonomic nerve dysfunction in AMI patients with fQRS. (2) Combined assessment with fQRS and sHRV enhances the predicting efficacy on outcome in AMI patients. To the best of our knowledge, this study is the first report describing the association between fQRS and cardiac autonomic function defined by sHRV, and the prediction efficacy of combined assessment of fQRS and sHRV on outcome of patients with AMI.The fQRS is caused by the inhomogeneous conduction of electrocardiographic activities in the infarcted and ischemic regions, reflecting the inhomogeneous electrical activity of ventricular depolarization after AMI . HRV is SDNN is a HRV index reflecting the total activity of the sympathetic and vagus nerves. VLF is a controversial indicator. Most studies showed that VLF was related to the sympathetic nerve , 22, whiSDNN with various cutoffs was used as a risk stratification factor in studies of MI patients. In some studies, SDNN  < 70 ms was used as a prognostic factor to predict all-cause death or cardiac death in AMI patients , 25. In p < 0.001), thus fQRS and sHRV were confounding factors for the development of MACE. The predictive effect of fQRS was weakened when sHRV was added to the Cox regression model (p = 0.052), and the predictive effect of sHRV was weakened when fQRS was added to the Cox regression model (p = 0.097) (Table r = 0.322) (Table p = 0.021), and patients in the sHRV + fQRS group had a sixfold higher risk of MACE . Cox regression analysis showed that sHRV + fQRS was also an independent predictor on MACE in LVEF ≥ 50% subgroup (p = 0.008) Below is the link to the electronic supplementary material.


Frequentist and Bayesian analysis suggested that the groups did not differ from each other in the SNARC effect. These results are the first to provide evidence for the SNARC effect in a relatively large sample of gifted  children. In sum, our study provides another piece of evidence for no direct link between the SNARC effect and mathematical ability in childhood.The SNARC  effect  is prevalent across the whole lifespan. Because the ability to relate numbers to space has been viewed as a cornerstone in the development of mathematical skills, the relationship between the SNARC effect and math skills has been frequently examined. The results remain largely inconsistent. Studies testing groups of people with very low or very high skill levels in math sometimes found relationships between SNARC and math skills. So far, however, studies testing such extreme math skills level groups were mostly investigating the SNARC effect in individuals revealing math difficulties. Groups with above average math skills remain understudied, especially in regard to children. Here, we investigate the SNARC effect in gifted children, as compared to normally developing children  contains supplementary material, which is available to authorized users. Spatial–Numerical Associations  effect  study by Schneider, Grabner and Paetsch  characteristics in a task measuring the SNARC effect are related to the SNARC effect itself: slower and more varied responses, longer mean RT, and larger intraindividual variability in RT, (SD)RT are linked to a stronger SNARC effect  have been tested, that has not been the case for child studies so far. The four child studies conducted, which showed the relationship between the SNARC effect and math skill, considered either typically developing children with typical levels of skill in math , and the relationship between the SNARC effect and math skills in children is not linear.Second, we wish to investigate the SNARC effect in the group of highly gifted children. As we have already discussed, based on the existing literature it is hard to come up with a direct prediction regarding the SNARC effect in the gifted children. Specifically, there is some evidence favouring all three of the following theoretically possible scenarios:t (163) =  − 0.495, p = 0.621; or in gender, χ2 (163) = 0.054, p = 0.816. Children for whom data on the Raven’s Standard Progressive Matrices (n = 10) and arithmetic task (n = 7) was missing were not included in analyses considering these measures.A total of 182 children (from six different classrooms) participated in this study. They were recruited from Grades 3 to 5, at the end of their second semester (July), in a gifted education school in Beijing. In this school there are separate classes for gifted and normal pupils. Children following normal curriculum were considered as a control group. Out of these, 17 participants had to be excluded from analyses: two of them due non-completion of both blocks of the parity judgment task and 15 for whom we did not have a valid reaction time within at least one cell . Thus, analyses were conducted on 165 children  was odd or even with a key press. Single-digit Arabic numbers from 0 to 9 were used. Numbers were presented until the participant’s response, and the next trial followed immediately. Presentation order was randomized.There were two experimental blocks (order fixed across participants). They were separated by another task  is defined as the number of items that the participant can actually answer without guessing .The parity judgment task and arithmetic task were implemented in a web-based application “Online Psychological Experiment System (OPES)”  choice RT; (2) non-symbolic comparison (version 1); (3) parity judgment block 1; (4) estimate arithmetic; (5) numerical Stroop task (version 1); (6) non-symbolic comparison (version 2); (7) parity judgment block 2; (8) exact arithmetic; (9) numerical Stroop task (version 2). Here we only consider RSPM, the parity judgment task and the exact arithmetic task. Both sessions were administered within two weeks.Despite having used the full range of single digit numbers (0–9), in the main analysis, we excluded numbers 0 and 5. First, the number zero was shown to have a specific status  were compared to children enrolled in the normal curriculum, whose scores fell within the 25th–75th percentile (n = 19); (2) Arithmetic performance-based analysis we compared pupils enrolled in the gifted curriculum, who scored within the top 5% in the arithmetic task (n = 13), to pupils enrolled in the normal curriculum, whose scores fell within 25th and 75th percentile (n = 47). Detailed information on participants considered in each of the analyses is presented in Table Subsequently, the groups were compared. To obtain the largest power, we based our main group comparison on the Chinese selection process for placement in the gifted or normal curriculum . As one might question the Chinese selection system, we conducted two additional analyses considering subgroups selected based on additional criteria in order to check the robustness of our results: 1)  Intelligt tests along with their Bayesian equivalents. BF01 are reported in the text; therefore, values above 1 show that the null hypothesis model is favoured over the alternative hypothesis model, and consequently, values < 1 indicate that alternative hypothesis model is favoured over the null hypothesis model. Typically, values > 3 and < 0.3 are considered as conclusive evidence. Nevertheless, there are no strict cutoff criteria, instead BF values can and should be treated in a continuous way  = − 4.97, p < 0.001, d = − 0.78, and their intraindividual variability in reaction times was smaller, t (163) = − 4.00, p < 0.001, d = − 0.63.t (153) = 5.82, p < 0.001, d = 0.94, and on the arithmetic task, t (156) = 3.12, p = 0.002, d = 0.50.Descriptive statistics of all measures are presented in Table A robust SNARC effect was observed at the whole sample level , but the analysis still favoured the null hypothesis model.On the other hand, the SNARC effect measurements were not correlated with arithmetic performance scores. Bayesian analyses provided support for lack of correlations, only in one case could the Bayesian evidence be considered inconclusive .To sum up, the correlational analysis did not provide evidence that the SNARC effect is related to math skills nor intelligence. Moreover, Bayesian analyses supported the null hypothesis models.The main between-group comparisons (cf. Bottom part of Table t (59) = 1.60, p = 0.116, d = 0.46, BF01 = 1.244; the ST-SNARC effect without 0 and 5, t (59) = 0.57, p = 0.573, d = 0.16, BF01 = 3.074; the SNARC effect with 0 to 9, t (59) = 0.82, p = 0.413, d = 0.24, BF01 = 2.668; the ST-SNARC effect with 0 to 9, t (59) = 0.17, p = 0.864, d = 0.05, BF01 = 3.469.6In the comparison considering the additional criterion of RSPM scores, there were no between group differences in any of the SNARC effect measures: the SNARC effect without 0 and 5, t (58) = 0.85, p = 0.402, d = 0.27, BF01 = 2.449; the ST-SNARC effect without 0 and 5, t (58) = 0.69, p = 0.495, d = 0.22, BF01 = 2.700; the SNARC effect with 0 to 9, t (58) = 0.90, p = 0.373, d = 0.28, BF01 = 2.366; the ST-SNARC effect with 0 to 9, t (58) = 0.90, p = 0.374, d = 0.28, BF01 = 2.366.7In the comparison considering additional criterion of arithmetic performance there was no between group difference either: the SNARC effect without 0 and 5, In the current study we explored the SNARC effect in gifted children in comparison with a control group. We found a robust SNARC effect in both groups. As expected, groups differed considerably in performance in RSPM and arithmetic performance, with the gifted group performing better. In the gifted group the RTs were also shorter and less variable, which was to be expected as RTs are related to fluid intelligence  as the between group comparisons. Contrary to results reported in the literature, the SNARC effect did not correlate with reaction time characteristics in our Chinese samples.Importantly, in virtually all cases conclusions from frequentist and Bayesian analyses converge to favour null hypothesis models . Additionally, the SNARC effect did not change significantly with age or grade. On one hand, it may be because the age range of our participants was relatively small  differences in automatic magnitude processing, and (2) representation abstractness. This idea was recently elaborated in a model framework by Cipora et al. . The modAs proposed by Hoffmann et al.  the diffImportantly, in most of the studies which reported a relationship between math skills and the SNARC effect at the age of our participants, tested samples of **individuals who had math difficulties/developmental disorders  relationship between the SNARC effect and math skills are quite divergent, one might argue that this is because some studies have found evidence for an effect and some of them simply failed to find such an evidence , which does not preclude that such an effect exist . We believe that this is not the case here and for several reasons, we discuss below.Firstly, several studies (including the current) utilized Bayesian statistics, which can provide an evidence for absence of the effect in question differentiating it from inconclusive data (which is not possible within frequentist framework). Second, studies not reporting the effect were not characterized by systematically smaller sample sizes than studies revealing the effect. Moreover, low power does not only decrease the probability of observing the effect, which actually exists, but it also increases chances of false positives  of Bejingers are higher than the average of the whole nation, and SES correlates with mathematical achievement  bias in psychological science, which is known to affect even basic sensory and cognitive effects , than psychological category  performance on (timed) calculation tasks; (2) standardized math tests , a relationship with arithmetic skill/intelligence seems to exist. In the broader sense, the current research is in line with the view that different SNAs must be distinguished and that this distinction is necessary already for children.Supplementary file1 (DOCX 22 kb)Below is the link to the electronic supplementary material.


Plasma samples were collected from cows at − 10 d relative to parturition. Body weight, hip and wither height, hip width and body length were measured at birth and weekly through weaning (42 d of age) and until 9 weeks of age. Calf blood samples were collected from the jugular vein at birth , 24 h after first colostrum and at 7, 21, 42 and 50 d of age. The data were subjected to ANOVA using the mixed procedure of SAS. The statistical model included day, BCS, and their interactions.Nutritional management in the dry period can alter body condition score (BCS) in dairy cows, a subjective measure of body fat. As such, differences in BCS during late-pregnancy not only mirror nutrient utilization by fat depots, but also can play important roles on the metabolic and hormonal environment. We investigated the association between cow BCS during late-pregnancy on developmental parameters and blood variables of neonatal calves. Forty-nine multiparous Holstein cows were retrospectively divided by prepartal BCS into normal BCS ≤3.25  in HighBCS cows. Plasma concentrations of fatty acids, ceruloplasmin, and nitric oxide were greater overall (P < 0.05) at d − 10 prior to calving in HighBCS cows, and they tended (P = 0.08) to have greater concentrations of reactive oxygen metabolites. Birth body weight was lower (P = 0.03) in calves born to dams with HighBCS. In addition, plasma concentrations of fatty acids, albumin and urea (P < 0.05) were greater in those calves. Although calves born to cows with HighBCS maintained a lower postnatal body weight (P = 0.04), hip and wither height, hip width, and body length, there was no difference (P > 0.05) in daily starter intake and average daily gain due to maternal BCS.Dry matter intake (kg/d or % of body weight) during the last 4 weeks of pregnancy was lower (Overall, results highlight an association between BCS during late-gestation on in utero calf development and postnatal growth. A high maternal BCS during late-gestation was associated with lower calf body weights, which could be due to lower maternal intakes and a state of inflammation and metabolic stress.The online version contains supplementary material available at 10.1186/s40104-021-00566-2. Maintenance of dairy cow body condition score (BCS) during the periparturient period and its relation to health and production is an ongoing area of research. Cows that calve at a high BCS during this period (classified as a score ≥ 3.5) tend to have lower intakes and milk yield and mobilize more adipose tissue . FurtherIn non-ruminants, it is well known that maternal diet, health and lifestyle are important factors that can affect fetal development and subsequent health, development and performance of the offspring . Of thesLate-gestation represents an important period in fetal development due to the rapidly increasing nutrient needs of the fetus. During this period in dairy cattle, circulating nutrients and blood variables can directly alter placental and fetal metabolism , renderiCurrent recommendations for dry cow management suggest feeding higher-energy diets during the last 3–4 weeks of gestation to support the rapid increase of fetal growth, and to prepare the cow for the sudden decrease in dry matter intake (DMI) around parturition . As in nWe hypothesized that calf growth and performance during postnatal life would be affected by maternal BCS prior to calving. To address the hypothesis, measures of growth and development were collected along with plasma concentrations of biomarkers related to energy metabolism, liver function, inflammation and oxidative stress throughout the postnatal period.n = 30; NormBCS) or high BCS ≥ 3.75  groups. In HighBCS cows, parity (mean ± SD) prior to calving averaged 2.86 ± 1.15 while in NormBCS it averaged 3.15 ± 1.45. Previous 305-d milk yield was 10,742 ± 2016 kg for HighBCS cows and 11,080 ± 2031 kg for NormBCS cows. Cows in HighBCS averaged (mean ± SD) 51.80 ± 4.08 d dry and NormBCS cows averaged 50.05 ± 5.10 d dry. Sires used (6 bulls) on these cows were positive for production, health, and fertility traits. Body condition score was measured from − 4 to − 1 weeks prior to calving on a 5-point scale  in increments of 0.25 point [All procedures for this study were conducted in accordance with a protocol approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Illinois (protocol #17168). Forty-nine multiparous Holstein cows were retrospectively divided by prepartal BCS at − 4 weeks relative to parturition into normal BCS ≤ 3.25  or HighBCS  were used. Immediately after birth calves were removed from their dam, and growth measurements were taken. All calves received 3.8 L of first colostrum from their respective dam, if voluntary intake of colostrum did not reach 3.8 L calves were force-fed via esophageal tube to reach the required amount of colostrum. All calves were managed in the same fashion. Outdoor individual hutches were used, and calves were fed with milk replacer  (06:00 and 18:00 h) until 35 d of age. From 36 d of age until weaning (42 d of age) calves were switched to once a day feeding at 6:00 h. Calves received 4.54 kg/d of milk replacer (0.59 kg of milk replacer in 3.95 L of water) from 1 to 10 d of age, 5.90 kg/d (0.77 kg of milk replacer in 5.13 L of water) from 11 to 20 d of age, 7.26 kg/d (0.94 kg of milk replacer in 6.32 L of water) from 21 to 35 d of age, and 3.63 kg/d (0.47 kg of milk replacer in 3.16 L of water) in a single feeding from 36 to 42 d of age. Calves had ad libitum access to a starter grain mix  at 08:00 h from d 1 until d 65 of age. Starter intake was recorded daily and body weight with other growth measurements were taken weekly and used to calculate average daily gain.In total, 49 calves born to dams with NormBCS  samples were collected once a week and the ration was adjusted accordingly to maintain DM ratios of the ingredients. Weekly samples of ingredients and TMR were frozen at − 20 °C and composited monthly for nutrient composition analysis by standard wet chemistry techniques at a commercial laboratory .g for 15 min and stored at − 80 °C until further analysis. Aspartate aminotransferase , bilirubinn , glucose , and urea , gamma-glutamyl transferase  in plasma were measured with kits purchased from Instrumentation Laboratory Spa . The method described by Skinner et al. [x), nitrites, and nitrates were determined using the methods described by Trevisi et al. [Cow blood samples were collected from the coccygeal vein on d − 10 ± 2 relative to expected calving date. Calf blood samples were collected from the jugular vein at birth , 24 h after first colostrum and at 7, 21, 42 and 50 d of age. Plasma was obtained by centrifugation of sodium heparin tubes, at 1900×r et al. . Activiti et al. . RetinolP ≤ 0.05.Data were analyzed with the Proc MIXED procedure of SAS 9.4 . Fixed effects in the model were maternal group (BCS), day (D), and their interaction (BCS × D). Random effect was calf within . The covariance structure SP (POW) for repeated measures was used for the analysis of plasma data over times. The covariate structure (AR) (1) was used for the analysis of growth measurements and daily starter intake. Additionally, Pearson correlation coefficients between concentrations of blood variables prior to calving in cows and their offspring body weight (BW) at birth were calculated using PROC CORR of SAS. Data are presented as least-square means with pooled SEMs. Significance was declared at P < 0.01;1.43 vs. 1.75 ± 0.07 DMI % of BW)  DMI during last 4 d of pregnancy in HighBCS cows. Prior to parturition, the HighBCS group had greater overall body weight  and BCS   to be greater in those cows.In plasma, overall concentrations of glucose, NEFA, ceruloplasmin, and NOP < 0.01), ceruloplasmin , and ROM  at − 10 d. In contrast, a positive correlation between urea  and BW at birth was detected in HighBCS calves.In HighBCS cows, a negative correlation Table  was obseP = 0.03; 42.52 vs. 44.67 ± 0.72 kg)  to be lower. Among the plasma biomarkers analyzed, total bilirubin and paraoxonase  in HighBCS calves. Plasma FRAP also tended to be lower in HighBCS calves throughout the postnatal period evaluated  prior to calving suggested that HighBCS cows might have experienced more pronounced inflammation, oxidative stress and lower liver function [One of the commonly-used blood variables of liver function in dairy cattle is cholesterol , 26, whifunction , 10, 24.In non-ruminant species, it is well-documented that maternal body condition, e.g. obesity and starvation, alter offspring size and growth. Specifically, obese women are more likely to give birth to “large-for-gestational age” babies that will have an increased risk of becoming obese later in life . In rumiThe greater birth weight and BW throughout the pre-weaning period in NormBCS vs. HighBCS calves could, at least in part, be explained by the greater DMI of NormBCS cows during the last 4 weeks of gestation. Maternal diets could alter placental transport of nutrients both in non-ruminant and ruminant species , 36. ThuBesides increased nutrient availability to the fetus and newborn, efficiency of nutrient utilization may have also been altered by maternal BCS. For instance, the greater concentrations of NEFA and BHBA at birth in calves born to NormBCS dams suggested they potentially had a more mature metabolism capable to readily use fat depots as a source of fatty acids for oxidation and ketogenesis. This hypothesis agrees with previous work indicating that neonates need to quickly activate processes such as lipolysis, glycogenolysis and gluconeogenesis to maintain normal blood glucose concentrations .It has been documented in dairy calves that lactate and amino acids (AA) account for 60% of gluconeogenesis during the first 3 d of life . Thus, aAlbumin is a negative acute-phase protein that is decreased during periods of inflammation . The grex as a major mechanism to kill bacterial pathogens [x was being produced and metabolized, suggesting that those calves had a better functioning  immune system [With regards to immune response, macrophages produce NOathogens . The nitathogens . In the e system .Ferric reducing ability of plasma (FRAP) is an important indicator of antioxidant power, as it is a measure of the reduction of ferric to ferrous ion . Thus, tIn addition to the temporal changes in growth, it is noteworthy to highlight the few differences in the concentrations of plasma variables of liver function throughout the first 9 weeks of life. For instance, paraoxonase is an enzyme produced by the liver that associates with high-density lipoproteins in blood to prevent oxidation . BilirubAs discussed earlier, changes in plasma NEFA and BHBA concentrations at birth suggested that NormBCS calves were utilizing more fatty acids a source of energy. While increased mitochondrial oxidation of fatty acids would be expected to improve liver function due to the reduction in fat storage in the liver, it would also enhance the production of reactive oxygen species. Clearly, further research could help better understand the physiological mechanisms that may be affected in the neonate as a result of high maternal body condition.Overall, calves born from dams with higher than normal BCS (3.83 ± 0.15) during the last ~ 4 weeks prepartum had lower BW at birth and throughout the first 9 weeks of life. At the whole-animal level these effects did not seem to be fully associated with differences in maternal DMI or feed intake by the calves. Thus, alterations in inflammatory and oxidative stress status along with nutrient utilization in utero likely played a role. Whether these encompass “programming” effects at the cellular level could not be discerned, but based on non-ruminant data it appears likely. Additional work clearly is needed to clarify the mechanisms by which maternal BCS along with other factors such as genetics, calving season, and previous lactation regulate immunometabolism in the developing fetus and neonatal calf.Additional file 1: Supplementary Table 1. Ingredient and nutrient composition of the diet fed during close-up dry period


Knowledge of age‐related DNA methylation changes in skeletal muscle is limited, yet this tissue is severely affected by ageing in humans.n = 908 muscle methylomes from men and women aged 18–89 years old). We explored the genomic context of age‐related DNA methylation changes in chromatin states, CpG islands, and transcription factor binding sites and performed gene set enrichment analysis. We then integrated the DNA methylation data with known transcriptomic and proteomic age‐related changes in skeletal muscle. Finally, we updated our recently developed muscle epigenetic clock (https://bioconductor.org/packages/release/bioc/html/MEAT.html).We conducted a large‐scale epigenome‐wide association study meta‐analysis of age in human skeletal muscle from 10 studies  performed similarly to the original version of the muscle clock (median of 4.4 vs. 4.6 years in age prediction error), suggesting that the original version of the muscle clock was very accurate.We identified 6710 differentially methylated regions at a stringent false discovery rate <0.005, spanning 6367 unique genes, many of which related to skeletal muscle structure and development. We found a strong increase in DNA methylation at Polycomb target genes and bivalent chromatin domains and a concomitant decrease in DNA methylation at enhancers. Most differentially methylated genes were not altered at the mRNA or protein level, but they were nonetheless strongly enriched for genes showing age‐related differential mRNA and protein expression. After adding a substantial number of samples from five datasets (+371), the updated version of the muscle clock .We provide here the most comprehensive picture of DNA methylation ageing in human skeletal muscle and reveal widespread alterations of genes involved in skeletal muscle structure, development, and differentiation. We have made our results available as an open‐access, user‐friendly, web‐based tool called  While human lifespan  has increased by ~3.5 years per decade since 1900,primary hallmarks of ageing, leading to dysregulated nutrient sensing, mitochondrial dysfunction, and cellular senescence, which ultimately results in stem cell exhaustion and altered intercellular communication.n = 10–50). Studies relying on a small sample size fail to detect small effect sizes and can be prone to large error, so larger initiatives are needed to identify the comprehensive list of CpG loci that change in methylation with age in human skeletal muscle. Meta‐analyses significantly increase statistical power and are more likely to identify robust age‐related methylation sites.Epigenetics are modifications of DNA that confer on the cell the ability to remember a past event.n = 908 samples from 10 datasets. Second, we performed enrichment analyses to unravel the potential functional consequences of these robust age‐related DNA methylation changes. Thirdly, we integrated age‐related methylome changes with transcriptome and proteome changes in skeletal muscle using two external, large‐scale studies. Finally, we updated our skeletal muscle epigenetic clockMetaMeth (https://sarah‐voisin.shinyapps.io/MetaMeth/), enabling users to look at age‐related changes in any gene of interest across the muscle methylome, transcriptome, and proteome.To address these gaps, we performed a large‐scale bioinformatics analysis of DNA methylation, and mRNA and protein changes with age in human skeletal muscle. We integrated original DNA methylation data from our laboratory (the Gene SMART cohort) with available open‐access data from multiple repositories and published studies. Firstly, we aimed to identify robust age‐related CpGs in skeletal muscle in an EWAS meta‐analysis of age, combining n = 908 skeletal muscle samples collected from men and women across the lifespan  for robustness. Samples from the Gene SMART cohort (n = 234) include two batches, and our recently published paperWe combined four datasets of genome‐wide DNA methylation in skeletal muscle . The CpGs associated with age at a stringent meta‐analysis false discovery rate (FDR) <0.005 were considered differentially methylated positions (DMPs). We then identified differentially methylated regions (DMRs) (i.e. clusters of DMPs with consistent DNA methylation change with age) using the dmrcate package, at a Fisher's multiple comparison statistic <0.005, a Stouffer score <0.005, and a harmonic mean of the individual component FDRs <0.005.dmrcate works by smoothing the test statistic of CpGs separated by a maximum of 1000 bp using a Gaussian kernel; then, it models the smoothed test statistics, computes and corrects P‐values, and finally aggregates adjacent CpGs that are significant and within 1000 bp of each other. We focused on the DMRs for all downstream analyses, as DMRs remove spatial redundancy  at different positions with respect to CpG islands, (ii) in different skeletal muscle chromatin states from the Roadmap Epigenomics Project,P‐value <0.005 was deemed significant.We used a missMethyl package.et al. of Illumina HumanMethylation arraysWe performed gene ontology (GO), KEGG, and Reactome enrichment on the age‐related DMRs using all tested CpGs as the background , thanks to the goregion function from the et al. that combined 2852 public gene expression arrays in skeletal muscle and identified 957 genes whose mRNA levels changed with age.et al. performed a large‐scale proteomics analysis of human skeletal muscle and identified 1265 genes whose protein levels were altered with age.χ2 test to see whether a disproportionate number of DMGs were also differentially expressed at the mRNA or protein level, and a P‐value <0.005 was deemed significant.Each gene with at least one DMR annotated to it was considered a differentially methylated gene (DMG). To gain insights into the functional consequences of DNA methylation changes with age in skeletal muscle, we compared DMGs with known differentially expressed genes at the transcriptomicSince the development of the original muscle clock that used 682 samples from 12 datasets to predict age from DNA methylation data,n = 908 samples from 601 individuals, TableFigureTables S2 and S3). We found slightly more hypomethylated than hypermethylated DMPs . The magnitude of age‐related DNA methylation changes was small and similar for both hypo‐DMPs and hyper‐DMPs: hypo‐DMPs lost an average of ~0.8% in methylation per decade of life, and hyper‐DMPs gained an average of ~0.6% in methylation per decade of life , so it is possible that the age‐related signal captured in this dataset was partially confounded by BMI. We repeated the meta‐analysis without GSE50498, but results were largely unchanged . DMRs' distribution in chromatin states was different from that of all tested CpGs . Including non‐coding genes, there were 6367 genes that harboured at least one DMR, hereinafter referred to as DMGs. A pathway enrichment on the DMRs revealed that DMGs were enriched for 48 GO terms (Table S4), all of which related to skeletal muscle structure development, muscle contraction, and calcium transporter regulation , but no significant enrichment was found for hypermethylated DMGs.Next, we integrated a comprehensive annotation of Illumina HumanMethylation arrayset al.et al.Figureχ2 test P‐value <2.2 × 10−16, FigureWe investigated the potential downstream effects of these age‐related DNA methylation changes on mRNA and protein expression in muscle. We utilized two external published studies: a transcriptomic meta‐analysis of age that combined 2852 public gene expression arrays in skeletal muscleFigureTable S6, FigureFigureNext, we investigated in more details the relationship between DNA methylation and mRNA or protein expression. This relationship is complex and depends on the genomic context, particularly the underlying chromatin stateFBXO32, TRIM63, MYOG, HDAC4, and HDAC5),CD36, GOT2, CPT1A, HADH, LPL, SLC27A1, SLC27A4, and UCP3),MYH6, MYH7, MYH1, MYL3, and MYH2).HDAC4 was mostly hypomethylated, and there was a corresponding increase in mRNA levels , but not necessarily the same HOX genes or in the same direction.HOXA cluster that were nearly all hypomethylated, one hypermethylated DMR at HOXB2/HOXB‐AS1, four hypermethylated DMRs in the HOXC cluster, and two hypermethylated DMRs in the HOXD cluster . The home page of the website provides a detailed list of instructions on how to visualize results and focus on specific CpGs, genes, or genomic regions of interest in a user‐friendly, interactive manner. To obtain forest plots for individual CpGs, users can enter the name of their CpG of interest (e.g. ‘cg11109027’) in the ‘Forest Plot’ tab, and the corresponding graph will appear, with the possibility to download the plot in jpg, png, or tif formats and at any resolution. To help with choosing CpGs to display, users can filter the list of CpGs based on their genomic location . To download summary statistics for DMPs or DMRs in a table format, users can go to the ‘Summary Tables’ tab and download the data as an excel or csv file, after optionally filtering data based on genomic location and statistics. Finally, we have also displayed the scatter plot of genes showing methylation, and mRNA and protein changes with age as an interactive graph: users simply need to hover their mouse on one point of the graph to be shown the name of the gene and the number of DMRs annotated to it. The code used to produce the website is available in open access on Sarah Voisin's GitHub account (https://github.com/sarah‐voisin/MetaMeth).We have made the results of the EWAS meta‐analysis of age in skeletal muscle available as an online webtool called n < 20)  uses DNA methylation at 156 CpGs to predict age, 73 of which were in common with MEAT  that we recently published, with the exception of datasets that were invariant in age and the datasets that were too small ( 20) see .9 The T Figure. We founhttps://sarah‐voisin.shinyapps.io/MetaMeth/), which allows researchers to visualize results in an interactive and user‐friendly manner. Finally, we updated our muscle clockTo paint a comprehensive picture of age‐related DNA methylation changes in human skeletal muscle, we conducted an EWAS meta‐analysis of age in human muscle across the lifespan, combining 908 samples from 10 independent datasets. In this study, we were able to demonstrate a profound effect of age on the muscle methylome. Additionally, we have provided a detailed account of the genomic context of age‐affected regions, reported putatively affected pathways, and integrated methylome changes with known transcriptome and proteome changes in muscle. To maximize the usefulness of this large‐scale EWAS meta‐analysis to the scientific community, we created a website named MetaMeth  and updated version (MEAT 2.0) of the clock for their analyses.Recently, we established an epigenetic clock for human skeletal muscle, using 682 samples from 12 datasets.The age‐related changes in the muscle methylome uncovered herein and the epigenetic age calculated from the MEAT clock reflect both intracellular changes in methylation levels and age‐related changes in muscle cell‐type composition. Older muscle tends to have a greater proportion of type IIx (hybrid) muscle fibres,MetaMeth (https://sarah‐voisin.shinyapps.io/MetaMeth/), largely inspired by the MetaMex tool developed by Pillon et al. for transcriptomic meta‐analysis of exercise training and inactivity in human skeletal muscle.To provide the scientific community with a tool to assess DNA methylation changes with age in skeletal muscle, we have created a user‐friendly, interactive, and transparent way to explore our results. We built a web‐based tool called S.V. and N.E. contributed in the conceptualization of the study; S.V. and S.H. in the methodology; S.V. in the investigation; S.V. in the formal analysis; M.J., S.L., N.R.H., L.M.H., L.R.G., S.G., M.O., M.J., K.J.A., J.‐L.M.T., A.G., C.J., R.C., H.V., V.R., F.P., P.F., S.B., M.T., A.P.S., A.S., M.R., S.H., and N.E. in the resources; S.V. in the software; S.V. and N.E. in writing of the original draft; S.V., M.J., S.L., S.G., M.O., M.J., K.J.A., A.G., C.J., F.P., J.M.C., S.B., M.T., A.P.S., A.S., M.R., S.H., and N.E. in writing of the review and editing; and S.V., L.R.G., and N.E. in funding acquisition.None declared.We are grateful for the support of the Australian National Health and Medical Research Council (NHMRC) via S.V.'s Early Career Research Fellowship (APP11577321) and N.E.'s Career Development Fellowship (APP1140644). We are also grateful for the support of the Jack Brockoff Foundation via S.V.'s medical grant. We also thank the Australian Research Council (ARC) for supporting this study (DP190103081 and DP200101830). The Gene SMART and LITER studies were both supported by the Collaborative Research Network for Advancing Exercise and Sports Science (201202) from the Department of Education and Training, Australia. N.R.H. and Ms J.‐L.M.T. were supported by a PhD stipend also provided by Bond University CRN‐AESS. This research was also supported by infrastructure purchased with Australian Government EIF Super Science Funds as part of the Therapeutic Innovation Australia—Queensland Node project (L.R.G.). A.P.S. was supported by GlaxoSmithKline, North Staffordshire Medical Institute, the Society fort Endocrinology, the Medical Research Council (MRC) and Engineering and Physical Sciences Research Council (EPSRC), UK Doctoral Training Centre, and the Norwegian School of Sport Sciences (Norges Idrettshøgskole). The work was also supported by the German Federal Ministry of Education and Research [Bundesministerium für Bildung und Forschung (BMBF): DZD Grant 82DZD00302] and the Brandenburg State (Germany). The EPIK study was supported by the Foundation Scientific Research—Flanders (FWO Grant F.0898.15).All studies used in this articleFigure S1. Age distribution in each of the 10 datasets included in the EWAS metahyphen;analysis, and database of origin. dbGAP = database of Genotypes and Phenotypes; GEO = Gene Expression Omnibus.Click here for additional data file.Figure S2. Quantile‐quantile plot of −log10 transformed P‐values for each of the 10 datasets included in the EWAS meta‐analysis. Right panel using uncorrected P‐values and left panel using bacon bias‐ and inflation‐corrected P‐values.Click here for additional data file.Figure S3. Comparison of results from the full meta‐analysis and from a meta‐analysis excluding GSE50498 (a), type 2 diabetes (T2D) patients (b), the ABOS cohort (c), or non‐Caucasian individuals (d). Each point is one of the 40,479 differentially methylated positions (DMPs) discovered in the full meta‐analysis. To compare results from the full and partial meta‐analyses, we plotted the effect size in the full meta‐analysis (x‐axis), against the effect size in the partial meta‐analysis (y‐axis). To show whether DMPs remained significant in the partial meta‐analysis, we coloured points according to the false discovery rate (FDR) in the partial meta‐analysis.Click here for additional data file.Table S1. Supporting informationClick here for additional data file.