Setdb1 protects genome integrity in murine muscle stem cells to allow for regenerative myogenesis and inflammation.

The histone H3 lysine 9 methyltransferase SETDB1 controls transcriptional repression to direct stem cell fate. Here, we show that Setdb1 expression by adult muscle stem cells (MuSCs) is required for skeletal muscle regeneration. We find that SETDB1 represses the expression of endogenous retroviruses (ERVs) in MuSCs. ERV de-repression in Setdb1-null MuSCs prevents their amplification following exit from quiescence and promotes cell death. Multi-omics profiling shows that chromatin decompaction at ERV loci activates the DNA-sensing cGAS-STING pathway, entailing cytokine expression by Setdb1-null MuSCs. This is followed by aberrant infiltration of inflammatory cells, including pathological macrophages. The ensuing histiocytosis is accompanied by myofiber necrosis, which, in addition to progressive MuSCs depletion, completely abolishes tissue repair. In contrast, loss of Setdb1 in fibro-adipogenic progenitors (FAPs) does not impact immune cells. In conclusion, genome maintenance by SETDB1 in an adult somatic stem cell is necessary for both its regenerative potential and adequate reparative inflammation.

The impact of the cost-of-living crisis on population health in the UK: rapid evidence review.

BACKGROUND: In the UK, unique and unforeseen factors, including COVID-19, Brexit, and Ukraine-Russia war, have resulted in an unprecedented cost of living crisis, creating a second health emergency. We present, one of the first rapid reviews with the aim of examining the impact of this current crisis, at a population level. We reviewed published literature, as well as grey literature, examining a broad range of physical and mental impacts on health in the short, mid, and long term, identifying those most at risk, impacts on system partners, including emergency services and the third sector, as well as mitigation strategies. METHODS: We conducted a rapid review by searching PubMed, Embase, MEDLINE, and HMIC (2020 to 2023). We searched for grey literature on Google and hand-searched the reports of relevant public health organisations. We included interventional and observational studies that reported outcomes of interventions aimed at mitigating against the impacts of cost of living at a population level. RESULTS: We found that the strongest evidence was for the impact of cold and mouldy homes on respiratory-related infections and respiratory conditions. Those at an increased risk were young children (0-4 years), the elderly (aged 75 and over), as well as those already vulnerable, including those with long-term multimorbidity. Further short-term impacts include an increased risk of physical pain including musculoskeletal and chest pain, and increased risk of enteric infections and malnutrition. In the mid-term, we could see increases in hypertension, transient ischaemic attacks, and myocardial infarctions, and respiratory illnesses. In the long term we could see an increase in mortality and morbidity rates from respiratory and cardiovascular disease, as well as increase rates of suicide and self-harm and infectious disease outcomes. Changes in behaviour are likely particularly around changes in food buying patterns and the ability to heat a home. System partners are also impacted, with voluntary sectors seeing fewer volunteers, an increase in petty crime and theft, alternative heating appliances causing fires, and an increase in burns and burn-related admissions. To mitigate against these impacts, support should be provided, to the most vulnerable, to help increase disposable income, reduce energy bills, and encourage home improvements linked with energy efficiency. Stronger links to bridge voluntary, community, charity and faith groups are needed to help provide additional aid and support. CONCLUSION: Although the CoL crisis affects the entire population, the impacts are exacerbated in those that are most vulnerable, particularly young children, single parents, multigenerational families. More can be done at a community and societal level to support the most vulnerable, and those living with long-term multimorbidity. This review consolidates the current evidence on the impacts of the cost of living crisis and may enable decision makers to target limited resources more effectively.

Criteria to achieve safe antimicrobial intravenous-to-oral switch in hospitalised adult populations: a systematic rapid review.

OBJECTIVES: This rapid review aimed to assess and collate intravenous-to-oral switch (IVOS) criteria from the literature to achieve safe and effective antimicrobial IVOS in the hospital inpatient adult population. DESIGN: The rapid review follows the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement. DATA SOURCES: OVID Embase and Medline databases. ELIGIBILITY CRITERIA: Articles of adult populations published globally between 2017 and 2021 were included. DATA EXTRACTION AND SYNTHESIS: An Excel spreadsheet was designed with specific column headings. IVOS criteria from UK hospital IVOS policies informed the framework synthesis. RESULTS: IVOS criteria from 45/164 (27%) local IVOS policies were categorised into a five-section framework: (1) timing of IV antimicrobial review, (2) clinical signs and symptoms, (3) infection markers, (4) enteral route and (5) infection exclusions. The literature search identified 477 papers, of which 16 were included. The most common timing for review was 48-72 hours from initiation of intravenous antimicrobial (n=5, 30%). Nine studies (56%) stated clinical signs and symptoms must be improving. Temperature was the most frequently mentioned infection marker (n=14, 88%). Endocarditis had the highest mention as an infection exclusion (n=12, 75%). Overall, 33 IVOS criteria were identified to go forward into the Delphi process. CONCLUSION: Through the rapid review, 33 IVOS criteria were collated and presented within five distinct and comprehensive sections. The literature highlighted the possibility of reviewing IVOS before 48-72 hours and of presenting heart rate, blood pressure and respiratory rate as a combination early warning score criterion. The criteria identified can serve as a starting point of IVOS criteria review for any institution globally, as no country or region limits were applied. Further research is required to achieve consensus on IVOS criteria from healthcare professionals that manage patients with infections. PROSPERO REGISTRATION NUMBER: CRD42022320343.

Influence of factors commonly known to be associated with health inequalities on antibiotic use in high-income countries: a systematic scoping review.

INTRODUCTION: Inappropriate antibiotic use contributes to antimicrobial resistance. High-income countries have high rates of antibiotic use, with a prevalence of health inequalities amongst populations. OBJECTIVES: To understand the influence of factors commonly known to be associated with health inequalities on antibiotic use in high-income countries. METHODS: Factors commonly known to be associated with health inequalities were defined as protected characteristics under UK's Equality Act (age, disability, gender reassignment, marriage and civil partnership, pregnancy and maternity, race/ethnicity, religion or belief, sex, sexual orientation), socioeconomic characteristics (income, insurance, employment status, deprivation, education), geography (urban versus rural, region) and vulnerable groups. The study followed PRISMA-ScR and, PRISMA-E statements. RESULTS: Fifty-eight of 402 identified studies met inclusion criteria. Fifty of those papers (86%) included one or more protected characteristics, 37 (64%) socioeconomic characteristics, 21 (36%) geography and 6 (10%) vulnerable groups. Adults in older age groups, especially those in residential care, had the highest antibiotic use. The influence of race or ethnicity and antibiotic use was particular to country context. Areas of high deprivation had higher antibiotic use compared with areas of no or low deprivation, and geographical variation existed within countries. When faced with health system barriers, migrants relied on alternative routes of antibiotic supply other than prescription. RECOMMENDATIONS FOR FUTURE RESEARCH: To investigate how factors and wider social determinants of health interplay and impact antibiotic use, using frameworks/approaches to reduce health inequalities such as England's Core20PLUS approach. Antimicrobial stewardship initiatives should equip healthcare professionals to review patients at the highest risk of antibiotic use.

GLI3 regulates muscle stem cell entry into GAlert and self-renewal.

Satellite cells are required for the growth, maintenance, and regeneration of skeletal muscle. Quiescent satellite cells possess a primary cilium, a structure that regulates the processing of the GLI family of transcription factors. Here we find that GLI3 processing by the primary cilium plays a critical role for satellite cell function. GLI3 is required to maintain satellite cells in a G0 dormant state. Strikingly, satellite cells lacking GLI3 enter the GAlert state in the absence of injury. Furthermore, GLI3 depletion stimulates expansion of the stem cell pool. As a result, satellite cells lacking GLI3 display rapid cell-cycle entry, increased proliferation and augmented self-renewal, and markedly enhanced regenerative capacity. At the molecular level, we establish that the loss of GLI3 induces mTORC1 signaling activation. Therefore, our results provide a mechanism by which GLI3 controls mTORC1 signaling, consequently regulating muscle stem cell activation and fate.

Acetylation of PAX7 controls muscle stem cell self-renewal and differentiation potential in mice.

Muscle stem cell function has been suggested to be regulated by Acetyl-CoA and NAD+ availability, but the mechanisms remain unclear. Here we report the identification of two acetylation sites on PAX7 that positively regulate its transcriptional activity. Lack of PAX7 acetylation reduces DNA binding, specifically to the homeobox motif. The acetyltransferase MYST1 stimulated by Acetyl-CoA, and the deacetylase SIRT2 stimulated by NAD +, are identified as direct regulators of PAX7 acetylation and asymmetric division in muscle stem cells. Abolishing PAX7 acetylation in mice using CRISPR/Cas9 mutagenesis leads to an expansion of the satellite stem cell pool, reduced numbers of asymmetric stem cell divisions, and increased numbers of oxidative IIA myofibers. Gene expression analysis confirms that lack of PAX7 acetylation preferentially affects the expression of target genes regulated by homeodomain binding motifs. Therefore, PAX7 acetylation status regulates muscle stem cell function and differentiation potential to facilitate metabolic adaptation of muscle tissue.

Mouse WIF1 Is Only Modified with O-Fucose in Its EGF-like Domain III Despite Two Evolutionarily Conserved Consensus Sites.

The Wnt Inhibitory Factor 1 (Wif1), known to inhibit Wnt signaling pathways, is composed of a WIF domain and five EGF-like domains (EGF-LDs) involved in protein interactions. Despite the presence of a potential O-fucosylation site in its EGF-LDs III and V, the O-fucose sites occupancy has never been demonstrated for WIF1. In this study, a phylogenetic analysis on the distribution, conservation and evolution of Wif1 proteins was performed, as well as biochemical approaches focusing on O-fucosylation sites occupancy of recombinant mouse WIF1. In the monophyletic group of gnathostomes, we showed that the consensus sequence for O-fucose modification by Pofut1 is highly conserved in Wif1 EGF-LD III while it was more divergent in EGF-LD V. Using click chemistry and mass spectrometry, we demonstrated that mouse WIF1 was only modified with a non-extended O-fucose on its EGF-LD III. In addition, a decreased amount of mouse WIF1 in the secretome of CHO cells was observed when the O-fucosylation site in EGF-LD III was mutated. Based on sequence comparison and automated protein modeling, we suggest that the absence of O-fucose on EGF-LD V of WIF1 in mouse and probably in most gnathostomes, could be related to EGF-LD V inability to interact with POFUT1.

The effectiveness of smoking cessation, alcohol reduction, diet and physical activity interventions in changing behaviours during pregnancy: A systematic review of systematic reviews.

BACKGROUND: Pregnancy is a teachable moment for behaviour change. Multiple guidelines target pregnant women for behavioural intervention. This systematic review of systematic reviews reports the effectiveness of interventions delivered during pregnancy on changing women's behaviour across multiple behavioural domains. METHODS: Fourteen databases were searched for systematic reviews published from 2008, reporting interventions delivered during pregnancy targeting smoking, alcohol, diet or physical activity as outcomes. Data on behaviour change related to these behaviours are reported here. Quality was assessed using the JBI critical appraisal tool for umbrella reviews. Consistency in intervention effectiveness and gaps in the evidence-base are described. RESULTS: Searches identified 24,388 results; 109 were systematic reviews of behaviour change interventions delivered in pregnancy, and 36 reported behavioural outcomes. All smoking and alcohol reviews identified reported maternal behaviours as outcomes (n = 16 and 4 respectively), whereas only 16 out of 89 diet and/or physical activity reviews reported these behaviours. Most reviews were high quality (67%) and interventions were predominantly set in high-income countries. Overall, there was consistent evidence for improving healthy diet behaviours related to increasing fruit and vegetable consumption and decreasing carbohydrate intake, and fairly consistent evidence for increase in some measures of physical activity (METs and VO2 max) and for reductions in fat intake and smoking during pregnancy. There was a lack of consistent evidence across reviews reporting energy, protein, fibre, or micronutrient intakes; smoking cessation, abstinence or relapse; any alcohol behaviours. CONCLUSIONS: The most consistent review evidence is for interventions improving dietary behaviours during pregnancy compared with other behaviours, although the majority of diet reviews prioritised reporting health-related outcomes over behavioural outcomes. Heterogeneity between reported behaviour outcomes limits ability to pool data in meta-analysis and more consistent reporting is needed. Limited data are available for alcohol interventions in pregnancy or interventions in low- or middle-income-countries, which are priority areas for future research.

MLL1 is required for PAX7 expression and satellite cell self-renewal in mice.

PAX7 is a paired-homeobox transcription factor that specifies the myogenic identity of muscle stem cells and acts as a nodal factor by stimulating proliferation while inhibiting differentiation. We previously found that PAX7 recruits the H3K4 methyltransferases MLL1/2 to epigenetically activate target genes. Here we report that in the absence of Mll1, myoblasts exhibit reduced H3K4me3 at both Pax7 and Myf5 promoters and reduced Pax7 and Myf5 expression. Mll1-deficient myoblasts fail to proliferate but retain their differentiation potential, while deletion of Mll2 had no discernable effect. Re-expression of PAX7 in committed Mll1 cKO myoblasts restored H3K4me3 enrichment at the Myf5 promoter and Myf5 expression. Deletion of Mll1 in satellite cells reduced satellite cell proliferation and self-renewal, and significantly impaired skeletal muscle regeneration. Pax7 expression was unaffected in quiescent satellite cells but was markedly downregulated following satellite cell activation. Therefore, MLL1 is required for PAX7 expression and satellite cell function in vivo. Furthermore, PAX7, but not MLL1, is required for Myf5 transcriptional activation in committed myoblasts.

EGFR-Aurka Signaling Rescues Polarity and Regeneration Defects in Dystrophin-Deficient Muscle Stem Cells by Increasing Asymmetric Divisions.

Loss of dystrophin expression in Duchenne muscular dystrophy (DMD) causes progressive degeneration of skeletal muscle, which is exacerbated by reduced self-renewing asymmetric divisions of muscle satellite cells. This, in turn, affects the production of myogenic precursors and impairs regeneration and suggests that increasing such divisions may be beneficial. Here, through a small-molecule screen, we identified epidermal growth factor receptor (EGFR) and Aurora kinase A (Aurka) as regulators of asymmetric satellite cell divisions. Inhibiting EGFR causes a substantial shift from asymmetric to symmetric division modes, whereas EGF treatment increases asymmetric divisions. EGFR activation acts through Aurka to orient mitotic centrosomes, and inhibiting Aurka blocks EGF stimulation-induced asymmetric division. In vivo EGF treatment markedly activates asymmetric divisions of dystrophin-deficient satellite cells in mdx mice, increasing progenitor numbers, enhancing regeneration, and restoring muscle strength. Therefore, activating an EGFR-dependent polarity pathway promotes functional rescue of dystrophin-deficient satellite cells and enhances muscle force generation.

Orienting Muscle Stem Cells for Regeneration in Homeostasis, Aging, and Disease.

Muscle stem cells, or satellite cells, are required for skeletal muscle maintenance, growth, and repair. Following satellite cell activation, several factors drive asymmetric cell division to generate a stem cell and a proliferative progenitor that forms new muscle. The balance between symmetric self-renewal and asymmetric division significantly impacts the efficiency of regeneration. In this Review, we discuss the relationship of satellite cell heterogeneity and the establishment of polarity to asymmetric division, as well as how these processes are impacted in homeostasis, aging, and disease. We also highlight therapeutic opportunities for targeting satellite cell polarity and self-renewal to stimulate muscle regeneration.

The Dystrophin Glycoprotein Complex Regulates the Epigenetic Activation of Muscle Stem Cell Commitment.

Asymmetrically dividing muscle stem cells in skeletal muscle give rise to committed cells, where the myogenic determination factor Myf5 is transcriptionally activated by Pax7. This activation is dependent on Carm1, which methylates Pax7 on multiple arginine residues, to recruit the ASH2L:MLL1/2:WDR5:RBBP5 histone methyltransferase complex to the proximal promoter of Myf5. Here, we found that Carm1 is a specific substrate of p38γ/MAPK12 and that phosphorylation of Carm1 prevents its nuclear translocation. Basal localization of the p38γ/p-Carm1 complex in muscle stem cells occurs via binding to the dystrophin-glycoprotein complex (DGC) through ß1-syntrophin. In dystrophin-deficient muscle stem cells undergoing asymmetric division, p38γ/ß1-syntrophin interactions are abrogated, resulting in enhanced Carm1 phosphorylation. The resulting progenitors exhibit reduced Carm1 binding to Pax7, reduced H3K4-methylation of chromatin, and reduced transcription of Myf5 and other Pax7 target genes. Therefore, our experiments suggest that dysregulation of p38γ/Carm1 results in altered epigenetic gene regulation in Duchenne muscular dystrophy.

Intermittent fasting interventions for treatment of overweight and obesity in adults: a systematic review and meta-analysis.

OBJECTIVE: To examine the effectiveness of intermittent energy restriction in the treatment for overweight and obesity in adults, when compared to usual care treatment or no treatment. INTRODUCTION: Intermittent energy restriction encompasses dietary approaches including intermittent fasting, alternate day fasting, and fasting for two days per week. Despite the recent popularity of intermittent energy restriction and associated weight loss claims, the supporting evidence base is limited. INCLUSION CRITERIA: This review included overweight or obese (BMI ≥25 kg/m) adults (≥18 years). Intermittent energy restriction was defined as consumption of ≤800 kcal on at least one day, but no more than six days per week. Intermittent energy restriction interventions were compared to no treatment (ad libitum diet) or usual care (continuous energy restriction ∼25% of recommended energy intake). Included interventions had a minimum duration of 12 weeks from baseline to post outcome measurements. The types of studies included were randomized and pseudo-randomized controlled trials. The primary outcome of this review was change in body weight. Secondary outcomes included: i) anthropometric outcomes (change in BMI, waist circumference, fat mass, fat free mass); ii) cardio-metabolic outcomes (change in blood glucose and insulin, lipoprotein profiles and blood pressure); and iii) lifestyle outcomes: diet, physical activity, quality of life and adverse events. METHODS: A systematic search was conducted from database inception to November 2015. The following electronic databases were searched: MEDLINE, Embase, CINAHL, Cochrane Library, ClinicalTrials.gov, ISRCTN registry, and anzctr.org.au for English language published studies, protocols and trials. Two independent reviewers evaluated the methodological quality of included studies using the standardized critical appraisal instruments from the Joanna Briggs Institute. Data were extracted from papers included in the review by two independent reviewers using the standardized data extraction tool from the Joanna Briggs Institute. Effect sizes were expressed as weighted mean differences and their 95% confidence intervals were calculated for meta-analyses. RESULTS: Six studies were included in this review. The intermittent energy restriction regimens varied across studies and included alternate day fasting, fasting for two days, and up to four days per week. The duration of studies ranged from three to 12 months. Four studies included continuous energy restriction as a comparator intervention and two studies included a no treatment control intervention. Meta-analyses showed that intermittent energy restriction was more effective than no treatment for weight loss (-4.14 kg; 95% CI -6.30 kg to -1.99 kg; p ≤ 0.001). Although both treatment interventions achieved similar changes in body weight (approximately 7 kg), the pooled estimate for studies that investigated the effect of intermittent energy restriction in comparison to continuous energy restriction revealed no significant difference in weight loss (-1.03 kg; 95% CI -2.46 kg to 0.40 kg; p = 0.156). CONCLUSIONS: Intermittent energy restriction may be an effective strategy for the treatment of overweight and obesity. Intermittent energy restriction was comparable to continuous energy restriction for short term weight loss in overweight and obese adults. Intermittent energy restriction was shown to be more effective than no treatment, however, this should be interpreted cautiously due to the small number of studies and future research is warranted to confirm the findings of this review.

Single EDL Myofiber Isolation for Analyses of Quiescent and Activated Muscle Stem Cells.

Adult satellite cells are quiescent, but are poised for activation in response to exercise, injury, or disease allowing adult muscle growth or repair. Once activated, satellite cells proliferate extensively to produce enough myogenic progenitors in order to regenerate the muscles. In order to self-renew, a subset of activated satellite cells can resist the myogenic differentiation and return to quiescence to replenish the satellite cell pool. These cellular processes that normally occur during skeletal muscle regeneration can be recapitulated ex vivo using isolated and cultured myofibers. Here, we describe a protocol to isolate single myofibers from the extensor digitorum longus muscle. Moreover, we detail experimental conditions for analyzing satellite cells in quiescence and progression through the myogenic lineage.

The myogenic regulatory factors, determinants of muscle development, cell identity and regeneration.

The Myogenic Regulatory Factors (MRFs) Myf5, MyoD, myogenin and MRF4 are members of the basic helix-loop-helix family of transcription factors that control the determination and differentiation of skeletal muscle cells during embryogenesis and postnatal myogenesis. The dynamics of their temporal and spatial expression as well as their biochemical properties have allowed the identification of a precise and hierarchical relationship between the four MRFs. This relationship establishes the myogenic lineage as well as the maintenance of the terminal myogenic phenotype. The application of genome-wide technologies has provided important new information as to how the MRFs function to activate muscle gene expression. Application of combined functional genomics technologies along with single cell lineage tracing strategies will allow a deeper understanding of the mechanisms mediating myogenic determination, cell differentiation and muscle regeneration.

Concise Review: Epigenetic Regulation of Myogenesis in Health and Disease.

Skeletal muscle regeneration is initiated by satellite cells, a population of adult stem cells that reside in the muscle tissue. The ability of satellite cells to self-renew and to differentiate into the muscle lineage is under transcriptional and epigenetic control. Satellite cells are characterized by an open and permissive chromatin state. The transcription factor Pax7 is necessary for satellite cell function. Pax7 is a nodal factor regulating the expression of genes associated with satellite cell growth and proliferation, while preventing differentiation. Pax7 recruits chromatin modifiers to DNA to induce expression of specific target genes involved in myogenic commitment following asymmetric division of muscle stem cells. Emerging evidence suggests that replacement of canonical histones with histone variants is an important regulatory mechanism controlling the ability of satellite cells and myoblasts to differentiate. Differentiation into the muscle lineage is associated with a global gene repression characterized by a decrease in histone acetylation with an increase in repressive histone marks. However, genes important for differentiation are upregulated by the specific action of histone acetyltransferases and other chromatin modifiers, in combination with several transcription factors, including MyoD and Mef2. Treatment with histone deacetylase (HDAC) inhibitors enhances muscle regeneration and is considered as a therapeutic approach in the treatment of muscular dystrophy. This review describes the recent findings on epigenetic regulation in satellite stem cells and committed myoblasts. The potential of epigenetic drugs, such as HDAC inhibitors, as well as their molecular mechanism of action in muscle cells, will be addressed.

Enhancement of C2C12 myoblast proliferation and differentiation by GASP-2, a myostatin inhibitor.

BACKGROUND: GASP-2 is a secreted multi-domain glycoprotein known as a specific inhibitor of myostatin and GDF-11. Here we investigate the role of GASP-2 on myogenesis and the effect of its glycosylation on its activity. METHODS: GASP-2 overexpression or knockdown by shRNAs were carried out on C2C12 myoblasts cells. In silico analysis of GASP-2 protein was performed to identify its glycosylation sites. We produced a mouse recombinant GASP-2 protein in a prokaryotic system to obtain a fully deglycosylated protein allowing us to study the importance of this post-translational modification on GASP-2 activity. RESULTS: Both mature and deglycosylated GASP-2 proteins increase C2C12 proliferation and differentiation by inhibiting the myostatin pathway. In silico and western-blot analyses revealed that GASP-2 presents one consensus sequence for N-glycosylation and six potential sites of mucin-type O-glycosylation. CONCLUSIONS: GASP-2 promotes myogenesis and thus independently of its glycosylation. GENERAL SIGNIFICANCE: This is the first report demonstrating that GASP-2 promotes proliferation and differentiation of myoblasts by inhibiting the canonical pathway of myostatin.