SMCHD1 activates the expression of genes required for the expansion of human myoblasts.

SMCHD1 is an epigenetic regulatory protein known to modulate the targeted repression of large chromatin domains. Diminished SMCHD1 function in muscle fibers causes Facioscapulohumeral Muscular Dystrophy (FSHD2) through derepression of the D4Z4 chromatin domain, an event which permits the aberrant expression of the disease-causing gene DUX4. Given that SMCHD1 plays a broader role in establishing the cellular epigenome, we examined whether loss of SMCHD1 function might affect muscle homeostasis through additional mechanisms.

A human immune/muscle xenograft model of FSHD muscle pathology.

Background: Facioscapulohumeral muscular dystrophy (FSHD) disease progression is associated with muscle inflammation, although its role in FSHD muscle pathology is unknown.

Methods: We have developed a novel humanized mouse strain, NSG-SGM3-W41, that supports the co- engraftment of human hematopoietic stem cells (HSCs) and muscle myoblasts as an experimental model to investigate the role of innate immunity in FSHD muscle pathology.

Results: The NSG-SGM3-W41 mouse supports the selective expansion of human innate immune cell lineages following engraftment of human HSCs and the co-engraftment and differentiation of patient-derived FSHD or control muscle myoblasts.

Flavones provide resistance to DUX4-induced toxicity via an mTor-independent mechanism.

Facioscapulohumeral muscular dystrophy (FSHD) is among the most common of the muscular dystrophies, affecting nearly 1 in 8000 individuals, and is a cause of profound disability. Genetically, FSHD is linked to the contraction and/or epigenetic de-repression of the D4Z4 repeat array on chromosome 4, thereby allowing expression of the DUX4 gene in skeletal muscle. If the DUX4 transcript incorporates a stabilizing polyadenylation site the myotoxic DUX4 protein will be synthesized, resulting in muscle wasting.

Addressing the dNTP bottleneck restricting prime editing activity.

Prime editing efficiency is modest in cells that are quiescent or slowly proliferating where intracellular dNTP levels are tightly regulated. MMLV-reverse transcriptase - the prime editor polymerase subunit - requires high intracellular dNTPs levels for efficient polymerization. We report that prime editing efficiency in primary cells and in vivo is increased by mutations that enhance the enzymatic properties of MMLV-reverse transcriptase and can be further complemented by targeting SAMHD1 for degradation.

Post-Translational Modifications of the DUX4 Protein Impact Toxic Function in FSHD Cell Models.

Objective: Facioscapulohumeral muscular dystrophy (FSHD) is caused by abnormal de-repression of the myotoxic transcription factor DUX4. Although the transcriptional targets of DUX4 are known, the regulation of DUX4 protein and the molecular consequences of this regulation are unclear. Here, we used in vitro models of FSHD to identify and characterize DUX4 post-translational modifications (PTMs) and their impact on the toxic function of DUX4.

Impaired mitochondrial oxidative metabolism in skeletal progenitor cells leads to musculoskeletal disintegration.

Although skeletal progenitors provide a reservoir for bone-forming osteoblasts, the major energy source for their osteogenesis remains unclear. Here, we demonstrate a requirement for mitochondrial oxidative phosphorylation in the osteogenic commitment and differentiation of skeletal progenitors. Deletion of Evolutionarily Conserved Signaling Intermediate in Toll pathways (ECSIT) in skeletal progenitors hinders bone formation and regeneration, resulting in skeletal deformity, defects in the bone marrow niche and spontaneous fractures followed by persistent nonunion.

Generation of iMyoblasts from Human Induced Pluripotent Stem Cells.

Skeletal muscle stem cells differentiated from human-induced pluripotent stem cells (hiPSCs) serve as a uniquely promising model system for investigating human myogenesis and disease pathogenesis, and for the development of gene editing and regenerative stem cell therapies. Here, we present an effective and reproducible transgene-free protocol for derivation of human skeletal muscle stem cells, iMyoblasts, from hiPSCs. Our two-step protocol consists of 1) small molecule-based differentiation of hiPSCs into myocytes, and 2) stimulation of differentiated myocytes with growth factor-rich medium to activate the proliferation of undifferentiated reserve cells, for expansion and cell line establishment.

DUX4 expression activates JNK and p38 MAP kinases in myoblasts.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by misexpression of the DUX4 transcription factor in skeletal muscle that results in transcriptional alterations, abnormal phenotypes and cell death. To gain insight into the kinetics of DUX4-induced stresses, we activated DUX4 expression in myoblasts and performed longitudinal RNA sequencing paired with proteomics and phosphoproteomics. This analysis revealed changes in cellular physiology upon DUX4 activation, including DNA damage and altered mRNA splicing.

Efficient Homology-Directed Repair with Circular Single-Stranded DNA Donors.

While genome editing has been revolutionized by the advent of CRISPR-based nucleases, difficulties in achieving efficient, nuclease-mediated, homology-directed repair (HDR) still limit many applications. Commonly used DNA donors such as plasmids suffer from low HDR efficiencies in many cell types, as well as integration at unintended sites. In contrast, single-stranded DNA (ssDNA) donors can produce efficient HDR with minimal off-target integration.

An acute naproxen dose does not affect core temperature or Interleukin-6 during cycling in a hot environment.

Non-steroidal anti-inflammatory drugs' anti-pyretic and anti-inflammatory effects has led some individuals to theorize these medications may blunt core body temperature (Tc) increases during exercise. We utilized a double-blind, randomized, and counterbalanced cross-over design to examine the effects of a 24-h naproxen dose (3-220 ​mg naproxen pills) and placebo (0 ​mg naproxen) on Tc and plasma interleukin-6 (IL-6) concentrations during cycling in a hot or ambient environment. Participants ( ​= ​11; 6 male, 5 female; age ​= ​27.

Outcome Measures in Facioscapulohumeral Muscular Dystrophy Clinical Trials.

Facioscapulohumeral muscular dystrophy (FSHD) is a debilitating muscular dystrophy with a variable age of onset, severity, and progression. While there is still no cure for this disease, progress towards FSHD therapies has accelerated since the underlying mechanism of epigenetic derepression of the double homeobox 4 () gene leading to skeletal muscle toxicity was identified. This has facilitated the rapid development of novel therapies to target expression and downstream dysregulation that cause muscle degeneration.

iMyoblasts for ex vivo and in vivo investigations of human myogenesis and disease modeling.

Skeletal muscle myoblasts (iMyoblasts) were generated from human induced pluripotent stem cells (iPSCs) using an efficient and reliable transgene-free induction and stem cell selection protocol. Immunofluorescence, flow cytometry, qPCR, digital RNA expression profiling, and scRNA-Seq studies identify iMyoblasts as a skeletal myogenic lineage with a fetal-like transcriptome signature, distinct from adult muscle biopsy myoblasts (bMyoblasts) and iPSC-induced muscle progenitors. iMyoblasts can be stably propagated for >12 passages or 30 population doublings while retaining their dual commitment for myotube differentiation and regeneration of reserve cells.

p38 MAPKs - roles in skeletal muscle physiology, disease mechanisms, and as potential therapeutic targets.

p38 MAPKs play a central role in orchestrating the cellular response to stress and inflammation and in the regulation of myogenesis. Potent inhibitors of p38 MAPKs have been pursued as potential therapies for several disease indications due to their antiinflammatory properties, although none have been approved to date. Here, we provide a brief overview of p38 MAPKs, including their role in regulating myogenesis and their association with disease progression.

A 24 hour naproxen dose on gastrointestinal distress and performance during cycling in the heat.

Using a double-blind, randomized and counterbalanced, cross-over design, we assessed naproxen's effects on gastrointestinal (GI) distress and performance in eleven volunteers (6 male, 5 female). Participants completed 4 trials: 1) placebo and ambient); 2) placebo and heat; 3) naproxen and ambient; and 4) naproxen and heat. Independent variables were one placebo or 220 mg naproxen pill every 8 h (h) for 24 h and ambient (22.

Identification of the hyaluronic acid pathway as a therapeutic target for facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is linked to epigenetic derepression of the germline/embryonic transcription factor DUX4 in skeletal muscle. However, the etiology of muscle pathology is not fully understood, as DUX4 misexpression is not tightly correlated with disease severity. Using a DUX4-inducible cell model, we show that multiple DUX4-induced molecular pathologies that have been observed in patient-derived disease models are mediated by the signaling molecule hyaluronic acid (HA), which accumulates following DUX4 induction.

Precise therapeutic gene correction by a simple nuclease-induced double-stranded break.

Current programmable nuclease-based methods (for example, CRISPR-Cas9) for the precise correction of a disease-causing genetic mutation harness the homology-directed repair pathway. However, this repair process requires the co-delivery of an exogenous DNA donor to recode the sequence and can be inefficient in many cell types. Here we show that disease-causing frameshift mutations that result from microduplications can be efficiently reverted to the wild-type sequence simply by generating a DNA double-stranded break near the centre of the duplication.

Contemporary changes to herding systems in China and effects on pasture quality: a case study in Gansu Province, 2000-2012.

Post-2000 efforts to protect China's pastoral areas are distinct from earlier efforts in that funding for the most recent round of policies and programs is commensurate with the task. Even with appropriate funding, however, effective methods of mitigating pasture degradation are widely disputed. The most controversial of the current policies include herding family resettlement, pasture fallow programs (herding "bans"), and the promotion of confined animal feeding operations (CAFOs).

Facioscapulohumeral Muscular Dystrophy.

Facioscapulohumeral Muscular Dystrophy is a common form of muscular dystrophy that presents clinically with progressive weakness of the facial, scapular, and humeral muscles, with later involvement of the trunk and lower extremities. While typically inherited as autosomal dominant, facioscapulohumeral muscular dystrophy (FSHD) has a complex genetic and epigenetic etiology that has only recently been well described. The most prevalent form of the disease, FSHD1, is associated with the contraction of the D4Z4 microsatellite repeat array located on a permissive 4qA chromosome.

Individual fluid plans versus ad libitum on hydration status in minor professional ice hockey players.

Background: Despite exercising in cool environments, ice hockey players exhibit several dehydration risk factors. Individualized fluid plans (IFPs) are designed to mitigate dehydration by matching an individual's sweat loss in order to optimize physiological systems and performance.

Methods: A randomized control trial was used to examine IFP versus ad libitum fluid ingestion on hydration in 11 male minor professional ice hockey players (mean age = 24.

Morpholino-mediated Knockdown of DUX4 Toward Facioscapulohumeral Muscular Dystrophy Therapeutics.

Derepression of DUX4 in skeletal muscle has emerged as a likely cause of pathology in facioscapulohumeral muscular dystrophy (FSHD). Here we report on the use of antisense phosphorodiamidate morpholino oligonucleotides to suppress DUX4 expression and function in FSHD myotubes and xenografts. The most effective was phosphorodiamidate morpholino oligonucleotide FM10, which targets the polyadenylation signal of DUX4.