Recent advances in base editing have created an exciting opportunity to precisely correct disease-causing mutations. However, the large size of base editors and their inherited off-target activities ...pose challenges for in vivo base editing. Moreover, the requirement of a protospacer adjacent motif (PAM) nearby the mutation site further limits the targeting feasibility. Here we modify the NG-targeting adenine base editor (iABE-NGA) to overcome these challenges and demonstrate the high efficiency to precisely edit a Duchenne muscular dystrophy (DMD) mutation in adult mice. Systemic delivery of AAV9-iABE-NGA results in dystrophin restoration and functional improvement. At 10 months after AAV9-iABE-NGA treatment, a near complete rescue of dystrophin is measured in mdx
mouse hearts with up to 15% rescue in skeletal muscle fibers. The off-target activities remains low and no obvious toxicity is detected. This study highlights the promise of permanent base editing using iABE-NGA for the treatment of monogenic diseases.
Duchenne muscular dystrophy is a severe inherited form of muscular dystrophy caused by mutations in the reading frame of the dystrophin gene disrupting its protein expression. Dystrophic ...cardiomyopathy is a leading cause of death in Duchenne muscular dystrophy patients, and currently no effective treatment exists to halt its progression. Recent advancement in genome editing technologies offers a promising therapeutic approach in restoring dystrophin protein expression. However, the impact of this approach on Duchenne muscular dystrophy cardiac function has yet to be evaluated. Therefore, we assessed the therapeutic efficacy of CRISPR (clustered regularly interspaced short palindromic repeats)-mediated genome editing on dystrophin expression and cardiac function in
mice after a single systemic delivery of recombinant adeno-associated virus.
To examine the efficiency and physiological impact of CRISPR-mediated genome editing on cardiac dystrophin expression and function in dystrophic mice.
Here, we packaged SaCas9 (clustered regularly interspaced short palindromic repeat-associated 9 from
) and guide RNA constructs into an adeno-associated virus vector and systemically delivered them to
neonates. We showed that CRIPSR-mediated genome editing efficiently excised the mutant exon 23 in dystrophic mice, and immunofluorescence data supported the restoration of dystrophin protein expression in dystrophic cardiac muscles to a level approaching 40%. Moreover, there was a noted restoration in the architecture of cardiac muscle fibers and a reduction in the extent of fibrosis in dystrophin-deficient hearts. The contractility of cardiac papillary muscles was also restored in CRISPR-edited cardiac muscles compared with untreated controls. Furthermore, our targeted deep sequencing results confirmed that our adeno-associated virus-CRISPR/Cas9 strategy was very efficient in deleting the ≈23 kb of intervening genomic sequences.
This study provides evidence for using CRISPR-based genome editing as a potential therapeutic approach for restoring dystrophic cardiomyopathy structurally and functionally.
Wound care is a major healthcare expenditure. Treatment of burns, surgical and trauma wounds, diabetic lower limb ulcers and skin wounds is a major medical challenge with current therapies largely ...focused on supportive care measures. Successful wound repair requires a series of tightly coordinated steps including coagulation, inflammation, angiogenesis, new tissue formation and extracellular matrix remodelling. Zinc is an essential trace element (micronutrient) which plays important roles in human physiology. Zinc is a cofactor for many metalloenzymes required for cell membrane repair, cell proliferation, growth and immune system function. The pathological effects of zinc deficiency include the occurrence of skin lesions, growth retardation, impaired immune function and compromised would healing. Here, we discuss investigations on the cellular and molecular mechanisms of zinc in modulating the wound healing process. Knowledge gained from this body of research will help to translate these findings into future clinical management of wound healing.
Zinc, an essential micronutrient, has a cancer preventive role. Zinc deficiency has been shown to contribute to the progression of esophageal cancer. Orai1, a store‐operated Ca2+ entry (SOCE) ...channel, was previously reported to be highly expressed in tumor tissues removed from patients with esophageal squamous cell carcinoma (ESCC) with poor prognosis, and elevation of its expression contributes to both hyperactive intracellular Ca2+ oscillations and fast cell proliferation in human ESCC cells. However, the molecular basis of cancer preventive functions of zinc and its association with Orai1‐mediated cell proliferation remains unknown. The present study shows that zinc supplementation significantly inhibits proliferation of ESCC cell lines and that the effect of zinc is reversible with N,N,N′,N′‐tetrakis (2‐pyridylmethyl) ethylenediamine, a specific Zn2+ chelator, whereas non‐ tumorigenic esophageal epithelial cells are significantly less sensitive to zinc treatment. Fluorescence live cell imaging revealed that extracellular Zn2+ exerted rapid inhibitory effects on Orai1‐mediated SOCE and on intracellular Ca2+ oscillations in the ESCC cells. Knockdown of Orai1 or expression of Orai1 mutants with compromised zinc binding significantly diminished sensitivity of the cancer cells to zinc treatment in both SOCE and cell proliferation analyses. These data suggest that zinc may inhibit cell proliferation of esophageal cancer cells through Orai1‐mediated intracellular Ca2+ oscillations and reveal a possible molecular basis for zinc‐induced cancer prevention and Orai1‐SOCE signaling pathway in cancer cells.—Choi, S., Cui, C., Luo, Y., Kim, S.‐H., Ko, J.‐K., Huo, X., Ma, J., Fu, L.‐W., Souza, R. F., Korichneva, I., Pan, Z. Selective inhibitory effects of zinc on cell proliferation in esophageal squamous cell carcinoma through Orai1. FASEB J. 32,404‐416 (2018). www.fasebj.org
Quantum confined materials have been extensively studied for photoluminescent applications. Due to intrinsic limitations of low biocompatibility and challenging modulation, the utilization of ...conventional inorganic quantum confined photoluminescent materials in bio-imaging and bio-machine interface faces critical restrictions. Here, we present aromatic cyclo-dipeptides that dimerize into quantum dots, which serve as building blocks to further self-assemble into quantum confined supramolecular structures with diverse morphologies and photoluminescence properties. Especially, the emission can be tuned from the visible region to the near-infrared region (420 nm to 820 nm) by modulating the self-assembly process. Moreover, no obvious cytotoxic effect is observed for these nanostructures, and their utilization for in vivo imaging and as phosphors for light-emitting diodes is demonstrated. The data reveal that the morphologies and optical properties of the aromatic cyclo-dipeptide self-assemblies can be tuned, making them potential candidates for supramolecular quantum confined materials providing biocompatible alternatives for broad biomedical and opto-electric applications.
Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disorder characterized by progressive weakness of almost all skeletal muscles, whereas extraocular muscles (EOMs) are comparatively ...spared. While hindlimb and diaphragm muscles of end-stage SOD1G93A (G93A) mice (a familial ALS mouse model) exhibit severe denervation and depletion of Pax7
satellite cells (SCs), we found that the pool of SCs and the integrity of neuromuscular junctions (NMJs) are maintained in EOMs. In cell sorting profiles, SCs derived from hindlimb and diaphragm muscles of G93A mice exhibit denervation-related activation, whereas SCs from EOMs of G93A mice display spontaneous (non-denervation-related) activation, similar to SCs from wild-type mice. Specifically, cultured EOM SCs contain more abundant transcripts of axon guidance molecules, including
, along with more sustainable renewability than the diaphragm and hindlimb counterparts under differentiation pressure. In neuromuscular co-culture assays, AAV-delivery of
to G93A-hindlimb SC-derived myotubes enhances motor neuron axon extension and innervation, recapitulating the innervation capacity of EOM SC-derived myotubes. G93A mice fed with sodium butyrate (NaBu) supplementation exhibited less NMJ loss in hindlimb and diaphragm muscles. Additionally, SCs derived from G93A hindlimb and diaphragm muscles displayed elevated expression of
and improved renewability following NaBu treatment in vitro. Thus, the NaBu-induced transcriptomic changes resembling the patterns of EOM SCs may contribute to the beneficial effects observed in G93A mice. More broadly, the distinct transcriptomic profile of EOM SCs may offer novel therapeutic targets to slow progressive neuromuscular functional decay in ALS and provide possible 'response biomarkers' in pre-clinical and clinical studies.
TRIM family proteins play integral roles in the innate immune response to virus infection. MG53 (TRIM72) is essential for cell membrane repair and is believed to be a muscle-specific TRIM protein. ...Here we show human macrophages express MG53, and MG53 protein expression is reduced following virus infection. Knockdown of MG53 in macrophages leads to increases in type I interferon (IFN) upon infection. MG53 knockout mice infected with influenza virus show comparable influenza virus titres to wild type mice, but display increased morbidity accompanied by more accumulation of CD45+ cells and elevation of IFNβ in the lung. We find that MG53 knockdown results in activation of NFκB signalling, which is linked to an increase in intracellular calcium oscillation mediated by ryanodine receptor (RyR). MG53 inhibits IFNβ induction in an RyR-dependent manner. This study establishes MG53 as a new target for control of virus-induced morbidity and tissue injury.
Kidney is a vital organ with high energy demands to actively maintain plasma hemodynamics, electrolytes and water homeostasis. Among the nephron segments, the renal tubular epithelium is endowed with ...high mitochondria density for their function in active transport. Acute kidney injury (AKI) is an important clinical syndrome and a global public health issue with high mortality rate and socioeconomic burden due to lack of effective therapy. AKI results in acute cell death and necrosis of renal tubule epithelial cells accompanied with leakage of tubular fluid and inflammation. The inflammatory immune response triggered by the tubular cell death, mitochondrial damage, associative oxidative stress, and the release of many tissue damage factors have been identified as key elements driving the pathophysiology of AKI. Autophagy, the cellular mechanism that removes damaged organelles via lysosome-mediated degradation, had been proposed to be renoprotective. An in-depth understanding of the intricate interplay between autophagy and innate immune response, and their roles in AKI pathology could lead to novel therapies in AKI. This review addresses the current pathophysiology of AKI in aspects of mitochondrial dysfunction, innate immunity, and molecular mechanisms of autophagy. Recent advances in renal tissue regeneration and potential therapeutic interventions are also discussed.
Tumorigenesis is associated with increased glucose consumption and lipogenesis, but how these pathways are interlinked is unclear. Here, we delineate a pathway in which EGFR signaling, by increasing ...glucose uptake, promotes N-glycosylation of sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) and consequent activation of SREBP-1, an ER-bound transcription factor with central roles in lipid metabolism. Glycosylation stabilizes SCAP and reduces its association with Insig-1, allowing movement of SCAP/SREBP to the Golgi and consequent proteolytic activation of SREBP. Xenograft studies reveal that blocking SCAP N-glycosylation ameliorates EGFRvIII-driven glioblastoma growth. Thus, SCAP acts as key glucose-responsive protein linking oncogenic signaling and fuel availability to SREBP-dependent lipogenesis. Targeting SCAP N-glycosylation may provide a promising means of treating malignancies and metabolic diseases.
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•SCAP acts as a glucose-responsive protein, linking fuel supply to SREBP activation•N-glycosylation of SCAP facilitates SCAP/SREBP trafficking from ER to the Golgi•EGFR activates SCAP/SREBP by promoting glucose import and SCAP N-glycosylation•Targeting SCAP N-glycosylation is a promising strategy to treat malignancies
Cheng et al. show that glucose controls N-glycosylation of SCAP, which is essential for SREBP-dependent lipogenesis, and that activated growth factor receptors increase glucose intake and SREBP activation. Inhibition of SCAP N-glycosylation inhibits the growth of glioblastoma cells with hyperactived EGFR.
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