A total of 71 patients with Lyme disease were identified for analysis in whom treatment with disulfiram was initiated between 15 March 2017 and 15 March 2020. Four patients were lost to follow-up, leaving 67 evaluable patients. Our retrospective review found patients to fall into a “high-dose” group (≥4 mg/kg/day) and a “low-dose” group (<4 mg/kg/day). In total, 62 of 67 (92.5%) patients treated with disulfiram were able to endorse a net benefit of the treatment with regard to their symptoms. Moreover, 12 of 33 (36.4%) patients who completed one or two courses of “high-dose” therapy enjoyed an “enduring remission”, defined as remaining clinically well for ≥6 months without further anti-infective treatment. The most common adverse reactions from disulfiram treatment in the high-dose group were fatigue (66.7%), psychiatric symptoms (48.5%), peripheral neuropathy (27.3%), and mild to moderate elevation of liver enzymes (15.2%). We observed that although patients on high dose experienced a higher risk for adverse reactions than those on a low dose, high-dose patients were significantly more likely to achieve enduring remission.

Formation of skeletal muscle is among the most striking examples of cellular plasticity in animal tissue development, and while muscle progenitor cells are reprogrammed by epithelial-mesenchymal transition (EMT) to migrate during embryonic development, the regulation of EMT in post-natal myogenesis remains poorly understood. Here, we demonstrate that the long noncoding RNA (lncRNA) Meg3 regulates EMT in myoblast differentiation and skeletal muscle regeneration. Chronic inhibition of Meg3 in C2C12 myoblasts induced EMT, and suppressed cell state transitions required for differentiation. Furthermore, adenoviral Meg3 knockdown compromised muscle regeneration, which was accompanied by abnormal mesenchymal gene expression and interstitial cell proliferation. Transcriptomic and pathway analyses of Meg3-depleted C2C12 myoblasts and injured skeletal muscle revealed a significant dysregulation of EMT-related genes, and identified TGFβ as a key upstream regulator. Importantly, inhibition of TGFβR1 and its downstream effectors, and the EMT transcription factor Snai2, restored many aspects of myogenic differentiation in Meg3-depleted myoblasts in vitro. We further demonstrate that reduction of Meg3-dependent Ezh2 activity results in epigenetic alterations associated with TGFβ activation. Thus, Meg3 regulates myoblast identity to facilitate progression into differentiation.

statement Muscle differentiation and regeneration are regulated by an evolutionarily conserved long noncoding RNA that restricts gene expression to coordinate cell state transitions Abstract Formation of skeletal muscle is among the most striking examples of cellular plasticity in animal tissue development, where mononucleated, lineage-restricted progenitor cells are reprogrammed by epithelial-mesenchymal transition (EMT) to produce multinucleated myofibers. While some mediators of EMT have been shown to function in muscle formation, the regulation of this process in this tissue remains poorly understood. The long noncoding RNA (lncRNA) Meg3 is processed from the >200 kb Dlk1-Dio3 polycistron that we have previously shown is involved in skeletal muscle differentiation and regeneration. Here, we demonstrate that Meg3 regulates EMT in myoblast differentiation and skeletal muscle regeneration. Chronic inhibition of Meg3 in C2C12 myoblasts promoted aberrant EMT activation, and suppressed cell state transitions required for fusion and myogenic differentiation. Furthermore, adenoviral Meg3 knockdown compromised muscle regeneration, which was accompanied by abnormal mesenchymal gene expression and interstitial cell proliferation in the regenerating milieu. Transcriptomic and pathway analyses of Meg3-depleted C2C12 myoblasts and injured skeletal muscle revealed a significant dysregulation of EMT-related genes, and identified TGFβ as a key upstream regulator. Importantly, chemical inhibition of TGFβR1, as well as its downstream effectors ROCK1/2 and p38 MAPK, restored many aspects of myogenic differentiation in Meg3depleted myoblasts in vitro. Thus, Meg3 regulates myoblast identity to maintain proper cell state for progression into differentiation. Benetatos L, Hatzimichael E, Londin E, Vartholomatos G, Loher P, Rigoutsos I, Briasoulis E. (2013) The microRNAs within the DLK1-DIO3 genomic region: involvement in disease pathogenesis. Cell Mol Life Sci. 70(5):795-814. Benetatos L, Vartholomatos G, Hatzimichael E (2014). DLK1-DIO3 imprinted cluster in induced pluripotency: landscape in the mist. Cell Mol Life Sci. 71(22):4421-30. Buckingham M, Rigby PW (2014). Gene regulatory networks and transcriptional mechanisms that control myogenesis. Dev Cell. 28(3):225-38. Burks TN, Cohn RD. (2011) Role of TGF-β signaling in inherited and acquired myopathies. Skelet Muscle. 1(1):19. Campbell K, Casanova J. (2016) A common framework for EMT and collective cell migration. Development. 143(23):4291-4300. Caretti G, Di Padova M, Micales B, Lyons GE, Sartorelli V. (2004) The Polycomb Ezh2 methyltransferase regulates muscle gene expression and skeletal muscle differentiation. Genes Dev. 18(21):2627-38. Castel D, Baghdadi MB, Mella S, Gayraud-Morel B, Marty V, Cavaillé J, Antoniewski C, Tajbakhsh S (2018). Small-RNA sequencing identifies dynamic microRNA deregulation during skeletal muscle lineage progression. Sci Rep. 8(1):4208. Chal J, Pourquié O (2017). Making muscle: skeletal myogenesis in vivo and in vitro. . (2018). MEG3 affects the pro...