A dual-color PAX7 and MYF5 in vivo reporter to investigate muscle stem cell heterogeneity in regeneration and aging.

Increasing evidence suggests that the muscle stem cell (MuSC) pool is heterogeneous. In particular, a rare subset of PAX7-positive MuSCs that has never expressed the myogenic regulatory factor MYF5 displays unique self-renewal and engraftment characteristics. However, the scarcity and limited availability of protein markers make the characterization of these cells challenging. Here, we describe the generation of StemRep reporter mice enabling the monitoring of PAX7 and MYF5 proteins based on equimolar levels of dual nuclear fluorescence. High levels of PAX7 protein and low levels of MYF5 delineate a deeply quiescent MuSC subpopulation with an increased capacity for asymmetric division and distinct dynamics of activation, proliferation, and commitment. Aging primarily reduces the MYF5Low MuSCs and skews the stem cell pool toward MYF5High cells with lower quiescence and self-renewal potential. Altogether, we establish the StemRep model as a versatile tool to study MuSC heterogeneity and broaden our understanding of mechanisms regulating MuSC quiescence and self-renewal in homeostatic, regenerating, and aged muscles.

Transanal Hemorrhoidal Dearterialization With Mucopexy Versus Vessel-Sealing Device Hemorrhoidectomy for Grade III to IV Hemorrhoids: Long-term Outcomes From the THDLIGA Randomized Controlled Trial.

BACKGROUND: Both transanal hemorrhoidal dearterialization and vessel-sealing device hemorrhoidectomy are safe for grade III to IV hemorrhoid treatment. Whether one of them is superior regarding long-term results remains unclear. OBJECTIVE: To compare long-term results after transanal hemorrhoidal dearterialization and vessel-sealing device hemorrhoidectomy. DESIGN: Multicenter randomized controlled trial. SETTINGS: This study was conducted at 6 centers. PATIENTS: Patients ≥18 years of age with grade III to IV hemorrhoids were included in the study. INTERVENTIONS: Patients were randomly assigned to transanal hemorrhoidal dearterialization (n = 39) or vessel-sealing device hemorrhoidectomy (n = 41). MAIN OUTCOME MEASURES: The primary outcome was hemorrhoid symptom recurrence assessed by a specific questionnaire 2 years postoperatively. Secondary outcomes included long-term complications, reoperations, fecal continence, and patient satisfaction and quality of life. RESULTS: Five of the 80 patients included in the study were lost to follow-up. Thirty-six patients randomly assigned to transanal hemorrhoidal dearterialization and 39 patients randomly assigned to vessel-sealing device hemorrhoidectomy were included in the long-term analysis. The differences between mean baseline and mean 2-year score in the 2 groups were similar (-11.0, SD 3.8 vs -12.5, SD 3.6; p = 0.080). Three patients in the transanal hemorrhoidal dearterialization group underwent supplementary procedures for hemorrhoid symptoms, compared with none in the vessel-sealing device hemorrhoidectomy group ( p = 0.106). Four patients in the vessel-sealing hemorrhoidectomy group and none in the transanal hemorrhoidal dearterialization group experienced chronic opened wound ( p = 0.116). LIMITATIONS: Lack of stratification for hemorrhoid grade and power calculation based on the main outcome trial but not on the end point of this long-term study. CONCLUSIONS: Transanal hemorrhoidal dearterialization with mucopexy is associated with hemorrhoid symptom recurrence similar to vessel-sealing device hemorrhoidectomy at 2 years. See Video Abstract at http://links.lww.com/DCR/B933 . REGISTRATION: Clinicaltrials.gov ; ID: NCT02654249. DESARTERIALIZACIN HEMORROIDAL TRANSANAL CON MUCOPEXIA VERSUS HEMORROIDECTOMA CON DISPOSITIVO DE SELLADO DE VASOS PARA HEMORROIDES DE GRADO IIIIV RESULTADOS A LARGO PLAZO DEL ENSAYO CLNICO ALEATORIZADO THDLIGARCT: ANTECEDENTES:Tanto la desarterialización hemorroidal transanal como la hemorroidectomía con dispositivo de sellado de vasos son seguras y bien toleradas para el tratamiento de las hemorroides de grado III-IV. La primera se asocia con una necesidad más breve de analgesia posoperatoria que la hemorroidectomía con dispositivo de sellado de vasos. No está claro si uno de ellos es superior con respecto a los resultados a largo plazo.OBJETIVO:El objetivo fue comparar los resultados a largo plazo después de la desarterialización hemorroidal transanal y la hemorroidectomía con dispositivo de sellado de vasos.DISEÑO:Se realizó un ensayo clínico aleatorizado multicéntrico.AJUSTE:Este estudio se realizó en 6 centros.PACIENTES:Se incluyeron en el estudio pacientes de ≥18 años con hemorroides de grado III-IV.INTERVENCIONES:Los pacientes fueron asignados al azar a desarterialización hemorroidal transanal (n = 39) o hemorroidectomía con dispositivo de sellado de vasos (n = 41).PRINCIPALES MEDIDAS DE RESULTADO:El resultado primario fue la recurrencia de los síntomas de hemorroides evaluada mediante un cuestionario específico 2 años después de la operación. Los resultados secundarios incluyeron complicaciones a largo plazo, reoperaciones, continencia fecal, satisfacción del paciente y calidad de vida.RESULTADOS:Cinco de los 80 pacientes incluidos en el estudio se perdieron durante el seguimiento. En el análisis a largo plazo se incluyeron 36 pacientes aleatorizados a desarterialización hemorroidal transanal y 39 aleatorizados a hemorroidectomía con dispositivo de sellado de vasos. Las diferencias entre la puntuación inicial media y la puntuación media a los 2 años en los dos grupos fueron similares (-11,0, DE 3,8 frente a -12,5, DE 3,6; p = 0,080). Tres pacientes en el grupo de desarterialización hemorroidal transanal se sometieron a procedimientos complementarios por síntomas de hemorroides, en comparación con ninguno en el grupo de hemorroidectomía con dispositivo de sellado de vasos (p = 0,106). Cuatro pacientes en el grupo de hemorroidectomía con sellado de vasos y ninguno en el grupo de desarterialización hemorroidal transanal experimentaron herida abierta crónica (p = 0,116). No se encontraron diferencias en cuanto a continencia fecal (p = 0,657), satisfacción del paciente (p = 0,483) y calidad de vida.LIMITACIONES:No hay estratificación para el grado de hemorroides ni el cálculo del poder basado en el resultado principal del ensayo, pero no en el criterio de valoración de este estudio a largo plazo.CONCLUSIONES:La desarterialización hemorroidal transanal con mucopexia se asocia con una recurrencia de síntomas de hemorroides similar a la hemorroidectomía con dispositivo de sellado de vasos a los dos años. See Video Abstract at http://links.lww.com/DCR/B933 . (Traducción- Dr. Francisco M. Abarca-Rendon )REGISTRO DE PRUEBA:Clinicaltrials.gov (NCT02654249).

Crosstalk between Drp1 phosphorylation sites during mitochondrial remodeling and their impact on metabolic adaptation.

Mitochondria constantly undergo fusion and fission events, referred as mitochondrial dynamics, which determine mitochondrial architecture and bioenergetics. Cultured cell studies demonstrate that mitochondrial dynamics are acutely regulated by phosphorylation of the mitochondrial fission orchestrator dynamin-related protein 1 (Drp1) at S579 or S600. However, the physiological impact and crosstalk of these phosphorylation sites is poorly understood. Here, we describe the functional interrelation between S579 and S600 phosphorylation sites in vivo and their role on mitochondrial remodeling. Mice carrying a homozygous Drp1 S600A knockin (Drp1 KI) mutation display larger mitochondria and enhanced lipid oxidation and respiratory capacities, granting improved glucose tolerance and thermogenic response upon high-fat feeding. Housing mice at thermoneutrality blunts these differences, suggesting a role for the brown adipose tissue in the protection of Drp1 KI mice against metabolic damage. Overall, we demonstrate crosstalk between Drp1 phosphorylation sites and provide evidence that their modulation could be used in the treatment and prevention of metabolic diseases.

Compound- and fiber type-selective requirement of AMPKγ3 for insulin-independent glucose uptake in skeletal muscle.

OBJECTIVE: The metabolic master-switch AMP-activated protein kinase (AMPK) mediates insulin-independent glucose uptake in muscle and regulates the metabolic activity of brown and beige adipose tissue (BAT). The regulatory AMPKγ3 isoform is uniquely expressed in skeletal muscle and potentially in BAT. Herein, we investigated the role that AMPKγ3 plays in mediating skeletal muscle glucose uptake and whole-body glucose clearance in response to small-molecule activators that act on AMPK via distinct mechanisms. We also assessed whether γ3 plays a role in adipose thermogenesis and browning. METHODS: Global AMPKγ3 knockout (KO) mice were generated. A systematic whole-body, tissue, and molecular phenotyping linked to glucose homeostasis was performed in γ3 KO and wild-type (WT) mice. Glucose uptake in glycolytic and oxidative skeletal muscle ex vivo as well as blood glucose clearance in response to small molecule AMPK activators that target the nucleotide-binding domain of the γ subunit (AICAR) and allosteric drug and metabolite (ADaM) site located at the interface of the α and ß subunit (991, MK-8722) were assessed. Oxygen consumption, thermography, and molecular phenotyping with a ß3-adrenergic receptor agonist (CL-316,243) treatment were performed to assess BAT thermogenesis, characteristics, and function. RESULTS: Genetic ablation of γ3 did not affect body weight, body composition, physical activity, and parameters associated with glucose homeostasis under chow or high-fat diet. γ3 deficiency had no effect on fiber-type composition, mitochondrial content and components, or insulin-stimulated glucose uptake in skeletal muscle. Glycolytic muscles in γ3 KO mice showed a partial loss of AMPKα2 activity, which was associated with reduced levels of AMPKα2 and ß2 subunit isoforms. Notably, γ3 deficiency resulted in a selective loss of AICAR-, but not MK-8722-induced blood glucose-lowering in vivo and glucose uptake specifically in glycolytic muscle ex vivo. We detected γ3 in BAT and found that it preferentially interacts with α2 and ß2. We observed no differences in oxygen consumption, thermogenesis, morphology of BAT and inguinal white adipose tissue (iWAT), or markers of BAT activity between WT and γ3 KO mice. CONCLUSIONS: These results demonstrate that γ3 plays a key role in mediating AICAR- but not ADaM site binding drug-stimulated blood glucose clearance and glucose uptake specifically in glycolytic skeletal muscle. We also showed that γ3 is dispensable for ß3-adrenergic receptor agonist-induced thermogenesis and browning of iWAT.

A reduced form of nicotinamide riboside defines a new path for NAD+ biosynthesis and acts as an orally bioavailable NAD+ precursor.

OBJECTIVE: A decay in intracellular NAD+ levels is one of the hallmarks of physiological decline in normal tissue functions. Accordingly, dietary supplementation with NAD+ precursors can prevent, alleviate, or even reverse multiple metabolic complications and age-related disorders in diverse model organisms. Within the constellation of NAD+ precursors, nicotinamide riboside (NR) has gained attention due to its potent NAD+ biosynthetic effects in vivo while lacking adverse clinical effects. Nevertheless, NR is not stable in circulation, and its utilization is rate-limited by the expression of nicotinamide riboside kinases (NRKs). Therefore, there is a strong interest in identifying new effective NAD+ precursors that can overcome these limitations. METHODS: Through a combination of metabolomics and pharmacological approaches, we describe how NRH, a reduced form of NR, serves as a potent NAD+ precursor in mammalian cells and mice. RESULTS: NRH acts as a more potent and faster NAD+ precursor than NR in mammalian cells and tissues. Despite the minor structural difference, we found that NRH uses different steps and enzymes to synthesize NAD+, thus revealing a new NRK1-independent pathway for NAD+ synthesis. Finally, we provide evidence that NRH is orally bioavailable in mice and prevents cisplatin-induced acute kidney injury. CONCLUSIONS: Our data identify a new pathway for NAD+ synthesis and classify NRH as a promising new therapeutic strategy to enhance NAD+ levels.

Endogenous nicotinamide riboside metabolism protects against diet-induced liver damage.

Supplementation with the NAD+ precursor nicotinamide riboside (NR) ameliorates and prevents a broad array of metabolic and aging disorders in mice. However, little is known about the physiological role of endogenous NR metabolism. We have previously shown that NR kinase 1 (NRK1) is rate-limiting and essential for NR-induced NAD+ synthesis in hepatic cells. To understand the relevance of hepatic NR metabolism, we generated whole body and liver-specific NRK1 knockout mice. Here, we show that NRK1 deficiency leads to decreased gluconeogenic potential and impaired mitochondrial function. Upon high-fat feeding, NRK1 deficient mice develop glucose intolerance, insulin resistance and hepatosteatosis. Furthermore, they are more susceptible to diet-induced liver DNA damage, due to compromised PARP1 activity. Our results demonstrate that endogenous NR metabolism is critical to sustain hepatic NAD+ levels and hinder diet-induced metabolic damage, highlighting the relevance of NRK1 as a therapeutic target for metabolic disorders.

Short-term Outcomes of Transanal Hemorrhoidal Dearterialization With Mucopexy Versus Vessel-Sealing Device Hemorrhoidectomy for Grade III to IV Hemorrhoids: A Prospective Randomized Multicenter Trial.

BACKGROUND: Transanal hemorrhoidal dearterialization with mucopexy and vessel-sealing device hemorrhoidectomy seem to reduce postoperative pain compared with classic excisional hemorrhoidectomy, but whether one of them is superior remains unclear. OBJECTIVE: We compared transanal hemorrhoidal dearterialization with mucopexy and vessel-sealing device hemorrhoidectomy. DESIGN: This was a multicenter, randomized controlled trial. SETTING: The study was conducted at 6 Spanish centers. PATIENTS: Patients aged ≥18 years with grade III to IV hemorrhoids were included. INTERVENTIONS: Patients were randomly assigned to transanal hemorrhoidal dearterialization with mucopexy (n = 39) or vessel-sealing device hemorrhoidectomy (n = 41). MAIN OUTCOME MEASURES: Primary outcome was the mean postoperative number of days in which patients needed nonsteroidal anti-inflammatory drugs. Secondary outcomes were postoperative pain, 30-day morbidity, patient satisfaction, Vaizey score, hemorrhoid symptoms score, return to work, and quality of life. RESULTS: More patients were still taking analgesia in the vessel-sealing device hemorrhoidectomy group during the second postoperative week compared with the transanal hemorrhoidal dearterialization with mucopexy group (87.8% vs 53.8%; p = 0.002). For the transanal hemorrhoidal dearterialization with mucopexy group, analgesia consumption continued until day 10.1 (mean; SD = 7.22 d), whereas in the vessel-sealing device hemorrhoidectomy group it continued until day 15.2 (mean; SD = 8.70 d; p = 0.006). The mean daily average pain was similar during the first (p = 0.900) and second postoperative weeks (p = 0.265). Mean operative time was higher for the transanal hemorrhoidal dearterialization with mucopexy group versus the vessel-sealing device hemorrhoidectomy group (45 min; range, 40-60 vs 20 min; range, 15-41 min; p < 0.001). Postoperative complications rate, use of laxatives, patient satisfaction, Vaizey score, hemorrhoids symptoms score, return to work, and quality of life at 1 month after surgery were similar between groups. LIMITATIONS: The main limitation of this study was that the 2 groups did not contain equal numbers of grade III and IV hemorrhoids. CONCLUSIONS: Transanal hemorrhoidal dearterialization with mucopexy is associated with a shorter need for postoperative analgesia compared with vessel-sealing device hemorrhoidectomy. See Video Abstract at http://links.lww.com/DCR/A915. TRIAL REGISTRATION: clinicaltrials.gov identifier: NCT02654249.

Clinical and pathologic predictors of outcome in thymoma-associated myasthenia gravis.

BACKGROUND: Myasthenia gravis is by far the most common paraneoplastic syndrome of thymomas. There is little information regarding the influence of clinical variables and thymoma-associated factors on biologic development of myasthenia gravis. The aim of the study was to determine independent predictors of clinical outcome in thymoma with myasthenia gravis. METHODS: We studied 108 patients with thymoma-associated myasthenia gravis undergoing removal of the mediastinal mass between 1967 and 2000. Clinical and pathologic variables associated with clinical outcome of myasthenia were assessed by multivariate Cox regression analysis. RESULTS: Patients were followed for a mean period of 10 years (9 months to 33 years). A total of 38 patients died (35.2%), in 14 cases (37%) because of myasthenia gravis and in 6 (16%) because of recurrence of thymoma. With respect to clinical outcome of myasthenia gravis, at the end of the follow-up period, the rate of remission was 16% (n = 17). Of the 91 patients in whom remission was not achieved, 55 had no symptoms with immunosuppressive medication and 36 had symptoms with medication. CONCLUSIONS: In patients with thymoma-associated myasthenia gravis, well-differentiated thymic carcinoma (Müller-Hermelink system), age more than 55 years, and interval from the onset of symptoms to thymectomy of less than 1 year were found to be independent predictors of nonremission of myasthenia gravis after thymectomy.