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Molecular screens comparing different disease states to identify candidate genes rely on the availability of fast, reliable and multiplexable systems to interrogate genes of interest. CRISPR/Cas9-based reverse genetics is a promising method to eventually achieve this. However, such methods are sorely lacking for multi-nucleated muscle fibers, since highly efficient nuclei editing is a requisite to robustly inactive candidate genes. Here, we couple Cre-mediated skeletal muscle fiber-specific Cas9 expression with myotropic adeno-associated virus-mediated sgRNA delivery to establish a system for highly effective somatic gene deletions in mice. Using well-characterized genes, we show that local or systemic inactivation of these genes copy the phenotype of traditional gene-knockout mouse models. Thus, this proof-of-principle study establishes a method to unravel the function of individual genes or entire signaling pathways in adult skeletal muscle fibers without the cumbersome requirement of generating knockout mice. Methods for somatic gene perturbation would offer advantages for screening multiple muscle gene candidates. Here the authors couple Cre-mediated skeletal muscle fiber-specific Cas9 expression with myotropic adeno-associated virus-mediated sgRNA delivery and report a system for effective somatic gene deletions in mice.

The neuromuscular junction (NMJ) is the synapse formed between motor neurons and skeletal muscle fibers. Its stability relies on the continued expression of genes in a subset of myonuclei, called NMJ myonuclei. Here, we use single-nuclei RNA-sequencing (snRNA-seq) to identify numerous NMJ-specific transcripts. To elucidate how the NMJ transcriptome is regulated, we also performed snRNA-seq on sciatic nerve transected, botulinum toxin injected, and Musk knockout muscles. The data show that NMJ gene expression is not only driven by agrin-Lrp4/MuSK signaling but is also affected by electrical activity and trophic factors other than agrin. By selecting the three NMJ genes Etv4 , Lrtm1 , and Pdzrn4 , we further characterize novel contributors to NMJ stability and function. AAV-mediated overexpression shows that Etv4 is sufficient to upregulate the expression of −50% of the NMJ genes in non-synaptic myonuclei, while AAV-CRISPR/Cas9-mediated muscle-specific knockout of Pdzrn4 induces NMJ fragmentation. Further investigation of Pdzrn4 revealed that it localizes to the Golgi apparatus and interacts with MuSK protein. Collectively, our data provide a rich resource of NMJ transcripts, highlight the importance of ETS transcription factors at the NMJ, and suggest a novel pathway for NMJ post-translational modifications. Here they use single nuclei RNA-seq to identify transcripts in skeletal muscle that maintain the neuromuscular junction, both under normal and denervated conditions, which allows them to characterize PDZRN4 as a novel MuSK interactor at the Golgi apparatus.

The oncogenic transcription factor Myc stimulates many growth processes including cell cycle progression and ribosome biogenesis. Myc expression is low in adult skeletal muscle, but is upregulated upon growth stimuli. Furthermore, muscle fiber Myc overexpression recapitulates many aspects of growth-related gene expression, leading to the hypothesis that Myc mediates pro-growth responses to anabolic stimuli, such as exercise. Here, we test this hypothesis by examining mouse models in which Myc is specifically eliminated or overexpressed in skeletal muscle fibers or muscle stem cells (MuSC). While muscle fiber Myc expression increased during muscle growth and Myc expression in MuSCs was required for successful muscle regeneration, muscle fiber Myc expression was dispensable for post-natal, mechanical overload or PKBα/Akt1-induced muscle growth in mice. Similarly, constitutive Myc expression did not promote skeletal muscle hypertrophy, but instead impaired muscle fiber structure and function within days. These data question the role of Myc in skeletal muscle growth. The transcription factor Myc stimulates growth in many cells. This study shows that Myc expression is essential in muscle stem cells to support muscle growth, but is neither sufficient nor necessary for increases in muscle fiber size.

Non invasive immunologic markers of virus-induced liver disease are unmet needs. We tested the clinical significance of quantitative total and IgM-anti-HBc in well characterized chronic-HBsAg-carriers. Sera (212) were obtained from 111 HBsAg-carriers followed-up for 52 months (28-216) during different phases of chronic-HBV-genotype-D-infection: 10 HBeAg-positive, 25 inactive-carriers (HBV-DNA≤2000IU/ml, ALT<30U/L), 66 HBeAg-negative-CHB-patients and 10 with HDV-super-infection. In 35 patients treated with Peg-IFN±nucleos(t)ide-analogues (NUCs) sera were obtained at baseline, end-of-therapy and week-24-off-therapy and in 22 treated with NUCs (for 60 months, 42-134m) at baseline and end-of-follow-up. HBsAg and IgM-anti-HBc were measured by Architect-assays (Abbott, USA); total-anti-HBc by double-antigen-sandwich-immune-assay (Wantai, China); HBV-DNA by COBAS-TaqMan (Roche, Germany). Total-anti-HBc were detectable in all sera with lower levels in HBsAg-carriers without CHB (immune-tolerant, inactive and HDV-superinfected, median 3.26, range 2.26-4.49 Log10 IU/ml) versus untreated-CHB (median 4.68, range 2.76-5.54 Log10 IU/ml), p<0.0001. IgM-anti-HBc positive using the chronic-hepatitis-cut-off\" (0.130-S/CO) were positive in 102 of 212 sera (48.1%). Overall total-anti-HBc and IgM-anti-HBc correlated significantly (p<0.001, r=0.417). Total-anti-HBc declined significantly in CHB patients with response to Peg-IFN (p<0.001) and in NUC-treated patients (p<0.001); the lowest levels (median 2.68, range 2.12-3.08 Log10 IU/ml) were found in long-term responders who cleared HBsAg subsequently. During spontaneous and therapy-induced fluctuations of CHB (remissions and reactivations) total- and IgM-anti-HBc correlated with ALT (p<0.001, r=0.351 and p=0.008, r=0.185 respectively). Total-anti-HBc qualifies as a useful marker of HBV-induced-liver-disease that might help to discriminate major phases of chronic HBV infection and to predict sustained response to antivirals.

Inter-instrument variations of liver stiffness measure (LSM) by transient elastography might be problematic in clinical practice. LSMs provided by 2 different systems were compared in outpatients with chronic liver disease (CLD). In 777 consecutive asymptomatic outpatients admitted at the Hepatology Unit of Pisa University Hospital the agreement of LSMs measured by FibroScan ® (Echosens, France) and FT9000 (Hisky Medical, China) (LSM Fibroscan /LSM FT9000 ) was tested using Pearson correlation and Bland-Altman analyses (BAA). Delta FT9000-FibroScan ® LSM (LSM-D) variations were analysed according to clinic-pathologic characteristics. ALT/AST/GGT and platelets-counts/portal-vein-caliber/spleen-bipolar-diameter were used as proxies of necro-inflammatory and portal hypertension, respectively. LSM FT9000 and LSM Fibroscan were highly correlated ( r  = 0.781, p  < 0.001) overall, but agreement varied according to BMI (normal-weight r  = 0.841, over-weight r  = 0.747, obese r  = 0.647, p  < 0.001). LSM showed a + 0.52 kPa bias ( p  = 0.01) with − 11/12 kPa as 95% Limit of agreement, with higher values measured by LSM FT9000 below 10 kPa and lower values above 20 kPa. In bivariate analysis LSM-D correlated with LSM Fibroscan and AST (β = 0.002, p  = 0.001 with significant interaction, p  = 0.018) and was associated with spleen-bipolar-diameter (β = 0.033, p  = 0.027) and platelets-count (β=-0.008, p  = 0.018) with significant interaction with LSM Fibroscan ( p  < 0.001). LSM Fibroscan and LSM FT9000 are strongly correlated overall with optimal agreement around 10 kPa but limited at ends of the dynamic range: LSM FT9000 provides higher values below 10 kPa whereas LSM Fibroscan higher values over 20 kPa with different measures distribution. LSM discrepancies associate with anthropometric characteristics, necro-inflammatory activity and portal hypertension proxies. These findings suggest the need of systems specific cut-offs in clinical practice.

The virus/host interplay mediates liver pathology in chronic HBV infection. MiRNAs play a pivotal role in virus/host interactions and are detected in both serum and HBsAg-particles, but studies of their dynamics during chronic infection and antiviral therapy are missing. We studied serum miRNAs during different phases of chronic HBV infection and antiviral treatment. MiRNAs were profiled by miRCURY-LNA-Universal-RT-miRNA-PCR (Exiqon-A/S) and qPCR-panels-I/II-739-miRNA-assays and single-RT-q-PCRs. Two cohorts of well-characterized HBsAg-carriers were studied (median follow-up 34-52 months): a) training-panel (141 sera) and HBsAg-particles (32 samples) from 61 HBsAg-carriers and b) validation-panel (136 sera) from 84 carriers. Thirty-one miRNAs were differentially expressed in inactive-carriers (IC) and chronic-hepatitis-B (CHB) with the largest difference for miR-122-5p, miR-99a-5p and miR-192-5p (liver-specific-miRNAs), over-expressed in both sera and HBsAg-particles of CHB (ANOVA/U-test p-values: <0.000001/0.000001; <0.000001/0.000003; <0.000001/0.000005, respectively) and significantly down-regulated during- and after-treatment in sustained-virological-responders (SVR). MiRNA-profiles of IC and SVR clustered in the heatmap. Liver-miRNAs were combined with miR-335, miR-126 and miR-320a (internal controls) to build a MiR-B-Index with 100% sensitivity, 83.3% and 92.5% specificity (-1.7 cut-off) in both training and validation cohorts to identify IC. MiR-B-Index (-5.72, -20.43/14.38) correlated with ALT (49, 10/2056 U/l, ρ = -0.497, p<0.001), HBV-DNA (4.58, undetectable/>8.3 Log10 IU/mL, ρ = -0.732, p<0.001) and HBsAg (3.40, 0.11/5.49 Log10 IU/mL, ρ = -0.883, p<0.001). At multivariate analysis HBV-DNA (p = 0.002), HBsAg (p<0.001) and infection-phase (p<0.001), but not ALT (p = 0.360) correlated with MiR-B-Index. In SVR to Peg-IFN/NUCs MiR-B-Index improved during-therapy and post-treatment reaching IC-like values (5.32, -1.65/10.91 vs 6.68, 0.54/9.53, p = 0.324) beckoning sustained HBV-immune-control earlier than HBsAg-decline. Serum miRNA profile change dynamically during the different phases of chronic HBV infection. We identified a miRNA signature associated with both natural-occurring and therapy-induced immune control of HBV infection. The MiR-B-Index might be a useful biomarker for the early identification of the sustained switch from CHB to inactive HBV-infection in patients treated with antivirals.

Treatment of neuromuscular diseases is still an unsolved problem. Evidence over the last years strongly indicates the involvement of malformation and dysfunction of neuromuscular junctions in the development of such medical conditions. Stabilization of NMJs thus seems to be a promising approach to attenuate the disease progression of muscle wasting diseases. An important pathway for the formation and maintenance of NMJs is the agrin/Lrp4/MuSK pathway. Here we demonstrate that the agrin biologic NT-1654 is capable of activating the agrin/Lrp4/MuSK system in vivo, leading to an almost full reversal of the sarcopenia-like phenotype in neurotrypsin-overexpressing (SARCO) mice. We also show that injection of NT-1654 accelerates muscle re-innervation after nerve crush. This report demonstrates that a systemically administered agrin fragment has the potential to counteract the symptoms of neuromuscular disorders.

miRNAs circulating in whole serum and HBsAg-particles are differentially expressed in chronic hepatitis B (CHB) and HBeAg-negative-HBV infection (ENI); their profiles are unknown in chronic hepatitis D (CHD). Serum- and HBsAg-associated miRNAs were analyzed in 75 subjects of 3 well-characterized groups (CHB 25, CHD 25, ENI 25) using next-generation sequencing (NGS). Overall miRNA profiles were consonant in serum and HBsAg-particles but significantly different according to the presence of hepatitis independently of Hepatitis D Virus (HDV)-co-infection. Stringent (Bonferroni Correction < 0.001) differential expression analysis showed 39 miRNAs upregulated in CHB vs. ENI and 31 of them also in CHD vs. ENI. miRNA profiles were coincident in CHB and CHD with only miR-200a-3p upregulated in CHB. Three miRNAs (miR-625-3p, miR-142-5p, and miR-223-3p) involved in immune response were upregulated in ENI. All 3 hepatocellular miRNAs of MiR-B-Index (miR-122-5p, miR-99a-5p, miR-192-5p) were overexpressed in both CHB and CHD patients. In conclusion, CHD and CHB patients showed highly similar serum miRNA profiling that was significantly different from that of individuals with HBeAg-negative infection and without liver disease.

Muscle size is controlled by the PI3K-PKB/Akt-mTORC1-FoxO pathway, which integrates signals from growth factors, energy and amino acids to activate protein synthesis and inhibit protein breakdown. While mTORC1 activity is necessary for PKB/Akt-induced muscle hypertrophy, its constant activation alone induces muscle atrophy. Here we show that this paradox is based on mTORC1 activity promoting protein breakdown through the ubiquitin-proteasome system (UPS) by simultaneously inducing ubiquitin E3 ligase expression via feedback inhibition of PKB/Akt and proteasome biogenesis via Nuclear Factor Erythroid 2-Like 1 (Nrf1). Muscle growth was restored by reactivation of PKB/Akt, but not by Nrf1 knockdown, implicating ubiquitination as the limiting step. However, both PKB/Akt activation and proteasome depletion by Nrf1 knockdown led to an immediate disruption of proteome integrity with rapid accumulation of damaged material. These data highlight the physiological importance of mTORC1-mediated PKB/Akt inhibition and point to juxtaposed roles of the UPS in atrophy and proteome integrity. Exploring the relationship between mTORC1 and the ubiquitin-proteasome system, light is shed on the paradox between mTORC1-mediated muscle hypertrophy induced by PKB/Akt and the muscle atrophy induced by mTORC1 alone.

Background: Hepatocellular carcinoma prognosis depends on both liver and tumor determinants, especially on maximum tumor diameter, multifocality, and presence of portal vein thrombosis, despite apparently complete tumor removal by resection or liver transplantation. Aims: To examine parameters of hepatocellular carcinoma aggressiveness as tumor size increases. Methods: A large hepatocellular carcinoma database was examined for trends in serum alpha-fetoprotein and the percentage of patients with macroscopic portal vein thrombosis or tumor multifocality. Results: A total of 13,016 hepatocellular carcinoma patients were identified having full tumor and survival data. Of these, 76.56% were male and 23.44% were female, with a median age of 64.4 years. We found that as the maximum tumor diameter increased, there was a significant trend for increased alpha-fetoprotein levels (P<0.001) and an increased percentage of patients with either portal vein thrombosis or tumor multifocality, each P<0.0001. Furthermore, the increases of both alpha-fetoprotein and portal vein thrombosis were proportionately greater than the related maximum tumor diameter increases. These trends of increased alpha-fetoprotein, portal vein thrombosis, and multifocality with increasing maximum tumor diameter had non-linear patterns. Within alpha-fetoprotein and multifocality trends, there were identifiable sub-trends associated with specific maximum tumor diameter ranges. Conclusions: The greater fold-increases in alpha-fetoprotein and portal vein thrombosis compared with increases in maximum tumor diameter imply that hepatocellular carcinoma characteristics may change with increasing size to a more aggressive phenotype, suggesting that follow-up tumor sampling might be useful, in addition to baseline tumor sampling, for optimal therapeutic choices to be made.