Serum MYCN as a predictive biomarker of prognosis and therapeutic response in the prevention of hepatocellular carcinoma recurrence.

The proto-oncogene MYCN expression marked a cancer stem-like cell population in hepatocellular carcinoma (HCC) and served as a therapeutic target of acyclic retinoid (ACR), an orally administered vitamin A derivative that has demonstrated promising efficacy and safety in reducing HCC recurrence. This study investigated the role of MYCN as a predictive biomarker for therapeutic response to ACR and prognosis of HCC. MYCN gene expression in HCC was analyzed in the Cancer Genome Atlas and a Taiwanese cohort (N = 118). Serum MYCN protein levels were assessed in healthy controls (N = 15), patients with HCC (N = 116), pre- and post-surgical patients with HCC (N = 20), and a subset of patients from a phase 3 clinical trial of ACR (N = 68, NCT01640808). The results showed increased MYCN gene expression in HCC tumors, which positively correlated with HCC recurrence in non-cirrhotic or single-tumor patients. Serum MYCN protein levels were higher in patients with HCC, decreased after surgical resection of HCC, and were associated with liver functional reserve and fibrosis markers, as well as long-term HCC prognosis (>4 years). Subgroup analysis of a phase 3 clinical trial of ACR identified serum MYCN as the risk factor most strongly associated with HCC recurrence. Patients with HCC with higher serum MYCN levels after a 4-week treatment of ACR exhibited a significantly higher risk of recurrence (hazard ratio 3.27; p = .022). In conclusion, serum MYCN holds promise for biomarker-based precision medicine for the prevention of HCC, long-term prognosis of early-stage HCC, and identification of high-response subgroups for ACR-based treatment.

Quality assessment of enzymatic methyl-seq library constructed using crude cell lysate.

Enzymatic methyl-seq (EM-seq), an enzyme-based method, identifies genome-wide DNA methylation, which enables us to obtain reliable methylome data from purified genomic DNA by avoiding bisulfite-induced DNA damage. However, the loss of DNA during purification hinders the methylome analysis of limited samples. The crude DNA extraction method is the quickest and minimal sample loss approach for obtaining useable DNA without requiring additional dissolution and purification. However, it remains unclear whether crude DNA can be used directly for EM-seq library construction. In this study, we aimed to assess the quality of EM-seq libraries prepared directly using crude DNA. The crude DNA-derived libraries provided appropriate fragment sizes and concentrations for sequencing similar to those of the purified DNA-derived libraries. However, the sequencing results of crude samples exhibited lower reference sequence mapping efficiencies than those of the purified samples. Additionally, the lower-input crude DNA-derived sample exhibited a marginally lower cytosine-to-thymine conversion efficiency and hypermethylated pattern around gene regulatory elements than the higher-input crude DNA- or purified DNA-derived samples. In contrast, the methylation profiles of the crude and purified samples exhibited a significant correlation. Our findings indicate that crude DNA can be used as a raw material for EM-seq library construction.

FLI1 is associated with regulation of DNA methylation and megakaryocytic differentiation in FPDMM caused by a RUNX1 transactivation domain mutation.

Familial platelet disorder with associated myeloid malignancies (FPDMM) is an autosomal dominant disease caused by heterozygous germline mutations in RUNX1. It is characterized by thrombocytopenia, platelet dysfunction, and a predisposition to hematological malignancies. Although FPDMM is a precursor for diseases involving abnormal DNA methylation, the DNA methylation status in FPDMM remains unknown, largely due to a lack of animal models and challenges in obtaining patient-derived samples. Here, using genome editing techniques, we established two lines of human induced pluripotent stem cells (iPSCs) with different FPDMM-mimicking heterozygous RUNX1 mutations. These iPSCs showed defective differentiation of hematopoietic progenitor cells (HPCs) and megakaryocytes (Mks), consistent with FPDMM. The FPDMM-mimicking HPCs showed DNA methylation patterns distinct from those of wild-type HPCs, with hypermethylated regions showing the enrichment of ETS transcription factor (TF) motifs. We found that the expression of FLI1, an ETS family member, was significantly downregulated in FPDMM-mimicking HPCs with a RUNX1 transactivation domain (TAD) mutation. We demonstrated that FLI1 promoted binding-site-directed DNA demethylation, and that overexpression of FLI1 restored their megakaryocytic differentiation efficiency and hypermethylation status. These findings suggest that FLI1 plays a crucial role in regulating DNA methylation and correcting defective megakaryocytic differentiation in FPDMM-mimicking HPCs with a RUNX1 TAD mutation.

Corn Oligopeptide Alleviates Nonalcoholic Fatty Liver Disease by Regulating the Sirtuin Signaling Pathway.

Nonalcoholic fatty liver disease (NAFLD) represents the most prevalent type of chronic liver disease, spanning from simple steatosis to nonalcoholic steatohepatitis (NASH). Corn oligopeptide (CP) is a functional peptide known for its diverse pharmacological effects on metabolism. In this study, we evaluated the protective activity of CP against fatty liver disease. Oral administration of CP significantly reduced body weight gain by 2.95%, serum cholesterol by 22.54%, and liver injury, as evidenced by a reduction of 32.19% in serum aspartate aminotransferase (AST) and 49.10% in alanine aminotransferase (ALT) levels in mice subjected to a high-fat diet (HFD). In a streptozotocin/HFD-induced NASH mouse model, CP attenuated body weight gain by 5.11%, liver injury (with a 34.15% decrease in AST and 11.43% decrease in ALT), and, to some extent, liver inflammation and fibrosis. Proteomic analysis revealed the modulation of oxidative phosphorylation and sirtuin (SIRT) signaling pathways by CP. Remarkably, CP selectively inhibited the hepatic expression of mitochondrial SIRT3 and SIRT5 in both HFD and NASH models. In summary, CP demonstrates a preventive effect against metabolic-stress-induced NAFLD progression by modulating oxidative stress and the SIRT signaling pathway, suggesting the potential of CP as a therapeutic agent for the treatment of NAFLD and advanced-stage NASH.

Transcriptome analysis of long non-coding RNAs in Mycobacterium avium complex-infected macrophages.

Mycobacterium avium complex (MAC) is a non-tuberculous mycobacterium widely distributed in the environment. Even though MAC infection is increasing in older women and immunocompromised patients, to our knowledge there has been no comprehensive analysis of the MAC-infected host-cell transcriptome-and particularly of long non-coding RNAs (lncRNAs). By using in vitro-cultured primary mouse bone-marrow-derived macrophages (BMDMs) and Cap analysis of gene expression, we analyzed the transcriptional and kinetic landscape of macrophage genes, with a focus on lncRNAs, during MAC infection. MAC infection of macrophages induced the expression of immune/inflammatory response genes and other genes similar to those involved in M1 macrophage activation, consistent with previous reports, although Nos2 (M1 activation) and Arg1 (M2 activation) had distinct expression profiles. We identified 31 upregulated and 30 downregulated lncRNA promoters corresponding respectively to 18 and 26 lncRNAs. Upregulated lncRNAs were clustered into two groups-early and late upregulated-predicted to be associated with immune activation and the immune response to infection, respectively. Furthermore, an Ingenuity Pathway Analysis revealed canonical pathways and upstream transcription regulators associated with differentially expressed lncRNAs. Several differentially expressed lncRNAs reported elsewhere underwent expressional changes upon M1 or M2 preactivation and subsequent MAC infection. Finally, we showed that expressional change of lncRNAs in MAC-infected BMDMs was mediated by toll-like receptor 2, although there may be other mechanisms that sense MAC infection. We identified differentially expressed lncRNAs in MAC-infected BMDMs, revealing diverse features that imply the distinct roles of these lncRNAs in MAC infection and macrophage polarization.

A small molecule iCDM-34 identified by in silico screening suppresses HBV DNA through activation of aryl hydrocarbon receptor.

IFN-alpha have been reported to suppress hepatitis B virus (HBV) cccDNA via APOBEC3 cytidine deaminase activity through interferon signaling. To develop a novel anti-HBV drug for a functional cure, we performed in silico screening of the binding compounds fitting the steric structure of the IFN-alpha-binding pocket in IFNAR2. We identified 37 compounds and named them in silico cccDNA modulator (iCDM)-1-37. We found that iCDM-34, a new small molecule with a pyrazole moiety, showed anti-HCV and anti-HBV activities. We measured the anti-HBV activity of iCDM-34 dependent on or independent of entecavir (ETV). iCDM-34 suppressed HBV DNA, pgRNA, HBsAg, and HBeAg, and also clearly exhibited additive inhibitory effects on the suppression of HBV DNA with ETV. We confirmed metabolic stability of iCDM-34 was stable in human liver microsomal fraction. Furthermore, anti-HBV activity in human hepatocyte-chimeric mice revealed that iCDM-34 was not effective as a single reagent, but when combined with ETV, it suppressed HBV DNA compared to ETV alone. Phosphoproteome and Western blotting analysis showed that iCDM-34 did not activate IFN-signaling. The transcriptome analysis of interferon-stimulated genes revealed no increase in expression, whereas downstream factors of aryl hydrocarbon receptor (AhR) showed increased levels of the expression. CDK1/2 and phospho-SAMHD1 levels decreased under iCDM-34 treatment. In addition, AhR knockdown inhibited anti-HCV activity of iCDM-34 in HCV replicon cells. These results suggest that iCDM-34 decreases the phosphorylation of SAMHD1 through CDK1/2, and suppresses HCV replicon RNA, HBV DNA, and pgRNA formation.