A circulating cell-free DNA methylation signature for the detection of hepatocellular carcinoma

To address the shortcomings of current hepatocellular carcinoma (HCC) surveillance tests, we set out to find HCC-specific methylation markers and develop a highly sensitive polymerase chain reaction (PCR)-based method to detect them in circulating cell-free DNA (cfDNA). The analysis of large methylome data revealed that Ring Finger Protein 135 (RNF135) and Lactate Dehydrogenase B (LDHB) are universally applicable HCC methylation markers with no discernible methylation level detected in any other tissue types. These markers were used to develop Methylation Sensitive High-Resolution Analysis (MS-HRM), and their diagnostic accuracy was tested using cfDNA from healthy, at-risk, and HCC patients. The combined MS-HRM RNF135 and LDHB analysis detected 57% of HCC, outperforming the alpha-fetoprotein (AFP) tests sensitivity of 45% at comparable specificity. Furthermore, when used with the AFP test, the methylation assay can detect 70% of HCC. Our findings suggest that the cfDNA methylation assay could be used for HCC liquid biopsy.This study was supported by grants from the National Research Foundation (NRF) funded by the Ministry of Science & ICT (No. 2017M3A9A7050614, No. 2020R1C1C1012749, and NRF-2017R1D1A1B06029547) and the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health & Welfare, Republic of Korea (No. HI22C0147

Down-Regulation of NF-κB Target Genes by the AP-1 and STAT Complex during the Innate Immune Response in Drosophila

The activation of several transcription factors is required for the elimination of infectious pathogens via the innate immune response. The transcription factors NF-κB, AP-1, and STAT play major roles in the synthesis of immune effector molecules during innate immune responses. However, the fact that these immune responses can have cytotoxic effects requires their tight regulation to achieve restricted and transient activation, and mis-regulation of the damping process has pathological consequences. Here we show that AP-1 and STAT are themselves the major inhibitors responsible for damping NF-κB–mediated transcriptional activation during the innate immune response in Drosophila. As the levels of dAP-1 and Stat92E increase due to continuous immune signaling, they play a repressive role by forming a repressosome complex with the Drosophila HMG protein, Dsp1. The dAP-1–, Stat92E-, and Dsp1-containing complexes replace Relish at the promoters of diverse immune effector genes by binding to evolutionarily conserved cis-elements, and they recruit histone deacetylase to inhibit transcription. Reduction by mutation of dAP-1, Stat92E, or Dsp1 results in hyperactivation of Relish target genes and reduces the viability of bacterially infected flies despite more efficient pathogen clearance. These defects are rescued by reducing the Relish copy number, thus confirming that mis-regulation of Relish, not inadequate activation of dAP-1, Stat92E, or Dsp1 target genes, is responsible for the reduced survival of the mutants. We conclude that an inhibitory effect of AP-1 and STAT on NF-κB is required for properly balanced immune responses and appears to be evolutionarily conserved

Identification and Functional Analysis of Antifungal Immune Response Genes in Drosophila

Essential aspects of the innate immune response to microbial infection appear to be conserved between insects and mammals. Although signaling pathways that activate NF-κB during innate immune responses to various microorganisms have been studied in detail, regulatory mechanisms that control other immune responses to fungal infection require further investigation. To identify new Drosophila genes involved in antifungal immune responses, we selected genes known to be differentially regulated in SL2 cells by microbial cell wall components and tested their roles in antifungal defense using mutant flies. From 130 mutant lines, sixteen mutants exhibited increased sensitivity to fungal infection. Examination of their effects on defense against various types of bacteria and fungi revealed nine genes that are involved specifically in defense against fungal infection. All of these mutants displayed defects in phagocytosis or activation of antimicrobial peptide genes following infection. In some mutants, these immune deficiencies were attributed to defects in hemocyte development and differentiation, while other mutants showed specific defects in immune signaling required for humoral or cellular immune responses. Our results identify a new class of genes involved in antifungal immune responses in Drosophila

Identification of Drosophila SOD3 and its protective role against phototoxic damage to cells

AbstractSuperoxide dismutase (SOD) is one of several major proteins that regulate removal of superoxide. Three isoforms of SOD exist in mammals. It has long been thought that Drosophila lacks the SOD3 gene. However, a putative SOD3 gene sequence (dSod3) in the Drosophila genome was reported recently. Thus we investigated whether dSod3 truly functions as a SOD3 homolog in Drosophila. We found that dSod3 not only retains SOD activity but also properties of secreted proteins, as do other SOD3s. In addition, the dSod3 protein alleviates ultraviolet-induced cellular damage. These results suggest that dSod3 functions as an extracellular SOD3

Requirement of Split ends for Epigenetic Regulation of Notch Signal-Dependent Genes during Infection-Induced Hemocyte Differentiation▿ †

Drosophila producing a mutant form of the putative transcription coregulator, Split ends (Spen), originally identified in the analysis of neuronal development, display diverse immune defects. In order to understand the role of Spen in the innate immune response, we analyzed the transcriptional defects associated with spen mutant hemocytes and their relationship to the Notch signaling pathways. Spen is regulated by the Notch pathway in the lymph glands and is required for Notch-dependent activation of a large number of genes involved in energy metabolism and differentiation. Analysis of the epigenetic marks associated with Spen-dependent genes indicates that Spen performs its function as a coactivator by regulating chromatin modification. Intriguingly, expression of the Spen-dependent genes was transiently downregulated in a Notch-dependent manner by the Dif activated upon recognition of pathogen-associated molecules, demonstrating the existence of cross talk between hematopoietic regulation and the innate immune response. Our observations reveal a novel connection between the Notch and Toll/IMD signaling pathways and demonstrate a coactivating role for Spen in activating Notch-dependent genes in differentiating cells