Inflammatory bowel disease (IBD) is a chronic intestinal disorder with recurrent inflammation for which effective therapeutic options remain limited. Probiotics from the Bifidobacterium genus have potential beneficial effects on the prevention of IBD by improving intestinal barrier integrity and modulating immune responses. However, whether these effects are mediated by the regulation of gut metabolism remains largely unclear. This study was designed to explore the protective effect of an infant-derived Bifidobacterium animalis subsp. lactis 832 (B. lactis 832) on dextran sulfate sodium (DSS)-induced colitis in mice and its underlying mechanism. B. lactis 832 treatment significantly alleviated colitis severity (p < 0.05), as evidenced by reduced weight loss, disease activity index (DAI), and colonic injury, accompanied by significantly decreased pro-inflammatory cytokine expression and increased Il10 expression (p < 0.05). It also improved intestinal barrier integrity and modulated gut microbiota composition by reducing potentially pathogenic bacteria while enriching beneficial taxa. Surprisingly, metabolomic analysis revealed that B. lactis 832 intervention enhanced intestinal phospholipid metabolism, particularly increasing phosphatidylethanolamine (PE) and phosphatidylcholine (PC) levels. Notably, PE or PC supplementation recapitulated the protective effects against DSS-induced colitis (p < 0.05). These findings suggest that B. lactis 832 alleviates colitis through microbiota-associated metabolic regulation, highlighting a key role for phospholipid metabolism in mediating probiotic effects.

2'-Fucosyllactose (2'-FL), an industrial breast milk oligosaccharide, is approved for use in infant formula and may reduce cow's milk protein allergenicity. To investigate whether glycosylation products of 2'-FL in dairy products (2'-FL-β-LG) increase its sensitization, we cross-linked β-LG with 2'-FL and used it to sensitize Balb/c mice, comparing it with nonglycosylated β-LG. Both 2'-FL-β-LG sensitization and oral 2'-FL intervention reduced allergic symptoms, specific antibodies (IgE, IgG, and IgG2a), inflammatory cytokine levels, and intestinal damage. 2'-FL also shifted T-cell differentiation, reduced cell surface expression of DC receptors, and enhanced gut microbial diversity. Oral 2'-FL showed the greatest efficacy, suggesting its potential for lowering milk allergenicity in formula.

Defining the mechanisms that promote development and progression of myeloproliferative neoplasms (MPNs) is important for understanding the mechanisms of malignant hematopoiesis and critical development of new treatment approaches. We provide evidence for a key and essential role of the kinase ULK1 in MPN pathophysiology. Our studies demonstrate that genetic or pharmacological targeting of ULK1 delays substantially disease development in Jak2V617F-mutant MPN models in vivo and establish that ULK1 activity is required for transcription of genes that control hematopoietic stem cell differentiation. Pharmacological targeting of ULK1 exhibits potent therapeutic effects, resulting in reduction of early stage erythroid progenitors in spleen and bone marrow, decreased levels of hemoglobin, and reduced spleen size in MPN mouse models in vivo. Taken together, these findings provide the first evidence for a novel protumorigenic role for ULK1 downstream of the hyperactive JAK2 signaling in MPNs and raise the potential of ULK1 as a new therapeutic target for the treatment of MPNs.

Cronobacter sakazakii (C. sakazakii) is a foodborne pathogen associated with severe neonatal infections with high mortality and morbidity. As an enteric pathogen, C. sakazakii primarily infects neonates via the ingestion of contaminated powdered infant formula. Following entry and colonization in the gastrointestinal tract (GIT), C. sakazakii evades the innate immune response by surviving and replicating in macrophages, which is the crucial step to establish the systemic infection in the host. However, the dominants contributing to this step have poorly studied. Here, we report a complete signal regulatory pathway where the small RNA, CsrO is activated by the SoxRS two-component system under reactive oxygen species (ROS). CsrO promotes the expression of osmY by facilitating the stability of osmY mRNA which provides a benefit of C. sakazakii to resist the hypertonic stress in macrophages, as deletion of osmY is more easily to occur plasmolysis within macrophage. This study reveals a host signaling-mediated virulence activation pathway in C. sakazakii, which plays a critical role in its intracellular survival within macrophages and systemic infection in rats. These findings provide insights into C. sakazakii pathogenesis and host-pathogen interaction.

Colorectal cancer (CRC) is one of the most lethal and prevalent malignancies. While the overexpression of pioneer factor GATA6 in CRC has been linked with metastasis, its role in genome-wide gene expression dysregulation remains unclear. Through studies of primary human CRC tissues and analysis of the TCGA data, we found that GATA6 preferentially binds at CRC-specific active enhancers, with enrichment at enhancer-promoter loop anchors. GATA6 protein also physically interacts with CTCF, suggesting its critical role in 3D genome organization. The ablation of GATA6 through AID and CRISPR systems severely impaired cancer cell clonogenicity and proliferation. Mechanistically, GATA6 knockout induced global loss of CRC-specific open chromatins and extensive alterations of critical enhancer-promoter interactions for CRC oncogenes. Last, we showed that GATA6 knockout greatly reduced tumor growth and improved survival in mice. Together, we revealed a previously unidentified mechanism by which GATA6 contributes to the pathogenesis of colorectal cancer.

Polycomb group (PcG) proteins are epigenetic gene silencers that have been implicated in stem cell maintenance and cancer development. PcG proteins exist in two protein complexes, Polycomb repressive complex 2 (PRC2) and Polycomb repressive complex 1 (PRC1). PRC2 methylates H3K27, while PRC1 ubiquitinates H2AK119. PRC1 also involves in modulating 3D chromatin structure. Bmi1 and Mel18 are two major homologs of the PCGF subunit within the canonical PRC1. While Bmi1 is a positive regulator of hematopoietic stem cell (HSC) and leukemia stem cell (LSC) self-renewal, the role of Mel18 is not fully understood.To determine the role of Mel18 in HSCs, we examined HSC behaviors in the Mel18 conditional knockout mice (Mel18f/f-Mx1Cre+). We found that acute deletion of Mel18 enhances the repopulating potential of HSCs and increases functional HSC numbers without affecting homing. Loss of Mel18 decreases the senescence rate in HSCs. In addition, increased frequencies of HSCs entering S phase and G2/M phase were observed.Mechanistically, loss of Mel18 increases the chromatin accessibility to genes important for HSC self-renewal and ex vivo expansion, including homeobox gene Hoxb4. By performing CUT&RUN, we discovered that loss of Mel18 reduces the H2AK119ub1 peaks at promoter regions of Cdk4 and Cdk6 genes, leading to the enhanced expressions of important cell cycle regulators in HSPCs. Besides that, we also observed a diminished chromatin looping event at the calcium binding protein S100a9 locus, a gene signature of senescence and inflammation, via Hi-C sequencing.Collectively, these findings demonstrated that Mel18 play distinct roles in regulating HSC behaviors than that of Bmi1. Orchestrating multiple layers of regulation in epigenome and higher order genomic structure, Mel18 represses HSC self-renewal and proliferation but promotes cellular senescence.

Three glycosylated proteins were prepared from 2 '-fucosyllactose (2 '-FL) with alpha-lactalbumins, 8-lactoglobulin and whey protein isolate, respectively, and their protein structures, glycosylation sites, digestion and transport capacities, and antibody binding capacities were determined. The findings indicate that following heating at 68 degrees C for 90 min, 2 '-FL can form stable bonds with amino acid residues in milk proteins, leading to enhanced antioxidant properties, reduced hydrophobicity, and modified secondary structure content of the proteins. The glycosylation sites were mainly located in the amino acid sequence of 8-lactoglobulin (K47, K91 and K135) and significantly reduce the affinity of its specific antibodies. 2 '-FL glycosylation did not have a significant effect on the digestibility of cow's milk proteins, but it could significantly reduce the affinity of their specific antibody, indicating that it could be used in producing hypoallergenic dairy product.

Vibrio cholerae causes cholera, an important cause of death worldwide. A fuller understanding of how virulence is regulated offers the potential for developing virulence inhibitors, regarded as efficient therapeutic alternatives for cholera treatment. Here we show using competitive infections of wild-type and mutant bacteria that the regulator of chitosan utilization, ChsR, increases V. cholerae virulence in vivo. Mechanistically, RNA sequencing, chromatin immunoprecipitation with sequencing and molecular biology approaches revealed that ChsR directly upregulated the expression of the virulence regulator, TcpP, which promoted expression of the cholera toxin and the toxin co-regulated pilus, in response to low O2 levels in the small intestine. We also found that chitosan degradation products inhibit the ChsR-tcpP promoter interaction. Consistently, administration of chitosan oligosaccharide, particularly when delivered via sodium alginate microsphere carriers, reduced V. cholerae intestinal colonization and disease severity in mice by blocking the chsR-mediated pathway. These data reveal the potential of chitosan oligosaccharide as supplemental therapy for cholera treatment and prevention.

Mitochondrial dynamics are critical in cellular energy production, metabolism, apoptosis, and immune responses. Pathogenic bacteria have evolved sophisticated mechanisms to manipulate host cells' mitochondrial functions, facilitating their proliferation and dissemination. Salmonella enterica serovar Typhimurium (S. Tm), an intracellular foodborne pathogen, causes diarrhea and exploits host macrophages for survival and replication. However, S. Tm-associated mitochondrial dynamics during macrophage infection remain poorly understood. In this study, we showed that within macrophages, S. Tm remodeled mitochondrial fragmentation to facilitate intracellular proliferation mediated by Salmonella invasion protein A (SipA), a type III secretion system effector encoded by Salmonella pathogenicity island 1. SipA directly targeted mitochondria via its N-terminal mitochondrial targeting sequence, preventing excessive fragmentation and the associated increase in mitochondrial reactive oxygen species, loss of mitochondrial membrane potential, and release of mitochondrial DNA and cytochrome c into the cytosol. Macrophage replication assays and animal experiments showed that mitochondria and SipA interact to facilitate intracellular replication and pathogenicity of S. Tm. Furthermore, we showed that SipA delayed mitochondrial fragmentation by indirectly inhibiting the recruitment of cytosolic dynamin-related protein 1, which mediates mitochondrial fragmentation. This study revealed a novel mechanism through which S. Tm manipulates host mitochondrial dynamics, providing insights into the molecular interplay that facilitates S. Tm adaptation within host macrophages.

Enterohemorrhagic Escherichia coli (EHEC) is an important foodborne pathogen that infects humans by colonizing the large intestine. The genome of EHEC O157:H7 contains 177 unique O islands (OIs). Certain OIs significantly contribute to the heightened virulence and pathogenicity exhibited by EHEC O157:H7. However, the function of most OI genes remains unknown. We demonstrated here that EHEC O157:H7 adherence to and colonization of the mouse large intestine are both dependent on OI-97. Z3495, which is annotated as a LysR-type transcriptional regulator and encoded in OI-97, contributes to this phenotype. Z3495 activated the locus of enterocyte effacement (LEE) gene expression, promoting bacterial adherence. Deletion of z3495 significantly decreased the transcription of ler and other LEE genes, the ability to adhere to the host cells, and colonization in the mouse large intestine. Furthermore, the ChIP-seq results confirmed that Z3495 can directly bind to the promoter region of rcsF, which is a well-known activator of Ler, and increase LEE gene expression. Finally, phylogenetic analysis revealed that Z3495 is a widespread transcriptional regulator in enterohemorrhagic and enteropathogenic Escherichia coli. As a result of this study, we have gained a deeper understanding of how bacteria control their virulence and provide another example of a laterally acquired regulator that regulates LEE gene expression in bacteria.

Intratumor heterogeneity (ITH) of bladder cancer (BLCA) contributes to therapy resistance and immune evasion affecting clinical prognosis. The molecular and cellular mechanisms contributing to BLCA ITH generation remain elusive. It is found that a TM4SF1-positive cancer subpopulation (TPCS) can generate ITH in BLCA, evidenced by integrative single cell atlas analysis. Extensive profiling of the epigenome and transcriptome of all stages of BLCA revealed their evolutionary trajectories. Distinct ancestor cells gave rise to low-grade noninvasive and high-grade invasive BLCA. Epigenome reprograming led to transcriptional heterogeneity in BLCA. During early oncogenesis, epithelial-to-mesenchymal transition generated TPCS. TPCS has stem-cell-like properties and exhibited transcriptional plasticity, priming the development of transcriptionally heterogeneous descendent cell lineages. Moreover, TPCS prevalence in tumor is associated with advanced stage cancer and poor prognosis. The results of this study suggested that bladder cancer interacts with its environment by acquiring a stem cell-like epigenomic landscape, which might generate ITH without additional genetic diversification.

Abstract Colorectal cancer (CRC) is one of the most lethal and prevalent malignancies with elusive molecular causes. While the abnormal activity of transcription factors has been reported as a vital component of cancer progression, the mechanism through which they function remains unclear. Here, through the analysis of primary human CRC tissue data, we identified GATA6 as a CRC specific regulator, whose expression upregulation is potentially associated with promoter hypomethylation. In the human genome, GATA6 showed preferential binding at tumor specific active enhancers. Through both auxin-inducible degron (AID) and CRISPR knockout, we showed that GATA6 depletion severely impaired clonogenicity and proliferation. GATA6 depletion caused the loss of CRC specific open chromatin, suggesting a specific regulatory role of GATA6 in CRC pathogenesis. Interestingly, we also discovered the co-occupancy of GATA6 with CTCF and its direct involvement in promoter enhancer loops, suggesting the possibility that GATA6 mediate transcriptional regulation through 3D genome. The loss of GATA6 led to alterations in E-P chromatin interactions of a panel of oncogenes, accompanied by the oncogene downregulation. Finally, we showed that GATA6 loss is detrimental to the xenograft tumor growth and thereby rescued mice survival. Taken together, we unrevealed a novel mechanism by which GATA6 contributes to CRC. Citation Format: Huijue Lyu, Xintong Chen, Yang Cheng, Te Zhang, Ping Wang, Josiah Hiu-yuen Wong, Juan Wang, Lena Stasiak, Leyu Sun, Guangyu Yang, Lu Wang, Feng Yue. GATA6 is a novel regulator of gene expression and 3D genome in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4401.

Although alpha s1-casein poses significant health risks to individuals with milk allergies, the availability of quantification methods for this allergen remains limited. In this study, we developed an immunomagnetic beads-based immunoassay (IMBs-ELISA) for the precise quantitative detection of bovine alpha s1-CN, specifically targeting epitope AA173-194. No cross-reactivity was observed with the other 7 food allergens including milk allergen. The linear detection range of the established IMBs-ELISA method was 0.125 mu g/mL-2.000 mu g/mL, with a limit of detection of 0.099 mu g/mL. The accuracy of this method was 1.048 %, and the intra-plate and inter-plate precision achieved 4.100 % and 6.777 %, respectively. Notably, the entire IMBs-ELISA process could be completed within 75 min, representing a substantial time-saving advantage over traditional ELISA methods. These results proved the reliability and rapidity of the IMBs-ELISA method for detecting alpha s1-CN in real food.

Polycomb group (PcG) proteins are epigenetic gene silencers that have been implicated in stem cell maintenance and cancer development. Genetic and biochemical studies indicate that Polycomb group proteins exist in at least two protein complexes, Polycomb repressive complex 2 (PRC2) and Polycomb repressive complex 1 (PRC1), that act in concert to initiate and maintain stable gene repression. Bmi1 and Mel18 are two major homologs of the PCGF subunit within the canonical PRC1 complex. While Bmi1 is a positive regulator of hematopoietic stem cell (HSC) and leukemia stem cell (LSC) self-renewal, the role of Mel18 in normal and malignant hematopoiesis is not fully understood. To further determine the role of Mel18 in hematopoiesis, we have generated Mel18 conditional knockout mice ( Mel18 f/f-Mx1Cre +). Acute deletion of Mel18 in the hematopoietic compartment did not affect the frequency and survival of hematopoietic stem and progenitor cells (HSPCs). To determine the impact of Mel18 deficiency on HSPC proliferation, we performed serial replating assays and found that loss of Mel18 increases the replating potential of HSPCs in vitro. To determine the role of Mel18 in HSC self-renewal, we first performed serial bone marrow (BM) transplantation assays and found that Mel18 null BM cells show increased engraftment in both primary and secondary transplantation assays. Limiting dilution transplantation showed that there were more functional HSCs in the BM of Mel18 knockout mice. We then performed HSC transplantation assays and found that loss Mel18 enhances HSC self-renewal. Thus, we demonstrated that Mel18 inhibits HSC self-renewal and decreases the number of functional HSCs in vivo. To understand the mechanism by which Mel18 inhibits HSC self-renewal and proliferation, we performed RNA-seq using Mel18 +/+ and Mel18 -/- HSCs. We then performed gene set enrichment analysis (GSEA) to identify potential Mel18 target genes important for HSC behavior. We found that HSC gene signatures are enriched in Mel18 -/- HSCs. We also found that leukemia stem cell gene signatures and CBF-mutant-AML genes are significantly enriched in Mel18 -/- HSCs, suggesting that loss of Mel18 may prime normal HSCs for leukemic transformation. Notably, our RNA-seq assay showed that senescence-related genes, including S100a9 and S100a8, were down-regulated in Mel18 -/- HSPCs. Importantly, loss of Mel18 significantly decreased the number of senescent HSPCs. To understand how Mel18 regulates gene transcription, we performed ATAC-seq assays in Mel18 +/+ and Mel18 -/- HSPCs. Mel18 null HSPCs showed decreased chromatin accessibility near both S100a8 and S100a9 genes. We confirmed that both S100a8 and S100a9 are downregulated in Mel18 -/- HSPCs compared to Mel18 +/+ HSPCs. Given that cell cycle arrest is a common feature of cellular senescence, we also examined the expression of cell cycle related genes in Mel18 -/- HSPCs. The expression of cell cycle regulators, including CDK4 and CCND2, is increased in Mel18 -/- HSPCs. Mel18 can bind to a specific DNA sequence, 5‘-GACTNGACT-3‘, thereby repressing the expression of target genes. Mel18 directly binds to the CCND2 gene locus as revealed by ChIP-seq assays in embryonic stem cells, suggesting that Mel18 may directly repress CCND2 expression in HSPCs. In summary, we demonstrate that Mel18 inhibits HSC self-renewal via repressing the expression of genes that are important for cellular senescence and proliferation.

Nearly 70% of Uterine fibroid (UF) tumors are driven by recurrent MED12 hotspot mutations. Unfortunately, no cellular models could be generated because the mutant cells have lower fitness in 2D culture conditions. To address this, we employ CRISPR to precisely engineer MED12 Gly44 mutations in UF-relevant myometrial smooth muscle cells. The engineered mutant cells recapitulate several UF-like cellular, transcriptional, and metabolic alterations, including altered Tryptophan/kynurenine metabolism. The aberrant gene expression program in the mutant cells is, in part, driven by a substantial 3D genome compartmentalization switch. At the cellular level, the mutant cells gain enhanced proliferation rates in 3D spheres and form larger lesions in vivo with elevated production of collagen and extracellular matrix deposition. These findings indicate that the engineered cellular model faithfully models key features of UF tumors and provides a platform for the broader scientific community to characterize genomics of recurrent MED12 mutations.

In recent years,milk allergy has attracted more and more attention because of its increasing incidence and in-depth research on it.The glycosylation modification of milk proteins not only can improve the functional properties of dairy products,but also reduce the allergenicity of dairy products by destroying or shielding the epitope of allergen proteins.This modification has found applications in various fields such as food flavor,antioxidants and carriers.This paper introduces common glycosylation reactions in the dairy industry,discusses their advantages and disadvantages and the pattern of changes in functional properties and allergenicity of milk proteins after glycosylation,and summarizes the methods to avoid the negative effects of glycosylation reactions.This review provides a reference for the research,development and application of glycosylation modification in milk allergy and dairy processing.

Objective Surveillance tools for early cancer detection are suboptimal, including hepatocellular carcinoma (HCC), and biomarkers are urgently needed. Extracellular vesicles (EVs) have gained increasing scientific interest due to their involvement in tumour initiation and metastasis; however, most extracellular RNA (exRNA) blood-based biomarker studies are limited to annotated genomic regions. Design EVs were isolated with differential ultracentrifugation and integrated nanoscale deterministic lateral displacement arrays (nanoDLD) and quality assessed by electron microscopy, immunoblotting, nanoparticle tracking and deconvolution analysis. Genome-wide sequencing of the largely unexplored small exRNA landscape, including unannotated transcripts, identified and reproducibly quantified small RNA clusters (smRCs). Their key genomic features were delineated across biospecimens and EV isolation techniques in prostate cancer and HCC. Three independent exRNA cancer datasets with a total of 479 samples from 375 patients, including longitudinal samples, were used for this study. Results ExRNA smRCs were dominated by uncharacterised, unannotated small RNA with a consensus sequence of 20 nt. An unannotated 3-smRC signature was significantly overexpressed in plasma exRNA of patients with HCC (p<0.01, n=157). An independent validation in a phase 2 biomarker case-control study revealed 86% sensitivity and 91% specificity for the detection of early HCC from controls at risk (n=209) (area under the receiver operating curve (AUC): 0.87). The 3-smRC signature was independent of alpha-fetoprotein (p<0.0001) and a composite model yielded an increased AUC of 0.93. Conclusion These findings directly lead to the prospect of a minimally invasive, blood-only, operator-independent clinical tool for HCC surveillance, thus highlighting the potential of unannotated smRCs for biomarker research in cancer.

近年来食物过敏发生率急剧增加,已是全球关注的公共卫生问题.美拉德反应作为食品加工和储存过程中最重要的化学反应之一,过敏原蛋白被修饰后由于结构发生了变化,必然引起致敏性改变,进而可能引发机体对于不同处理条件的抗原免疫耐受不一致,导致过敏反应.本文首先介绍了美拉德反应修饰蛋白质的过程及机制,进而解析不同影响因素调节美拉德反应终产物对致敏性的影响,最后阐述影响肠道菌群及其免疫应答机制的相关研究进行综述,并对美拉德反应作为一种尚未被完全开发和应用的加工方式进行展望.

Abstract Background State‐of‐art non‐invasive diagnosis processes for bladder cancer (BLCA) harbour shortcomings such as low sensitivity and specificity, unable to distinguish between high‐ (HG) and low‐grade (LG) tumours, as well as inability to differentiate muscle‐invasive bladder cancer (MIBC) and non‐muscle‐invasive bladder cancer (NMIBC). This study investigates a comprehensive characterization of the entire DNA methylation (DNAm) landscape of BLCA to determine the relevant biomarkers for the non‐invasive diagnosis of BLCA. Methods A total of 304 samples from 224 donors were enrolled in this multi‐centre, prospective cohort study. BLCA‐specific DNAm signature discovery was carried out with genome‐wide bisulfite sequencing in 32 tumour tissues and 12 normal urine samples. A targeted sequencing assay for BLCA‐specific DNAm signatures was developed to categorize tumour tissue against normal urine, or MIBC against NMIBC. Independent validation was performed with targeted sequencing of 259 urine samples in a double‐blinded manner to determine the clinical diagnosis and prognosis value of DNAm‐based classification models. Functions of genomic region harbouring BLCA‐specific DNAm signature were validated with biological assays. Concordances of pathology to urine tumour DNA (circulating tumour DNA [ctDNA]) methylation, genomic mutations or other state‐of‐the‐art diagnosis methods were measured. Results Genome‐wide DNAm profile could accurately classify LG tumour from HG tumour (LG NMIBC vs. HG NMIBC: p = .038; LG NMIBC vs. HG MIBC, p = .00032; HG NMIBC vs. HG MIBC: p = .82; Student's t‐test). Overall, the DNAm profile distinguishes MIBC from NMIBC and normal urine. Targeted‐sequencing‐based DNAm signature classifiers accurately classify LG NMIBC tissues from HG MIBC and could detect tumours in urine at a limit of detection of less than .5%. In tumour tissues, DNAm accurately classifies pathology, thus outperforming genomic mutation or RNA expression profiles. In the independent validation cohort, pre‐surgery urine ctDNA methylation outperforms fluorescence in situ hybridization (FISH) assay to detect HG BLCA (n = 54) with 100% sensitivity (95% CI: 82.5%–100%) and LG BLCA (n = 26) with 62% sensitivity (95% CI: 51.3%–72.7%), both at 100% specificity (non‐BLCA: n = 72; 95% CI: 84.1%–100%). Pre‐surgery urine ctDNA methylation signature correlates with pathology and predicts recurrence and metastasis. Post‐surgery urine ctDNA methylation (n = 61) accurately predicts recurrence‐free survival within 180 days, with 100% accuracy. Conclusion With the discovery of BLCA‐specific DNAm signatures, targeted sequencing of ctDNA methylation outperforms FISH and DNA mutation to detect tumours, predict recurrence and make prognoses.