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Abstract Background The high mutation rate throughout the entire melanoma genome presents a major challenge in stratifying true driver events from the background mutations. Numerous recurrent non-coding alterations, such as those in enhancers, can shape tumor evolution, thereby emphasizing the importance in systematically deciphering enhancer disruptions in melanoma. Results Here, we leveraged 297 melanoma whole-genome sequencing samples to prioritize highly recurrent regions. By performing a genome-scale CRISPR interference (CRISPRi) screen on highly recurrent region-associated enhancers in melanoma cells, we identified 66 significant hits which could have tumor-suppressive roles. These functional enhancers show unique mutational patterns independent of classical significantly mutated genes in melanoma. Target gene analysis for the essential enhancers reveal many known and hidden mechanisms underlying melanoma growth. Utilizing extensive functional validation experiments, we demonstrate that a super enhancer element could modulate melanoma cell proliferation by targeting MEF2A, and another distal enhancer is able to sustain PTEN tumor-suppressive potential via long-range interactions. Conclusions Our study establishes a catalogue of crucial enhancers and their target genes in melanoma growth and progression, and illuminates the identification of novel mechanisms of dysregulation for melanoma driver genes and new therapeutic targeting strategies.

Abstract Metal-organic frameworks (MOFs) are a sort of promising peroxidase-like nanozyme but face the challenge that the inorganic nodes in most of the MOF structures are generally blocked by the organic linkers. Further enhancement or activation of their peroxidase-like activity plays an important role in developing MOF-based nanozymes. Herein, a multimetallic nanoparticle (NP) decorated-MOF, Cu/Au/Pt NP decorated-Cu-TCPP(Fe) nanozyme (CuAuPt/Cu-TCPP(Fe)) was synthesized in situ and served as a peroxidase-like nanozyme. The peroxidase-like activity of this stable CuAuPt/Cu-TCPP(Fe) nanozyme was enhanced due to the decreased potential barriers for *OH generation in the catalytic process. Owing to the remarkable peroxidase-like activity, a CuAuPt/Cu-TCPP(Fe)-based colorimetric assay was established for the sensitive determination of H2O2 and glucose with the limit of detection (LOD) of 9.3 µM and 4.0 µM, respectively. In addition, a visual point-of-care testing (POCT) device was developed by integrating the CuAuPt/Cu-TCPP(Fe)-based test strips with a smartphone and was employed for a portable test of 20 clinical serum glucose samples. The results determined by this method agree well with the values deduced by clinical automatic biochemical analysis. This work not only represents an inspiration for the usage of MNP/MOF composite as a novel nanozyme for POCT diagnosis, but also provides a deeper insight and understanding into the enhanced enzyme-mimic effect of MNP-hybrid MOF composites, which in turn will guide the engineering of MOF-based functional nanomaterials. Graphical Abstract

Abstract Thrombocytopenia is a major complication in a subset of patients with multiple myeloma (MM). However, little is known about its development and significance during MM. Here, we show thrombocytopenia is linked to poor prognosis in MM. In addition, we identify serine, which is released from MM cells into the bone marrow microenvironment, as a key metabolic factor that suppresses megakaryopoiesis and thrombopoiesis. The impact of excessive serine on thrombocytopenia is mainly mediated through the suppression of megakaryocyte (MK) differentiation. Extrinsic serine is transported into MKs through SLC38A1 and downregulates SVIL via SAM-mediated tri-methylation of H3K9, ultimately leading to the impairment of megakaryopoiesis. Inhibition of serine utilization or treatment with TPO enhances megakaryopoiesis and thrombopoiesis and suppresses MM progression. Together, we identify serine as a key metabolic regulator of thrombocytopenia, unveil molecular mechanisms governing MM progression, and provide potential therapeutic strategies for treating MM patients by targeting thrombocytopenia.

Here the authors used an evidence-based strategy to prioritize causal pleiotropic variants of autoimmune diseases, and revealed that rs4728142 modulates aberrant IRF5 alternative promoter usage by ZBTB3-mediated chromatin looping.

ABSTRACTGut microbiota plays an essential role in nonalcoholic fatty liver disease (NAFLD). However, the contribution of individual bacterial strains and their metabolites to childhood NAFLD pathogenesis remains poorly understood. Herein, the critical bacteria in children with obesity accompanied by NAFLD were identified by microbiome analysis. Bacteria abundant in the NAFLD group were systematically assessed for their lipogenic effects. The underlying mechanisms and microbial-derived metabolites in NAFLD pathogenesis were investigated using multi-omics and LC-MS/MS analysis. The roles of the crucial metabolite in NAFLD were validated in vitro and in vivo as well as in an additional cohort. The results showed that Enterococcus spp. was enriched in children with obesity and NAFLD. The patient-derived Enterococcus faecium B6 (E. faecium B6) significantly contributed to NAFLD symptoms in mice. E. faecium B6 produced a crucial bioactive metabolite, tyramine, which probably activated PPAR-γ, leading to lipid accumulation, inflammation, and fibrosis in the liver. Moreover, these findings were successfully validated in an additional cohort. This pioneering study elucidated the important functions of cultivated E. faecium B6 and its bioactive metabolite (tyramine) in exacerbating NAFLD. These findings advance the comprehensive understanding of NAFLD pathogenesis and provide new insights for the development of microbe/metabolite-based therapeutic strategies.

The bone tumour microenvironment plays an essential role in multiple myeloma (MM) development. Here, the authors show that bone collagen degradation provides glycine to support MM progression through glutathione and purine synthesis.

Microcystin-LR (MC-LR) contamination is a worldwide environmental problem that poses a grave threat to the water ecosystem and public health. Exposure to MC-LR has been associated with the development of intestinal injury, but there are no effective treatments for MC-LR-induced intestinal disease. Probiotics are “live microorganisms that are beneficial to the health of the host when administered in sufficient quantities”. It has been demonstrated that probiotics can prevent or treat a variety of human diseases; however, their ability to mitigate MC-LR-induced intestinal harm has not yet been investigated. The objective of this study was to determine whether probiotics can mitigate MC-LR-induced intestinal toxicity and its underlying mechanisms. We first evaluated the pathological changes in colorectal tissues using an animal model with sub-chronic exposure to low-dose MC-LR, HE staining to assess colorectal histopathologic changes, qPCR to detect the expression levels of inflammatory factors in colorectal tissues, and WB to detect the alterations on CSF1R signaling pathway proteins in colorectal tissues. Microbial sequencing analysis and screening of fecal microorganisms differential to MC-LR treatment in mice. To investigate the role of microorganisms in MC-LR-induced colorectal injury, an in vitro model of MC-LR co-treatment with microorganisms was developed. Our findings demonstrated that MC-LR treatment induced an inflammatory response in mouse colorectal tissues, promoted the expression of inflammatory factors, activated the CSF1R signaling pathway, and significantly decreased the abundance of Lactobacillus. In a model of co-treatment with MC-LR and Lactobacillus fermentum (L. fermentum), it was discovered that L. fermentum substantially reduced the incidence of the colorectal inflammatory response induced by MC-LR and inhibited the protein expression of the CSF1R signaling pathway. This is the first study to suggest that L. fermentum inhibits the CSF1R signaling pathway to reduce the incidence of MC-LR-induced colorectal inflammation. This research may provide an excellent experimental foundation for the development of strategies for the prevention and treatment of intestinal diseases in MC-LR.

Microcystin-LR (MC-LR), a potent hepatotoxin can cause liver damages. However, research on hepatic lipid metabolism caused by long-term exposure to environmental concentrations MC-LR is limited. In the current study, mice were exposed to various low concentrations of MC-LR (0, 1, 30, 60, 90, 120 μg/L in the drinking water) for 9 months. The general parameters, serum and liver lipids, liver tissue pathology, lipid metabolism-related genes and proteins of liver were investigated. The results show that chronic MC-LR exposure had increased the levels of triglyceride (TG) and total cholesterol (TC) in serum and liver. In addition, histological observation revealed that hepatic lobules were disordered with obvious inflammatory cell infiltration and lipid droplets. More importantly, the mRNA and proteins expression levels of lipid synthesis-related nuclear sterol regulatory element binding protein-1c (nSREBP-1c), SREBP-1c, cluster of differentiation 36 (CD36), acetyl-CoA-carboxylase1 (ACC1), stearoyl-CoA desaturase1 (SCD1) and fatty acid synthase (FASN) were increased in MC-LR treated groups, the expression levels of fatty acids β-oxidation related genes peroxisomal acyl-coenzyme A oxidase 1 (ACOX1) was decreased after exposure to 60–120 μg/L MC-LR. Furthermore, the inflammatory factors interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) were higher than that in the control group. All the findings indicated that mice were exposed to chronic low concentrations MC-LR caused liver inflammation and hepatic lipid metabolism disorder .

Summary: Precise identification of context-specific transcriptional regulators (TRs) cooperation facilitates the understanding of complex gene regulation. However, previous methods are highly reliant on the availability of ChIPped TRs. Here, we provide a protocol for running 3DCoop, a pipeline for computational inference of cell type-specific TR cooperation in 3D chromatin by integrating TR motifs, open chromatin profiles, gene expression, and chromatin loops. 3DCoop provides a feasible solution to study the potential interplay among TRs across multiple human or mouse tissue/cell types.For complete details on the use and execution of this protocol, please refer to Yi et al. (2021).

Background: Fisetin, a natural potent flavonoid, has various beneficial, pharmacological activities. In this study, we investigated expression changes of the fisetin regulating genes in lipopolysaccharide (LPS)-treated RAW264.7 cells and explored the role of fisetin in inflammation and autophagy. Methods and results: Microarray analysis identified 1,071 genes that were regulated by fisetin in LPS-treated RAW264.7 cells, and these genes were mainly related to the process of immune system response. Quantitative real-time polymerase chain reaction and Bio-Plex analysis indicated that fisetin decreased the expression and secretion of several inflammatory cytokines in cells administered with LPS. Western blot analysis and immunofluorescence assay showed that fisetin decreased microtubule-associated protein 1 light-chain 3B (LC3B) and lysosome-associated membrane protein 1 (LAMP1) expression in LPS-treated cells, while the autophagy inhibitor chloroquine (CQ) could partially reverse this effect. In addition, fisetin reduced the elevated expression of p-PI3K, p-AKT and p-mTOR induced by LPS in a concentration-dependent manner. Conclusions: Fisetin diminished the expression and secretion of inflammatory cytokines and facilitated autophagosome-lysosome fusion and degradation in LPS-treated RAW264.7 cells via inhibition of the PI3K/AKT/mTOR signaling pathway. Overall, the results of this study provide new clues for the anti-inflammatory mechanism of fisetin and explain the crosstalk between autophagy and inflammation to some extent.

Abstract It has been previously shown that (never in mitosis gene A)‐related kinase 2 (NEK2) is upregulated in multiple myeloma (MM) and contributes to drug resistance. However, the mechanisms behind this upregulation remain poorly understood. In this study, it is found that amplification of NEK2 and hypermethylation of distal CpG islands in its promoter correlate strongly with increased NEK2 expression. Patients with NEK2 amplification have a poor rate of survival and often exhibit TP53 deletion, which is an independent prognostic factor in MM. This combination of TP53 knockout and NEK2 overexpression induces asymmetric mitosis, proliferation, drug resistance, and tumorigenic behaviors in MM in vitro and in vivo. In contrast, delivery of wild type p53 and suppression of NEK2 in TP53−/− MM cell lines inhibit tumor formation and enhance the effect of Bortezomib against MM. It is also discovered that inactivating p53 elevates NEK2 expression genetically by inducing NEK2 amplification, transcriptionally by increased activity of cell cycle‐related genes like E2F8 and epigenetically by upregulating DNA methyltransferases. Dual defects of TP53 and NEK2 may define patients with the poorest outcomes in MM with p53 inactivation, and NEK2 may serve as a novel therapeutic target in aggressive MM with p53 abnormalities.

Summary: Context-specific activities of transcription regulators (TRs) in the nucleus modulate spatiotemporal gene expression precisely. Using the largest ChIP-seq data and chromatin loops in the human K562 cell line, we initially interrogated TR cooperation in 3D chromatin via a graphical model and revealed many known and novel TRs manipulating context-specific pathways. To explore TR cooperation across broad tissue/cell types, we systematically leveraged large-scale open chromatin profiles, computational footprinting, and high-resolution chromatin interactions to investigate tissue/cell type-specific TR cooperation. We first delineated a landscape of TR cooperation across 40 human tissue/cell types. Network modularity analyses uncovered the commonality and specificity of TR cooperation in different conditions. We also demonstrated that TR cooperation information can better interpret the disease-causal variants identified by genome-wide association studies and recapitulate cell states during neural development. Our study characterizes shared and unique patterns of TR cooperation associated with the cell type specificity of gene regulation in 3D chromatin.

NEK2 is associated with drug resistance in multiple cancers. Our previous studies indicated that high NEK2 confers inferior survival in multiple myeloma (MM); thus, a better understanding of the mechanisms by which NEK2 induces drug resistance in MM is required. In this study, we discovered that NEK2 enhances MM cell autophagy, and a combination of autophagy inhibitor chloroquine (CQ) and chemotherapeutic bortezomib (BTZ) significantly prevents NEK2‐induced drug resistance in MM cells. Interestingly, NEK2 was found to bind and stabilize Beclin‐1 protein but did not affect its mRNA expression and phosphorylation. Moreover, autophagy enhanced by NEK2 was significantly prevented by knockdown of Beclin‐1 in MM cells, suggesting that Beclin‐1 mediates NEK2‐induced autophagy. Further studies demonstrated that Beclin‐1 ubiquitination is decreased through NEK2 interaction with USP7. Importantly, knockdown of Beclin‐1 sensitized NEK2‐overexpressing MM cells to BTZ in vitro and in vivo. In conclusion, we identify a novel mechanism whereby autophagy is activated by the complex of NEK2/USP7/Beclin‐1 in MM cells. Targeting the autophagy signaling pathway may provide a promising therapeutic strategy to overcome NEK2‐induced drug resistance in MM.

The global prevalence of nonalcoholic fatty liver disease (NAFLD) has been increasing rapidly in recent years, which is now estimated to be over 25%. NAFLD is one of the most common chronic liver diseases in the world. At present, with the rapid development of economy and industrialization, many chemicals are released into the environment. These chemical contaminants in the environment might cause harm to human health and result in lipid metabolism disorder during long-term exposure. Moreover, the incentive of many NAFLD cases is unknown, and the environmental risk factors of NAFLD need to be urgently identified. Hence, we focus on the impacts of several popular environmental contaminants in water environment on the development and progression of NAFLD. These contaminants mainly include microcystins (MCs), disinfection by-products (DBPs), heavy metals (HMs), dioxins and polychlorinated biphenyls (PCBs). Through analyzing a great many epidemiological and toxicological studies, we have found positive associations between NAFLD and chronic exposure to these contaminants at the environmental levels. This review may enhance the understanding of liver damage caused by environmental pollutants, which are considered as tangible environmental risk factors for NAFLD.

Obesity, a metabolic disease caused by excessive fat accumulation in the body, has attracted worldwide attention. Microcystin-LR (MC-LR) is a hepatotoxic cyanotoxin which has been reportedly to cause lipid metabolism disorder. In this study, C57BL/6J mice were fed a high-fat diet (HFD) for eight weeks to build obese an animal model, and subsequently, the obese mice were fed MC-LR for another eight weeks, and we aimed to determine how MC-LR exposure affects the liver lipid metabolism in high-fat-diet-induced obese mice. The results show that MC-LR increased the obese mice serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT), indicating damaged liver function. The lipid parameters include serum triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-c), and liver TG, which were all increased, whilst the high-density lipoprotein cholesterol (HDL-c) was decreased. Furthermore, after MC-LR treatment, histopathological observation revealed that the number of red lipid droplets increased, and that steatosis was more severe in the obese mice. In addition, the lipid synthesis-related genes were increased and the fatty acid β-oxidation-related genes were decreased in the obese mice after MC-LR exposure. Meanwhile, the protein expression levels of phosphorylation phosphatidylinositol 3-kinase (p-PI3K), phosphorylation protein kinase B (p-AKT), phosphorylation mammalian target of rapamycin (p-mTOR), and sterol regulatory element binding protein 1c (SREBP1-c) were increased; similarly, the p-PI3K/PI3K, p-AKT/AKT, p-mTOR/mTOR, and SREBP1/β-actin were significantly up-regulated in obese mice after being exposed to MC-LR, and the activated PI3K/AKT/mTOR/SREBP1 signaling pathway. In addition, MC-LR exposure reduced the activity of superoxide dismutase (SOD) and increased the level of malondialdehyde (MDA) in the obese mice’s serum. In summary, the MC-LR could aggravate the HFD-induced obese mice liver lipid metabolism disorder by activating the PI3K/AKT/mTOR/SREBP1 signaling pathway to hepatocytes, increasing the SREBP1-c-regulated key enzymes for lipid synthesis, and blocking fatty acid β-oxidation.

Pneumonia accounts for a significant cause of morbidity and mortality in multiple myeloma (MM) patients. It has been previously shown that intestinal Klebsiella pneumonia (K. pneumonia) enriches in MM and promotes MM progression. However, what role the altered gut microbiota plays in MM with pneumonia remains unknown. Here, we show that intestinal K. pneumonia is significantly enriched in MM with pneumonia. This enriched intestinal K. pneumonia links to the incidence of pneumonia in MM, and intestinal colonization of K. pneumonia contributes to pneumonia in a 5TGM1 MM mice model. Further targeted metabolomic assays reveal the elevated level of glutamine, which is consistently increased with the enrichment of K. pneumonia in MM mice and patients, is synthesized by K. pneumonia, and leads to the elevated secretion of TNF-α in the lung normal fibroblast cells for the higher incidence of pneumonia. Inhibiting glutamine synthesis by establishing glnA-mutated K. pneumonia alleviates the incidence of pneumonia in the 5TGM1 MM mice model. Overall, our work proposes that intestinal K. pneumonia indirectly contributes to pneumonia in MM by synthesizing glutamine. Altogether, we unveil a gut–lung axis in MM with pneumonia and establish a novel mechanism and a possible intervention strategy for MM with pneumonia.

Identifying causal regulatory variants and their target genes from the majority of non-coding disease-associated genetic loci is the main challenge in post-Genome-Wide Association Studies (GWAS) functional studies. Although chromosome conformation capture (3C) and its derivative technologies have been successfully applied to nominate putative causal genes for non-coding variants, many GWAS target genes have not been identified yet. This study generated a high-resolution contact map from epithelial ovarian cancer (EOC) cells with two H3K27ac-HiChIP libraries and analyzed the underlying gene networks for 15 risk loci identified from the largest EOC GWAS. By combinatory analysis of 4,021 fine-mapped credible variants of EOC GWAS and high-resolution contact map, we obtained 162 target genes that mainly enriched in cancer related pathways. Compared with GTEx eQTL genes in ovarian tissue and annotated proximal genes, 132 HiChIP targets were first identified for EOC causal variants. More than half of the credible variants (CVs) involved interactions that were over 185 kb in distance, indicating that long-range transcriptional regulation is an important mechanism for the function of GWAS variants in EOC. We also found that many HiChIP gene targets showed significantly differential expressions between normal ovarian and EOC tumor samples. We validated one of these targets by manipulating the rs9303542 located region with CRISPR-Cas9 deletion and dCas9-VP64 activation experiments and found altered expression of HOXB7 and HOXB8 at 17q21.32. This study presents a systematic analysis to identify putative target genes for causal variants of EOC, providing an in-depth investigation of the mechanisms of non-coding regulatory variants in the etiology and pathogenesis of ovarian cancer.

Microcystin-LR (MC-LR), a cyanotoxin produced by cyanobacteria, induces oxidative stress in various types of cells. Prodigiosin, a red linear tripyrrole pigment, has been recently reported to have antimicrobial, antioxidative, and anticancer properties. How prodigiosin reacts to reactive oxygen species (ROS) induced by MC-LR is still undetermined. This study aimed to examine the effect of prodigiosin against oxidative stress induced by MC-LR in HepG2 cells. Ros was generated after cells were treated with MC-LR and was significantly inhibited with treatment of prodigiosin. In prodigiosin-treated cells, the levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and Nrf2-related phase II enzyme heme oxygenase-1 (HO-1) were increased. Besides, prodigiosin contributed to enhance nuclear Nrf2 level and repressed ubiquitination. Furthermore, prodigiosin promoted Nrf2 protein level and inhibited ROS in Nrf2 knocked down HepG2 cells. Results indicated that prodigiosin reduced ROS induced by MC-LR by enhancing Nrf2 translocation into the nucleus in HepG2 cells. The finding presents new clues for the potential clinical applications of prodigiosin for inhibiting MC-LR-induced oxidative injury in the future.

Microcystin-LR is a cyclic heptapeptide hepatotoxin produced by harmful cyanobacteria. A panel of microRNAs containing miR-451a were found to be significantly changed in normal human liver cells HL7702 after exposure to microcystin-LR (MC-LR) in our previous study. However, the functions of miR-451a in hepatotoxicity induced by MC-LR remained unclear. The study aimed to investigate the impacts of miR-451a in HL7702 cells following treatment with 5 or 10 μM MC-LR. The comet assay indicated that MC-LR can influence Olive tail moment (OTM) in HL7702 cells. Furthermore, increase of miR-451a significantly repressed DNA damage and the protein expression level of γ-H2AX induced by MC-LR. Moreover, over-expression of miR-451a inhibited the expression level of p-AKT1 protein in cells following treatment by MC-LR. These results showed that miR-451a may protect from MC-LR-induced DNA damage by down-regulating the expression of p-AKT1, which provides new clues for the diagnosis and therapy policies for liver damage induced by MC-LR.

Fisetin, a dietary flavonoid, is reported to have cellular antioxidant activity with an unclear mechanism. In this study, we investigated the effect of fisetin on the nuclear factor, erythroid 2-like 2 (Nrf2) signaling pathway in HepG2 cells to explore the cellular antioxidant mechanism. Fisetin upregulated the mRNA expression of heme oxygenase-1 (HO-1), glutamate-cysteine ligase catalytic subunit (GCLC), glutamate-cysteine ligase modifier subunit (GCLM), and NAD(P)H quinone oxidoreductase-1 (NQO1), and induced the protein of HO-1 but had no significant effect on the protein of GCLC, GCLM and NQO1. Moreover, nuclear accumulation of Nrf2 was clearly observed by immunofluorescence analysis and western blotting after fisetin treatment, and an enhanced luciferase activity of antioxidant response element (ARE)-regulated transactivation was obtained by dual-luciferase reporter gene assays. In addition, fisetin upregulated the protein level of Nrf2 and downregulated the protein level of Kelch-like ECH-associated protein 1 (Keap1). However, fisetin had no significant effect on Nrf2 mRNA expression. When protein synthesis was inhibited with cycloheximide (CHX), fisetin prolonged the half-life of Nrf2 from 15 min to 45 min. When blocking Nrf2 degradation with proteasome inhibitor MG132, ubiquitinated proteins were enhanced, and fisetin reduced ubiquitination of Nrf2. Taken together, fisetin translocated Nrf2 into the nucleus and upregulated the expression of downstream HO-1 gene by inhibiting the degradation of Nrf2 at the post-transcriptional level. These data provide the molecular mechanism to understand the cellular antioxidant activity of fisetin.

We examined the promoter hypermethylation of tumorsuppressor genes RASSF1A, TSLC1, quantitated EBV DNA load in nasopharyngeal carcinoma (NPC) tissues (T tissues), matched tumor-adjacent tissues outside 0.5 cm (P tissues), outside 1.0 cm (Z tissues) to evaluate the role of promoter hypermethylation of RASSF1A, TSLC1 as well as viral load in the pathogenesis of NPC. Methylation-specific polymerase chain reaction (PCR) for RASSF1A, TSLC1, quantitative real-time PCR analysis of EBV DNA were performed on matched T, P, Z tissues (n = 28) as well as chronic nasopharyngitis tissues (n = 8). Hypermethylated RASSF1A was frequently detected in the T (82%), P tissues (75%), but less frequently in Z tissues (46%). The average quantities of EBV DNA (copies/μg DNA) in matched T, P, Z tissues were 673,000, 90,000, 7000. The differences of promoter hypermethylation of RASSF1A, EBV viral load among T, P, Z tissues were statistically significant, with more frequent methylation, higher viral load detected when tissues examined were nearer to the NPC tissues. Our results suggest that aberrant hypermethylation of RASSF1A, high EBV load might be important events in NPC pathogenesis, they may be useful molecular diagnostic markers for this cancer.