Precise prediction of phase-separation key residues by machine learning.

Understanding intracellular phase separation is crucial for deciphering transcriptional control, cell fate transitions, and disease mechanisms. However, the key residues, which impact phase separation the most for protein phase separation function have remained elusive. We develop PSPHunter, which can precisely predict these key residues based on machine learning scheme. In vivo and in vitro validations demonstrate that truncating just 6 key residues in GATA3 disrupts phase separation, enhancing tumor cell migration and inhibiting growth. Glycine and its motifs are enriched in spacer and key residues, as revealed by our comprehensive analysis. PSPHunter identifies nearly 80% of disease-associated phase-separating proteins, with frequent mutated pathological residues like glycine and proline often residing in these key residues. PSPHunter thus emerges as a crucial tool to uncover key residues, facilitating insights into phase separation mechanisms governing transcriptional control, cell fate transitions, and disease development.

The transcriptomic profile shows the protective effects of celecoxib on cirrhotic splenomegaly.

BACKGROUND: Splenomegaly can exacerbate liver cirrhosis and portal hypertension. We have previously demonstrated that cyclooxygenase-2 (COX-2) inhibitor can attenuate cirrhotic splenomegaly. However, the mechanism of cirrhotic splenomegaly remains unclear, thus becoming the focus of the present study. MATERIALS AND METHODS: Thioacetamide (TAA) intraperitoneal injection was used to induce cirrhotic splenomegaly. Rats were randomized into the control, TAA and TAA + celecoxib groups. Histological analysis and high-throughput RNA sequencing of the spleen were conducted. Splenic collagen III, α-SMA, Ki-67, and VEGF were quantified. RESULTS: A total of 1461 differentially expressed genes (DEGs) were identified in the spleens of the TAA group compared to the control group. The immune response and immune cell activation might be the major signaling pathways involved in the pathogenesis of cirrhotic splenomegaly. With its immunoregulatory effect, celecoxib presents to ameliorate cirrhotic splenomegaly and liver cirrhosis. Furthermore, 304 coexisting DEGs were obtained between TAA vs. control and TAA + celecoxib vs. TAA. Gene ontology (GO) and KEGG analyses collectively indicated that celecoxib may attenuate cirrhotic splenomegaly through the suppression of splenic immune cell proliferation, inflammation, immune regulation, and fibrogenesis. The impacts on these factors were subsequently validated by the decreased splenic Ki-67-positive cells, macrophages, fibrotic areas, and mRNA levels of collagen III and α-SMA. CONCLUSIONS: Celecoxib attenuates cirrhotic splenomegaly by inhibiting splenic immune cell proliferation, inflammation, and fibrogenesis. The current study sheds light on the therapeutic strategy of liver cirrhosis by targeting splenic abnormalities and provides COX-2 inhibitors as a novel medical treatment for cirrhotic splenomegaly.

Chloroquine Alleviates Atherosclerosis by Modulating Regulatory T Cells Through the ATM/AMPK/mTOR Signaling Pathway in ApoE -/- Mice.

BACKGROUND: Clinical observation suggests the atheroprotective effect of chloroquine and its derivatives, while its mechanism remains unclear. This study aimed to observe the protective effect of chloroquine against atherosclerosis and explore the underlying mechanism. METHODS: Ataxia telangiectasia mutated (ATM) wild-type or haploinsufficient apolipoprotein-E-knockout (ATM+/+ApoE-/- or ATM+/-ApoE-/-) mice were treated with different dosages of chloroquine. Anti-CD25 antibody was used to deplete natural Tregs in ATM+/+ApoE-/- mice. The atherosclerotic burden in different groups of mice was comprehensively evaluated by H&E staining and Masson staining. The effect of chloroquine on the regulatory T cells (Tregs) was assessed in vivo and in vitro by flow cytometry and immunohistochemical staining. The expression of related proteins was detected by real-time polymerase chain reaction and western blotting. RESULTS: In ATM+/+ApoE-/- mice, chloroquine alleviated atherosclerotic lesions, stabilized the plaque, and increased Treg counts in the atherosclerotic lesions and spleens. However, in ATM haploinsufficient mice (ATM+/-ApoE-/-), chloroquine no longer prevented atherosclerosis or impacted Treg counts. Abolishing Treg cells using an anti-CD25 antibody in vivo abrogated the atheroprotective effect of chloroquine. In vitro, chloroquine promoted the differentiation of Tregs from naïve T cells, which was accompanied by enhanced ATM/AMP-activated protein kinase (AMPK) activity and reduced downstream mammalian target of rapamycin (mTOR) activity. DISCUSSION: These findings suggest that chloroquine ameliorates atherosclerosis and stabilizes plaque by modulating Tregs differentiation through the regulation of the ATM/AMPK/mTOR pathway.

DASES: a database of alternative splicing for esophageal squamous cell carcinoma.

Esophageal carcinoma ranks as the sixth leading cause of cancer-related mortality globally, with esophageal squamous cell carcinoma (ESCC) being particularly prevalent among Asian populations. Alternative splicing (AS) plays a pivotal role in ESCC development and progression by generating diverse transcript isoforms. However, the current landscape lacks a specialized database focusing on alternative splicing events (ASEs) derived from a large number of ESCC cases. Additionally, most existing AS databases overlook the contribution of long non-coding RNAs (lncRNAs) in ESCC molecular mechanisms, predominantly focusing on mRNA-based ASE identification. To address these limitations, we deployed DASES (http://www.hxdsjzx.cn/DASES). Employing a combination of publicly available and in-house ESCC RNA-seq datasets, our extensive analysis of 346 samples, with 93% being paired tumor and adjacent non-tumor tissues, led to the identification of 257 novel lncRNAs in esophageal squamous cell carcinoma. Leveraging a paired comparison of tumor and adjacent normal tissues, DASES identified 59,094 ASEs that may be associated with ESCC. DASES fills a critical gap by providing comprehensive insights into ASEs in ESCC, encompassing lncRNAs and mRNA, thus facilitating a deeper understanding of ESCC molecular mechanisms and serving as a valuable resource for ESCC research communities.

Disruption of cholangiocyte-B cell crosstalk by blocking the CXCL12-CXCR4 axis alleviates liver fibrosis.

B cells can promote liver fibrosis, but the mechanism of B cell infiltration and therapy against culprit B cells are lacking. We postulated that the disruption of cholangiocyte-B-cell crosstalk could attenuate liver fibrosis by blocking the CXCL12-CXCR4 axis via a cyclooxygenase-2-independent effect of celecoxib. In wild-type mice subjected to thioacetamide, celecoxib ameliorated lymphocytic infiltration and liver fibrosis. By single-cell RNA sequencing and flow cytometry, CXCR4 was established as a marker for profibrotic and liver-homing phenotype of B cells. Celecoxib reduced liver-homing B cells without suppressing CXCR4. Cholangiocytes expressed CXCL12, attracting B cells to fibrotic areas in human and mouse. The proliferation and CXCL12 expression of cholangiocytes were suppressed by celecoxib. In CXCL12-deficient mice, liver fibrosis was also attenuated with less B-cell infiltration. In the intrahepatic biliary epithelial cell line HIBEpiC, bulk RNA sequencing indicated that both celecoxib and 2,5-dimethyl-celecoxib (an analog of celecoxib that does not show a COX-2-dependent effect) regulated the TGF-ß signaling pathway and cell cycle. Moreover, celecoxib and 2,5-dimethyl-celecoxib decreased the proliferation, and expression of collagen I and CXCL12 in HIBEpiC cells stimulated by TGF-ß or EGF. Taken together, liver fibrosis can be ameliorated by disrupting cholangiocyte-B cell crosstalk by blocking the CXCL12-CXCR4 axis with a COX-2-independent effect of celecoxib.

Atypical cholangiocytes derived from hepatocyte-cholangiocyte transdifferentiation mediated by COX-2: a kind of misguided liver regeneration.

BACKGROUND: Hepatocyte-cholangiocyte transdifferentiation (HCT) is a potential origin of proliferating cholangiocytes in liver regeneration after chronic injury. This study aimed to determine HCT after chronic liver injury, verify the impacts of HCT on liver repair, and avoid harmful regeneration by understanding the mechanism. METHODS: A thioacetamide (TAA)-induced liver injury model was established in wild-type (WT-TAA group) and COX-2 panknockout (KO-TAA group) mice. HCT was identified by costaining of hepatocyte and cholangiocyte markers in vivo and in isolated mouse hepatocytes in vitro. The biliary tract was injected with ink and visualized by whole liver optical clearing. Serum and liver bile acid (BA) concentrations were measured. Either a COX-2 selective inhibitor or a ß-catenin pathway inhibitor was administered in vitro. RESULTS: Intrahepatic ductular reaction was associated with COX-2 upregulation in chronic liver injury. Immunofluorescence and RNA sequencing indicated that atypical cholangiocytes were characterized by an intermediate genetic phenotype between hepatocytes and cholangiocytes and might be derived from hepatocytes. The structure of the biliary system was impaired, and BA metabolism was dysregulated by HCT, which was mediated by the TGF-ß/ß-catenin signaling pathway. Genetic deletion or pharmaceutical inhibition of COX-2 significantly reduced HCT in vivo. The COX-2 selective inhibitor etoricoxib suppressed HCT through the TGF-ß-TGFBR1-ß-catenin pathway in vitro. CONCLUSIONS: Atypical cholangiocytes can be derived from HCT, which forms a secondary strike by maldevelopment of the bile drainage system and BA homeostasis disequilibrium during chronic liver injury. Inhibition of COX-2 could ameliorate HCT through the COX-2-TGF-ß-TGFBR1-ß-catenin pathway and improve liver function.

C-C motif chemokine receptor 2 inhibition reduces liver fibrosis by restoring the immune cell landscape.

The accumulation of extracellular matrix (ECM) proteins in the liver leads to liver fibrosis and end-stage liver cirrhosis. C-C motif chemokine receptor 2 (CCR2) is an attractive target for treating liver fibrosis. However, limited investigations have been conducted to explore the mechanism by which CCR2 inhibition reduces ECM accumulation and liver fibrosis, which is the focus of this study. Liver injury and liver fibrosis were induced by carbon tetrachloride (CCl4) in wild-type mice and Ccr2 knockout (Ccr2-/-) mice. CCR2 was upregulated in murine and human fibrotic livers. Pharmacological CCR2 inhibition with cenicriviroc (CVC) reduced ECM accumulation and liver fibrosis in prevention and treatment administration. In single-cell RNA sequencing (scRNA-seq), CVC was demonstrated to alleviate liver fibrosis by restoring the macrophage and neutrophil landscape. CVC administration and CCR2 deletion can also inhibit the hepatic accumulation of inflammatory FSCN1+ macrophages and HERC6+ neutrophils. Pathway analysis indicated that the STAT1, NFκB, and ERK signaling pathways might be involved in the antifibrotic effects of CVC. Consistently, Ccr2 knockout decreased phosphorylated STAT1, NFκB, and ERK in the liver. In vitro, CVC could transcriptionally suppress crucial profibrotic genes (Xaf1, Slfn4, Slfn8, Ifi213, and Il1ß) in macrophages by inactivating the STAT1/NFκB/ERK signaling pathways. In conclusion, this study depicts a novel mechanism by which CVC alleviates ECM accumulation in liver fibrosis by restoring the immune cell landscape. CVC can inhibit profibrotic gene transcription via inactivating the CCR2-STAT1/NFκB/ERK signaling pathways.

STING mediates hepatocyte pyroptosis in liver fibrosis by Epigenetically activating the NLRP3 inflammasome.

The activation of stimulator of interferon genes (STING) and NOD-like receptor protein 3 (NLRP3) inflammasome-mediated pyroptosis signaling pathways represent two distinct central mechanisms in liver disease. However, the interconnections between these two pathways and the epigenetic regulation of the STING-NLRP3 axis in hepatocyte pyroptosis during liver fibrosis remain unknown. STING and NLRP3 inflammasome signaling pathways are activated in fibrotic livers but are suppressed by Sting knockout. Sting knockout ameliorated hepatic pyroptosis, inflammation, and fibrosis. In vitro, STING induces pyroptosis in primary murine hepatocytes by activating the NLRP3 inflammasome. H3K4-specific histone methyltransferase WD repeat-containing protein 5 (WDR5) and DOT1-like histone H3K79 methyltransferase (DOT1L) are identified to regulate NLRP3 expression in STING-overexpressing AML12 hepatocytes. WDR5/DOT1L-mediated histone methylation enhances interferon regulatory transcription factor 3 (IRF3) binding to the Nlrp3 promoter and promotes STING-induced Nlrp3 transcription in hepatocytes. Moreover, hepatocyte-specific Nlrp3 deletion and downstream Gasdermin D (Gsdmd) knockout attenuate hepatic pyroptosis, inflammation, and fibrosis. RNA-sequencing and metabolomics analysis in murine livers and primary hepatocytes show that oxidative stress and metabolic reprogramming might participate in NLRP3-mediated hepatocyte pyroptosis and liver fibrosis. The STING-NLRP3-GSDMD axis inhibition suppresses hepatic ROS generation. In conclusion, this study describes a novel epigenetic mechanism by which the STING-WDR5/DOT1L/IRF3-NLRP3 signaling pathway enhances hepatocyte pyroptosis and hepatic inflammation in liver fibrosis.

Proangiogenic role of circRNA-007371 in liver fibrosis.

Circular RNAs (circRNAs) are crucially involved in cancers as competing endogenous RNA (ceRNA) or microRNA (miRNA) sponges. However, the function and mechanism of circRNAs in liver fibrosis remain unknown and are the focus of this study. Murine fibrotic models were induced by thioacetamide (TAA) or carbon tetrachloride (CCl4 ). Increased angiogenesis is accompanied by liver fibrosis in TAA- and CCl4 -induced murine fibrotic livers. circRNA microarray and argonaute 2 (AGO2)-RNA immunoprecipitation (RIP) sequencing (AGO2-RIP sequencing) were performed in murine livers to screen for functional circRNAs. Compared to control livers, 86 differentially expressed circRNAs were obtained in TAA-induced murine fibrotic livers using circRNA microarray. In addition, 551 circRNAs were explored by AGO2-RIP sequencing of murine fibrotic livers. The circRNA-007371 was then selected and verified for back-spliced junction, resistance to RNase R, and loop formation. In vitro, murine hemangioendothelioma endothelial (EOMA) cells were transfected with circRNA-007371 overexpressing plasmid or empty plasmid. circRNA-007371 overexpression promoted tube formation, migration, and cell proliferation of EOMA cells. RNA sequencing and miRNA sequencing were then performed to explore the mechanism of the proangiogenic effects of circRNA-007371. circRNA-007371 promotes liver fibrosis via miRNA sponges or ceRNA mechanisms. Stag1, the parent gene of circRNA-007371, may play a significant role in proangiogenic progression. In conclusion, circRNA-007371 enhances angiogenesis via a miRNA sponge mechanism in liver fibrosis. The antiangiogenic effect of circRNA-007371 inhibition may provide a new strategy for treating patients with liver cirrhosis.

Liver-Targeted Delivery of Small Interfering RNA of C-C Chemokine Receptor 2 with Tetrahedral Framework Nucleic Acid Attenuates Liver Cirrhosis.

Liver cirrhosis is the end stage of chronic liver diseases without approved clinical drugs. In this study, a new strategy that uses a C-C chemokine receptor 2 (CCR2) small interfering RNA silencing (siCcr2)-based therapy by loading multivalent siCcr2 with tetrahedron framework DNA nanostructure (tFNA) vehicle (tFNA-siCcr2) was established to attenuate liver fibrosis. tFNA-siCcr2 was successfully synthesized without changing the physiochemical properties of tFNA. Compared to the naked siCcr2 molecule, the tFNA-siCcr2 complex altered the accumulation from the kidney to the liver after the intraperitoneal injection. The tFNA-siCcr2 complex also prolonged hepatic retention and mainly colocalized within macrophages and endothelial cells. tFNA-siCcr2 efficiently silenced CCR2 and significantly ameliorated liver fibrosis in prevention and treatment interventions. Single-cell RNA sequencing followed by experimental validation suggested that tFNA-siCcr2 can restore the immune cell landscape and construct an antifibrotic niche by inhibiting profibrotic macrophage and neutrophil accumulation in the murine fibrotic liver. Molecularly, the tFNA-siCcr2 complex reduced inflammatory mediator production by inactivating the NF-κB signaling pathway. In conclusion, the tFNA-based liver-targeted tFNA-siCcr2 delivery complex efficiently ameliorated liver fibrosis by restoring the immune cell landscape and constructing an antifibrotic niche, which makes the tFNA-siCcr2 complex a potential therapeutic candidate for the clinical treatment of liver cirrhosis.

Portal vein thrombosis associates with high platelet-fibrin clot strength and platelet activation in decompensated cirrhosis: A retrospective study.

BACKGROUND AND AIMS: Alteration of platelet status associates with decompensation and death in cirrhosis, while its effect on portal vein thrombosis (PVT) remains unclear. We aimed to retrospectively investigate whether PVT associates with platelet-fibrin clot strength and platelet activation in decompensated cirrhosis. METHODS: Platelet-fibrin clot strength (G) was measured by thromboelastography (TEG). Platelet activation was reflected by plasma concentrations of soluble p-selectin (sPs) and a platelet aggregation test adjusted for platelet counts. RESULTS: Among 166 patients, 45 had PVT. The platelet count was significantly lower in PVT. While the G value was positively correlated with platelet count (ρ = 0.74, P < 0.01), increased G was associated with PVT after adjusting for platelet count in the logistic regression (P = 0.04). The normalized G value according to the linear relation with platelet count was calculated as follows: Gplatelet = [(G - 2622)/platelet count]. This coefficient had no correlation with platelet count and was an independent risk factor of PVT (OR = 1.03, CI95%: 1.01-1.05, P = 0.012). In two subanalyses, the collagen-induced platelet aggregation (n = 37, P = 0.029) and plasma concentration of sPs (n = 56, P = 0.001) adjusted for platelet count were significantly higher in PVT. CONCLUSION: This study showed a positive correlation of high platelet-fibrin clot strength detected via TEG and platelet activation with PVT in decompensated cirrhosis.

Inflammasomes and Pyroptosis of Liver Cells in Liver Fibrosis.

Inflammasomes are multiprotein complexes that can sense danger signals and activate caspase-1 to mediate pro-inflammatory cytokines release and pyroptotic cell death. There are two main canonical and non-canonical signaling pathways that trigger inflammasome activation. Inflammasomes are expressed and assembled in parenchymal and nonparenchymal cells in response to liver injury in the liver. Additionally, the hepatocytes, biliary epithelial cells (cholangiocytes), hepatic stellate cells (HSCs), hepatic macrophages, and liver sinusoidal endothelial cells (LSECs) contribute to liver fibrosis via different mechanisms. However, the underlying mechanism of the inflammasome and pyroptosis in these liver cells in liver fibrosis remains elusive. This review summarizes the activation and function of inflammasome complexes and then discusses the association between inflammasomes, pyroptosis, and liver fibrosis. Unlike other similar reviewers, we will focus on the effect of inflammasome activation and pyroptosis in the various liver cells during the development of liver fibrosis. We will also highlight the latest progress of pharmacological intervention in inflammasome-mediated liver fibrosis.

Role of Immune Cells in Biliary Repair.

The biliary system is comprised of cholangiocytes and plays an important role in maintaining liver function. Under normal conditions, cholangiocytes remain in the stationary phase and maintain a very low turnover rate. However, the robust biliary repair is initiated in disease conditions, and different repair mechanisms can be activated depending on the pathological changes. During biliary disease, immune cells including monocytes, lymphocytes, neutrophils, and mast cells are recruited to the liver. The cellular interactions between cholangiocytes and these recruited immune cells as well as hepatic resident immune cells, including Kupffer cells, determine disease outcomes. However, the role of immune cells in the initiation, regulation, and suspension of biliary repair remains elusive. The cellular processes of cholangiocyte proliferation, progenitor cell differentiation, and hepatocyte-cholangiocyte transdifferentiation during biliary diseases are reviewed to manifest the underlying mechanism of biliary repair. Furthermore, the potential role of immune cells in crucial biliary repair mechanisms is highlighted. The mechanisms of biliary repair in immune-mediated cholangiopathies, inherited cholangiopathies, obstructive cholangiopathies, and cholangiocarcinoma are also summarized. Additionally, novel techniques that could clarify the underlying mechanisms of biliary repair are displayed. Collectively, this review aims to deepen the understanding of the mechanisms of biliary repair and contributes potential novel therapeutic methods for treating biliary diseases.

Mechanotransduction-induced glycolysis epigenetically regulates a CXCL1-dominant angiocrine signaling program in liver sinusoidal endothelial cells in vitro and in vivo.

BACKGROUND & AIMS: Liver sinusoidal endothelial cells (LSECs) are ideally situated to sense stiffness and generate angiocrine programs that potentially regulate liver fibrosis and portal hypertension. We explored how specific focal adhesion (FA) proteins parlay LSEC mechanotransduction into stiffness-induced angiocrine signaling in vitro and in vivo. METHODS: Primary human and murine LSECs were placed on gels with incremental stiffness (0.2 kPa vs. 32 kPa). Cell response was studied by FA isolation, actin polymerization assay, RNA-sequencing and electron microscopy. Glycolysis was assessed using radioactive tracers. Epigenetic regulation of stiffness-induced genes was analyzed by chromatin-immunoprecipitation (ChIP) analysis of histone activation marks, ChIP sequencing and circularized chromosome conformation capture (4C). Mice with LSEC-selective deletion of glycolytic enzymes (Hk2fl/fl/Cdh5cre-ERT2) or treatment with the glycolysis inhibitor 3PO were studied in portal hypertension (partial ligation of the inferior vena cava, pIVCL) and early liver fibrosis (CCl4) models. RESULTS: Glycolytic enzymes, particularly phosphofructokinase 1 isoform P (PFKP), are enriched in isolated FAs from LSECs on gels with incremental stiffness. Stiffness resulted in PFKP recruitment to FAs, which paralleled an increase in glycolysis. Glycolysis was associated with expansion of actin dynamics and was attenuated by inhibition of integrin ß1. Inhibition of glycolysis attenuated a stiffness-induced CXCL1-dominant angiocrine program. Mechanistically, glycolysis promoted CXCL1 expression through nuclear pore changes and increases in NF-kB translocation. Biochemically, this CXCL1 expression was mediated through spatial re-organization of nuclear chromatin resulting in formation of super-enhancers, histone acetylation and NF-kB interaction with the CXCL1 promoter. Hk2fl/fl/Cdh5cre-ERT2 mice showed attenuated neutrophil infiltration and portal hypertension after pIVCL. 3PO treatment attenuated liver fibrosis in a CCl4 model. CONCLUSION: Glycolytic enzymes are involved in stiffness-induced angiocrine signaling in LSECs and represent druggable targets in early liver disease. LAY SUMMARY: Treatment options for liver fibrosis and portal hypertension still represent an unmet need. Herein, we uncovered a novel role for glycolytic enzymes in promoting stiffness-induced angiocrine signaling, which resulted in inflammation, fibrosis and portal hypertension. This work has revealed new targets that could be used in the prevention and treatment of liver fibrosis and portal hypertension.

Inhibition of Transketolase Improves the Prognosis of Colorectal Cancer.

Colorectal cancer (CRC) remains a heavy health burden worldwide. Transketolase (TKT) is a crucial enzyme in the non-oxidative phase of the Pentose Phosphate Pathway (PPP), and is up-regulated in multiple cancer types. However, the role of TKT in the prognosis of CRC remains unclear. We aimed to explore whether TKT expression is altered in CRC, how TKT is associated with the prognosis of CRC, and whether the regulation of TKT might have an impact on CRC. Differentially expressed genes (DEGs) were identified using bioinformatics analysis. TKT expression was examined in the human colon adenocarcinoma tissue microarray and xenografts. Cell viability, proliferation, migration, and apoptosis assays in vitro were applied to evaluate the protumoral effects of TKT on CRC. TKT was found to be a risk factor for the poor prognosis of CRC by bioinformatics analysis among the DEGs. TKT was significantly up-regulated in colon adenocarcinoma tissues compared with normal colon tissues in patients. Moreover, similar results were found in HCT116 and RKO human colon adenocarcinoma xenografts in nude mice. TKT expression was positively associated with advanced TNM stage, positive lymph nodes, and poor 5 or 10-year overall survival of CRC patients. In vitro, inhibition of TKT reduced cell viability, proliferation, and migration, and induced cell apoptosis. In addition, inhibition of TKT decreased the protein levels of NICD and Hes1. In conclusion, high TKT expression was associated with the poor prognosis of CRC patients. The protumoral effects of downregulating TKT may be realized by suppressing the Notch signaling pathway. TKT may be a new prognostic biomarker and therapeutic target for CRC.