The success of replantation for amputated limbs is limited by the short safe preservation time of static cold storage and issues like ischemia-reperfusion injury. Normothermic machine perfusion (NMP) represents a revolutionary advancement with the potential to overcome these limitations, demonstrating considerable potential in extending preservation time, improving preservation quality, and enabling viability assessment. This study comprehensively reviews the technical principles and theoretical advantages of NMP. Through comparative analysis with static cold storage and hypothermic oxygenated machine perfusion, its unique value is elucidated. The review then focuses on summarizing experimental research findings from large animal models and human amputated limb models. Finally, it discusses future research directions, such as perfusate optimization, assessment system development, and exploration of NMP as a therapeutic platform, along with prospects for clinical application.

Background: Post-hepatectomy liver failure (PHLF) is the most serious complication after extended hepatectomy, with a high perioperative mortality rate. Matricellular protein tenascin-C (TNC) has been related to blood flow change and inflammation. We aimed to explore the hepatic changes of TNC and its relationship with PHLF. Materials and Methods: Rats were divided into seven groups: Sham operation, 90% partial hepatectomy (90PH) as a PHLF model, 85% (85PH), and 68% (68PH) hepatectomy without or with lipopolysaccharide (LPS) treatment. The distribution of TNC protein, the mRNA expression of TNC and other endothelial response genes, and the cellular TNC located in the rat livers were evaluated by immunostaining, quantitative real-time polymerase chain reaction, and RNAscope assay. Results: TNC mRNA in the liver tissue was significantly upregulated after hepatectomy and reached the highest level in the 90PH rats compared with those in the 85PH and 68PH rats. This upregulation was closely correlated with the changes of severe liver injury, inhibited liver regeneration, and upregulation of KLF2, ET-1, eNOS, and TM-1 in 90PH rat livers. We found a robust disturbance of hepatic TNC protein distribution in 90PH rat livers with an obvious reduction in hepatic sinusoids but an increase around large blood vessels, which were consistent with the severe liver injury of the 90PH rats. It was also found for the first time that treatment with the endotoxin LPS tremendously exacerbated the above changes of TNC. Conclusion: The distribution and expression of TNC mRNA changed dramatically and contributed to the occurrence of PHLF.

Immune checkpoint inhibitors (ICIs) combined with anti-angiogenic agents manifest improved survival in advanced hepatocellular carcinoma (HCC), but responses remain heterogeneous. Although high PIVKA-II expression correlates with advanced disease stage, early recurrence, shorter survival, and may predict resistance to anti-PD-1 plus lenvatinib therapy, the tumor microenvironment (TME) and resistance mechanisms in HCC with high PIVKA-II expression remain unclear. Clinical data from 156 resected HCC patients and 104 patients treated with anti-PD-1 plus lenvatinib are analyzed to correlate PIVKA-II expression with clinical features and outcomes. Single-cell RNA sequencing (scRNA-seq) is performed on tumors from 15 untreated and 7 treated patients. Mechanistic findings are validated in vitro and in vivo. High PIVKA-II expression is associated with advanced disease stage, increased microvascular invasion (MVI), early recurrence, and poor response to therapy. ScRNA-seq revealed an immunosuppressive TME enriched with regulatory T cells (Tregs), exhausted CD8⁺ T cells, and SPP1⁺ tumor-associated macrophages (TAMs). Mechanistically, tumors with high PIVKA-II expression upregulated NQO1, which stabilized p65 by inhibiting ubiquitination, activating the NF-κB/CXCL12 axis, and recruiting Tregs. This pathway mediated therapeutic resistance. Plerixafor, a CXCL12 inhibitor, disrupted this axis and significantly enhanced anti-tumor efficacy when combined with anti-PD-1 plus lenvatinib in vivo. PIVKA-II is a potentially effective biomarker for predicting resistance to anti-PD-1 plus lenvatinib therapy. Its high expression denotes an immunosuppressive TME. Targeting the NQO1/CXCL12/Tregs axis with Plerixafor may restore sensitivity and improve outcomes.

To investigate the mechanism of oral sodium taurocholate on liver regeneration after portal vein ligation. A rat model of 70% portal vein ligation (PVL) was established. The rats were randomly divided into a sodium taurocholate intervention group (PVL treatment, PVLT) and a normal diet control group (PVL control, PVLC). Histological damage and bile duct hyperplasia were assessed by H&E and CK19 staining. Liver regeneration capacity was evaluated by measuring the ratio of non-ligated liver lobe weight to total liver weight and the expression of Ki67 protein. Liver function was assessed by measuring serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBil), total bile acids (TBA), and hepatic TBA levels. Transcriptome analysis was performed using bulk RNA sequencing, combined with qPCR validation of gene expression related to bile acid metabolism. In the non-ligated lobes of both PVLT and PVLC groups, we found mild bile duct hyperplasia confined to the portal areas (p > 0.05), with no evident histological damage. Ki67 expression peaked on the second day after surgery in both groups, with more significant liver regeneration observed in the PVLT group. Sodium taurocholate administration led to bile acid accumulation and concomitant liver function injury. Transcriptome analysis revealed that differentially expressed genes were significantly enriched in the bile acid secretion pathway, and Gene Set Enrichment Analysis (GSEA) suggested activation of the Hippo signaling pathway. Sodium taurocholate promotes liver regeneration after portal vein ligation by regulating bile acid metabolism and the Hippo signaling pathway.

Background & Aims: Only a minority of patients could benefit from systemic therapy owing to the high heterogeneity of HCC. Therefore, a deeper understanding of the pathogenesis of HCC is essential for precision therapy. Genomic and proteomic studies of HCC have enhanced our understanding of HCC. However, phosphoproteomic characterization of HCC remains poorly understood. Approach & Results: We conducted an in-depth analysis of a clinical cohort of HCC using high-coverage phosphoproteomic. Effective therapeutic targets were validated using liver cancer cell lines and HCC patient-derived xenograft (PDX) mouse models that correspond to the phosphoproteomic subtypes of HCC. Phosphoproteomic analysis classified HCC into three subtypes, A, B, and C, with increasing malignancy and correlation with clinical features, including patient prognosis, tumor staging, serum alpha fetoprotein (AFP) levels, tumor thrombus, and tumor size. Phosphoproteomic subtyping deeply reflected the biological characteristics and clinical features of HCC patients. The profiles of HCC dysregulated kinase activities inferred from the different phosphoproteomic subtypes, consistently identifying increased kinase activity related to cell proliferation. Subtype-C HCC patients showed the most significant dysregulation, indicating a potential therapeutic target. The corresponding drug, bosutinib, demonstrated efficacy in inhibiting the growth of subtype C tumors in liver cancer cell lines and HCC patient-derived xenograft (PDX) mouse models representative of the phosphoproteomic HCC subtypes. Conclusions: Our study provides a comprehensive exploration of the phosphoproteomic landscape of HCC, establishing new subtypes that match clinical features and identifying potential therapeutic targets for the most malignant C subtype.

Globally, pancreatic cancer is recognized as one of the deadliest malignancies that lacks effective targeted therapies. This study aims to explore the role of cyclin I-like protein (CCNI2), a homolog of cyclin I (CCNI), in the progression of pancreatic cancer, thereby providing a theoretical basis for its treatment. Firstly, the expression of CCNI2 in pancreatic cancer tissues was determined through immunohistochemical staining. The biological role of CCNI2 in pancreatic cancer cells was further assessed using both in vitro and in vivo loss/gain-of-function assays. Our data revealed that CCNI2 expression was abnormally elevated in pancreatic cancer, and clinically, increased CCNI2 expression generally correlated with reduced overall survival. Functionally, CCNI2 contributed to the malignant progression of pancreatic cancer by promoting the proliferation and migration of tumor cells. Consistently, in vivo experiments verified that CCNI2 knockdown impaired the tumorigenic ability of pancreatic cancer cells. Moreover, the addition of phosphatidylinositol 3-kinase (PI3K) inhibitors could partially reverse the promoting effect of CCNI2 on the malignant phenotypes of pancreatic cancer cells. CCNI2 promoted pancreatic cancer through PI3K/protein kinase B (AKT) signaling pathway, indicating its potential as a prognostic marker and therapeutic target for pancreatic cancer.

This study aims to investigate the interplay between tumor-associated endothelial cells (TECs) and immune cells within the tumor microenvironment (TME) and its impact on tumor prognosis. We conducted single-cell RNA sequencing (scRNA-seq) of tumor, normal, and lymph node tissues obtained from intrahepatic cholangiocarcinoma (ICC) patients to reveal the role of TECs in tumor angiogenesis and their significant heterogeneity. Meanwhile, we identified genes highly expressed in TECs and constructed TEC signatures (TEC.Sig). Next, we calculated TEC scores of samples based on TEC.Sig. Patients with higher TEC scores exhibited a higher frequency of KRAS mutations, which was associated with increased infiltration of neutrophils and immature dendritic cells (iDCs), and decreased numbers of natural killer (NK), CD4 + T, and CD8 + T effector memory (Tem) cells, indicating an inflammation-dominated immunosuppressive phenotype. In contrast, BAP1 mutations and CXCL12 overexpression showed a contrasting trend. Spatial transcriptomics analysis and histological experiments further confirmed that TECs interacted with various tumor-killing immune cells through the CXCL12/CXCR4 axis. Multiple tumor immunotherapy datasets confirmed that the TEC.Sig could predict patient responses to immunotherapy. The TEC score is a promising and reliable biomarker for predicting genetic mutations and prognosis in ICC patients. Enhancing the regulation of the CXCL12/CXCR4 signaling pathway may represent a potential novel therapeutic target for ICC treatment.

Inflammation plays a critical role in tumor development. Inflammatory cell infiltration and inflammatory mediator synthesis cause changes in the tumor microenvironment (TME) in several cancers, especially in intrahepatic cholangiocellular carcinoma (ICC). However, methods to ascertain the inflammatory state of patients using reliable biomarkers are still being explored. We retrieved the RNA sequencing and somatic mutation analyses results and the clinical characteristics of 244 patients with ICC from published studies. We performed consensus clustering to identify the molecular subtypes associated with inflammation. We compared the prognostic patterns, clinical characteristics, somatic mutation profiles, and immune cell infiltration patterns across inflammatory subtypes. We performed quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) to confirm gene expression. We performed logistic regression analyses to construct a nomogram predicting the inflammatory status of patients with ICC. Our results confirmed that ICC can be categorized into an inflammation-high subtype (IHS) and an inflammation-low subtype (ILS). Patients from each group had distinct prognosis, clinical characteristics, and TME composition. Patients with ICC in the IHS group showed poorer prognosis owing to the immunosuppressive microenvironment and high frequency of KRAS and TP53 mutations. Cancer-associated fibroblast (CAF)-derived COLEC11 reduced myeloid inflammatory cell infiltration and attenuated inflammatory responses. The results of qRT-PCR and IHC experiments confirmed that COLEC11 expression levels were significantly reduced in tumor tissues compared to those in paracancerous tissues. Patients with ICC in the IHS group were more likely to respond to treatment with immune checkpoint inhibitors (ICIs) owing to their higher tumor mutational burden (TMB) scores, tumor neoantigen burden (TNB) scores, neoantigen counts, and immune checkpoint expression levels. Finally, we developed a nomogram to effectively predict the inflammatory status of patients with ICC based on their clinical characteristics and inflammatory gene expression levels. We evaluated the calibration, discrimination potential, and clinical utility of the nomogram. The inflammatory response in IHS is primarily induced by myeloid cells. COLEC11 can reduce the infiltration level of this group of cells, and myeloid inflammatory cells may be a novel target for ICC treatment. We developed a novel nomogram that could effectively predict the inflammatory state of patients with ICC, which will be useful for guiding individualized treatment plans.

Combination therapy of anti-programmed cell death protein-1 (PD-1) antibodies and tyrosine kinase inhibitors (TKIs) has significantly improved the prognosis for hepatocellular carcinoma (HCC), but many patients still have unsatisfactory outcomes. CD8 T cells are known to exert a pivotal function in the immune response against tumors. Nevertheless, most CD8 T cells in HCC tissues are in a state of exhaustion, losing the cytotoxic activity against malignant cells. Cytokines, mainly secreted by immune cells, play an important role in the occurrence and development of tumors. Here, we demonstrated the changes in exhausted CD8T cells during combination therapy by single-cell RNA sequencing (scRNA-seq) analysis on tumor samples before and after treatment. Combination therapy exerted a substantial impact on the exhausted CD8T cells, particularly in terms of cytokine expression. CCL5 was the most abundantly expressed cytokine in CD8T cells and exhausted CD8T cells, and its expression increased further after treatment. Subsequently, we discovered the CCL5/CCR5/CYP1A1 pathway through RNA sequencing (RNA-seq) on CCL5-stimulated Huh7 cells and verified through a series of experiments that this pathway can mediate the resistance of liver cancer cells to lenvatinib. Tissue experiments showed that after combination therapy, the CCL5/CCR5/CYP1A1 pathway was activated, which can benefit the residual tumor cells to survive treatment. Tumor-bearing mouse experiments demonstrated that bergamottin (BGM), a competitive inhibitor of CYP1A1, can enhance the efficacy of both lenvatinib and combination therapy. Our research revealed one mechanism by which hepatoma cells can survive the combination therapy, providing a theoretical basis for the refined treatment of HCC.

Liver fibrosis is characterized by excessive synthesis and deposition of extracellular matrix (ECM) in liver tissues. However, it still has been lacking of early detection and diagnosis methods. The collagen hybridizing peptide (CHP) is a novel synthetic peptide that enables detection of collagen damage and tissue remodeling. Here, we showed that obvious CHP-positive staining could be detected in the liver while given CCl4 for only 3 days, which was significantly enhanced while given CCl4 for 7 days. However, H & E staining showed no significant changes in fibrous tissue, and sirius red-positive staining could only be observed while given CCl4 for 14 days. Moreover, CHP-positive staining enhanced initially at portal area which further extended into the hepatic lobule, which was increased more significantly than sirius red-positive staining in the model of 10 and 14 days. Further proteomic analysis of CHP-positive staining revealed that pathways associated with ECM remodeling were significantly increased, while retinol metabolism was downregulated. Meanwhile, proteins enriched in cellular gene transcription and signal transduction involved in fibrogenesis were also upregulated, suggesting that fibrosis occurred in CHP-positive staining. Our study provided evidence that CHP could detect the collagen damage in liver, which might be an efficient indicator for the diagnosis of liver fibrosis at a very early stage.

Objectives An acute injury is often accompanied by tissue regeneration. In this process, epithelial cells show a tendency of cell proliferation under the induction of injury stress, inflammatory factors, and other factors, accompanied by a temporary decline of cellular function. Regulating this regenerative process and avoiding chronic injury is a concern of regenerative medicine. The severe coronavirus disease 2019 (COVID-19) has posed a significant threat to people's health caused by the coronavirus. Acute liver failure (ALF) is a clinical syndrome resulting from rapid liver dysfunction with a fatal outcome. We hope to analyze the two diseases together to find a way for acute failure treatment. Methods COVID-19 dataset (GSE180226) and ALF dataset (GSE38941) were downloaded from the Gene Expression Omnibus (GEO) database, and the "Deseq2" package and "limma" package were used to identify differentially expressed genes (DEGs). Common DEGs were used for hub genes exploration, Protein-Protein Interaction (PPI) network construction, Gene Ontology (GO) functional enrichment, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. The real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) was used to verify the role of hub genes in liver regeneration during in vitro expansion of liver cells and a CCl4-induced ALF mice model. Results: The common gene analysis of the COVID-19 and ALF databases revealed 15 hub genes from 418 common DEGs. These hub genes, including CDC20, were related to cell proliferation and mitosis regulation, reflecting the consistent tissue regeneration change after the injury. Furthermore, hub genes were verified in vitro expansion of liver cells and in vivo ALF model. On this basis, the potential therapeutic small molecule of ALF was found by targeting the hub gene CDC20. Conclusion We have identified hub genes for epithelial cell regeneration under acute injury conditions and explored a new small molecule Apcin for liver function maintenance and ALF treatment. These findings may provide new approaches and ideas for treating COVID-19 patients with ALF.

BACKGROUND & AIMS:Liver fibrosis is an intrinsic wound-healing response to chronic injury and the major cause of liver-related morbidity and mortality worldwide. However, no effective diagnostic or therapeutic strategies are available, owing to its poorly characterized molecular etiology. We aimed to elucidate the mechanisms underlying liver fibrogenesis. METHODS:We performed a quantitative proteomic analysis of clinical fibrotic liver samples to identify dysregulated proteins. Further analyses were performed on the sera of 164 patients with liver fibrosis. Two fibrosis mouse models and several biochemical experiments were used to elucidate liver fibrogenesis. RESULTS:We identified cathepsin S (CTSS) up-regulation as a central node for extracellular matrix remodeling in the human fibrotic liver by proteomic screening. Increased serum CTSS levels efficiently predicted liver fibrosis, even at an early stage. Secreted CTSS cleaved collagen 18A1 at its C-terminus, releasing endostatin peptide, which directly bound to and activated hepatic stellate cells via integrin α5β1 signaling, whereas genetic ablation of Ctss remarkably suppressed liver fibrogenesis via endostatin reduction in vivo. Further studies identified macrophages as the main source of hepatic CTSS, and splenectomy effectively attenuated macrophage infiltration and CTSS expression in the fibrotic liver. Pharmacologic inhibition of CTSS ameliorated liver fibrosis progression in the mouse models. CONCLUSIONS:CTSS functions as a novel profibrotic factor by remodeling extracellular matrix proteins and may represent a promising target for the diagnosis and treatment of liver fibrosis.

ObjectivesThe exhausted CD8+T (Tex) cells are a unique cell population of activated T cells that emerges in response to persistent viral infection or tumor antigens. Tex cells showed the characteristics of aging cells, including weakened self-renewal ability, effector function inhibition, sustained high expression of inhibitory receptors including PD-1, TIGIT, TIM-3, and LAG-3, and always accompanied by metabolic and epigenetic reprogramming. Tex cells are getting more and more attention in researching immune-related diseases and tumor immunotherapy. However, studies on Tex-related models for tumor prognosis are still lacking. We hope to establish a risk model based on Tex-related genes for HCC prognosis.MethodsTex-related GEO datasets from different pathologic factors (chronic HBV, chronic HCV, and telomere shortening) were analyzed respectively to acquire differentially expressed genes (DEGs) by the ‘limma’ package of R. Genes with at least one intersection were incorporated into Tex-related gene set. GO, KEGG, and GSEA enrichment analyses were produced. Hub genes and the PPI network were established and visualized by the STRING website and Cytoscape software. Transcription factors and targeting small molecules were predicted by the TRUST and CLUE websites. The Tex-related HCC prognostic model was built by Cox regression and verified based on different datasets. Tumor immune dysfunction and exclusion (TIDE) and SubMap algorithms tested immunotherapy sensitivity. Finally, qRT-PCR and Flow Cytometry was used to confirm the bioinformatic results.ResultsHub genes such as AKT1, CDC6, TNF and their upstream transcription factor ILF3, Regulatory factor X-associated protein, STAT3, JUN, and RELA/NFKB1 were identified as potential motivators for Tex. Tex-related genes SLC16A11, CACYBP, HSF2, and ATG10 built the HCC prognostic model and helped with Immunotherapy sensitivity prediction.ConclusionOur study demonstrated that Tex-related genes might provide accurate prediction for HCC patients in clinical decision-making, prognostic assessment, and immunotherapy. In addition, targeting the hub genes or transcription factors may help to reverse T cell function and enhance the effect of tumor immunotherapy.

BACKGROUND AIMS:Sepsis is a potentially life-threatening disease that results from a severe systemic inflammatory response due to infection. Mesenchymal stromal cell-derived small extracellular vesicles (MSC sEVs) are able to transfer bioactive molecules and have been demonstrated to play an important role in the pathophysiological process of sepsis. Herein the authors aimed to investigate the potential role and downstream molecular mechanism of MSC sEVs in sepsis.METHODS:MSC sEVs were acquired by ultracentrifugation and then injected into a cecal ligation and puncture mouse model. The efficacy of MSC sEVs in both in vitro and in vivo models of sepsis was evaluated.RESULTS:MSC sEV therapy improved survival, reduced sepsis-induced inflammation, attenuated pulmonary capillary permeability and improved liver and kidney function in septic mice. In addition, the authors found that microRNA-21a-5p (miR-21a-5p) was highly enriched in MSC sEVs, could be transferred to recipient cells, inhibited inflammation and increased survival in septic mice. Furthermore, the authors demonstrated that MSC sEV miR-21a-5p suppressed inflammation by targeting toll-like receptor 4 and programmed cell death 4. The therapeutic efficacy of MSC sEVs was partially abrogated by transfection with miR-21a-5p inhibitors.CONCLUSIONS:Collectively, the authors' data suggest that miR-21a-5p-bearing MSC sEVs may be a prospective and effective sepsis therapeutic strategy.

Bioadhesives are revolutionizing wound closure procedures. However, traditional bioadhesives lack functions to promote wound healing with a higher wound dehiscence rate than those treated with sutures. As a result, creating multifunctional bioadhesives to improve wound healing and facilitate wound care is extremely desirable in clinical settings. This study develops a series of multifunctional bioadhesives using succinic anhydride‐modified hyaluronic acid (HASA) and plant‐derived tannic acid (TA) with centrifuging and freeze‐drying. By varying the ratio of HASA to TA, HASATA 1:2, 1:7, and 1:14 are fabricated with tunable adhesive strength from 39.6 ± 8.2 to 63.0 ± 12.0 kPa. The bioadhesives exhibit self‐healing adhesive strength due to the reversible cohesion network developed by HASA and TA, enabling 25 to 50 times repeated wound closure. The animal studies reveal that the developed bioadhesives accelerate wound healing in the first week with a higher collagen content, more M2 macrophage upregulation and less oxidative stress. Moreover, the plant‐derived TA endows the bioadhesives with antioxidant properties against four kinds of free radicals, and antibacterial activity against both gram positive and negative bacteria. Altogether, these bioadhesives may open new avenues for noninvasive wound closure in the clinical setting.

OBJECTIVE:Compared to portal vein ligation (PVL), simultaneous bile duct and portal vein ligation (BPL) can significantly enhance hypertrophy of the intact liver. This study aimed to investigate whether BPL could improve survival after extended hepatectomy independently of an increased remnant liver.METHODS:We adopted rat models of 90% BPL or 90% PVL. To investigate the role of bile acids (BAs) the BA pools in the PVL and BPL groups were altered by the diet. Staged resection preserving 10% of the estimated liver weight was performed 3 days after BPL; PVL; or sham operation. Histology, canalicular network (CN) continuity; and hepatocyte polarity were evaluated.RESULTS:At 3 days after BPL; PVL; or sham operation when the volumetric difference of the intended liver remained insignificant, the survival rates after extended hepatectomy were 86.7%, 47%, and 23.3%, respectively (P<0.01). BPL induced faster restoration of canalicular integrity along with an intensive but transient BA overload. Staged hepatectomy after BPL shortened the duration of the bile CN disturbance and limited BA retention. Decreasing the BA pools in the rats that underwent BPL could compromise these effects, whereas increasing the BA pools of rats that underwent PVL could induce similar effects. The changes in CN restoration were associated with activation of LKB1.CONCLUSION:In addition to increasing the future remnant liver, BPL shortened the duration of the spatial disturbance of the CN and could significantly improve the tolerance of the hypertrophied liver to staged resection. BPL may be a safe and efficient future option for patients with an insufficient remnant liver.

Background Immunotherapy has been the first-line treatment option in advanced Hepatocellular Carcinoma(HCC); but now, there are no established molecular markers that can predict immunotherapy response. Estrogen has a crucial role in the development of a variety of liver illnesses, including liver fibrosis, Nonalcoholic fatty liver disease (NAFLD), and HCC. Nonetheless, the significance of estrogen-related genes in HCC immunotherapy and the underlying molecular mechanisms are not yet fully understood. Method In this study, we constructed a novel estrogen-related gene prognostic signature (ERGPS) by analyzing bulk RNA sequencing data from 365 HCC patients. Based on the median risk score, we divided 365 HCC patients into low- and high-risk groups. Tumor mutation burden (TMB), Microsatellite instability (MSI), T cell receptor (TCR) richness, B cell receptor (BCR) richness, single-nucleotide variants (SNV) Neoantigens, Cancer Testicular Antigens (CTA) scores, and Tumour Immune Dysfunction and Exclusion (TIDE) scores were used to evaluate the magnitude of immunotherapy response. Multiple external datasets validate the validity and robustness of the prognostic signature. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to validate estrogen-related gene overexpression in HCC tissue samples. Results ERGPS is an independent risk factor affecting the prognosis of HCC patients and is superior to other clinical variables in predicting patient survival and immunotherapy response. Multiple independent external datasets confirmed the superior predictive efficacy of the prognostic signature. The prognostic signature was positively correlated with TMB score, MSI score, TCR richness, BCR richness, SNV Neoantigens score, CTA score, expression levels of immune checkpoint-related genes, and TIDE score. Patients with HCC in the high-risk group identified by the prognostic signature were likely to be more responsive to immunotherapy and more suitable for immunotherapy. qRT-PCR confirmed that estrogen-related genes of the construct signature were highly expressed in HCC tumor tissues. Conclusion Estrogen-related genes are overexpressed in HCC tissues. Our novel prognostic signature can accurately predict not only the prognosis but also the immunotherapy response of HCC patients. In the future, prognostic signatures will be a useful tool for clinicians to screen patients with HCC who are suitable for immunotherapy.

Due to the lack of a suitable model, research on biliary biology is far behind that on other organs. A mouse model of common bile duct (CBD) dilation (BDD) was first established and compared with CBD ligation mice (BDL). Then, in a transplantation experiment, the dilated CBD of recipient BDD mice was injured by making an elliptical incision and repaired by transplanting a bile duct patch from donor BDD mice. Biochemical and histological changes were analyzed and cell proliferation of the bile duct grafts was determined. Slightly dilated and unblocked CBD with a diameter of 2.89±0.76 mm was obtained in BDD mice, while the CBD diameter was 0.51±0.08 mm in the Sham group and 4.71±0.64 mm in the BDL group on day 14 after surgery. The liver damage was very mild in BDD mice compared with BDL mice, proving that the BDD model could be further used for bile duct transplantation. By cross transplanting the bile duct patch from enhanced green fluorescence protein and wild-type BDD mice, it was found that the CBD injury was well repaired and the cells of the bile duct patch were completely replaced by recipient-derived cells at 12 week after the repair operation. α Smooth muscle actin, Ki67 and cytokeratin 19 immunofluorescence staining showed that the proliferation of bile duct epithelial cells and abundant active fibroblasts were found within the bile duct patch during the regeneration process. Therefore, a reliable new mouse model of bile duct injury and repair was successfully established and can be used in the study of biliary repair mechanisms and tissue engineering of biliary ducts.

Background Hepatocellular carcinoma (HCC) is characterized by a high recurrence rate and poor prognosis. In recent years, the therapeutic regimen of programmed cell death protein 1 (PD-1) antibody combined with multi-targeted tyrosine kinase inhibitors (mTKIs) has achieved better results in the clinical application of hepatocellular carcinoma. Whole-exome sequencing can reflect the mutational characteristics of patients' exons and guide the clinical selection of molecular targeting drugs more accurately, which is in line with the concept of precision medicine. Methods We performed exome sequencing on 63 patients with HCC treated with radical surgery at our hospital and collected their clinical indexes and postoperative follow-up data. Using machine learning, a prediction model for recurrence within 1 year was constructed and the model was presented in a nomogram. Patients treated with PD-1 antibodies in combination with mTKIs after relapse were grouped by prognosis, and the valuable mutated genes were screened according to whole-exome sequencing data. The tumor tissue immune cells were analyzed using the UCSC Xena database. The expressions of target proteins were verified by Polymerase Chain Reaction (PCR) and Immunohistochemistry (IHC), respectively, on commercial HCC cell lines and pathological specimens of hepatocellular carcinoma collected clinically. Results The proportion of patients who relapsed within a year was 41% and the prognosis of those patients was poor. The characteristic exon mutation profile with a high frequency of variants in multiple mucin genes was present in Chinese HCC patients. Multiple nidi and 30 exon variants were brought into the prediction model with an area under the curve (AUC) = 0.94. MUC6 gene mutation was obvious in patients with an early recurrence, and MUC3A and MUC4 gene mutations were evident in patients with poorer responses to PD-1 antibodies combined with mTKIs. Those three mucins were negatively correlated with immune infiltrating cells. Conclusions We depicted the exon characteristics of hepatocellular carcinoma in the Chinese population and established a predictive model for recurrence within 1 year after radical surgical treatment. Moreover, we found that mucins were worthy targets of hepatocellular carcinoma.

Objective:To determine the risk factors of drainage time longer than 1 day in patients with selective abdominal drainage after laparoscopic cholecystectomy.Methods:The clinical data related to patients with selective abdominal drainage undergoing laparoscopic cholecystectomy from November 2009 to November 2019 at Chinese PLA General Hospital were retrospectively analyzed. Of 233 patients enrolled into this study, there were 147 males and 86 females, with a median aged 59.0 (47.5, 65.5) years old. The patients were divided into drainage time 1 day group of 65 patients and longer than 1 day group of 168 patients according to postoperative drainage time. The baseline data and perioperative data were collected, the risk factors correlated with drainage time longer than 1 day were analyzed.Results:The drainage time was 1 in the 1 day group and 2~8 in another group. Among the 233 patients, there was one with biliary leakage and 14 patients had abdominal bleeding, all of them healed after 2~3 days. All of the 233 patients were recovered when discharged. Independent risk factors related to drainage time longer than 1 day include BMI≥28 kg/m 2 ( OR=3.443, 95% CI: 1.411-8.405, P=0.007), operation time ≥65 min ( OR=2.570, 95% CI: 1.310-5.045, P=0.006), thickness of gallbladder wall ≥0.5 cm ( OR=12.720, 95% CI: 1.350-5.478, P=0.005), postoperative stomachache ( OR=13.537, 95% CI: 1.685-108.748, P=0.014) and postoperative fever ( OR=8.156, 95% CI: 1.035-64.249, P=0.046). Conclusion:For patients undergoing selective abdominal drainage after laparoscopic cholecystectomy with BMI ≥28 kg/m 2, operation time ≥65 min, gallbladder wall thickness ≥0.5 cm, postoperative abdominal pain and fever, clinicians should appropriately prolong the drainage time to ensure medical safety.