In recent years， clinical studies on targeted therapy and immunotherapy for advanced hepatocellular carcinoma used alone or in combination have provided abundant evidence on efficacy and safety for the selection of first-line therapies. However， no consensus has been reached on the selection of second-line therapies in various clinical guidelines for hepatocellular carcinoma， which is caused by the fact that existing evidence is limited to the options after failure of sorafenib and that there is still a lack of high-level evidence for new first-line therapies such as second-line therapies after resistance to targeted therapy and immunotherapy for hepatocellular carcinoma. This article reviews the results of current clinical trials and summarizes the studies on second-line therapies for hepatocellular carcinoma after resistance to first-line targeted therapy and immunotherapy for hepatocellular carcinoma based on the different mechanisms of action of drugs， as well as the research advances in recent years. For hepatocellular carcinoma patients with resistance to first-line targeted therapy and immunotherapy， targeted combination therapy and dual-immune therapy are expected to improve treatment outcome and survival， and more prospective clinical studies are needed in the future to provide effective and safe treatment regimens for hepatocellular carcinoma patients with resistance to targeted therapy and immunotherapy.

AIM:To explore the effects of CD38 on lysosome reformation and cholesterol efflux in macro-phages.METHODS:Bone marrow-derived macrophages from low-density lipoprotein(LDL)receptor knockout(LDLr-/-)mice were cultured as cell model.Live cell imaging system was applied to evaluate the effect of nicotinic acid adenine di-nucleotide phosphate(NAADP)on lysosome number.ELISA was conducted to measure NAADP level in macrophages.After the cells were treated with nicotinic acid(NA),RT-qPCR was conducted to detect CD38 mRNA expression,and Western blot was conducted to observe CD38 protein expression and phosphorylated transcription factor EB(TFEB)level.Laser scanning confocal microscopy was applied to evaluate the influence of CD38/NAADP signaling on lysosome number and cholesterol egression.RESULTS:NAADP remarkably increased lysosome number(P<0.05),and this effect was significantly inhibited by NAADP antagonist NED-19,Ca2+ chelator BAPTA,and calcineurin inhibitor CsA(P<0.05).CD38 markedly enhanced NAADP synthesis in macrophages(P<0.05).NAADP synthetic substrate NA prominently ele-vated the expression of CD38 mRNA and protein(P<0.05).NA significantly decreased the phosphorylated TFEB level;this effect was also attenuated by NED-19,BAPTA and CsA(P<0.05).Disrupting CD38/NAADP signaling pathway markedly inhibited NA-induced enhancement of lysosome number,lysosomal free cholesterol and cytosol cholesterol ester efflux in macrophages(P<0.05).NA-induced enhancement of lysosome number,lysosomal free cholesterol and cytosol cholesterol ester efflux abolished in LDLr/CD38 DKO macrophages(P<0.05),whereas these effects induced by NA were recovered after CD38 gene rescue.CONCLUSION:CD38 triggers lysosome reformation via TFEB and consequently pro-motes the efflux of lysosomal free cholesterol and cytosol cholesterol ester.

Objective To explore the sufentanil by adjusting the adenosine monosodium phosphate activa-ted protein kinase(AMPK)/thioredoxin interacting proteins(TXNIP)/nucleotide-binding oligomerization do-main-like receptor protein 3(NLRP3)signaling pathways of the alveolar epithelial cells induced by lipopo-lysaccharide to the dead.Methods Human alveolar epithelial type Ⅱ cell(AECⅡ)were cultured to logarith-mic phase.Then they were divided into control group(normal glucose culture),lipopolysaccharide group(li-popolysaccharide induced injury by 50 μg/mL),low concentration sufentanil group(20 μmol/L),high concen-tration sufentanil group(40 μmol/L),high concentration sufentanil(40 μmol/L)+AMPK-IN-3 group(50μmol/L AMPK inhibitors).MTT assay was used to detect cell proliferation.Pyroptosis was detected by Ho-echst 33342 and propidium iodide(PI)double staining.The levels of interleukin(IL)-1β,IL-18 and tumor nec-rosis factor-α(TNF-α)in cell supernatant were detected by enzyme-linked immunosorbent assay.Quantitative real-time PCR was used to detect the mRNA expression of focal death related factors cysteinyl proteinase-1(Caspase-1),apoptosis-associated speck-like protein(ASC),and gasdermin D(GSDMD).The protein expres-sion of AMPK,phosphorylated adenosine monosodium phosphate activated protein kinase(p-AMPK),TXNIP and NLRP3 were detected by Western blot.Results Compared with the control group,the survival rate of AEC Ⅱ cells in the lipopolysaccharide group decreased,the number of PI+positive cells increased,the release of inflammatory factors IL-1β,IL-18 and TNF-α increased,the expression of Caspase-1,ASC and GSDMD mR-NA increased,the expression of p-AMPK/AMPK decreased,and the expression of TXNIP and NLRP3 in-creased(P＜0.05).Compared with the lipopolysaccharide group,the survival rate of AEC Ⅱ cells in the low concentration sufentanil group and the high concentration sufentanil group increased,PI+positive cells de-creased,the release of inflammatory factors IL-1β,IL-18 and TNF-α decreased,the expression of Caspase-1,ASC and GSDMD mRNA decreased,the expression of p-AMPK/AMPK increased,and the expression of TX-NIP and NLRP3 decreased(P＜0.05).Compared with the high concentration sufentanil group,the survival rate of AEC Ⅱ cells in the high concentration sufentanil+AMPK-IN-3 group decreased,the number of PI+positive cells increased,the release of inflammatory factors IL-1β,IL-18 and TNF-α increased,the expression of Caspase-1,ASC and GSDMD mRNA increased,the expression of p-AMPK/AMPK decreased,and the ex-pression of TXNIP and NLRP3 increased(P＜0.05).Conclusion Sufentanil may improve lipopolysaccharide induced alveolar epithelial cell pyroptosis by regulating AMPK/TXNIP/NLRP3 signaling pathway.

Objective To construct recombinant human butyrylcholinesterase(rhBChE)knock-in goat fetal fibroblast cell lines(GFFs)by using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)-mediated homology-directed repair mechanism for subsequent production of goat expressing rhBChE.Methods The efficient sgRNA sites targeting goat[3-casein(CSN2)gene were designed and screened,and the targeting efficiency of the sgRNA in goat mammary epithelial cells(GMECs)was confirmed by electro-transfection,flow sorting,and sequencing of PCR products.The red fluorescent reporter gene homology repair vector(P2A-mCherry)targeting the sgRNA was constructed,and then the integration and expression efficiency was detected by flow cytometry.The rhBChE homology repair vector(P2A-rhBChE)targeting the sgRNA of CSN2 gene was constructed in GFFs,the rhBChE positive cell clones were obtained via electro-transfection and flow sorting,and the rhBChE knock-in cell lines was identified by sequencing of PCR products.Results The sgRNA4 was identified as an efficient target of goat CSN2 gene,which could be also used for targeted integration of other genes.Three rhBChE knock-in cell lines were successfully constructed.Conclusion The rhBChE knock-in GFFs targeting goat CSN2 gene lays the foundation for the production of mammary bioreactors expressing rhBChE.

Objective: To investigate the risk factors for yersiniosis, so as to provide insights into prevention of yersiniosis. Methods: The patients with yersiniosis admitted to the clinics in the surveillance site of Chengbei Township of Jin'an District and Chengnan Township of Yu'an District in Lu'an City from 2013 to 2021 were included as the case group, and the healthy family members matched to cases were selected as the family control group, while normal residents with a 1︰2 match in the same village, gender, and age difference within 5 years were included in the community control group. Participants' demographics, hand-washing and eating habits, living environment hygiene, poultry and livestock feeding were collected using questionnaire surveys, and factors affecting yersiniosis were identified using a multivariable conditional logistic regression model. Results: There were 43 cases in the case group, with a median (interquartile range) age of 45 (34) years, 91 cases in the family control group, with a median (interquartile range) age of 36 (36) years and 86 cases in the community control group, with a median (interquartile range) age of 46 (34) years. Multivariable conditional logistic regression analysis showed that compared with the family control group, the habit of drinking unboiled water (OR=6.721, 95%CI: 1.765-25.588), and direct consumption of food stored in the refrigerator (OR=7.089, 95%CI: 1.873-26.829) were risk factors for yersiniosis in the case group; and compared with the community control group, not washing hands after contacting with poultry and livestock (OR=50.592, 95%CI: 2.758-927.997), habit of eating raw vegetables and fruits (OR=5.340, 95%CI: 1.022-27.887), direct consumption of food stored in the refrigerator (OR=19.973, 95%CI: 2.118-188.336), and unclean refrigerator (OR=12.692, 95%CI: 1.992-80.869) were risk factors for yersiniosis in the case group. Compared with the family and community control groups, not washing hands after contacting with poultry and livestock (OR=4.075, 95%CI: 1.427-11.637), habit of drinking unboiled water (OR=4.153, 95%CI: 1.331-12.957), habit of eating raw vegetables and fruits (OR=4.744, 95%CI: 1.609-13.993), and direct consumption of food stored in the refrigerator (OR=5.051, 95%CI: 1.773-14.395) were risk factors for yersiniosis in the control group. Conclusion Unhealthy habits such as eating raw vegetables and fruits, drinking unboiled water, direct consumption of food stored in the refrigerator, unclean refrigerator, and not washing hands after contacting poultry and livestock may increase the risk of yersiniosis.

In recent years, the risk of perioperative concurrent venous thromboembolism (venous thromboembolism, VTE) has been significantly increased in patients with pancreatic cancer. Early prediction of VTE and strengthening the quality evaluation and clinical management of pancreatic surgery are the key to promote the prevention and treatment of VTE in pancreatic cancer patients. In view of the particularity of pancreatic surgery and the risk of related complications, clinicians should not only focus on the prevention of VTE, but also take into account the occurrence of serious complications such as postoperative bleeding, and strive to balance the two. Based on the relevant guidelines and the clinical experience of the center, the author's team elaborated on the early prediction of VTE in patients with pancreatic cancer, the indication and timing of perioperative VTE prevention and treatment in pancreatic surgery, in order to provide beneficial reference for the clinicians of VTE prevention and treatment in such patients.

The step-up approach is the most important modality in the treatment of infected pancreatic necrosis (IPN) and has been recommended by several national and international guidelines. Screening patients with low success rates of percutaneous drainage for timely treatment using the step-up approach and selecting appropriate escalation approach based on IPN staging are expected to improve the overall cure rate of IPN. The open debridement in the step-up approach should be carried out under reasonable indications and timing. When the patient's overall condition is poor and the condition of disease is complex, it is not necessary to adhere to a fixed treatment mode and choose a leapfrogging treatment strategy in a timely manner after thorough evaluation.When following the step-up approach in the treatment of IPN, endoscopic and surgical interventions are advocated in parallel, and escalating and leapfrogging strategies are promoted to establish an integrated, disease-centric, multidisciplinary treatment platform, with the aim of improving clinical prognosis. The authors review relevant literature and combine with team's treatment experience to explore the escalating strategies of surgical intervention for IPN, with a view to further improving the overall cure rate of IPN patients.

Objective:To compare the safety and efficacy of the " step-up approach" versus the " step-jump approach" in treatment of infected pancreatic necrosis (IPN).Method:The clinical data of IPN patients who underwent step-up strategy or step-jump strategy treatment at the Department of Pancreatic and Biliary Surgery of the First Affiliated Hospital of Harbin Medical University from December 2018 to November 2022 were analyzed retrospectively. Propensity score matching (PSM) was done based on the nearest neighbor matching method (1: 1 ratio). After matching the baseline data (the caliper value was 0.01), a total of 62 patients with IPN were included, including 41 males and 21 females, aged (41.1±13.1) years old. Patients who were treated with the step-up strategy were included in the step-up group, while patients who were treated with the step-jump strategy were included in the step-jump group. There were 31 patients in each group after PSM, and the treatment effect of the two groups were compared.Results:Of the 62 patients with IPN, 43 received surgical intervention, and 19 were managed successfully using symptomatic anti-inflammatory treatment or percutaneous catheter drainage. The total hospitalization cost of patients in the step-jump group was significantly higher than that in the step-up group [122 000 (73 000, 179 000) yuan vs. 88 000 (46 000, 144 000) yuan, P=0.034]. The overall cure rate of IPN patients in the step-jump group was 93.5%(29/31). The 2 patients who died had type Ⅲ IPN. In the IPN patients in the step-up group were all cured, and the overall cure rate was 100%(31/31), with no death. There were no statistical differences between the two groups in the rates of death, postoperative complications, residual infection, debridement ≥2 times, and positive bacterial culture in blood or drainage fluid (all P>0.05). A total of 19.4% (12/62) patients had postoperative complications, including 4 patients with abdominal bleeding, 3 patients with new organ dysfunction, 2 patients with gastrointestinal bleeding, 2 patients with gastrointestinal fistula, and 1 patient with venous thrombosis in both lower limbs. Conclusion:Both the step-up treatment strategy and the step-jump treatment strategy were safe and effective for treatment of IPN patients.

Objective:To screen T-cell exhaustion-related signature genes as the prognostic marker for osteosarcoma and establish a prognostic model for osteosarcoma patients based on weighted gene co-expression network analysis(WGCNA)and Least absolute shrinkage and selection operator(LASSO)-COX regression analysis. Methods:GSE21257 dataset was downloaded from Gene Expression Omnibus(GEO)database for the establishment of the prognostic model for osteosarcoma.4 T-cell exhaustion-related gene sets were downloaded from The Molecular Signatures Database(MisgDB)and their enrichment scores in GSE21257 samples were calculated by single sample gene set enrichment analysis(ssGSEA).WGCNA was carried out to screen the gene module that is highly associated with T-cell exhaustion based on ssGSEA results followed by GO(Gene Ontology)and KEGG(Kyoto Encyclopedia of Genes and Genomes)analysis of the biological processes and signaling transduction pathways that those genes are involved in.The signature genes that are highly associated with the prognosis of osteosarcoma patients were obtained through LASSO-COX regression and a prognostic model was established based on these signature genes.Osteosarcoma-related expression profile data from the GSE21257 and TAEGET datasets on XENA were downloaded from the Gene Expression Omnibus.Clinical information for the training and validation sets was obtained.T-cell exhaustion-related genes were screened using a weighted correlation network analysis.Realtime fluorescence quantitative PCR,COX regression analysis,external dataset and nomogram were used to evaluate the reliability and accuracy of the prognostic model.A immunotherapy-related dataset was used to assess the efficacy of this prognostic model for the prediction of patients'responses to immunotherapy. Results:Analysis results based on the ssGSEA scores showed that T-cell exhaustion-related genes were related to the metastasis and age of osteosarcoma patients.Many T-cell exhaustion-related genes were found to be differentially expressed in metastatic and non-metastatic osteosarcoma patients.1 256 T-cell exhaustion-related genes were identified through WGCNA and these candidate markers were mainly distributed in structures like secretory granule membranes and endocytic vesicles and were involved in T-cell activation.COX regression analysis screened 68 significant prognostic markers out of the 1 256 genes,and 12 signature genes were further confirmed with LASSO-COX regression analysis.A prognostic model was established based on the 12 signature genes.Results of real-time fluorescence quantitative PCR showed a similar trend in the expression of most of the signature genes in different osteosarcoma cell lines.COX regression analysis of the internal and external datasets verified that the risk score calculated with the prognostic model was an independent prognostic factor for osteosarcoma patients,and high-risk score was associated with poor prognosis of the patients.Receiver operating characteristic(ROC)curves demonstrated excellent prognostic efficacy of the model.Nomogram analysis verified the prognostic model is highly accurate and reliable in predicting the prognosis of osteosarcoma patients.Analysis using the immunotherapy-related dataset indicated that this prognostic model could also be used to predict patients'responses to immunotherapy. Conclusion:The 12 signature gene(CD300LB,TRO,SNX3,VENTX,PPM1M,DOT1L,CDC37,NAT9,TRMT1,PPP1R3C,CHTF18 and NSUN5)-based prognostic model can effectively predict the prognosis and responses to immune check-point inhibitors for osteosarcoma patients,which may provide evidence for the prediction of prognosis as well as the selection of immunotherapy plans in clinical practice.

Objective:To study the serological and molecular biological characteristics and differences of occult hepatitis B infection (OBI) between blood donors and chronic hepatitis B (CHB) patients.Methods:Nineteen OBI samples from blood donors and Nineteen OBI samples from CHB patients were collected, and named as group A and group B, respectively. Chemiluminescence method was used for hepatitis B five items detection. Real-time PCR method was used for HBV DNA quantification, and S gene was amplified by nested PCR and sequenced by DNA sequencing.Results:The level of HBV DNA in group A was significantly lower than that in group B ( P<0.05), and there were no significant differences in the serological result between the two groups. The mutation rate in the " a" determinant of S gene in group B was significantly higher than that in group A ( P<0.05). Conclusions:Compared with CHB patients, OBI blood donors have lower levels of viral replication and less chance of S gene mutation, and the use of highly sensitive nucleic acid detection reagents and method during blood screening can maximize the safety of blood transfusion.