OBJECTIVE To provide reference for rational clinical use of immune checkpoint inhibitor （ICI）. METHODS Electronic medical record information of patients who received ICI treatment from January 1st 2020 to December 31st 2023 at a certain hospital was collected. Patients were divided into thyroid immune-related adverse event （irAE） group （subdivided into clinical hypothyroidism， clinical hyperthyroidism， subclinical hypothyroidism， and subclinical hyperthyroidism subgroups） and non- thyroid irAE group based on whether they experienced immune-induced thyroid irAE. Univariate and multivariate Logistic regression analyses were employed to analyze the influencing factors of ICI-related thyroid adverse events. RESULTS A total of 382 patients who received ICI treatment were included， with 137 cases in the thyroid irAE group （accounting for 35.9%） and 245 cases in the non-thyroid irAE group （accounting for 64.1%）. Multivariate Logistic regression analysis， following univariate screening， revealed that ICI combined with radiotherapy was positively associated with the occurrence of thyroid irAE [odds ratio （OR）＝2.157， 95% confidence interval （CI） （1.144， 4.066）， P＜0.05]， while lung squamous cell carcinoma was negatively associated with the occurrence of thyroid irAE [OR＝0.600， 95%CI （0.369， 0.975）， P＜0.05]. Among various thyroid irAE， nasopharyngeal malignancy was positively associated with the occurrence of immune-related clinical hyperthyroidism [OR＝4.678， 95%CI （1.149， 19.042）， P＜0.05]； ICI combined with radiotherapy [OR＝2.622， 95%CI （1.227， 5.603）， P＜0.05] and lung adenocarcinoma [OR＝2.013， 95%CI （1.078， 3.759）， P＜0.05] were positively associated with the occurrence of immune-related subclinical hyperthyroidism. Age was negatively associated with the occurrence of immune-related clinical hypothyroidism [OR＝0.944， 95%CI （0.896， 0.995）， P＜0.05]； age [OR＝0.963， 95%CI （0.932， 0.994）， P＜0.05] and ICI combined with chemotherapy [OR＝0.332， 95%CI （0.137， 0.802）， P＜0.05] were negatively associated with the occurrence of immune-related subclinical hypothyroidism. CONCLUSIONS Among patients receiving ICI treatment， younger patients are more prone to thyroid irAE. Patients receiving ICI combined with chemotherapy are less likely to experience subclinical hypothyroidism， while ICI combined with radiotherapy significantly increases the risk of thyroid adverse events.

Triple-negative breast cancer (TNBC) is aggressive, with high recurrence rates and poor prognosis. Paclitaxel (PTX) remains a key chemotherapeutic agent for TNBC, but its efficacy diminishes due to the emergence of drug resistance, largely driven by cancer stem-like cells (CSCs), vasculogenic mimicry (VM) formation and tumor immunosuppressive microenvironment (TIME). Pyruvate kinase M2 (PKM2) is highly expressed in TNBC, and is a potential target for TNBC treatment. In this study, we developed a biomimetic codelivery system using albumin nanoparticles (termed S/P NP) to co-encapsulate PTX and shikonin (SHK), a natural inhibitor of PKM2. By inhibiting PKM2, SHK suppressed β-Catenin signaling, thereby reversing CSC stemness and preventing VM formation. The S/P NP system exhibited tumor-targeting delivery effect and significantly inhibited TNBC growth and lung metastasis. Mechanistically, the treatment reversed epithelial-mesenchymal transition (EMT) and stem-like properties of TNBC cells, suppressed VM formation, and remodeled the TIME. It reduced immunosuppressive cells (M2 macrophages, MDSCs) while promoting anti-tumor immunity (M1 macrophages, dendritic cells, cytotoxic T cells, and memory T cells). This dual-action strategy holds promise for improving TNBC therapy by targeting CSCs, VM, and the immune microenvironment, and for overcoming PTX resistance and reducing metastasis.

The global prevalence of obesity continues to rise, accompanied by various metabolic complications. While metabolic surgery has significant efficacy, there are still problems, such as suboptimal clinical outcomes and recurrent weight gain. The superior metabolic regulatory of glucagon-like peptide-1 (GLP-1) receptor agonists has made them a new option for bariatric treatment. This article systematically explored the clinical application framework of combining metabolic surgery with GLP-1 receptor agonists, including target populations (e.g., patients with postoperative weight regain, unremitted diabetes, or comorbid complications), intervention timing, drug selection strategies, and multidisciplinary collaboration pathways. Although combination therapy holds broad prospects, it still faces challenges, such as optimal treatment strategies, long-term safety, and cost-effectiveness. We should emphasize multidisciplinary collaboration and individualized plans to optimize the long-term effective management of obesity in the future.

Objective:To discuss the effect of exosomes(Exos)loaded with microRNA-520a-5p(miR-520a-5p)on the pregnancy outcomes in fetal mice with intrauterine growth restriction(FGR),and to clarify its mechanism.Methods:The mouse placental mesenchymal stem cells(MSCs)were cultured in vitro and transfected with miR-520a-5p adenovirus vector(Ad-miR-520a-5p)to obtain the Exos with high miR-520a-5p load(miR-520a-5p-MSCs-Exos),which were then identified.The C57BL/6 mice were mated in cages at a female∶male ratio of 2∶1 to achieve successful pregnancy.Forty pregnant mice were divided into control group,FGR group,NC-MSCs-Exos group,and miR-520a-5p-MSCs-Exos group,with 10 mice in each group.Except for control group,the mice in other groups were exposed to excessive dexamethasone(DEX)during pregnancy to induce FGR models in the pregnant mice.The body weights of the fetal mice at birth and at 1,2,3,and 4 weeks after birth were detected;real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of miR-520a-5p,DNA methyltransferase 3b(DNMT3b),and vascular endothelial growth factor(VEGF)mRNA in placenta tissue of the mice in various groups;Western blotting method was used to detect the expression levels of DNMT3b and VEGF proteins in placenta tissue of the mice in various groups;methylation-specific PCR(MSP)was used to analyze the methylation rates of VEGF promoter in placenta tissue of the mice in various groups;dual-luciferase reporter gene assay was used to verify the targeting relationship between miR-520a-5p and DNMT3b.Results:The results of transmission electron microscope(TEM)and nanoparticle tracking analysis(NTA)showed that the Exos were spherical with particle size concentrated near 100 nm;the Western blotting method results showed that the surface biomarkers CD63 and CD81 of Exos were positively expressed.The RT-qPCR results showed that compared with NC-MSCs-Exos and Ad-NC-MSCs-Exos,the expression level of miR-520a-5p in Ad-miR-520a-5p-MSCs-Exos was increased(P＜0.001).The differences in birth body weight and the body weights at 1,2,and 3 weeks after birth of the fetal mice among four groups were statistically significant(F=36.084,F=19.851,F=77.755,F=103.223;P＜0.001).Compared with control group,the birth body weight and the body weights at 1,2,and 3 weeks after birth of the fetal mice in FGR group were decreased(P＜0.05);compared with FGR group and NC-MSCs-Exos group,the birth body weight and the body weights at 1,2,and 3 weeks after birth of the fetal mice in miR-520a-5p-MSCs-Exos group were increased(P＜0.05).The One-way ANOVA results showed that the differences in the expression levels of miR-520a-5p,DNMT3b,and VEGF mRNA in placenta tissue of the mice among four groups were statistically significant(F=103.224,F=856.460,F=214.563;P＜0.001).The pairwise comparison between groups showed that compared with control group,the expression levels of miR-520a-5p and VEGF mRNA in placenta tissue of the mice in FGR group were decreased(P＜0.05),and the expression level of DNMT3b mRNA was increased(P＜0.05);compared with FGR group and NC-MSCs-Exos group,the expression levels of miR-520a-5p and VEGF mRNA in placenta tissue of the mice in miR-520a-5p-MSCs-Exos group were increased(P＜0.05),and the expression level of DNMT3b mRNA was decreased(P＜0.05).The One-way ANOVA results showed that the differences in the expression levels of DNMT3b and VEGF proteins in placenta tissue of the mice among four groups were statistically significant(F=245.601,F=149.360;P＜0.001).The pairwise comparison between groups showed that compared with control group,the expression level of DNMT3b protein in placenta tissue of the mice in FGR group was increased(P＜0.05),and the expression level of VEGF protein was decreased(P＜0.05);compared with FGR group and NC-MSCs-Exos group,the expression level of DNMT3b protein in placenta tissue of the mice in miR-520a-5p-MSCs-Exos group was decreased(P＜0.05),and the expression level of VEGF protein was increased(P＜0.05).The One-way ANOVA results showed that the difference in the methylation rate of VEGF promoter in placenta tissue of the mice among four groups was statistically significant(F=687.096,P＜0.001).The pairwise comparison between groups showed that compared with control group,the methylation rate of VEGF promoter in placenta tissue of the mice in FGR group was increased(P＜0.05);compared with FGR group and NC-MSCs-Exos group,the methylation rate of VEGF promoter in placenta tissue of the mice in miR-520a-5p-MSCs-Exos group was decreased(P＜0.05).Dual-luciferase reporter gene assay results showed that compared with miR-NC group,the luciferase activity in the cells containing DNMT3b-WT reporter vector in miR-520a-5p group was decreased(P＜0.05);compared with miR-NC group,the luciferase activity in the cells containing DNMT3b-MUT reporter vector in miR-520a-5p group had no change,no significant difference was observed(P＞0.05).Conclusion:The MSCs-derived Exos highly loaded with miR-520a-5p may improve the pregnancy outcomes of FGR fetal mice by targeting and down-regulating the expression of DNMT3b,inhibiting VEGF methylation,and promoting VEGF expression.

Objective: To construct a Family with sequence similarity 50 member A(FAM50A) gene knockout mouse insulinoma pancreatic β-cell line Beta-TC-6 using CRISPR/Cas9 gene editing technology and to prepare polyclonal antibodies specifically recognizing FAM50A. Methods: Two guide RNAs(sgRNAs) targeting the FAM50A gene were designed,and a recombinant plasmid expressing blue fluorescent protein(BFP) was constructed for gene knockout.The successfully constructed plasmid was transfected into Beta-TC-6 cells,and BFP-positive single cells were isolated for clonal expansion.The expanded monoclonal cell lines were genotyped by Sanger sequencing,and FAM50A protein expression was assessed by Western blot.Purified human recombinant FAM50A protein was used to immunize New Zealand rabbits for the preparation of a polyclonal antibody.The specificity of the prepared antibody was then validated using the successfully established FAM50A knockout cell line. Results: A monoclonal cell line with a successful knockout of the FAM50A gene was identified.Sanger sequencing confirmed base deletions at the target site.Western blot analysis showed a complete absence of FAM50A protein expression in this cell line.The prepared polyclonal antibody successfully recognized endogenous murine FAM50A protein in wild-type Beta-TC-6 cells and in hTERT-RPE1 cells overexpressing human FAM50A-GFP fusion protein,while no signal was detected in the FAM50A knockout cells. Conclusion This study successfully established a FAM50A gene knockout Beta-TC-6 cell model and generated a FAM50A polyclonal antibody,providing powerful tools for future research.

Objective To investigate the expression of inositol polyphosphate 4-phosphatase type Ⅱ B(INPP4B)in colorectal cancer(CRC)and the relevant clinical significance,to determine the relationship between INPP4B and matrix metallopeptidase 7(MMP7)in CRC cells,and to make preliminary exploration of the effects of INPP4B on the proliferation and migration of CRC cells and mechanisms involved.Methods The TIMER2.0 and GEPIA2 databases were used to analyze the differences in INPP4B expression between cancer and para-cancerous tissues and the effects of such differences on the prognosis of CRC.The expression of INPP4B in 102 surgically resected CRC tumors was determined by immunohistochemistry(IHC),and the correlation between INPP4B and clinical pathological indicators was analyzed.In CRC cells with overexpressed/knocked-down INPP4B,the expression of INPP4B and MMP7 were examined by real time fluorogenic quantitative PCR,the protein expression of INPP4B was assessed by Western blot,cell proliferation was determined using the CellTiter 96? AQueous One assay,and cell migration and invasion were assessed using wound healing assay and real-time label-free dynamic cell analysis(RTCA).The LinkedOmics database was used to analyze signaling pathways related to INPP4B function,and the role of potential key molecules was validated at the cellular level.Results Analysis with the TIMER2.0 database and GEPIA2 database showed elevated INPP4B expression(colon adenocarcinoma[COAD]:2.30,rectal adenocarcinoma[READ]:2.33)in CRC compared to normal tissue(COAD:1.91,READ:1.89).IHC testing confirmed that INPP4B was upregulated in clinical CRC tissues and paracancerous tissues(P＜0.001).Cox regression model analysis showed that INPP4B(hazards ratio[HR]=1.457,95％confidence interval[CI]:1.003-2.115)affected the prognosis of CRC,and the Kaplan-Meier curve showed that patients with high INPP4B expression had shorter overall survival(P＜0.05).x2 test was performed to analyze the relationship between INPP4B expression and clinicopathological indexes,and it was found that high expression of INPP4B was correlated with lymph node metastasis(x2=3.997,P=0.046)and neural invasion(x2=8.511,P=0.004).In in vitro experiments,CRC cells overexpressing INPP4B showed a significantly increased cell proliferation and migration compared to the cells in the control group(P＜0.05).Analysis using the LinkedOmics database showed that INPP4B was correlated with extracellular matrix remodeling and cell migration.Pearson's correlation analysis showed that MMP7 was positively correlated with INPP4B(r=0.3782,P＜0.001).INPP4B overexpression or knockdown in vitro also led to the upregulation or the downregulation of MMP7 expression in CRC cells.Conclusion INPP4B is highly expressed in CRC tissues and significantly correlated with lymph node metastasis,neural invasion,and patient prognosis.MMP7 may mediate the role of INPP4B in promoting CRC cell migration and invasion.

OBJECTIVE To study the immunotherapeutic effect of chimeric virus-like particles(VLPs) based on hepatitis E virus(HEV) against human papillomavirus type 16(HPV 16) tumor. METHODS HPV16 E7 was inserted into the p239 protein of HEV to form the recombinant chimeric protein p239-HPV16 E7. The constructed recombinant protein was expressed by Escherichia coli, purified, and then refolded, and the protein was detected by electron microscopy and dynamic light scattering to confirm size and shape. Then, the C57B/L mice were immunized with the protein grain, and the lymphocyte differentiation of mouse spleen was detected by flow cytometry and enzyme-linked immune spot immunoassay; in addition, TC-1 tumor cells were used to construct tumor models in C57B/L mice to evaluate the anti-tumor immune effect of protein particles in mice. RESULTS After refold in vitro, the structure of chimeric protein was observed under electron microscopy, and the size of particle was 22.80 nm. The obtained protein particles induced favorable specific cellular immune response in C57B/L mice. Compared with the control group, the proportions of CD3+/CD4+ and CD3+/CD8+ in spleen lymphocytes of experimental groups were significantly different(P＜0.05), and effector T cells secreting IFN-γ interferon were also increased remarkably. At the same time, the obtained protein particles could effectively inhibit the growth of tumor cells in TC-1 tumor-bearing mice, and the mice did not die during the experimental period, while the tumors in the control mice grew rapidly and all died after 6 weeks. CONCLUSION Chimeric protein p239-HPV16E7 which was expressed in prokaryotes can form virus-like particles and effectively induce anti-tumor immunity against HPV16.

Background In recent years, the incidence of metabolic syndrome (MS) is increasing significantly in China. Some studies have found that temperature is related to single metabolic index, but there is a lack of research on associated mechanism and identifying path of the influence of temperature on MS. Objective Based on the data of Guangdong Province, to investigate the effect of temperature on MS and its pathway. Methods A total of 8524 residents were enrolled by multi-stage random sampling from October 2015 to January 2016 in Guangdong. Basic characteristics, behavioral characteristics, health status, and physical activity level were obtained through questionnaires and physical examinations, and meteorological data were obtained from meteorological monitoring sites. We matched individual data both with the temperature data of the physical examination day and of a lag of 14 d. A generalized additive model was used to explore the exposure-effect relationship between temperature and MS and its indexes, calculate effect values, and explore the effects of single-day lag temperature. Based on the literature and the results of generalized additive model analysis, a path analysis was conducted to explore the pathways of temperature influencing MS. Results The association between daily average temperature on the current day or lag 14 day and MS risk was not statistically significant. When daily average temperature increased by 1 ℃, the change values of fasting blood-glucose (FBG), systolic blood pressure (SBP), diastolic blood pressure (DBP), and high density lipoprotein cholesterol (HDL-C) were −0.033 (95%CI: −0.040-−0.026) mmol·L⁻¹, −0.662 (95%CI: −0.741-−0.583) mmHg, −0.277 (95%CI: −0.323-−0.230) mmHg, and −0.005 (95%CI: −0.007-−0.004) mmol·L⁻¹ respectively. The effects of average daily temperature on FBG, blood pressure, HDL-C, and waist circumference lasted until lag 14 day. The effects of daily average temperature on SBP and DBP were the largest on the current day. Daily average temperature of current day had direct and indirect effects on FBG and SBP. Temperature had an indirect effect on TG, and the intermediate variables were waist circumference and FBG, with an indirect effect value of −0.011 (95%CI: −0.020-−0.002). The indirect effects of daily average temperature on SBP, FBG, and TG were weak. Conclusion There is no significant correlation between temperature and risk of MS, and daily average temperature of current day could significantly affected blood pressure and FBG with a lag effect. Daily average temperature of current day has indirect effects on FBG and TG.

Objective:To investigate the postoperative changes in levels of 25-hydroxy vitamin D (25-[OH]D], parathyroid hormone (PTH) and other relevant biomarkers in obese patients receiving metabolic surgery and analyze the dynamic changes in relevant biomarkers in the short term (after 3-6 months) and the long term (after 12-24 months).Methods:A total of 96 obese patients who underwent metabolic surgery and received follow-up examinations from January 2018 to January 2020 were included. Baseline and postoperative data were collected, including anthropometric data (height, weight, neck circumference, waistline and hipline) and laboratory test results (fasting glucose, glycated hemoglobin, 25-[OH)D, PTH, serum calcium and serum phosphorus). Body mass index (BMI) and waist-hip ratio were calculated. The anthropometric data were analyzed by repeated measures analysis of variance and laboratory data were compared between groups using t test and Kruskal-Wallis test. Results:96 patients (33 males and 63 females) were included, of whom 49 were complicated with diabetes. Prior to surgery, 79 (79.17%) of the patients had 25-(OH)D deficiency (< 20 μg/L), 16 (16.67%) had 25-(OH)D insufficiency (≥ 20 μg/L and < 30 μg/L) and 23 (23.96%) had high PTH levels (> 70 ng/L). After the surgery, 25-(OH)D level was transiently increased in the short term ( P = 0.01) but declined thereafter in the long term ( P < 0.01) to levels lower than baseline ( P = 0.023). Long-term PTH level was higher than baseline ( P = 0.012), with 11 patients showing PTH levels higher than normal (> 70 ng/L). Serum phosphorus level was increased in both the short term and the long term ( P < 0.01). Conclusions:Obese patients have 25-(OH)D deficiency/insufficiency before metabolic surgery and experience further decrease in the long term after surgery, despite a transient increase. Secondary increase in PTH level occurs in some of the patients after surgery. Long-term nutritional supplements and comprehensive nutritional management play important roles in postoperative management of obese patient.

Objective:To investigate the value of minimal residual disease (MRD) in prediction of prognosis in acute lymphoblastic leukemia (ALL) patients with or above complete remission 2 (CR2) underwent.Methods:A retrospective analysis was performed on 201 ALL patients who received allogeneic stem cell transplantation (allo-SCT) and pretransplant disease status ≥CR2 in Peking University People′s Hospital from January 2009 to December 2018. MRD was measured by multi-parameter flow cytometry at 1 month before transplantation and 1 month, 2 months, 3 months, 4 months, 6 months, 9 months or 12 months after transplantation. To investigate the influence of dynamic changes of MRD before and after transplantation on prognosis.Results:201 ALL patients, including 126 males and 75 females, with a median age of 18 years. The 3-year cumulative incidence of relapse (CIR), non-relapse mortality (NRM), leukemia-free survival (LFS) and overall survival (OS) of all cases were 34%, 16%, 50%, and 56%, respectively. Positive pre-SCT MRD patients with higher 3-year CIR (47% vs 26%, P=0.003), lower 3-year LFS (40% vs 55%, P=0.047) and OS (42% vs 60%, P=0.065) than those with negative one. Subjects with positive post-MRD had higher 3-year CIR (73% vs 22%, P<0.001) and lower 3-year LFS (28% vs 56%, P=0.005) and OS (32% vs 60%, P=0.040) compared with those with negative one. Multivariate analysis showed that both pre-MRD and post-MRD were associated with higher CIR ( HR=1.823, P=0.018; HR=3.474, P<0.001), lower LFS ( HR=1.779, P=0.007; HR=2.185, P=0.001) and OS ( HR=1.609, P=0.034; HR=1.970, P=0.001). Negative pre-and post-SCT MRD group had lower 3-year CIR (17%, 42%, 82%; P<0.001) and higher 3-year LFS (61%, 44%, 18%; P<0.001) and OS (63%, 47%, 27%; P<0.001) compared with those unrisen post-SCT MRD group, and increased post-SCT MRD group. Multivariate analysis showed that pre-and post-SCT MRD dynamics were associated with CIR, LFS and OS ( P<0.01 for all) independently. The pre-and post-SCT MRD dynamics could better distinguish CIR (C=0.669) from that of pre-SCT MRD (C=0.587) and post-SCT MRD (C=0.629). Conclusion:Our data suggest that pre-SCT MRD, post-SCT MRD and the dynamic peri-SCT MRD could be used to predict transplant outcome of ALLpatients with or above CR2 who underwent allo-SCT.