The purpose of this study is to investigate the effect of TPCK trypsin on the proliferation pattern of porcine epidemic diarrhea virus in Vero cells.The TPCK trypsin and conventional tryp-sin were added for virus proliferation,and RT-qPCR technology was used to analyze the changes in virus adsorption and invasion in Vero cells.The replication ability of porcine epidemic diarrhea vi-rus in Vero cells was explored through growth curve drawing,IFA identification,and cell activity detection.The results showed that the optimal concentrations of TPCK trypsin and conventional trypsin were 1 mg/L and 6 mg/L,respectively.The virus showed a decreasing trend with the pro-longation of TPCK trypsin and conventional trypsin pretreatment time.Adding different pancreatic enzymes during the virus proliferation process did not promote the virus invasion in Vero cells.Af-ter 4 h of invasion,the virus particles of each group gradually increased.By plotting the growth curve,it was found that the virus content of the TPCK trypsin group reached its highest level at 24 h(lgTCID50=(6.30±0.14)/0.1 mL),followed by a decreasing trend at 36 h,and the fluorescence intensity produced at 24 h was higher than that of conventional trypsin.In summary,TPCK trypsin has a better promoting effect on the proliferation of porcine epidemic diarrhea virus in Vero cells.It provided theoretical basis for further research on the mechanism of TPCK trypsin affecting porcine epidemic diarrhea virus proliferation,and also provided data support for the isola-tion and purification of porcine epidemic diarrhea virus epidemic strains.

To rapidly and accurately detect Mycobacterium avium subsp.paratuberculosis(MAP),this study designed and screened primers and probes using its specific gene F57 as the detection target,established a recombinant enzyme-mediated isothermal amplification(RAA)fluorescence detection method,and applied this method to detect 116 clinical samples from cattle and sheep.The results showed that using the primer and probe combination B12F/B2R(0.4 μmol/L)+Probe B(0.12 μmol/L),MAP could be detected at a constant temperature of 42 ℃ within 20 min;this de-tection method had no cross-reaction with 11 common pathogens such as Escherichia coli,Clos-tridium,and bovine viral diarrhea in sheep and cattle;the lowest detection limit was 1.0×102 cop-ies/μL;the coefficient of variation was 3.77％—5.29％;24 clinical samples were positive,with a co-incidence rate of 88.89％with GBT27637-2011.In summary,this study established a fluorescent RAA detection method for MAP,which is simple,rapid,highly specific,sensitive,reproducible,and has a high coincidence rate with national standards,making it suitable for clinical detection and epi-demiological studies.

Swine enteric coronaviruses(SeCoV),such as porcine epidemic diarrhea virus(PEDV),transmissible gastroenteritis virus(TGEV),and porcine delta coronavirus(PDCoV),cause severe diarrhea in piglets,resulting in substantial losses in pig farming.In this study we establish a triple fluorescence reverse transcription-quantitative PCR(RT-qPCR)method for the simultaneous de-tection of PEDV,TGEV,and PDCoV.The specific primers and probes for each target virus were designed based on conserved sequences from the PEDV M gene,the TGEV ORF 1b gene,and the PDCoV ORF 1b gene respectively.Following the optimization of parameters and conditions,a triple RT-qPCR method was successfully established to simultaneously detect PEDV,TGEV,and PD-CoV.The developed assay exhibits strong specificity for these three pathogens without any cross-reaction with other common porcine viruses like CSFV,PCV2,PoRVA,PRV,and PRRSV.The de-tection limit of linear templates for pTOPO-PEDV 128,pTOPO-TGEV 116,and pTOPO-PDCoV 125 recombinant plasmids were 16.835,17.610 and 17.020 copies/μL,respectively.The intra group and inter group coefficients of variation were less than 5％,with no significant differences observed(P＞0.05).Moreover,the detection consistency rate of the developed RT-qPCR was compared with standard method and showed 100％agreement.Out of 35 small intestine tissue samples,17 tested positive for PEDV,resulting in a positive rate of 48.57％(17/35).The tests for TGEV and PDCoV yielded negative results,and no mixed infections were detected.Based on the above results,the tri-ple RT-qPCR method established is specific,sensitive,stable,and rapid,and can be used for clinical detection and differential diagnosis of PEDV,TGEV,and PDCoV simultaneously,providing a method for the detection and epidemiological investigation of porcine diarrhea coronaviruses.

Objective To develop a core outcome set(COS)for clinical effectiveness trials of heart failure with preserved ejection fraction(HFpEF).Methods Outcome measures were collected through database literatures search,clinical experts questionnaire survey and semi-structured patients interview.Then,the outcome measures pool was constructed and domains were divided.Candidate outcome measures of COS were screened through two rounds of Delphi survey.Finally,a consensus meeting was held to determine COS and reach a consensus.Results A total of 317 outcome measures which could be divided into 6 domains were collected through literature research,questionnaire survey and semi-structured interview.15 candidate outcome measures of COS were screened through two rounds of Delphi survey.Finally,the consensus meeting reached consensus on a COS with 6 entries.Conclusion In this study,a COS for clinical effectiveness trials of HFpEF was developed,which is conducive to the standardization of efficacy evaluation.

Objective:To investigate whether herb cake-insulated moxibustion affects the expression of cholesterol metabolism-related protein 3-hydroxy-3-methylglutaryl coenzyme A reductase(HMGR)and inhibits the development of atherosclerosis by regulating the exosomal miR-223 expression.Methods:Thirty-six male New Zealand rabbits were randomly assigned to a normal group,a model group,and an herb cake-insulated moxibustion group,with 12 rabbits in each group.The model and the herb cake-insulated moxibustion groups were fed a high-fat diet for 8 weeks to induce an atherosclerosis model.Following successful modeling,the herb cake-insulated moxibustion group was subjected to bundling and herb cake-insulated moxibustion intervention,while the other two groups were subjected only to bundling without moxibustion.After 8 weeks of intervention,hematoxylin-eosin(HE)staining was used to observe aortic morphology;the serum levels of total cholesterol(TC),triglyceride(TG),and low-density lipoprotein cholesterol(LDL-C)were detected using an automatic biochemical analyzer in each group.Exosome morphology was observed using the transmission electron microscope;Western blotting(WB)was used to detect the protein levels of serum exosomal CD63 and CD9 markers,as well as liver HMGR;additionally,real-time fluorescence quantitative polymerase chain reaction was used to quantify serum exosomal miR-223.Results:HE staining showed thickened aortic intima,lipid infiltration,foam cell aggregation,and structural damage to the arterial wall in the model group.Meanwhile,after modeling,the serum levels of LDL-C,TC,and TG increased significantly in the model and herb cake-insulated moxibustion groups compared to the normal group(P<0.05),suggesting successful atherosclerosis rabbit model preparation.The serum exosomes of rabbits in the model group exhibited a saucer-like or semi-concave spherical shape with diameters of 120-150 nm.WB detection results showed positive expression of the exosomal markers CD63 and CD9.After 8 weeks of intervention,the miR-223 level in the model group was significantly lower than that in the normal group(P<0.01).In contrast,the herb cake-insulated moxibustion group demonstrated significantly reduced serum levels of TC,TG,and LDL-C(P<0.05),increased miR-223 expression(P<0.01),and decreased relative liver HMGR protein expression(P<0.05)compared to the model group.Conclusion:Herb cake-insulated moxibustion may alleviate the progression of atherosclerosis by up-regulating exosomal miR-223 expression and down-regulating HMGR protein expression,thereby inhibiting cholesterol anabolic metabolism.

Objective:To investigate the value of tumor and adipose tissue metabolic parameters from baseline 18F-FDG PET/CT in predicting the efficacy and prognosis of immunotherapy in patients with advanced nasopharyngeal carcinoma (NPC). Methods:From February 2019 to February 2022, 112 patients (91 males, 21 females, age 21-73 years) with advanced NPC who were treated with programmed death-1 (PD-1) inhibitors at Sun Yat-Sen University Cancer Center were retrospectively included. All patients underwent baseline PET/CT examination. Tumor and adipose tissue metabolic parameters were measured and calculated. Patients were divided into clinical benefit and non-clinical benefit groups, and Mann-Whitney U test or χ2 test was used to assess the differences between groups. Prognostic analysis of progression-free survival (PFS) was performed using multivariate Cox proportional hazards regression model and a prognostic stratification system was constructed. Results:Of the 112 patients, 85 were in the clinical benefit group and 27 were in the non-clinical benefit group. In non-clinical benefit group and clinical benefit group, the metabolic tumor volume (MTV) of primary tumor (PT-MTV) were 47.7(7.7, 81.2) and 14.0(5.7, 27.1)cm 3, total lesion glycolysis (TLG) of primary tumor (PT-TLG) were 228.9(27.4, 492.8) and 72.7(20.4, 165.5)g, whole-body MTV (WB-MTV) were 94.2(45.9, 215.4) and 61.3(31.6, 104.3)cm 3, whole-body TLG (WB-TLG) were 605.5(214.1, 1 402.5) and 319.2(172.4, 632.8)g, SUV max of visceral adipose tissue (SUV max-VAT) were 0.77(0.55, 0.91) and 0.62(0.48, 0.76), respectively ( Z values: from -2.72 to -1.96, all P<0.05). The proportion of patients with lung metastasis in non-clinical benefit group was higher than that in clinical benefit group (44.4%(12/27) vs 23.5%(20/85); χ2=4.39, P=0.036). PT-MTV (hazard ratio ( HR)=2.807, 95% CI: 1.540-5.118, P=0.001) and the presence of lung metastases ( HR=1.691, 95% CI: 1.012-2.823, P=0.045) were independent predictive factors for PFS in multivariate analysis. The prognostic prediction model based on the two predictive factors was able to significantly differentiate the prognosis in patients. Conclusions:Baseline tumor metabolic parameters and SUV max-VAT are associated with the efficacy of immunotherapy in patients with advanced NPC. PT-MTV and lung metastasis can independently predict PFS. The constructed prediction model can stratify patients′ prognosis.

Cholera toxin B subunit(CTB)can enhance antigen presentation and promote T cell pro-liferation,B cell differentiation and B cell isotype conversion.Moreover,woodchuck hepatitis virus post transcriptional regulatory element(WPRE)can enhance gene expression efficiency by optimi-zing RNA polyadenylation,denuclearization and/or translation.In order to construct porcine epi-demic diarrhea virus like particles(VLPs)chimerized with CTB and WPRE and evaluate their im-munogenicity,the G Ⅱ type PEDV S gene,combined with the elements promoting the protein ex-pression and enhancing immune effects,was synthesized by the company and cloned into pET32a(+).Af-ter double enzyme digestion and gel recovery,the gene named as TSCW was cloned into pFastBacl to construct the recombinant plasmid pFastBac-TSCW.pFastBac-TSCW was further transformed into DH10Bac competent cells to obtain recombinant bacmid Bacmid-TSCW.Subsequently,the Bacmid-TSCW was transfected into sf9 cells to obtain recombinant baculovirus BV-TSCW.After co-infection of BV-TSCW and BV-M into sf9 cells,viral like particles VLP-TSCW was obtained and used to immunize mice to evaluate its immunogenicity.The results showed that the recombi-nant plasmid pFastBac-TSCW and bacmid Bacmid-TSCW were successfully constructed.After transfection of sf9 cells with recombinant baculovirus,significant cytopathic effects were observed.PCR and Western blot results showed that the recombinant baculoviruses existed stably in sf9 cells and the target proteins was also expressed stably.In addition,the electron microscopy results showed that BV-TSCW and BV-M successfully assembled into viral like particles VLP-TSCW.Furthermore,ELISA results indicated that VLP-TSCW induced high level specific antibodies.The above results laid the foundation for further optimization,design and development of PEDV VLPs subunit vaccines.

Objective:To study the impact of early blood concentrations of tacrolimus on the survival of patients after liver transplantation.Methods:Clinical data of 159 patients with liver diseases undergoing classic orthotopic liver transplantation at the Department of Hepatobiliary Surgery, the 900th Hospital of the Joint Logistics Support Force between January 2010 and December 2019 were retrospectively analyzed, including 123 males and 36 females, aged (48.0±12.2) years. According to survival status, patients were divided into the surviving group ( n=108) and death group ( n=51). Inverse probability of treatment weighting (IPTW) was applied to adjust for confounding factors by weighting covariates between the two groups. Univariate and multivariate Cox regression analysis were used to examine the relationship between early tacrolimus concentrations and mortality, and restrict cubic spline (RCS) curves were employed to assess the nonlinear relationship further. Results:After IPTW weighting, multivariate Cox regression analysis indicated that early tacrolimus concentration ( HR=2.479, 95% CI: 1.354-4.537, P<0.001) and preoperative international normalized ratio ( HR=0.358, 95% CI: 0.162-0.792, P=0.011) levels were risk factors for post-transplant survival. The RCS curve revealed that the optimal thresholds for early tacrolimus concentration were 6.30 ng/ml and 8.28 ng/ml ( P<0.001). Patients were therefore divided into the optimal concentration group ( n=60) and the non-optimal concentration group ( n=99). After IPTW weighting, the optimal concentration group comprised 102 cases, and the non-optimal concentration group included 212 cases. The 1-year, 3-year and 5-year survival rates in the optimal concentration group and the non-optimal concentration group were 97.06%, 81.37% and 75.49%, and 86.32%, 64.62% and 50.94%, respecitvely ( χ2=8.37, P<0.001). Conclusion:Early tacrolimus concentration is an independent risk factor for post-transplant survival. A tacrolimus concentration >8.28 ng/ml or <6.30 ng/ml is associated with a relatively higher mortality rate.

Objective:To elucidate the prognostic differences and risk factors associated with different patterns of recurrence and metastasis following pancreatic cancer (PC) resection.Methods:This is a retrospective case series study. Clinicopathological data and follow-up information were retrospectively collected from 210 patients who underwent surgery for PC at the Department of Hepatobiliary and Pancreatic Surgery, Peking University First Hospital, between January 2014 and December 2023. There were 114 males and 96 females; the age was (64.5±10.3) years (range: 29 to 89 years). Survival functions based on different times to recurrence and metastasis and different patterns of recurrence and metastasis were estimated using the Kaplan-Meier method, and survival differences among groups were compared using the Log-rank test. Identifying the optimal cutoff for time to postoperative recurrence/metastasis predicting overall survival (OS) in pancreatic cancer patients via the minimum p-value approach. Univariate and multivariate Cox regression analyses were performed to identify independent risk factors affecting OS following pancreatectomy.Results:A total of 210 patients met the inclusion and exclusion criteria. Among them, 71 patients remained free of recurrence and metastasis, while 139 developed recurrence and metastasis. The patterns included local recurrence ( n=34), liver metastasis ( n=39), lung metastasis ( n=11), peritoneal metastasis ( n=13), multiple sites metastasis ( n=38), bone metastasis ( n=3), and kidney metastasis ( n=1). OS was significantly shorter in the group with postoperative recurrence and metastasis compared to the group without recurrence/metastasis (23.07 months vs.not reached, P<0.01). The optimal cut-off time distinguishing early from late recurrence and metastasis was 13 months. There was a significant difference in post-recurrence survival between patients with early and late recurrence and metastasis (16.03 months vs. 52.40 months, P=0.009). The Kaplan-Meier survival curve showed that different postoperative recurrence and metastasis patterns had different impacts on OS, with lung metastasis showing the best prognosis compared to local recurrence, liver metastasis, peritoneal metastasis, and multiple sites metastasis ( P<0.01). Multivariate Cox analysis revealed that Eastern Cooperative Oncology Group (ECOG) score 1, postoperative carcinoembryonic antigen (CEA) ≥15 μg/L, poor tumor differentiation, postoperative local recurrence, liver metastasis, peritoneal metastasis, and multiple sites metastases are independent risk factors for postoperative recurrence and metastasis (all P<0.05). Conclusions:Considerable prognostic heterogeneity exists in postoperative PC patients depending on the site and pattern of recurrence or metastasis. Specifically, lung metastasis portends a significantly more favorable prognosis than liver metastasis, peritoneal metastasis, local recurrence, or multiple sites metastases. ECOG score 1, postoperative CEA≥15 μg/L, poor tumor differentiation, postoperative local recurrence, liver metastasis, peritoneal metastasis, and multiple sites metastases are independent risk factors for OS in postoperative PC patients.

Objective:To explore the safety and feasibility of circular-stapled anastomosis using purse-string suture device with multi-functional seal cap in totally laparoscopic total gastrectomy esophagojejunostomy.Methods:This technique is based on a new purse-string suture device and multi-functional seal cap. After laparoscopic total gastrectomy and D2 dissection, the purse-string suture device was inserted into the abdominal cavity through the multi-functional sealing cover and placed in a proper position along the distal esophagus for formation of purse-string suture. Anvils were introduced inside the purse-string after its fire, and a 25-mm cicular stapler was inserted to the abdominal cavity through the multi-functional seal cap, following laparoscopic end-to-side circular-stapled esophagojejunostomy.Results:From November 2024 to December 2024, three patients with gastric cancer underwent laparoscopic total gastrectomy with D2 lymphadenectomy and successfully accepted laparoscopic esophagojejunostomy based on the purse-string suture device with multi-functional seal cap. The operation time was 180 minutes, 260 minutes and 240 minutes, respectively with the time of anvil pacement of 4 minutes,4 minutes, 4.5 minutes for the three cases. The bleeding volume of each was 100 ml respectively. The proximal esophageal margins were 2 cm from the tumor, and the margins were negative for tumor. No extravasation of contrast agent was seen in the postoperative oral contrast anastomosis. All three patients were followed up for 33, 30, and 25 days after the operation, and no anastomotic bleeding, leakage, or other related complications were found.Conclusion:The circular anastomosis using purse-string suture device with multi-functional seal cap in totally laparoscopic total gastrectomy esophagojejunostomy is safe and feasible, which is a promising novel choice for laparoscopic radical treatment of gastric cancer with total gastrectomy oesophagojejunostomy.