Objective To prepare the recombinant Echinococcus granulosus Polo-like kinase 2 (rEgPLK2) protein and evaluate its immunoprotective efficacy against cystic echinococcosis, so as to provide insights into research and development of novel vaccines against echinococcosis. Methods The Polo-like kinase (PLK) protein sequences were retrieved from 12 species in the NCBI protein database, including E. granulosus and E. multilocularis. Multiple sequence alignment was performed using the Clustal Omega program, and structural visualization and homology analysis were conducted using the ESPript 3.2 program. The recombinant plasmid pET-30a-EgPLK2 was transformed into BL21(DE3) competent cells. Protein expression was induced with isopropyl-β-D-thiogalactoside (IPTG), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to characterize the expression and molecular weight of the rEgPLK2 protein. The purified rEgPLK2 protein was thoroughly emulsified with Freund’s complete adjuvant at a 1 : 1 volume ratio. Two New Zealand white rabbits were immunized with multipoint subcutaneous injection on the back at a dose of 300 μg per rabbit for primary immunization. For booster immunizations, the protein was emulsified with Freund’s incomplete adjuvant at a 1 : 1 volume ratio and administered on days 14, 28, and 42 after the primary immunization at a dose of 150 μg per rabbit. Serum was sampled from the rabbit ear vein on day 7 after the final immunization to yield anti-rEgPLK2 polyclonal antibodies. Antibody titer was determined by indirect enzyme-linked immunosorbent assay (ELISA), and antibody specificity was verified by Western blotting. The tissue localization of the EgPLK2 protein was detected in E. granulosus protoscoleces and adult worms using immunofluorescence assay (IFA). Eighteen 6- to 8-week-old female SPF-grade BALB/c mice were randomly divided into three groups, including the blank control group, rEgPLK2-ISA immunization group, and PBS-ISA adjuvant control group, of 6 mice each group. Mice in the rEgPLK2-ISA immunization group and PBSISA group received three primary immunizations via intramuscular injection, and animals in the rEgPLK2-ISA immunization group was inoculated with immunogens prepared by emulsifying rEgPLK2 protein with ISA 201 adjuvant at a 1 : 1 volume ratio (6 μg per mouse), while mice in the PBS-ISA adjuvant control group received an equal volume of PBS emulsified with ISA adjuvant at a 1 : 1 volume ratio. A fourth booster immunization was administered via intraperitoneal injection. Mice in the rEgPLK2-ISA immunization group received a booster immunization with 8 μg of rEgPLK2 protein per mouse, and animals in the PBS-ISA group received an equal volume of PBS, with immunizations given at 2-week intervals. Mice in the blank control group were given no treatment, and housed under standard conditions. Tail vein blood was collected from all mice 7 days after the final immunization, and levels of specific anti-rEgPLK2 IgG antibody and its subclasses (IgG1, IgG2a, IgG2b, IgG3) were measured by indirect ELISA. E. granulosus infection was modelled in mice through injection with 1 000 E. granulosus protoscoleces via intrahepatic portal vein in the rEgPLK2-ISA immunization group and PBS-ISA adjuvant control group 2 weeks after the last immunization. All mice were sacrificed and dissected. The number of cysts was counted in mouse livers, and the cyst reduction rate was calculated. Liver tissues were processed for paraffin sectioning and stained with hematoxylin and eosin (HE), and histopathological changes were examined under a light microscope. Results Sequence analysis revealed that EgPLK2 shared a high amino acid sequence homology with E. multilocularis PLK2 (EmPLK2) and contained the typical domains of the Polo-like kinase family, including the serine/threonine protein kinase catalytic domain (STKc) and Polo-box. The IPTG-induced rEgPLK2 protein was mainly expressed in the form of inclusion bodies, and the purified rEgPLK2 protein showed a relative molecular mass of approximately 70 kDa. The prepared rabbit anti-rEgPLK2 polyclonal antibody had a titer of 1 : 256 000, and Western blotting assay showed that this anti-body specifically recognized the rEgPLK2 protein with a relative molecular mass of approximately 70 kDa. Immunofluorescence assay showed that the EgPLK2 protein was localized in the excretory bladder and rostellum of E. granulosus protoscoleces, as well as the tegument, suckers, and inter-proglottid junctions of adult worms. Immunoprotective assay showed that the serum levels of specific anti-rEgPLK2 IgG, IgG1, IgG2a, and IgG2b antibodies were 2.92 ± 0.49, 0.33 ± 0.10, 0.31 (0.36), and 3.12 (1.73) in mice in the rEgPLK2-ISA immunization group, which were all significantly higher than those in the PBS-ISA adjuvant control group (0.14 ± 0.04, 0.07 ± 0.01, 0.12 ± 0.04, and 0.11 ± 0.04, respectively) (t = 19.28 and 8.46, Z = 3.75 and 4.15; all P values < 0.001); however, there was no significant difference in the serum anti-IgG3 antibody level between the rEgPLK2-ISA immunization group and the PBS-ISA adjuvant control group [0.07 (0.01) vs. 0.073 (0.07); Z = 0.69, P > 0.05)]. In the mouse model of E. granulosus infections, the area of hepatic lesions was reduced and the inflammatory infiltration was alleviated in the rEgPLK2-ISA immunization group than in the PBS-ISA adjuvant control group, and the number of hepatic cysts was higher in the PBS-ISA adjuvant control group than in the rEgPLK2-ISA immunization group [8.00 (2.00) vs. 1.00 (0.75); Z = −2.93, P < 0.01], with a cyst reduction rate of 80.40%. Indirect ELISA assay measured higher serum levels of specific anti-rEgPLK2 IgG (3.28 ± 0.48 vs. 0.11 ± 0.04; t = 15.86, P < 0.01), IgG1 (0.29 ± 0.02 vs. 0.09 ± 0.01; t = 15.67, P < 0.01), IgG2a [3.71 (1.09) vs. 0.08 (0.03); Z = 2.88, P < 0.01], and IgG2b antibodies [3.34 (1.01) vs. 0.08 (0.03); Z = 2.88, P < 0.01] in the rEgPLK2-ISA immunization group than in the PBS-ISA adjuvant control group, and there was no significant difference in the serum level of the specific anti-rEgPLK2 IgG3 antibody between the rEgPLK2-ISA immunization group and the PBS-ISA adjuvant control group (0.07 ± 0.01 vs. 0.07 ± 0.01; t = 1.29, P > 0.05). Conclusions The prokaryotic expression system has been successfully constructed for the EgPLK2 gene and the anti-rEgPLK2 polyclonal antibody has been obtained. The rEgPLK2 protein exhibits a high immunogenicity, and is effective to protect against E. granulosus infection, and inhibits cyst development, which is a promising candidate vaccine target against cystic echinococcosis.

Objective:To observe the clinical efficacy and safety of Jianpi Bushen Jiedu Prescription combined with 5-fluorouracil (5-FU)-based chemotherapy and targeted therapy for the treatment of advanced colorectal cancer patients with liver and kidney yin deficiency combined with spleen deficiency pattern.Methods:A randomized controlled trial was conducted. A total of 72 hospitalized patients with advanced colorectal cancer treated at the Department of Oncology, Nanjing Hospital of Traditional Chinese Medicine Affiliated to Nanjing University of Chinese Medicine from October 2022 to January 2024 were enrolled as study subjects. Using a random number table method, they were allocated into two groups, with 36 patients in each group. The control group received the mFOLFOX6/FOLFIRI combined with bevacizumab regimen, while the treatment group was administered additional oral Jianpi Bushen Jiedu Prescription on the basis of the control group. Two weeks was a cycle in both groups, with a total of 6 cycles of treatment. Serum levels of carcinoembryonic antigen (CEA), carbohydrate antigen 199 (CA199), and carbohydrate antigen 724 (CA724) were detected using electrochemiluminescence; the Karnofsky Performance Status (KPS) scale was utilized to evaluate patients' functional status; vital signs were continuously monitored, and adverse reactions were recorded. The short-term efficacy and TCM syndrome efficacy of patients were evaluated.Results:The treatment group demonstrated higher objective response rate (ORR) [31.25% (10/32) vs. 21.88% (7/32), χ2=0.72] and disease control rate (DCR) [84.38% (27/32) vs. 71.88% (23/32), χ2=1.46] compared to the control group, without statistical significance ( P>0.05). Post-treatment levels of CEA [4.09 (3.31,8.57) μg/L vs. 10.07 (4.55,22.35) μg/L, Z=-2.10] and CA72-4 [4.54 (2.04,10.99) mU/L vs. 9.48 (4.34,18.95) mU/L, Z=-2.52] in the treatment group were significantly lower than those in the control group ( P<0.05). The total effective rate of TCM syndrome was significantly higher in the treatment group [78.13% (25/32)] compared with the control group [50.00% (16/32)], with statistical significance ( χ2=5.50, P=0.019). Post-treatment KPS scores in the treatment group [80.0 (80.0, 80.0) vs. 70.0 (62.5, 80.0), Z=-2.76] were significantly higher compared with the control group ( P<0.01). During the treatment period, the treatment group showed statistical significance compared with the control group in the incidence of hemoglobin decrease ( χ2=4.66), leukopenia decrease ( χ2=4.27), and peripheral neuropathy ( χ2=3.93), with statistical significance ( P<0.05). Conclusion:The addition of Jianpi Bushen Jiedu Prescription to 5-FU-based chemotherapy combined with targeted therapy demonstrates significant clinical benefits in advanced colorectal cancer patients, including reducing tumor marker levels, alleviating clinical symptoms, improving quality of life, and mitigating treatment-related toxicities, with a good safety.

Objective To explore the value of artificial intelligence-assisted compressed sensing(ACS)-MR cine imaging(Cine)for assessing biventricular function and left ventricular myocardial strain compared with conventional cardiac Cine and united compressed sensing(uCS)-Cine.Methods A total of 30 subjects who underwent conventional Cine,uCS-Cine and ACS-Cine were prospectively enrolled.Based on a 5-point scale,subjective scoring of image quality was performed and compared among 3 sequences.Biventricular function parameters and myocardial strain parameters of the left ventricle,including left ventricular end-diastolic volume(LVEDV),left ventricular end-systolic volume(LVESV),left ventricular ejection fraction(LVEF),left ventricular end-diastolic mass(LVEDM),right ventricular end-diastolic volume(RVEDV),right ventricular end-systolic volume(RVESV),right ventricular ejection fraction(RVEF),left ventricular global radial strain(LVGRS),left ventricular global circumferential strain(LVGCS)and left ventricular global longitudinal strain(LVGLS)were measured and compared among sequences.Results The subjective image quality scores of all 3 sequences were≥3,of ACS-Cine and conventional Cine were not significantly different(adjusted P=0.306)but both slightly higher than that of uCS-Cine(both adjusted P＜0.05).LVEDV,LVEF and LVEDM based on ACS-Cine and uCS-Cine were not significantly different(all adjusted P＞0.05),but all lower than those of conventional Cine(all adjusted P＜0.05).No significant difference of LVESV was found between each two sequences(all adjusted P＞0.05).RVEDV based on ACS-Cine was lower than that based on conventional Cine,RVESV based on ACS-Cine was higher than that based on uCS-Cine,and RVEF based on ACS-Cine was lower than that based on uCS-Cine and conventional Cine(all adjusted P＜0.05),while no significant difference of right ventricular functional parameters was observed between uCS-Cine and conventional Cine(all adjusted P＞0.05).Both ACS-Cine and uCS-Cine had lower LVGRS and LVGCS than conventional Cine(all adjusted P＜0.05),with no significant difference was found between uCS-Cine and ACS-Cine(both adjusted P＞0.05).No significant difference of LVGLS was observed among 3 sequences(P＞0.05).Conclusion ACS-Cine could be used as appropriate supplement to conventional Cine for clinical assessment of biventricular function and left ventricular myocardial strain.

Multi-conjugate vaccine and polyvalent vaccine can reduce the number of vaccinations, improve vaccination efficiency, and provide wider protection against diseases, and can not only brings convenience to recipients but also reduce healthcare costs, making it a key focus in modern vaccine development. However, even if the components of the vaccine are derived from already approved monovalent vaccines, it must still be considered as a new vaccine and undergo randomized controlled clinical trials to evaluate their safety and efficacy in humans. Due to the inclusion of multiple antigens, clinical evaluation must consider the potential interactions between or among the components, as well as the impacts of adjuvants, preservatives, and other ingredients on the vaccines' safety and efficacy. These factors introduce certain specific challenges in the clinical evaluation of multi-conjugate vaccine and polyvalent vaccine. This article summarizes the key elements and methods of clinical study design for multi-conjugate vaccine and polyvalent vaccine in terms of safety, immunogenicity, and protective efficacy, and discuss the problems and challenges exisitng in vaccine clinical evaluation to provide reference for the standardization of clinical study design of multi-conjugate vaccine and polyvalent vaccine.

This study was a single-center cross-sectional investigation aimed at identifying risk factors for cognitive dysfunction in patients undergoing maintenance hemodialysis (MHD), with the goal of providing a basis for improving patient prognosis. Patients receiving MHD in the Blood Purification Center of Hainan Provincial People's Hospital from June 1 to June 30, 2023, were enrolled. Cognitive function was assessed using the Mini-Mental State Examination (MMSE), and potential risk factors for cognitive impairment were analyzed by using Logistic regression. A total of 278 patients were included, 69 patients (24.8%) of whom had cognitive impairment. Multivariate Logistic regression analysis indicated that the history of cerebrovascular disease ( OR=3.109, 95% CI 1.310-7.378, P=0.010), old age ( OR=1.077, 95% CI 1.040-1.115, P<0.001), low dialysis frequency ( OR=0.270, 95% CI 0.120-0.606, P=0.001), low academic qualification (using college/university as the control group: primary school group OR=26.960, 95% CI 7.519-96.673, P<0.001; Junior high school/technical secondary school group OR=4.264, 95% CI 1.330-13.650, P=0.015; High school group OR=9.554, 95% CI 2.861-31.904, P<0.001), high β2-microglobulin ( OR=1.609, 95% CI 1.044-2.480, P=0.031) and high C-reactive protein/albumin ( OR=2.672, 95% CI 1.226-5.826, P=0.013) were independent risk factors for cognitive impairment in MHD patients.

Autogenous arteriovenous fistula (AVF), compared to other vascular access routes, offers the advantages of stable blood flow, fewer complications, and a longer service life, making it the primary type of vascular access and the first choice for hemodialysis patients. The failure rate of AVF maturation is as high as 20% to 60%, primarily due to intimal hyperplasia following AVF surgery. Currently, the origin, function, and differentiation status of neointimal cells are not well understood, and most knowledge about neointimal cells was inferred from arterial neointima in non-AVF systems. Understanding the source of neointimal cells in AVF is crucial for experimental design and effectively providing strategies to reduce intimal hyperplasia. This article reviews the situation of venous intimal hyperplasia in patients with stage 5 chronic kidney disease, the relationship between changes in venous vessel wall and neointimal cells after AVF surgery, and the effects of outer membrane fibroblasts, local smooth muscle cells, and circulating bone marrow progenitor cells on AVF intimal hyperplasia. It is hoped that this will provide a reference for the treatment of intimal hyperplasia and experimental research.

The UL54 protein is a nonstructural protein of the pseudorabies virus(PRV).Electron microscopy observations showed that most of particles of recombinant PRV(rPRV-△UL54)with a deletion of the UL54 gene had only a nucleocapsid and lacked an intact vesicle structure.The in-complete structure of the viral particles led to a significant reduction in the infection efficiency of the virus,which could not be applied in the study of the gene function and mechanism of action of UL54.In this study,the UL54 gene was cloned into the lentiviral vector pLVX-IRES-Puro,and af-ter lentiviral transduction,the UL54 gene was integrated into the PK-15 cell genome.A cell line(PK-15-UL54)overexpressing UL54 protein was successfully constructed and characterized by PCR,indirect immunofluorescence assay and protein immunoblotting.Replication was assayed in the PK-15-UL54 cell line after inoculation with UL54 gene deletion virus(rPRV-△UL54-Re).Elec-tron microscopy observation showed that the rPRV-△UL54-Re virus had a complete viral particle structure.Fluorescence microscopy observations showed that rPRV-△UL54-Re virus could normal-ly infect cells and replicate.The results of quantitative PCR assay showed that the replication efficiency of rPRV-△UL54-Re virus increased.Therefore,PK-15-UL54 cell line is an important tool to improve the replication efficiency of UL54 gene deletion recombinant pseudorabies virus and plays an important role in exploring the mechanistic study of UL54 gene regulation of PRV repli-cation.

Objective To analyze the correlation between the Patellotrochlear index at different knee flexion angles and recurrent patellar dislocation,and to find the optimal knee flexion angle and the best diagnostic cut-off value for predicting recurrent patellar dislocation.Methods A total of 35 patients clinically diagnosed with recurrent patellar dislocation(study group)were selected.The lengths of the central sagittal plane of the patellar cartilage and the corresponding femoral trochlear cartilage were measured at knee flexion angles ranging from 0° to 70°,in 10° increments.The Patellotrochlear index was calculated and compared with the MRI data from 55 healthy volunteers(control group).Results The Patellotrochlear index in the study group and the control group showed a positive correlation with the knee flexion angle(r=0.935 and 0.909,P＜0.001).Statistically significant differences were found between the two groups at knee flexion angles of 0°,10°,20°,30°,and 40°(t=7.480,5.742,5.200,3.558,4.271,P＜0.05).The predictive efficacy of the Patellotrochlear index for recurrent patellar dislocation varied at different knee flexion angles,with the area under the curve(AUC)and the best diagnostic cut-off values of 0.891,0.834,0.778,0.710,0.741 and 0.195,0.323,0.486,0.606,0.712,respectively.Conclusion There are significant differences in the Patellotrochlear index at different knee flexion angles.A knee flexion angle of 0° is the optimal knee flexion angle for the Patellotrochlear index to predict recurrent patellar dislocation,with a recommended best diagnostic cut-off value of 0.195.

Multi-conjugate vaccine and polyvalent vaccine can reduce the number of vaccinations, improve vaccination efficiency, and provide wider protection against diseases, and can not only brings convenience to recipients but also reduce healthcare costs, making it a key focus in modern vaccine development. However, even if the components of the vaccine are derived from already approved monovalent vaccines, it must still be considered as a new vaccine and undergo randomized controlled clinical trials to evaluate their safety and efficacy in humans. Due to the inclusion of multiple antigens, clinical evaluation must consider the potential interactions between or among the components, as well as the impacts of adjuvants, preservatives, and other ingredients on the vaccines' safety and efficacy. These factors introduce certain specific challenges in the clinical evaluation of multi-conjugate vaccine and polyvalent vaccine. This article summarizes the key elements and methods of clinical study design for multi-conjugate vaccine and polyvalent vaccine in terms of safety, immunogenicity, and protective efficacy, and discuss the problems and challenges exisitng in vaccine clinical evaluation to provide reference for the standardization of clinical study design of multi-conjugate vaccine and polyvalent vaccine.

Objective The study aims to investigate the immunological functions of the nucleocapsid (N) protein of the novel coronavirus Omicron (BA.1, BA.2) and evaluate the differences among different N proteins of mutant strains in immunogenicity. Methods By aligning sequences, the mutation sites of the Omicron (BA.1, BA.2) N protein relative to prototype strain of the novel coronavirus (Wuhan-Hu-1) were determined. The pET-28a-N-Wuhan-Hu-1 plasmid was used as template to construct pET-28a-BA.1/BA.2-N through single point mutation or homologous recombination. The three kinds of N protein were expressed in prokaryotic system, purified through Ni-NTA affinity chromatography, and then immunized into mice. The titer and reactivity of the polyclonal antibody, as well as the expression level of IL-1β and IFN-γ in mouse spleen cells, were detected using indirect ELISA and Western blot assay. Results The constructed prokaryotic expression plasmids were successfully used to express the Wuhan-Hu-1 N, BA.1 N, and BA.2 N proteins in E.coli BL21(DE3) at 37 DegreesCelsius for 4 hours. The indirect ELISA test showed that the titers of polyclonal antibody prepared by three N proteins were all 1:51 200. All three N proteins can increase the expression of IFN-γ and IL-1β cytokines, but the effect of Omicron N protein in activing two cytokines was more obvious than that of Wuhan-Hu-1 N protein. Conclusion The study obtained three new coronavirus N proteins and polyclonal antibodies, and confirmed that mutations in the amino acid sites of the N protein can affect its immunogenicity. This provides a basis for developing rapid diagnostic methods targeting N protein of different novel coronavirus variants.
Animals ; Mice ; SARS-CoV-2/genetics* ; Coronavirus Nucleocapsid Proteins/immunology* ; Nucleocapsid Proteins/isolation & purification* ; COVID-19/immunology* ; Antibodies, Viral/immunology* ; Mice, Inbred BALB C ; Interferon-gamma/metabolism* ; Interleukin-1beta/metabolism* ; Female ; Escherichia coli/metabolism* ; Mutation ; Humans