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

Objective:To evaluate the efficacy and safety of transhepatic arterial chemoembolization (TACE) combined with tyrosine kinase inhibitors (TKI) and programmed death-1 (PD-1) inhibitors in the treatment of patients with initially unresectable hepatocellular carcinoma.Methods:The clinical data of 42 patients with initially unresectable hepatocellular carcinoma who were admitted to the Department of Hepatobiliary and Pancreatic Surgery of the First Affiliated Hospital of Zhengzhou University from January 2020 to December 2022 were included. There were 31 males and 11 females, with a median age of 56 years old (range, 45-72 years old). All patients received TACE+ TKI+ PD-1 inhibitor combined treatment. The systemic treatment cycles were calculated by the regimen of immunotherapy. The timing of local treatment depends on tumor size, blood supply and treatment response. Patients were followed up through hospitalization, outpatient visits and telephone review. The Kaplan-Meier curves were obtained for survival analysis.Results:The dosing cycle to achieve optimal imaging response in the patients was 4 (3, 7) [ M( Q1, Q3)], with a systemic treatment time of 141 (65, 194) d [ M( Q1, Q3)] and 2 (1, 3) times [ M( Q1, Q3)] of local treatments. All patients were evaluated by modified response evaluation criteria in solid tumors criteria after treatment, including nine patients with complete response (CR), 21 with partial response, eight with stable disease, and four with progressive disease. Objective response rate and disease control rate were 71.4% (30/42) and 90.5% (38/42), respectively. Treatment-related adverse reactions occurred in 85.7% (36/42) of patients and grade Ⅲ or Ⅳ adverse reactions occurred in 16.7% (7/42). There was no level Ⅳ adverse reactions. All adverse reactions were controlled after dose reduction and symptomatic treatment. Thirteen patients (31.0%, 13/42) redeemed resectable after treatment and underwent radical surgery. Seven patients had pathological CR after surgery. In two patients, the pathological residual cancer tissue was less than 10%. The cumulative overall survival rates of the 42 patients at 6 months, 1 year, 1.5 years after treatment were 100%, 91.7%, and 65.0%, respectively. The postoperative 1-year survival rate of patients undergoing surgery after successful conversion was 83.3%. Conclusion:This study preliminarily showed the safety and efficacy of TACE, TKI, and PD-1 inhibitor combined therapy in patients with initially unresectable hepatocellular carcinoma.

Objective: This study aimed to explore the feasibility of functional evaluation of the lateral pterygoid muscle (LPM) using diffusion tensor imaging (DTI) in patients with temporomandibular joint disorders (TMDs). Materials and Methods: A total of 119 patients with TMD (23 male and 96 female; mean age ± standard deviation, 41 ± 15 years; 58 bilateral and 61 unilateral involvements for a total of 177 joints) and 20 healthy volunteers (9 male and 11 female;40 ± 13 years; 40 joints) were included in this prospective study. Based on DTI of the jaw in the resting state, the diffusion parameters, apparent diffusion coefficient (ADC), fractional anisotropy (FA), λ1, λ2, and λ3 of the superior and inferior heads of the LPM (SHLPM and IHLPM) were measured. Patients with TMD with normal disc position (ND), anterior disc displacement with reduction (ADWR), and anterior disc displacement without reduction (ADWOR) were compared. Results: Patients with TMD overall, and ADWR and ADWOR subgroups had significantly higher ADC, λ1, λ2, and λ3 in both the SHLPM and IHLPM than those in volunteers (p < 0.05 for all), whereas the ND subgroup only had significantly higher ADC and λ1 (p < 0.001). Meanwhile, significant differences in FA in the SHLPM and IHLPM were found between volunteers and ADWOR (p = 0.014 and p = 0.037, respectively). Among the three TMD subgroups, except for λ3 and FA in the ADWR subgroup, ADWR and ADWOR subgroups had significantly higher ADC, λ1, λ2, and λ3 and lower FA than those in the ND group (p < 0.050). There was no significant difference in diffusion variables between ADWR and ADWOR. In ADWOR, the osteoarthritis group had significantly higher λ3 and lower FA values in the IHLPM than those in the non-osteoarthritis group. Conclusion DTI successfully detected functional changes in the LPM in patients with TMD. The unsynchronized diffusivity changes in the LPM in different subgroups of TMD signified the possibility of using diffusion parameters as indicators to identify the severity of LPM hyperfunction at various stages of TMD.