Objective To analyze the expression levels of matrix metalloproteinase-10(MMP-10)and Toll-like receptor 2(TLR2)in serum of patients underwent decompression surgery(DC)for severe traumatic brain injury(sTBI),and to explore their relationship with disease outcome.Methods From April 2021 to April 2024,sTBI patients(n=94)who received DC treatment in a single center were collected as the observation group.Another 90 healthy volunteers who underwent physical examinations at our hospital were selected as the control group.Six months after surgery,sTBI patients were assigned into the good group(n=53)and the adverse group(n=41)according to the Glasgow Outcome Scale(GOS).Data was collected from each group and their differences were compared.Enzyme linked immunosorbent assay(ELISA)was used to measure serum levels of MMP-10 and TLR2.Spearman method was used to analyze the correlation between MMP-10,TLR2 levels and disease outcomes.Logistic regression model used to analyze influencing factors of disease outcomes in sTBI patients after DC.The receiver operating characteristic(ROC)curve was applied to evaluate the predictive value of serum MMP-10 and TLR2 levels for disease outcome in sTBI patients after DC.Results Compared with the control group,the expression levels of serum MMP-10 and TLR2 were prominently higher in the observation group(P＜0.05).Compared with the good group,the proportions of sTBI patients with cerebral herniation,multiple brain contusions and lacerations,and serum levels of MMP-10 and TLR2 were significantly higher in the adverse group,while Glasgow Coma Scale(GCS)score was significantly lower(P＜0.05).Serum levels of MMP-10 and TLR2 in sTBI patients were positively correlated with poor prognosis after DC(P＜0.05).Elevated levels of serum MMP-10 and TLR2,and the increased proportions of patients with cerebral herniation and multiple brain contusions were risk factors affecting the disease outcome after DC in sTBI patients,while elevated GCS score was a protective factor(P＜0.05).The area under the curve(AUC)for predicting disease outcome in sTBI patients after DC using serum MMP-10 and TLR2 alone and in combination was 0.839(95％CI:0.749-0.907),0.847(95％CI:0.758-0.913)and 0.925(95％CI:0.852-0.969),respectively.The combined detection was superior to the individual detections(Zcombination-MMP-10=2.199,Zcombination-TLR2=2.377,both P＜0.05).Conclusion The expression levels of serum MMP-10 and TLR2 in sTBI patients are significantly elevated,and both are prominently correlated with disease outcome after DC.

A long-acting feline ω-interferon fusion protein(FSA-FeIFNω)was designed and its bio-logical function validated.According to the optimization of the sequence of feline serum albumin and feline ω interferon in NCBI,the recombinant plasmid pET-30a(+)-FSA-FeIFNω was con-structed,which was transformed into E.coli BL21(DE3)competent cells,the expression of re-combinant protein FSA-FeIFNω was induced by IPTG,and the expressed inclusion body protein was identified by Western blot,the refolding product was purified by Ni-NTA affinity chromatog-raphy,and the concentration of dialysis and concentrated protein after purification was determined by BCA method.The antiviral activity of recombinant protein was detected by micro-cytopathic in-hibition method in the CRFK/VSV system,the in vitro half-life was detected by 50％mouse plas-ma method,the tumor cell proliferation inhibition activity was detected by MTT method,and anti-tumor activity was detected by mouse melanoma model.The pET-30a(+)-FeIFNω and pET-30a(+)-FSA-FeIFNω expression vectors were successfully constructed,and 87 kDa recombinant FSA-FeIFNω protein was obtained in E.coli,with a purified protein purity of 95％,with a concen-tration of 1 g/L,and the biological activity was 2.56 × 106 IU/mg,the plasma half-life was significantly prolonged(＞24 h),and the half-inhibitory concentration IC50 of B16-F10 in mouse melanoma cells was 56.01 mg/L.The FSA-FeIFNω group significantly inhibited tumor growth,and the treatment effect was better than that of the control group and other experimental groups.The recombinant FSA-FeIFNω protein obtained in this study had long-acting effect and good biological activity.

This study aims to construct a recombinant vaccine containing the tetanus toxin C frag-ment(HC)with a built-in adjuvant of metallothionein 3(MT-3)and evaluate its immune effect in mice.The gene sequences of MT-3 and HC were fused via a linker to create the pET30a-MT-3-HC recombinant plasmid,which was then transformed into E.coli BL21(DE3)competent cells.The re-combinant plasmid was confirmed through double enzyme digestion.The M3C recombinant protein expression was induced,identified by SDS-PAGE and Western blot,and purified using Ni-NTA af-finity chromatography.Five groups of vaccines,including PBS,HC,TT(tetanus toxoid),M3C,and M3C+CpG(composite adjuvant),were administered to mice via intramuscular injection at 7-day intervals for three immunizations.Blood samples were periodically collected from the tail vein.ELISA was used to measure changes in specific IgG antibody titers in the serum,and on day 28,antibody subtypes(IgG1,IgG2a,IgG2b,IgG3,and IgM)and cytokine levels(IL-4,IFN-γ)were also measured.The results demonstrated that the pET30a-MT-3-HC recombinant plasmid was cor-rectly constructed,and the M3C recombinant protein was highly expressed in the supernatant fol-lowing ultrasonic disruption of the induced bacterial culture,with a single band observed post-puri-fication.ELISA results showed that the titer of specific IgG antibodies in the M3C+CpG group peaked at 3.54×105 14 days after the third immunization,which was 141 times,70 times,and 6.6 times higher than that in HC,TT,and TT groups,respectively.Antibody subtype analysis revealed significantly higher specific antibody subtype titers in the M3C and M3C+CpG groups compared to the PBS,HC,and TT groups(P＜0.05),with the M3 C group showing an IgG1/IgG2a ratio greater than 1,and the M3C+CpG group having an IgG1/IgG2a ratio of approximately 1.Serum concentrations of IFN-γ and IL-4 in the M3C and M3C+CpG groups were also significantly higher than those in the other experimental groups(P＜0.05).These results showed that the recombinant antigen containing MT-3 built-in adjuvant and tetanus toxin C fragment was successfully expressed and showed strong immunogenicity,which laid the experimental foundation for the development of this recombinant vaccine.

To develop a novel immunocastration vaccine for animals,researchers designed and syn-thesized the recombinant plasmid pET-30a-SF which could express the recombinant protein SF.This protein was then conjugated in vitro with the synthetic peptide STGP to prepare the SF-STGP nanoparticle vaccine,and its immunocastration effect on mice was studied.The Spy Catcher and ferritin amino acid sequences were connected via GGGGS,and after codon optimization for E.coli,the recombinant plasmid pET-30a-SF was constructed and transformed into E.coli for in-duced expression.The recombinant protein SF was purified using Ni-column affinity chromatogra-phy and characterized.The peptide STGP,composed of Spy Tag,GnRH,and PADRE connected by GGGS,was conjugated with the recombinant protein SF in vitro.The self-assembled nanoparticles were observed using transmission electron microscopy(TEM)and dynamic light scattering(DLS).The prepared SF-STGP nanoparticles were mixed with MONTANIDE ISA 206 VG at a 1∶1 ratio to form the vaccine,which was then subcutaneously injected into male BALB/c mice for immunocastration evaluation.The recombinant protein SF showed the highest soluble expression when induced at 18 ℃ with 0.25 mmol/L IPTG for 14 h,and the maximum conjugation efficiency with STGP was achieved at a 1∶8 molar ratio.TEM and DLS analyses revealed that both the re-combinant protein SF and SF-STGP could self-assemble into nanoparticles with average diameters of 16.2 nm and 17.8 nm,respectively.Mouse immunization results demonstrated that the SF-STGP nanoparticle vaccine generated specific GnRH antibodies after the first immunization,with the spe-cific antibody D45o reaching its peak at the 10 th week.The SF-STGP+ISA 206 immunization group showed a peak D450 value of 2.8,and the specific antibody levels in all immunization groups were significantly higher than those in the control group(P＜0.05).Additionally,the SF-STGP nanoparticle vaccine effectively reduced serum testosterone levels in mice,with the testosterone concentration in the immunization groups being significantly lower than that in the control group(P＜0.05).Compared to the control group,the immunization group exhibits testicular atrophy.The constructed SF-STGP nanoparticle vaccine proves to be a highly effective immunogen,capable of inducing testicular atrophy and reducing gonadal hormone concentrations,demonstrating excellent castration effects.This study provides new insights into immunocastration vaccines for mammals.

Objective To analyze the expression levels of matrix metalloproteinase-10(MMP-10)and Toll-like receptor 2(TLR2)in serum of patients underwent decompression surgery(DC)for severe traumatic brain injury(sTBI),and to explore their relationship with disease outcome.Methods From April 2021 to April 2024,sTBI patients(n=94)who received DC treatment in a single center were collected as the observation group.Another 90 healthy volunteers who underwent physical examinations at our hospital were selected as the control group.Six months after surgery,sTBI patients were assigned into the good group(n=53)and the adverse group(n=41)according to the Glasgow Outcome Scale(GOS).Data was collected from each group and their differences were compared.Enzyme linked immunosorbent assay(ELISA)was used to measure serum levels of MMP-10 and TLR2.Spearman method was used to analyze the correlation between MMP-10,TLR2 levels and disease outcomes.Logistic regression model used to analyze influencing factors of disease outcomes in sTBI patients after DC.The receiver operating characteristic(ROC)curve was applied to evaluate the predictive value of serum MMP-10 and TLR2 levels for disease outcome in sTBI patients after DC.Results Compared with the control group,the expression levels of serum MMP-10 and TLR2 were prominently higher in the observation group(P＜0.05).Compared with the good group,the proportions of sTBI patients with cerebral herniation,multiple brain contusions and lacerations,and serum levels of MMP-10 and TLR2 were significantly higher in the adverse group,while Glasgow Coma Scale(GCS)score was significantly lower(P＜0.05).Serum levels of MMP-10 and TLR2 in sTBI patients were positively correlated with poor prognosis after DC(P＜0.05).Elevated levels of serum MMP-10 and TLR2,and the increased proportions of patients with cerebral herniation and multiple brain contusions were risk factors affecting the disease outcome after DC in sTBI patients,while elevated GCS score was a protective factor(P＜0.05).The area under the curve(AUC)for predicting disease outcome in sTBI patients after DC using serum MMP-10 and TLR2 alone and in combination was 0.839(95％CI:0.749-0.907),0.847(95％CI:0.758-0.913)and 0.925(95％CI:0.852-0.969),respectively.The combined detection was superior to the individual detections(Zcombination-MMP-10=2.199,Zcombination-TLR2=2.377,both P＜0.05).Conclusion The expression levels of serum MMP-10 and TLR2 in sTBI patients are significantly elevated,and both are prominently correlated with disease outcome after DC.

A long-acting feline ω-interferon fusion protein(FSA-FeIFNω)was designed and its bio-logical function validated.According to the optimization of the sequence of feline serum albumin and feline ω interferon in NCBI,the recombinant plasmid pET-30a(+)-FSA-FeIFNω was con-structed,which was transformed into E.coli BL21(DE3)competent cells,the expression of re-combinant protein FSA-FeIFNω was induced by IPTG,and the expressed inclusion body protein was identified by Western blot,the refolding product was purified by Ni-NTA affinity chromatog-raphy,and the concentration of dialysis and concentrated protein after purification was determined by BCA method.The antiviral activity of recombinant protein was detected by micro-cytopathic in-hibition method in the CRFK/VSV system,the in vitro half-life was detected by 50％mouse plas-ma method,the tumor cell proliferation inhibition activity was detected by MTT method,and anti-tumor activity was detected by mouse melanoma model.The pET-30a(+)-FeIFNω and pET-30a(+)-FSA-FeIFNω expression vectors were successfully constructed,and 87 kDa recombinant FSA-FeIFNω protein was obtained in E.coli,with a purified protein purity of 95％,with a concen-tration of 1 g/L,and the biological activity was 2.56 × 106 IU/mg,the plasma half-life was significantly prolonged(＞24 h),and the half-inhibitory concentration IC50 of B16-F10 in mouse melanoma cells was 56.01 mg/L.The FSA-FeIFNω group significantly inhibited tumor growth,and the treatment effect was better than that of the control group and other experimental groups.The recombinant FSA-FeIFNω protein obtained in this study had long-acting effect and good biological activity.

This study aims to construct a recombinant vaccine containing the tetanus toxin C frag-ment(HC)with a built-in adjuvant of metallothionein 3(MT-3)and evaluate its immune effect in mice.The gene sequences of MT-3 and HC were fused via a linker to create the pET30a-MT-3-HC recombinant plasmid,which was then transformed into E.coli BL21(DE3)competent cells.The re-combinant plasmid was confirmed through double enzyme digestion.The M3C recombinant protein expression was induced,identified by SDS-PAGE and Western blot,and purified using Ni-NTA af-finity chromatography.Five groups of vaccines,including PBS,HC,TT(tetanus toxoid),M3C,and M3C+CpG(composite adjuvant),were administered to mice via intramuscular injection at 7-day intervals for three immunizations.Blood samples were periodically collected from the tail vein.ELISA was used to measure changes in specific IgG antibody titers in the serum,and on day 28,antibody subtypes(IgG1,IgG2a,IgG2b,IgG3,and IgM)and cytokine levels(IL-4,IFN-γ)were also measured.The results demonstrated that the pET30a-MT-3-HC recombinant plasmid was cor-rectly constructed,and the M3C recombinant protein was highly expressed in the supernatant fol-lowing ultrasonic disruption of the induced bacterial culture,with a single band observed post-puri-fication.ELISA results showed that the titer of specific IgG antibodies in the M3C+CpG group peaked at 3.54×105 14 days after the third immunization,which was 141 times,70 times,and 6.6 times higher than that in HC,TT,and TT groups,respectively.Antibody subtype analysis revealed significantly higher specific antibody subtype titers in the M3C and M3C+CpG groups compared to the PBS,HC,and TT groups(P＜0.05),with the M3 C group showing an IgG1/IgG2a ratio greater than 1,and the M3C+CpG group having an IgG1/IgG2a ratio of approximately 1.Serum concentrations of IFN-γ and IL-4 in the M3C and M3C+CpG groups were also significantly higher than those in the other experimental groups(P＜0.05).These results showed that the recombinant antigen containing MT-3 built-in adjuvant and tetanus toxin C fragment was successfully expressed and showed strong immunogenicity,which laid the experimental foundation for the development of this recombinant vaccine.

To develop a novel immunocastration vaccine for animals,researchers designed and syn-thesized the recombinant plasmid pET-30a-SF which could express the recombinant protein SF.This protein was then conjugated in vitro with the synthetic peptide STGP to prepare the SF-STGP nanoparticle vaccine,and its immunocastration effect on mice was studied.The Spy Catcher and ferritin amino acid sequences were connected via GGGGS,and after codon optimization for E.coli,the recombinant plasmid pET-30a-SF was constructed and transformed into E.coli for in-duced expression.The recombinant protein SF was purified using Ni-column affinity chromatogra-phy and characterized.The peptide STGP,composed of Spy Tag,GnRH,and PADRE connected by GGGS,was conjugated with the recombinant protein SF in vitro.The self-assembled nanoparticles were observed using transmission electron microscopy(TEM)and dynamic light scattering(DLS).The prepared SF-STGP nanoparticles were mixed with MONTANIDE ISA 206 VG at a 1∶1 ratio to form the vaccine,which was then subcutaneously injected into male BALB/c mice for immunocastration evaluation.The recombinant protein SF showed the highest soluble expression when induced at 18 ℃ with 0.25 mmol/L IPTG for 14 h,and the maximum conjugation efficiency with STGP was achieved at a 1∶8 molar ratio.TEM and DLS analyses revealed that both the re-combinant protein SF and SF-STGP could self-assemble into nanoparticles with average diameters of 16.2 nm and 17.8 nm,respectively.Mouse immunization results demonstrated that the SF-STGP nanoparticle vaccine generated specific GnRH antibodies after the first immunization,with the spe-cific antibody D45o reaching its peak at the 10 th week.The SF-STGP+ISA 206 immunization group showed a peak D450 value of 2.8,and the specific antibody levels in all immunization groups were significantly higher than those in the control group(P＜0.05).Additionally,the SF-STGP nanoparticle vaccine effectively reduced serum testosterone levels in mice,with the testosterone concentration in the immunization groups being significantly lower than that in the control group(P＜0.05).Compared to the control group,the immunization group exhibits testicular atrophy.The constructed SF-STGP nanoparticle vaccine proves to be a highly effective immunogen,capable of inducing testicular atrophy and reducing gonadal hormone concentrations,demonstrating excellent castration effects.This study provides new insights into immunocastration vaccines for mammals.

0.05).The apoptotic percentage was increased significantly after X-rays irradiation with the dose of 6 Gy(P

Objective To study the effect of small heterocyclic compound 8-oxo-3-thiomorpholino-8H-acenaphtho pyrrole-9-carbonitrile(S1) on apoptosis of human androgen independent prostatic carcinoma cells line(PC3) and its mechanism.Methods PC3 cells were cultivated,and divided into different groups: 10.00,5.00,1.00,0.50,0.10,0.05 and 0.01 ?mol?L-1 S1 groups,meanwhile,PC3 control group and cyclophosphamide group were set up.MTT was used to detect the inhibitory rate of PC3 cell proliferation.Flow cytometry was used to detect the inducing effect of S1 on apoptosis of PC3 cells.Caspase 3,8,9 kits were used to detect apoptosis route.Results The inhibitory rates of PC3 cells induced by 0.10-10.00 ?mol?L-1 S1 were significantly higher than that in cyclophosphamide group(P