A recombinant adenovirus(rAd)for expression of Mycobacterium tuberculosis(M.tb)c-di-AMP phosphodiesterase CnpB was constructed,and its induced humoral immune response was detected.The codon-optimized gene of M.tb CnpB was cloned into the adenoviral plasmid pcADV.The recombinant plasmid pcADV-CnpB was transfected into HEK293T cells,and expression was detected with Western blot.The recombinant plasmid pcADV-CnpB and the backbone plasmid were co-transfected into HEK293T cells to obtain the recombinant adenovirus rAd-CnpB.rAd-CnpB was amplified in HEK293T cells,and the target protein expression of rAd-CnpB was detected with Western blot and immunofluorescence.Mice were immunized with rAd-CnpB intranasally,and their sera and bronchoalveolar lavage fluid(BALF)were collected.ELISA was used to detect levels of antigen-specific antibodies.Restriction enzyme digestion and sequencing indicated that the recombinant plasmid pcADV-CnpB was successfully constructed and led to protein expression in eukaryotic cells.rAd-CnpB was packaged and produced in HEK293T cells.After amplification and purification,rAd-CnpB with a titer of 5.53×1010 PFU/mL was obtained.rAd-CnpB led to CnpB expression in HEK293T cells.Intranasal immunization with rAd-CnpB increased levels of IgG and secretory IgA in BALF and led to high levels of IgG in sera.rAd-CnpB,the recombinant adenovirus for expression of c-di-AMP phosphodiesterase CnpB was successfully constructed,and was found to induce antigen-specific humoral and mucosal immune responses through mucosal immunization.Thus,rAd-CnpB may be used in further research on new TB vaccine strategies.

Objective:To establish a mouse model of liver cancer resistant to PD-1 monoclonal antibody and analyze the changes in its metabolomics to explore the potential mechanism of drug resistance.Methods:BALB/c mice were randomly divided into control and treatment groups after being loaded with tumor,and a normal group was additionally set up.The normal and control groups were injected with saline,and the treatment group was injected with PD-1 monoclonal antibody,after which the mice in the treatment group were screened for drug resistant and response groups.Observed the drug-resistant situation,body mass,tumor growth and survival rate of mice in each group,calculate the spleen index.The pathological features of tumor tissues were observed by HE staining method.Serum metabolites were detected by non-targeted metabolomics.Finally,a bivariate Pearson correlation analysis was conducted between the differential serum metabolites and tumor size.Results:The tumor-bearing mouse model with PD-1 monoclonal antibody resistance was successfully established,and the drug resistance rate of the mice was 50％.Compared with the normal and response groups,mice in the resistant group showed an increase in body weight,a significant increase in tumor volume,a decrease in survival rate,and a significant increase in splenic index.There was less lymphocyte infiltration in the tumor tissue.Metabolomics analysis showed that the serum levels of glutamic acid and aspartic acid increased and malic acid decreased in the resistant mice compared with the response group,and these changes were closely related to the arginine biosynthesis pathway.Conclusions:The tumor-bearing mouse model with PD-1 monoclonal antibody resistance was successfully established.The changes in its peripheral serum metabolomics mainly involve arginine metabolism and the related changes of aspartate,malate and glutamate.

The application of mechanics in clinical wound healing has a long history;however,the systematic underlying mechanisms remain unclear.With recent advancements in biomechanics and mechanobiology,the principles regarding how mechanical factors influence the formation,progression,and healing of wounds have gradually been elucidated.Herein,based on progress in theories,technologies,and clinical practices concerning the interplay between mechanics and wound healing,this study introduces the concept of wound-repairing mechanomedicine.Relevant research is systematically reviewed from the perspectives of biomechanics,mechanobiology,and mechanotherapy.Additionally,potential future development directions are prospectively analyzed to provide novel insights into wound care and strategies for preventing scar formation.

Objective:To establish a mouse model of liver cancer resistant to PD-1 monoclonal antibody and analyze the changes in its metabolomics to explore the potential mechanism of drug resistance.Methods:BALB/c mice were randomly divided into control and treatment groups after being loaded with tumor,and a normal group was additionally set up.The normal and control groups were injected with saline,and the treatment group was injected with PD-1 monoclonal antibody,after which the mice in the treatment group were screened for drug resistant and response groups.Observed the drug-resistant situation,body mass,tumor growth and survival rate of mice in each group,calculate the spleen index.The pathological features of tumor tissues were observed by HE staining method.Serum metabolites were detected by non-targeted metabolomics.Finally,a bivariate Pearson correlation analysis was conducted between the differential serum metabolites and tumor size.Results:The tumor-bearing mouse model with PD-1 monoclonal antibody resistance was successfully established,and the drug resistance rate of the mice was 50％.Compared with the normal and response groups,mice in the resistant group showed an increase in body weight,a significant increase in tumor volume,a decrease in survival rate,and a significant increase in splenic index.There was less lymphocyte infiltration in the tumor tissue.Metabolomics analysis showed that the serum levels of glutamic acid and aspartic acid increased and malic acid decreased in the resistant mice compared with the response group,and these changes were closely related to the arginine biosynthesis pathway.Conclusions:The tumor-bearing mouse model with PD-1 monoclonal antibody resistance was successfully established.The changes in its peripheral serum metabolomics mainly involve arginine metabolism and the related changes of aspartate,malate and glutamate.

The application of mechanics in clinical wound healing has a long history;however,the systematic underlying mechanisms remain unclear.With recent advancements in biomechanics and mechanobiology,the principles regarding how mechanical factors influence the formation,progression,and healing of wounds have gradually been elucidated.Herein,based on progress in theories,technologies,and clinical practices concerning the interplay between mechanics and wound healing,this study introduces the concept of wound-repairing mechanomedicine.Relevant research is systematically reviewed from the perspectives of biomechanics,mechanobiology,and mechanotherapy.Additionally,potential future development directions are prospectively analyzed to provide novel insights into wound care and strategies for preventing scar formation.

Objective To prepare a stable rat model of chronic liver failure to provide a tool for basic research.Methods Sixty-six SPF SD rats were divided into a normal group(n=18)and a modeling group(n=48).Rats in the modeling group received an intraperitoneal injection of 50％CCl4 olive oil solution(1.5 mL/kg,twice a week).Multidimensional assessment was performed at 8,16,and 24 weeks,respectively,including ultrasonic examination of liver morphology,hardness,portal vein diameter,and ascites,and collection of serum,plasma,and liver tissue to detect liver function,coagulation function,and blood ammonia levels.Liver tissue injury and fibrosis were observed by hematoxylin-eosin(HE)and Masson staining.Cognitive function was assessed using the water maze test.Survival were recorded simultaneously.Results Rats in the model group showed decreased activity and appetite,yellow urine,and increased abdominal circumference compared with the normal group.Ultrasound showed enhanced liver parenchyma echo in the model group that thickened with time,secondary ascites formation,portal vein dilation,and portal hypertension.Water maze and blood ammonia tests confirmed cognitive decline(memory and orientation loss)and hepatic encephalopathy in the model group.Gross observation showed that the liver in the model group was atrophied and appeared rough and uneven.HE staining showed hepatocyte swelling,steatosis,and necrosis,and Masson staining confirmed fibrosis progression with pseudolobule formation.The liver function indexes AST,ALT,TBIL and blood ammonia continued to increase,and coagulation dysfunction(prolonged PT and increased INR)gradually increased with the modeling process.Conclusions Intraperitoneal injection of 50％CCl4 olive oil solution(1.5 mL/kg,every week)for 24 weeks can stably simulate persistent chronic liver injury in rats and lead to the typical pathological changes and complications of chronic liver failure,based on the decompensation stage of cirrhosis.This model replicates the pathological evolution of human hepatitis from liver fibrosis → liver cirrhosis compensation → decompensation → chronic liver failure,providing a reliable modeling reference for the study of the mechanism of chronic liver failure.

Objective To develop and validate a transgenic mouse model enabling specific and inducible optogenetic activation of oligodendrocyte precursor cells(OPCs).Methods A conditional allele for the photosensitive opsin chicken opsin 5(cOpn5)(Rosa26-LSL-cOpn5)was generated using CRISPR/Cas9 technology.These mice were subsequently crossed with NG2-CreERT transgenic mice to produce NG2-CreERT;cOpn5 animals.In this model,tamoxifen administration induces Cre-mediated recombination,leading to specific expression of cOpn5 in NG2-positive OPCs.The specificity and efficiency of cOpn5 expression in OPCs were confirmed by immunofluorescent staining.Functional validation of light-induced OPC activation was performed by using calcium imaging in acute brain slices after stimulation with 470 nm blue light.Results Immunofluorescence analysis confirmed robust and specific expression of cOpn5 within NG2-positive OPCs in the brains of tamoxifen-treated NG2-CreERT;cOpn5 mice.Crucially,calcium imaging of acute brain slices from these mice demonstrated a significant increase in intracellular calcium levels in cOpn5-expressing OPCs upon stimulation with 470 nm blue light,indicating successful optogenetic activation.Conclusion We have successfully generated and validated a novel transgenic mouse model(NG2-CreERT;cOpn5)that permits specific and inducible optogenetic activation of OPCs.This model provides a novel tool for subsequent in vivo studies of the role and regulating mechanisms of OPCs in the central nervous system.

Objective To investigate the expression of NOP2/Sun RNA methyltransferase family member 2(NSUN2)and protein arginine methyltransferase 5(PRMT5)in retinoblastoma(Rb)tissue and their correlation with the survival prognosis of Rb patients.Methods From February 2019 to February 2021,84 patients with Rb(Rb group)were selected from Beijing Anzhen Hospital Affiliated to Capital Medical University Nanchong Hospital,and 50 normal retinal tissues were used as control group.Real-time fluorescence quantitative PCR(qRT-PCR)was used to detect the levels of NSUN2 mRNA and PRMT5 mRNA in retinal tissues.Kaplan-Meier curve was used to analyze the effect of NSUN2 mRNA and PRMT5 mRNA on the prognosis of Rb patients.COX regression analysis was used to analyze the factors affecting the prognosis of Rb.Results The expression of NSUN2 mRNA(3.11±0.42)and PRMT5 mRNA(2.84±0.39)in Rb cancer tissues were higher than that in the control group(0.80±0.23,0.76±0.20),and the differences were statistically significant(t=35.935,35.033,all P<0.001).The expression of NSUN2 mRNA and PRMT5 mRNA in Rb cancer tissues with tumor diameter≥20mm,undifferentiated and ⅡRC stage D～E were higher than that in tumor diameter<20mm,differentiated tissues and ⅡRC stage A～C cancer tissues,and the differences were statistically significant(t=18.297,141.770,16.693;18.663,139.144,39.947,均P<0.001).The 3-year progression-free survival rate in the high expression group of NSUN2 mRNA was lower than that in the low expression group(60.00%vs 88.64%).The 3-year progression-free survival rate in the high expression group of PRMT5 mRNA was lower than that in the low expression group(56.10%vs 93.02%)(Log rank χ2=13.440,19.501,all P<0.001).Undifferentiated type,ⅡRC stage D-E stage,NSUN2 mRNA high,PRMT5 mRNA high were risk factors affecting the prognosis of Rb patients(Waldχ2=5.923～7.161,all P<0.001).Conclusion The expression of NSUN2 mRNA and PRMT5 mRNA in Rb tissues is increased,which is related to the maximum diameter of tumor,pathological classification and ⅡRC stage,and can be used as a tumor marker to evaluate the survival prognosis of Rb.

Objective To develop and validate a transgenic mouse model enabling specific and inducible optogenetic activation of oligodendrocyte precursor cells(OPCs).Methods A conditional allele for the photosensitive opsin chicken opsin 5(cOpn5)(Rosa26-LSL-cOpn5)was generated using CRISPR/Cas9 technology.These mice were subsequently crossed with NG2-CreERT transgenic mice to produce NG2-CreERT;cOpn5 animals.In this model,tamoxifen administration induces Cre-mediated recombination,leading to specific expression of cOpn5 in NG2-positive OPCs.The specificity and efficiency of cOpn5 expression in OPCs were confirmed by immunofluorescent staining.Functional validation of light-induced OPC activation was performed by using calcium imaging in acute brain slices after stimulation with 470 nm blue light.Results Immunofluorescence analysis confirmed robust and specific expression of cOpn5 within NG2-positive OPCs in the brains of tamoxifen-treated NG2-CreERT;cOpn5 mice.Crucially,calcium imaging of acute brain slices from these mice demonstrated a significant increase in intracellular calcium levels in cOpn5-expressing OPCs upon stimulation with 470 nm blue light,indicating successful optogenetic activation.Conclusion We have successfully generated and validated a novel transgenic mouse model(NG2-CreERT;cOpn5)that permits specific and inducible optogenetic activation of OPCs.This model provides a novel tool for subsequent in vivo studies of the role and regulating mechanisms of OPCs in the central nervous system.

Objective:To investigate the gut microbiota and metabolic profiles of patients with neuromyelitis optica spectrum disorder (NMOSD) and myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD), and to identify potential microbial biomarkers with diagnostic values.Methods:A total of 16 NMOSD patients, 6 MOGAD patients, and 22 age- and sex-matched healthy controls were recruited from Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology since June 2024. Fecal samples were subjected to metagenomic sequencing and untargeted metabolomics. Differential microbes were identified using LEfSe (linear discriminant analysis effect size), and receiver operating characteristic curve analysis was performed to evaluate diagnostic potential. Spearman correlation analysis was used to assess relationships between key microbes, metabolites, and serum antibody titers.Results:Distinct alterations in gut microbiota were observed in both disease groups compared with healthy controls. Ligilactobacillus salivarius was significantly enriched in both NMOSD and MOGAD patients and exhibited robust diagnostic accuracy (area under the curve=0.779 P=0.005). Metabolomics revealed that levels of ethosuximide and lysine-proline were elevated, while free fatty acids (15∶1) and 5, 6-dihydrothymine were reduced in the disease groups. Analysis results indicated that Ligilactobacillus salivarius abundance was positively correlated with aquaporin 4 antibody titers in NMOSD patients ( r=0.522, P=0.046). Conclusions:Patients with NMOSD and MOGAD have characteristic alterations in gut microbial and metabolic profiles.