ObjectiveTo express and purify the endolysin PlyL from the Bacillus anthracis prophage LambdaBa02 in prokaryotic cells, and to explore its role in the detection of Bacillus anthracis, so as to provide a novel strategy for the specific and rapid detection of Bacillus anthracis.MethodsAccording to the sequence of Bacillus anthracis vaccine strain CVCC40205 registered in GenBank, the plyL gene was amplified by PCR with plyL gene-specific primers and inserted into the prokaryotic expression vector pET28a(+) to construct the recombinant expression plasmid pET28a(+)-His-plyL, which was then transformed into E.coli BL21(DE3) and induced by IPTG. The induced expression conditions were optimized to enhance soluble expression, and the recombinant PlyL protein was purified via nickel-affinity chromatography. The lytic activity of recombinant PlyL protein was assessed by measuring absorbance values, and an ELISA method was established to evaluate the sensitivity and specificity of Bacillus anthracis detection.ResultsThe recombinant expression plasmid pET28a(+)-His-plyL was confirmed to be correctly constructed by NdeⅠ and XhoⅠ double digestion and sequencing. The optimal induction conditions were 0. 5 mmol/L IPTG at 16 ℃ for 16 h. The relative molecular mass of the expressed recombinant PlyL protein was about 28 600. After purification and concentration, the final concentration of the protein was2. 55 mg/mL, and the purity reached 80. 6%. The purified recombinant PlyL protein could reduce the A_(600)value of Bacillus anthracis culture solution. The sensitivity of the preliminarily established ELISA method for detecting Bacillus anthracis was1 × 10~3 CFU/mL, and there was no cross-reactivity against closely related strains such as Bacillus cereus and Bacillus thuringiensis(P < 0. 000 1).ConclusionThe recombinant PlyL protein was successfully expressed and purified in prokaryotic cells, which exhibits both efficient lytic activity and species specificity, making it a promising novel tool for the rapid detection of Bacillus anthracis.

Anxiety disorder is a highly prevalent psychological illness, and research has shown that obesity is a significant risk factor for its development. This study explored the ameliorative effects and mechanisms of saponins from Panax japonicus(SPJ) on anxiety disorder in mice fed a high-fat diet(HFD). Fifty C57BL/6J mice were randomly divided into normal control diet(NCD) group, HFD group, and low-and high-dose SPJ groups. At week 12, six mice from the HFD group were further divided into a control group(treated with DMSO) and an exogenous fibroblast growth factor 21(FGF21) group(administered rFGF21). The anxiety-like behavior of the mice was assessed using the open field test and elevated plus maze test. Hematoxylin-eosin(HE) staining and oil red O staining were performed to observe pathological changes in the liver and adipose tissue. Glucose metabolism was evaluated through the glucose tolerance test(GTT) and insulin tolerance test(ITT). Western blot analysis was performed to detect the expression of FGF21 and its downstream-related proteins in the liver and cortex, along with the expression of brain-derived neurotrophic factor(BDNF), disks large homolog 4(DLG4), and synaptophysin(SYP) in the cortex. Real-time quantitative fluorescent PCR(qPCR) was used to detect the expression of FGF21 and its receptor genes in the liver and cortex. Immunofluorescence staining was employed to examine the expression of neuronal activator c-Fos, FGF21, and the FGF21 co-receptor β-klotho in the cerebral cortex. The results showed that SPJ significantly improved the frequency of activity in the open arms of the elevated plus maze and the central area of the open field in HFD mice, up-regulated the expression of BDNF, DLG4, and SYP, and effectively alleviated anxiety-like behaviors in HFD mice. Compared with the NCD group, HFD mice exhibited up-regulated expression of FGF21 in the liver and cerebral cortex, while the expression of fibroblast growth factor receptor 1(FGFR1) and β-klotho was significantly down-regulated, suggesting that HFD mice exhibited FGF21 resistance. SPJ markedly up-regulated the β-klotho levels in HFD mice, reversing FGF21 resistance. Further comparison with exogenously administered FGF21 revealed that SPJ activates brain cortical regions in a consistent manner, and additionally, SPJ promotes the number and colocalization of c-Fos and β-klotho positive cells in the brain cortex. In summary, SPJ effectively alleviates anxiety-like behaviors in HFD mice. Its mechanism is associated with up-regulation of β-klotho expression in the brain, reversal of FGF21 resistance, and subsequent activation of neurons in the cerebral cortex and amygdala.
Animals ; Diet, High-Fat/adverse effects* ; Fibroblast Growth Factors/genetics* ; Mice ; Male ; Panax/chemistry* ; Mice, Inbred C57BL ; Anxiety/etiology* ; Saponins/administration & dosage* ; Brain-Derived Neurotrophic Factor/genetics* ; Humans ; Liver/metabolism* ; Drugs, Chinese Herbal/administration & dosage*

Objective To screen the anti-CD22-specific nanobodies to provide a basis for immunotherapy agents. Methods The naive phage nanobody library was constructed and its diversity was analyzed. Three rounds of biotinylated streptavidin liquid phase screening were performed by using biotinylated CD22 antigen as the target, and the sequence of nanobodies against CD22 were identified by ELISA and gene sequencing. Results The capacity of the constructed naive phage nanobody library was 3.89×10⁹ CFU/mL, and the insertion of effective fragments was higher than 85%. Based on this library, seven anti-human CD22 nanobodies were screened, and the amino acid sequence comparison results showed that the overall similarity was 70.34%, and all of them were hydrophilic proteins. The results of protein-protein complex docking prediction showed that the mimetic proteins of the five nanobody sequences could be paired and linked to CD22, and the main forces were hydrophobic interaction and hydrogen bonding. Conclusion This study provided a basis for the study of chimeric antigen receptor T cells targeting CD22, successfully constructed the natural phage nanobody library and obtaining five anti-CD22-specific nanobodies.
Camelus/immunology* ; Single-Domain Antibodies/chemistry* ; Peptide Library ; Humans ; Animals ; Sialic Acid Binding Ig-like Lectin 2/genetics* ; Amino Acid Sequence ; Molecular Docking Simulation

The cellular and molecular components of the tumor immune microenvironment (TIME) and their information exchange processes significantly influence the trends of anti-tumor immunity. In recent years, numerous studies have begun to evaluate TIME in the context of previous cancer treatment strategies. This review will systematically summarize the compositional characteristics of TIME and, based on this foundation, explore the impact of current cancer therapies on the remodeling of TIME, aiming to provide new insights for the development of innovative immune combination therapies that can convert TIME into an anti-tumor profile.
Tumor Microenvironment/immunology* ; Humans ; Neoplasms/therapy* ; Immunotherapy/methods* ; Animals

Objective To prepare monoclonal antibodies against bovine CD4 and identify their basic biological characteristics. Methods Recombinant bovine CD4 (rHis-BoCD4 and rGST-BoCD4) was successfully expressed and purified by constructing a prokaryotic plasmid of bovine CD4 gene. The bovine CD4 monoclonal antibody was produced using hybridoma technology. The subtype and potency of the monoclonal antibody were identified and analyzed by ELISA, while specificity was analyzed through indirect immunofluorescence assay (IFA) and Western-blot. Results Four hybridoma cell lines, namely, 1H4, 6A10, 3F9 and 4G10, stably secreting monoclonal antibodies against BoCD4 were successfully obtained. The subclasses of the monoclonal antibodies subclass 6A10 was IgG2b and the rest of the monoclonal antibodies were of IgM type. Western-blot results showed that the four anti-bovine CD4 mAb strains were able to specifically bind to the bovine CD4 protein expressed in vitro. Indirect immunofluorescence assay showed that four monoclonal antibodies were able to specifically recognize the natural bovine CD4 protein. Flow cytometry assay showed that 3F9 was best to recognize bovine natural CD4 molecules. Conclusion Four monoclonal antibody strains with high specificity to natural bovine CD4 protein were successfully prepared, which lays the foundation for the subsequent studies on the function of bovine CD4 and diagnosis and treatment of bovine T-lymphocyte diseases.
Animals ; Antibodies, Monoclonal/isolation & purification* ; Cattle ; CD4 Antigens/genetics* ; Hybridomas/immunology* ; Antibody Specificity/immunology* ; Mice ; Mice, Inbred BALB C ; Enzyme-Linked Immunosorbent Assay ; Fluorescent Antibody Technique, Indirect

Objective To explore the effect of the knockdown of transmembrane 9 superfamily protein member 2 (TM9SF2) on the replication of vesicular stomatitis virus (VSV), and investigate its role in the mechanism of antiviral innate immunity. Methods Small interfering RNA (siRNA) was used to knock down the TM9SF2 gene in human non-small cell lung cancer A549 cells. The CCK-8 method was used to assess cell proliferation. A VSV-green fluorescent protein (VSV-GFP) infected cell model was established. The plaque assay was used to measure the viral titer in the supernatant. RT-qPCR and Western blotting were employed to quantify the mRNA and protein levels of VSV genome replication in A549 cells following VSV infection, as well as the expression of interferon β (IFN-β) mRNA and interferon regulatory factor 3 (IRF3) protein phosphorylation following polyinosinic-polycytidylic acid (poly(I:C)) stimulation. Results Compared to the negative control, the knockdown of TM9SF2 exhibited a significant effect, with no observed impact on A549 cell proliferation. The VSV-GFP infected A549 cell model was successfully established. After viral stimulation, fluorescence intensity was reduced following TM9SF2 knockdown, and the mRNA and protein levels of VSV were significantly downregulated. The viral titer of VSV was decreased. After poly(I:C) stimulation, TM9SF2 knockdown significantly upregulated the mRNA level of IFN-β and the phosphorylation level of IRF3 protein. Conclusion The knockdown of TM9SF2 inhibits the replication of vesicular stomatitis virus, and positively regulates the type I interferon signaling pathway, thus enhancing the host's antiviral innate immune response.
Humans ; Virus Replication/genetics* ; Signal Transduction ; Membrane Proteins/metabolism* ; A549 Cells ; Vesiculovirus/physiology* ; Interferon-beta/metabolism* ; Interferon Regulatory Factor-3/genetics* ; Interferon Type I/metabolism* ; Vesicular Stomatitis/immunology* ; Gene Knockdown Techniques ; Vesicular stomatitis Indiana virus/physiology* ; RNA, Small Interfering/genetics*

Objective To explore macrophage subpopulations in ischemic stroke (IS) by using single-cell RNA sequencing (scRNA-seq) data analysis and High-Dimensional Weighted Gene Co-Expression Network Analysis (hdWGCNA). Methods Based on single-cell sequencing data, transcriptomic information for different cell types was obtained, and macrophages were selected for subpopulation identification. hdWGCNA, cell-cell communication, and pseudotime trajectory analysis were used to explore the characteristics of macrophage subpopulations following IS. Key genes related to IS were identified using microarray data and validated for diagnostic potential through Receiver Operating Characteristic (ROC) analysis. Gene Set Enrichment Analysis (GSEA) was conducted to investigate the potential functions of these genes. Results The scRNA-seq data analysis revealed significant changes in macrophage subpopulation composition after IS. A specific macrophage subpopulation enriched in the stroke group was identified and designated as MCAO-specific macrophages (MSM). Pseudotime trajectory analysis indicated that MSM cells were in an intermediate stage of macrophage differentiation. Cell-cell communication analysis uncovered complex interactions between MSM cells and other cells, with the CCL6-CCR1 signaling axis potentially playing a crucial role in neuroinflammation. Two gene modules associated with MSM were identified via hdWGCNA, significantly enriched in pathways related to NOD-like receptors and antigen processing. By integrating differentially expressed MSM genes with conventional transcriptomic data, three IS-related hub genes were identified: Arg1, CLEC4D, and CLEC4E. Conclusion This study reveals the characteristics and functions of macrophage subpopulations following IS and identifies three hub genes with potential diagnostic value, providing novel insights into the pathological mechanisms of IS.
Macrophages/metabolism* ; Computational Biology/methods* ; Single-Cell Analysis/methods* ; Transcriptome ; Ischemic Stroke/metabolism* ; Animals ; Gene Regulatory Networks ; Gene Expression Profiling ; Humans ; Male

Objective To investigate prokaryotic expression of the antigen sequence (amino acids 59-145) of mouse leukemia-related protein 16 (LRP16) protein and preparation of rabbit anti-mouse LRP16 polyclonal antibody. Methods The prokaryotic expression plasmid pLS962-LRP16 was constructed by the molecular cloning method and transferred into E.coli Rosetta to express LRP16 protein induced by IPTG. The recombinant protein was purified using Ni-NTA affinity columns followed by gel filtration chromatography. New Zealand white rabbits were immunized with the purified antigen to generate polyclonal antiserum, with antibody titer quantified by ELISA. Antigen-specific IgG was affinity-purified using Sepharose-coupled LRP16 and validated through Western blot and immunofluorescence assays. Results SDS-PAGE analysis confirmed insoluble expression of the LRP16 fusion protein as inclusion bodies. ELISA demonstrated exceptional antiserum titer (1:256 000). Western blot and immunofluorescence verified that the polyclonal antibody could specifically recognize endogenous LRP16 in murine tissues. Conclusion The prokaryotic expression of the LRP16 gene is successfully achieved, and the rabbit anti-mouse LRP16 polyclonal antibody exhibiting high specificity is prepared. This lays the foundation for further studies on the function of the LRP16 gene.
Animals ; Rabbits ; Mice ; Antibodies/immunology* ; Escherichia coli/metabolism* ; Enzyme-Linked Immunosorbent Assay ; Blotting, Western ; Antibody Specificity

Objective To explore the mechanism of ubiquitin specific peptidase 21 (USP21) increasing the stability of forkhead box protein M1 (FOXM1) and promoting M2 polarization of macrophages in endometriosis (EM). Methods Eutopic endometrial stromal cells (EESC) collected from patients and normal endometrial stromal cells (NESC) from routine health examiners were cultured in vitro, and the expression levels of USP21 and FOXM1 were detected using RT-qPCR and Western blot. EESCs were co-cultured with macrophages. M1 polarization markers of interleukin 6 (IL-6) and CXC chemokine ligand 10 (CXCL10) and M2 polarization markers of CD206 and fibronectin 1 (FN1) were tested using RT-qPCR. M2 marker CD206 was further detected by flow cytometry. IL-6, tumor necrosis factor-alpha (TNF-α), IL-10, and transforming growth factor-beta (TGF-β) levels in cell supernatant were detected by ELISA. Co-immunoprecipitation was used to assess the interaction between USP21 and FOXM1, and the ubiquitination level of FOXM1. FOXM1 protein stability was detected through cycloheximide (CHX) assay. Results USP21 and FOXM1 expression levels in the EESC group were significantly increased compared with those in the NESC group; compared with the NESC + M0 group, the EESC + M0 group showed no significant difference in the expression of M1 polarization markers (IL-6 and CXCL10), but increased expression of M2 polarization markers (CD206 and FN1), along with notably increased number of M2 macrophages; there was no significant difference in IL-6 and TNF-α levels, but increased levels of IL-10 and TGF-β in the cell supernatant. The above findings indicated that the deubiquitinase USP21 was highly expressed in EM, promoting M2 polarization of macrophages. Knocking down USP21 or FOXM1 can inhibit M2 polarization of EM macrophages. USP21 interacted with FOXM1 in EESC, leading to a decrease in FOXM1 ubiquitination level and an increase in FOXM1 protein stability. Overexpression of FOXM1 reversed the inhibitory effect of knocking down USP21 on M2 polarization of EM macrophages. Conclusion The deubiquitinase USP21 interacts with FOXM1 to increase the stability of FOXM1 and promote M2 polarization of EM macrophages.
Humans ; Forkhead Box Protein M1/genetics* ; Female ; Macrophages/cytology* ; Endometriosis/genetics* ; Ubiquitin Thiolesterase/genetics* ; Cells, Cultured ; Endometrium/metabolism* ; Ubiquitination ; Adult ; Interleukin-10/metabolism* ; Interleukin-6/metabolism* ; Protein Stability ; Stromal Cells/metabolism*

Objective To investigate the effect of FUNDC1 tyrosine phosphorylation site mutations on mitophagy in H9c2 myocardial cells by constructing tyrosine site mutant plasmids (Y11 and Y18) of the FUN14 domain-containing protein 1 (FUNDC1). Methods The mutant plasmids constructed by whole-gene synthesis were transfected into rat myocardial H9c2 cells and divided into five groups: empty plasmid group, FUNDC1 overexpression group, Y11 mutant group, Y18 mutant group, and Y11 combined with Y18 mutant group. The viability of H9c2 cells was assessed using the CCK-8 assay. Additionally, tetramethylrhodamine ethyl ester (TMRE) staining was utilized to detect mitochondrial membrane potential. The protein expression levels of FUNDC1, translocase of the outer mitochondrial membrane 20 (TOM20), and cytochrome c oxidase IV (COX IV) were detected by Western blot analysis. Confocal microscopy was used to evaluate transfection efficiency as well as the co-localization of mitochondria and lysosomes. Results The FUNDC1 overexpression, Y11, Y18, and Y11 combined with Y18 mutant plasmids were successfully constructed. After plasmid transfection, widespread GFP fluorescence expression was observed under confocal microscopy. Compared with the empty plasmid group, FUNDC1 protein expression levels were significantly increased in the FUNDC1 overexpression group, Y11 mutation group, Y18 mutation group, and Y11 combined with Y18 mutation group, while cell viability and mitochondrial membrane potential showed no significant changes. Compared to the empty plasmid group, cells transfected with Y18 and Y11 combined with Y18 mutant plasmids showed increased TOM20 and COX IV expression levels and decreased mitochondrial-lysosomal co-localization. Conclusion Transfection with FUNDC1 Y18 or Y11 combined with Y18 mutant plasmids inhibited mitophagy in H9c2 myocardial cells.
Animals ; Rats ; Mitophagy/genetics* ; Myocytes, Cardiac/cytology* ; Mitochondrial Proteins/metabolism* ; Mutation ; Phosphorylation ; Tyrosine/genetics* ; Cell Line ; Membrane Proteins/metabolism* ; Membrane Potential, Mitochondrial