This study aims to investigate the effect of Chaijin Jieyu Anshen Formula on the neuroinflammation of rats with insomnia complicated with depression through the regulation of triggering receptor expressed on myeloid cells 2(TREM2)/complement protein C1q signaling pathway. Rats were randomly divided into a normal group, a model group, a positive drug group, as well as a high, medium, and low-dose groups of Chaijin Jieyu Anshen Formula, with 10 rats in each group. Except for the normal group, the other groups were injected with p-chlorophenylalanine and exposed to chronic unpredictable mild stress to establish the rat model of insomnia complicated with depression. The sucrose preference experiment, open field experiment, and water maze test were performed to evaluate the depression in rats. Enzyme-linked immunosorbent assay was employed to detect serum 5-hydroxytryptamine(5-HT), dopamine(DA), and norepinephrine(NE) levels. Hematoxylin and eosin staining and Nissl staining were used to observe the damage in nucleus accumbens neurons. Western blot and immunofluorescence were performed to detect TREM2, C1q, postsynaptic density 95(PSD-95), and synaptophysin 1(SYN1) expressions in rat nucleus accumbens, respectively. Golgi-Cox staining was utilized to observe the synaptic spine density of nucleus accumbens neurons. The results show that, compared with the model group, Chaijin Jieyu Anshen Formula can significantly increase the sucrose preference as well as the distance and number of voluntary activities, shorten the immobility time in forced swimming test and the successful incubation period of positioning navigation, and prolong the stay time of space exploration in the target quadrant test. The serum 5-HT, DA, and NE contents in the model group are significantly lower than those in the normal group, with the above contents significantly increased after the intervention of Chaijin Jieyu Anshen Formula. In addition, Chaijin Jieyu Anshen Formula can alleviate pathological damages such as swelling and loose arrangement of tissue cells in the nucleus accumbens, while increasing the Nissl body numbers. Chaijin Jieyu Anshen Formula can improve synaptic damage in the nucleus accumbens and increase the synaptic spine density. Compared to the normal group, the expression of C1q protein was significantly higher in the model group, while the expression of TREM2 protein was significantly lower. Compared to the model group, the intervention with Chaijin Jieyu Anshen Formula significantly downregulated the expression of C1q protein and significantly upregulated the expression of TREM2. Compared with the model group, the PSD-95 and SYN1 fluorescence intensity is significantly increased in the groups receiving different doses of Chaijin Jieyu Anshen Formula. In summary, Chaijin Jieyu Anshen Formula can reduce the C1q protein expression, relieve the TREM2 inhibition, and promote the synapse-related proteins PSD-95 and SNY1 expression. Chaijin Jieyu Anshen Formula improves synaptic injury of the nucleus accumbens neurons, thereby treating insomnia complicated with depression.
Animals ; Male ; Rats ; Nucleus Accumbens/metabolism* ; Drugs, Chinese Herbal/administration & dosage* ; Depression/complications* ; Membrane Glycoproteins/genetics* ; Rats, Sprague-Dawley ; Sleep Initiation and Maintenance Disorders/complications* ; Neurons/metabolism* ; Receptors, Immunologic/genetics* ; Signal Transduction/drug effects* ; Synapses/metabolism*

Objective Currently, there is no commercially available quantitative detection kit for the main Felis domestic allergen (Fel d 1) in China. To establish a rapid detection method for Fel d 1, this study aims to prepare monoclonal antibodies against Fel d 1 protein. Methods The codon preference of Escherichia coli was utilized to optimize and synthesize the Fel d 1 gene. The prokaryotic expression plasmid pET-28a-Fel d 1 was constructed and used to express and purify the recombinant Fel d 1 protein. Subsequently, the recombinant protein was immunized into BALB/c mice and monoclonal antibodies (mAbs) were prepared by the hybridoma technique. An indirect ELISA was established using the recombinant Fel d 1 as the coating antigen, and hybridoma cell lines were screened for positive clones. The specificity and antigenic epitopes of the mAbs were confirmed by Western blot analysis. Finally, the selected hybridoma cells were injected into the peritoneal cavities of BALB/c mice for large-scale monoclonal antibody production. Results The recombinant plasmid pET-28a-Fel d 1 was successfully constructed, and soluble Fel d 1 protein was obtained after optimizing the expression conditions. Western blot and antibody titer assays confirmed the successful isolation of two hybridoma cell lines, 7D11 and 5H4, which stably secreted mAbs specific to Fel d 1. Antibody characterization revealed that the 5H4 mAb was of the IgG2a subtype and could recognize the amino acid region 105-163 of Fel d 1, while the 7D11 mAb was the IgG1 subtype and could recognize the amino acid region 1-59. Conclusion The high-purity recombinant Fel d 1 protein produced in this study provides a promising alternative for clinical immunotherapy of cat allergies. Furthermore, the monoclonal antibody prepared in this experiment lays a material foundation for the in-depth study of the biological function of Fel d 1 and the development of ELISA detection.
Animals ; Antibodies, Monoclonal/biosynthesis* ; Mice, Inbred BALB C ; Cats ; Mice ; Allergens/genetics* ; Glycoproteins/genetics* ; Enzyme-Linked Immunosorbent Assay ; Hybridomas/immunology* ; Recombinant Proteins/genetics* ; Female ; Antibody Specificity

OBJECTIVE: To reveal the effects and potential mechanisms by which synaptic vesicle glycoprotein 2A (SV2A) influences the distribution of amyloid precursor protein (APP) in the trans-Golgi network (TGN), endolysosomal system, and cell membranes and to reveal the effects of SV2A on APP amyloid degradation. METHODS: Colocalization analysis of APP with specific tagged proteins in the TGN, ensolysosomal system, and cell membrane was performed to explore the effects of SV2A on the intracellular transport of APP. APP, β-site amyloid precursor protein cleaving enzyme 1 (BACE1) expressions, and APP cleavage products levels were investigated to observe the effects of SV2A on APP amyloidogenic processing. RESULTS: APP localization was reduced in the TGN, early endosomes, late endosomes, and lysosomes, whereas it was increased in the recycling endosomes and cell membrane of SV2A-overexpressed neurons. Moreover, Arl5b (ADP-ribosylation factor 5b), a protein responsible for transporting APP from the TGN to early endosomes, was upregulated by SV2A. SV2A overexpression also decreased APP transport from the cell membrane to early endosomes by downregulating APP endocytosis. In addition, products of APP amyloid degradation, including sAPPβ, Aβ _1-42, and Aβ _1-40, were decreased in SV2A-overexpressed cells. CONCLUSION These results demonstrated that SV2A promotes APP transport from the TGN to early endosomes by upregulating Arl5b and promoting APP transport from early endosomes to recycling endosomes-cell membrane pathway, which slows APP amyloid degradation.
Amyloid beta-Protein Precursor/genetics* ; Membrane Glycoproteins/genetics* ; Animals ; Protein Transport ; Nerve Tissue Proteins/genetics* ; Humans ; Mice ; Endosomes/metabolism* ; trans-Golgi Network/metabolism*

Glycosylation modification is an important post-translational modification of proteins, which participates in regulating protein half-life, biological activity, and immunogenicity, thereby affecting their functions. Glycoproteins expressed in Pichia pastoris predominantly carry high-mannose type glycans, primarily composed of mannose residues, which starkly contrasts with the complex-type glycans synthesized by mammalian cells. This study aims to transform the high mannose glycosylation modification of P. pastoris into a hybrid glycosylation modification similar to that of mammalian cells through genetic engineering technology. We introduced the mannosidase Ⅰ gene (MDSⅠ) from Trichoderma viride and the human β-1,2-N-acetylglucosaminyltransferase I gene (GnTⅠ) into a previously constructed P. pastoris strain (∆och1) capable of producing Man₈GlcNAc₂ glycans. To precisely regulate the expression of MDSⅠ and GnTⅠ, we designed various promoter combinations, including the strong inducible AOX promoter and the constitutive GAP promoter. The receptor-binding domain (RBD, residues 377-588) of the spike protein from the Middle East respiratory syndrome coronavirus (MERS-CoV) was selected as the reporter protein for this investigation (MERS-RBD). The N-glycosylation profile of MERS-RBD was systematically analyzed using PNGase F digestion coupled with mass spectrometry. The results showed that after the knockout of och1 and the introduction of MDSⅠ and GnTⅠ genes with different promoter combinations, P. pastoris strains capable of producing GlcNAcMan₅GlcNAc₂ glycans were successfully generated. When the AOX promoter was used to control the MDSⅠ gene and the GAP promoter was used to control the GnTⅠ gene, the engineered strain exhibited the highest proportion of hybrid-type GlcNAcMan₅GlcNAc₂ glycans, which accounted for 68.38% of the total N-glycosylation. In conclusion, we successfully engineered a P. pastoris strain capable of synthesizing hybrid-type GlcNAcMan₅GlcNAc₂ glycans, establishing a foundation for subsequent research on the biosynthesis of complex-type N-glycans in P. pastoris.
Glycosylation ; Glycoproteins/genetics* ; Polysaccharides/metabolism* ; N-Acetylglucosaminyltransferases/metabolism* ; Pichia/metabolism* ; Humans ; Mannosidases/metabolism* ; Genetic Engineering ; Trichoderma/genetics* ; Recombinant Proteins/genetics* ; Saccharomycetales

Vaccination is the most effective measure for reducing and preventing influenza and related complications. In this study, we analyzed the mutation trend and the antigen dominant site changes of the amino acid sequence of hemagglutinin subunit 1 (HA1) of human influenza A virus (IAV) in the northern hemisphere from 2012 to 2022. According to the HA1 sequences of A/Darwin/6/2021 (H3N2) and A/Wisconsin/588/2019 (H1N1) recommended by the World Health Organization in the 2022 influenza season in northern hemisphere, we employed the mosaic algorithm to design three Mosaic-HA1 antigens through stepwise substitution. Mosaic-HA1 was expressed and purified in 293F cells and then mixed with the alum adjuvant at a volume ratio of 1:1. The mixture was used to immunize BALB/c mice, and the immunogenicity was evaluated. Enzyme-linked immunosorbent assay showed that Mosaic-HA1 induced the production of IgG targeting two types of HA1, the specific IgG titers for binding to H3 protein and H1 protein reached 10⁵ and 10³ respectively. The challenge test showed that Mosaic-HA1 protected mice from H3N2 or H1N1. This study designs the vaccines by recombination of major antigenic sites in different subtypes of IAV, giving new insights into the development of multivalent subunit vaccines against influenza.
Animals ; Influenza A Virus, H1N1 Subtype/genetics* ; Influenza A Virus, H3N2 Subtype/genetics* ; Mice, Inbred BALB C ; Mice ; Influenza Vaccines/genetics* ; Hemagglutinin Glycoproteins, Influenza Virus/genetics* ; Humans ; Antibodies, Viral/blood* ; Antigens, Viral/genetics* ; Immunoglobulin G/immunology* ; Female ; Orthomyxoviridae Infections/prevention & control* ; HEK293 Cells

OBJECTIVE: To analyze the correlation between the mRNA levels of breast cancer resistance protein (BCRP) and lung-specific X protein (LUNX) genes with pathological types and stages of patients with non-small cell lung cancer (NSCLC) and their significance for prognosis. METHODS: Eighty nine patients with NSCLC admitted to Huaihe Hospital of Henan University between June 2015 and June 2018 were recruited, with 55 patients with benign lung lesions admitted during the same period of time selected as the control group. The mRNA levels of BCRP and LUNX genes were detected in the peripheral blood samples from the two groups, and their correlation with the clinicopathological characteristics and prognosis of the patients was analyzed. RESULTS: The expression rates of BCRP and LUNX mRNA in the NSCLC group were significantly higher compared with the control group (P < 0.05). The level of BCRP mRNA of the NSCLC patients has correlated with the degree of differentiation and TNM staging (P < 0.05), but not with gender, age, smoking, pathological types and lymph node metastasis (P > 0.05). The level of LUNX mRNA of them has correlated with the degree of differentiation, TNM staging and lymph node metastasis (P < 0.05), but not with gender, age, smoking, and pathological types (P > 0.05). Compared with those with no expression, the overall survival rate of patients with BCRP and LUNX expression was significantly lower (P < 0.05). The degree of differentiation, TNM staging, lymph node metastasis, and expression of the BCRP and LUNX mRNA may all affect the prognosis of the patients. CONCLUSION The levels of BCRP and LUNX mRNA in the peripheral blood of patients with NSCLC are significantly increased. The expression of BCRP mRNA is correlated with the degree of differentiation and TNM staging, whilst the expression of LUNX mRNA is correlated with the differentiation degree, TNM staging and lymph node metastasis. Both may be used as independent predictors for the prognosis of patients with NSCLC.
Humans ; ATP Binding Cassette Transporter, Subfamily G, Member 2/genetics* ; Biomarkers, Tumor/genetics* ; Carcinoma, Non-Small-Cell Lung/pathology* ; Glycoproteins/genetics* ; Lung Neoplasms/pathology* ; Lymphatic Metastasis ; Neoplasm Proteins/genetics* ; Phosphoproteins/genetics* ; Prognosis ; RNA, Messenger/genetics*

A boy, aged 6 years, attended the hospital due to global developmental delay for 6 years and recurrent fever and convulsions for 5 years. The boy was found to have delayed mental and motor development at the age of 3 months and experienced recurrent fever and convulsions since the age of 1 year, with intermittent canker sores and purulent tonsillitis. During the fever period, blood tests showed elevated white blood cell count, C-reactive protein, and erythrocyte sedimentation rate, which returned to normal after the fever subsides. Electroencephalography showed epilepsy, and genetic testing showed compound heterozygous mutations in the GPAA1 gene. The boy was finally diagnosed with glycosylphosphatidylinositol biosynthesis deficiency 15 (GPIBD15) and periodic fever. The patient did not respond well to antiepileptic treatment, but showed successful fever control with glucocorticoid therapy. This article reports the first case of GPIBD15 caused by GPAA1 gene mutation in China and summarizes the genetic features, clinical features, diagnosis, and treatment of this disease, which provides a reference for the early diagnosis and treatment of GPIBD15.
Humans ; Male ; Fever ; Glycosylphosphatidylinositols/genetics* ; Membrane Glycoproteins/genetics* ; Mutation ; Rare Diseases ; Seizures ; Child

The aim of this study was to clone the chicken zp1 gene encoding zona pellucida 1 (Zp1) and investigate its tissues expression profile and its effect on osteoblast mineralization. The expression level of zp1 was quantified in various tissues of laying hens and in the tibia of the pre- and post-sexual maturity by RT-qPCR. Zp1 overexpressed vector was transfected into chicken calvarial osteoblasts which were induced differentiation for 8 days, and the extracellular mineral and the expression of mineralization-related genes were detected. The full-length chicken zp1 gene is 3 045 bp, encoding 958 amino acids residuals, and has two N-glycosylation sites. The highest expression level of the zp1 gene was found in the liver, followed by the tibia and yolk membrane, while no expression was detected in the heart and eggshell gland. Compared with the pre-sexual maturity hens, the concentration of estrogen (E2) in plasma, the content of glycosaminoglycan (GAG) and the expression level of the zp1 gene in the tibia with post-sexual maturity were higher. The extracellular matrix and the level of osteoblast mineralization-related genes showed a significantly upregulated expression in chicken calvarial osteoblasts with Zp1 overexpressed and addition of estrogen. The expression of the zp1 gene is tissue-specific and positively regulated osteoblast mineralization under the action of estrogen, laying the foundation for elucidating the functional properties of Zp1 in chicken bones during the egg production period.
Female ; Animals ; Zona Pellucida Glycoproteins ; Membrane Glycoproteins/metabolism* ; Chickens/genetics* ; Egg Proteins/metabolism* ; Receptors, Cell Surface ; Estrogens

The conserved hemagglutinin (HA) stem region of avian influenza virus (AIV) is an important target for designing broad-spectrum vaccines, therapeutic antibodies and diagnostic reagents. Previously, we obtained a monoclonal antibody (mAb) (5D3-1B5) which was reactive with the HA stem epitope (aa 428-452) of H7N9 subtype AIV. To systematically characterize the mAb, we determined the antibody titers, including the HA-binding IgG, hemagglutination-inhibition (HI) and virus neutralizing (VN) titers. In addition, the antigenic epitope recognized by the antibody as well as the sequence and structure of the antibody variable region (VR) were also determined. Moreover, we evaluated the cross-reactivity of the antibody with influenza virus strains of different subtypes. The results showed that the 5D3-1B5 antibody had undetectable HI and VN activities against H7N9 virus, whereas it exhibited strong reactivity with the HA protein. Using the peptide-based enzyme-linked immunosorbent assay and biopanning with a phage-displayed random peptide library, a motif with the core sequence (⁴³¹W-⁴³³Y-⁴³⁷L) in the C-helix domain in the HA stem was identified as the epitope recognized by 5D3-1B5. Moreover, the mAb failed to react with the mutant H7N9 virus which contains mutations in the epitope. The VR of the antibody was sequenced and the complementarity determining regions in the VR of the light and heavy chains were determined. Structural modeling and molecular docking analysis of the VR verified specific binding between the antibody and the C-helix domain of the HA stem. Notably, 5D3-1B5 showed a broad cross-reactivity with influenza virus strains of different subtypes belonging to groups 1 and 2. In conclusion, 5D3-1B5 antibody is a promising candidate in terms of the development of broad-spectrum virus diagnostic reagents and therapeutic antibodies. Our findings also provided new information for understanding the epitope characteristics of the HA protein of H7N9 subtype AIV.
Animals ; Antibodies, Monoclonal ; Antibodies, Viral ; Hemagglutinin Glycoproteins, Influenza Virus/genetics* ; Hemagglutinins ; Influenza A Virus, H7N9 Subtype ; Influenza in Birds ; Molecular Docking Simulation

Influenza B virus is one of the causes for seasonal influenza, which can account for serious illness or even death in some cases. We tested the expression of extracellular domain of hemagglutinin (HA-ecto) of influenza B viruses in mammalian cells, and then determined the immunogenicity of HA-ecto in mice. The gene sequence encoding influenza B virus HA-ecto, foldon sequence, and HIS tag was optimized and inserted into pCAGGS vector. The opening reading frame (ORF) of neuraminidase was also cloned into pCAGGS. The pCAGGS-HA-ecto and pCAGGS-NA were co-transfected into 293T cells using linear polyethylenimine. Cell supernatant after transfection was collected after 96 h, and the secreted trimmeric HA-ecto protein was purified by nickel ion affinity chromatography and size exclusion chromatography. Subsequently, the mice were immunized with HA-ecto protein, and the corresponding antibody titers were detected by ELISA and hemagglutination inhibition (HAI) assays. The results showed that soluble trimeric HA-ecto protein could be obtained using mammalian cell expression system. Moreover, trimeric HA-ecto protein, in combination with the adjuvant, induced high levels of ELISA and HAI antibodies against homogenous and heterologous antigens in mice. Thus, the soluble HA-ecto protein expressed in mammalian cells could be used as a recombinant subunit vaccine candidate for influenza B virus.
Animals ; Hemagglutinin Glycoproteins, Influenza Virus/genetics* ; Hemagglutinins/genetics* ; Influenza B virus/metabolism* ; Influenza Vaccines/genetics* ; Mammals/metabolism* ; Mice ; Mice, Inbred BALB C