Bio-mineralization has emerged as a promising strategy to enhance vaccine immunogenicity. This study optimized the calcium phosphate (CaP) mineralization process of foot-and-mouth disease virus-like particles (FMD VLPs) to achieve high mineralization efficiency and scalability. Key parameters, including concentrations of Ca²⁺, HPO₄^2-, NaCl, and VLPs, as well as stirring speed, were systematically optimized. Stability of the scaled-up reaction system and immunogenicity of the mineralized vaccine were evaluated. Optimal conditions [25.50 mmol/L Ca(NO₃)₂, 15 mmol/L Na₂HPO₄, 300 mmol/L NaCl, 0.75 mg/mL VLPs, and 1 500 r/min] yielded CaP-mineralized VLPs (VLPs-CaP) with high mineralization efficiency, uniform morphology, and a favorable particle size. Scaling up the reaction by 25 folds maintained consistent mineralization efficiency and particle characteristics. Immunization in mice demonstrated that VLPs-CaP induced higher titers of specific antibodies and neutralizing antibodies than unmineralized VLPs (P < 0.05). Higher IgG2a/IgG1 ratio and enhanced IFN-γ secretion (P < 0.05) further indicated robust cellular immune responses. We establish a stable and scalable protocol for VLPs-CaP, providing a theoretical and technical foundation for developing high-efficacy VLPs-CaP vaccines.
Vaccines, Virus-Like Particle/immunology* ; Immunogenicity, Vaccine ; Calcium Phosphates/chemistry* ; Foot-and-Mouth Disease Virus ; Biomineralization ; Particle Size ; Animals ; Mice ; Antibodies, Neutralizing/blood* ; Antibodies, Viral/blood* ; Immunity, Cellular

Vaccination is a crucial strategy for the prevention and control of infectious diseases. Virus-like particles (VLPs), composed of structural proteins, have garnered significant attention as a novel type of vaccine due to their excellent safety and immunogenicity. However, similar to most vaccine antigens, VLPs exhibit insufficient thermal stability, which not only restricts the widespread application of vaccines but also increases the risk of vaccine inactivation. This study aims to enhance the stability and shelf life of VLPs derived from type A foot-and-mouth disease virus (FMDV) by employing vacuum freeze-drying technology. The optimal lyoprotectant formulation was determined through single-factor and combinatorial screening. Subsequently, the correlation between the immunogenicity of the freeze-dried vaccine and the content of FMDV VLPs was evaluated via a mouse model. The stability of FMDV VLPs before and after freeze-drying was further assessed by storing them at 4, 25, and 37 ℃ for varying time periods. Results indicated that the lyoprotectant formulation No.1, composed of 7.5% trehalose, 0.1% Tween 80, 50 mmol/L glycine, 1% sodium glutamate, and 3% polyvinylpyrrolidone (PVP), effectively preserved the content of FMDV VLPs during the vacuum freeze-drying process. The immunization trial in mice revealed that the levels of specific antibodies, immunoglobulin G1 (IgG1), interleukin-4 (IL-4), and neutralizing antibodies induced by freeze-dried FMDV VLPs were comparable to those induced by non-freeze-dried FMDV VLPs. The heat treatment results showed that the storage periods of freeze-dried FMDV VLPs at 4, 25, and 37 ℃ were significantly longer than those of non-freeze-dried FMDV VLPs. In conclusion, the selected lyoprotectant formulation effectively improved the stability of FMDV VLPs vaccines. This study provides valuable insights for enhancing the stability of novel subunit vaccines.
Freeze Drying/methods* ; Animals ; Foot-and-Mouth Disease Virus/immunology* ; Mice ; Vaccines, Virus-Like Particle/chemistry* ; Foot-and-Mouth Disease/immunology* ; Vacuum ; Drug Stability ; Mice, Inbred BALB C ; Viral Vaccines/immunology*

ObjectiveTo study whether Chaihu Longgu Mulitang can inhibit hypothalamic inflammation， mitigate anxiety-like behavior， and alleviate anxiety symptoms by regulating the p38 mitogen-activated protein kinase/nuclear factor-κB （p38 MAPK/NF-κB） signaling pathway in the rat model of generalized anxiety disorder （GAD）. MethodTwelve out of 74 Wistar rats were randomly selected as the blank group， and the remaining rats were subjected to chronic restraint stress for the modeling of GAD. The open field test （OFT） and elevated Porteus maze test （PMT） were conducted 14 days after modeling to detect the anxiety-like behaviors. Sixty successfully modeled rats were selected and randomized into model， low-， medium-， and high-dose （6， 12， and 24 g·kg^-1， respectively） Chaihu Longgu Mulitang， and diazepam （1 mg·kg^-1） groups （n=12） and administrated with corresponding drugs for 14 consecutive days. OFT and PMT were then carried out to examine the anxiety-like behaviors of the rats. The levels of tumor necrosis factor-α （TNF-α）， interleukin-6 （IL-6）， and interleukin-1β （IL-1β） in the hypothalamus and serum of rats were determined by the enzyme-linked immunosorbent assay （ELISA）. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）was conducted to determine the mRNA levels of p38 MAPK， NF-κB p65， nuclear factor κB inhibitor α （IκBα）， and ionized calcium binding adaptor molecule 1 （Iba-1）. The protein levels of p38 MAPK， phosphorylated （p）-p38 MAPK， NF-κB p65， p-NF-κB p65， and IκBα in the hypothalamus of rats were determined by Western blot. The expression of Iba-1 in the hypothalamic microglia was detected by immunofluorescence assay. ResultCompared with the blank group， the model group had decreased body weight， scattered dark yellow fur， increased irritability， and preference to hibernation in the corner. In addition， the modeled rats showed increased edge movement distance and time in OFT （P<0.01） and decreased movement distance and time and the number of entries in the open arm in PMT （P<0.01）. The modeling increased the fluorescence intensity of Iba-1 in paraventricular nucleus of hypothalamus （P<0.01）， elevated the levels of IL-1β， IL-6， and TNF-α in the serum and hypothalamus （P<0.01）， up-regulated the protein and mRNA levels of p38 MAPK， NF-κB p65， p-p38 MAPK， p-NF-κB p65， and Iba-1 （P<0.05， P<0.01）， and down-regulated the protein and mRNA levels of IκBα （P<0.01） in the hypothalamus. Compared with the model group， medium- and high-dose Chaihu Longgu Mulitang and diazepam increased the body weight， improved the fur and behaviors， decreased the edge movement distance and time in OFT （P<0.05， P<0.01）， and increased the movement distance and time in the open arm in PMT （P<0.05， P<0.01）. Furthermore， they decreased the fluorescence intensity of Iba-1 in hypothalamic microglia （P<0.05， P<0.01）， lowered the levels of IL-1β， IL-6， and TNF-α in the serum and hypothalamic tissue （P<0.05， P<0.01）， down-regulated the mRNA and protein levels of p38 MAPK， NF-κB p65， p-p38 MAPK， p-NF-κB p65， and Iba-1 （P<0.05， P<0.01）， and up-regulated the mRNA and protein levels of IκBα （P<0.05， P<0.01） in the hypothalamus. ConclusionChaihu Longgu Mulitang can mitigate anxiety-like behaviors and relieve anxiety in GAD rats by inhibiting the p38 MAPK/NF-κB signaling pathway and reducing the activation of microglia and the levels of pro-inflammatory cytokines in the hypothalamus.

Objective To investigate the safety and efficacy of totally no tube three-port thoracoscopic surgery (TNTT) for thymic tumor via lateral thoracic approach. Methods The clinical data of patients with thymoma admitted to the Department of Thoracic Surgery of the General Hospital of Northern Theater Command from November 2021 to May 2022 were retrospectively analyzed. The patients were divided into a TNTT group and a single utility port video-assisted thoracic surgery (SVATS) group according to different surgical methods. The clinical data were compared between the two groups. Results A total of 111 patients were collected. There were 44 patients in the TNTT group, including 20 males and 24 females, with an average age of 60.11±8.64 years, and 67 patients in the SVATS group, including 30 males and 37 females, with an average age of 62.40±7.92 years. There was no significant difference between the two groups in the baseline data (P>0.05). The postoperative hospital stay and intraoperative blood loss were shorter or less in the TNTT group (P<0.05), and the visual analogue scale score 48 hours after the operation was smaller in the SVATS group (P<0.05). Conclusion TNTT has a good surgical safety, and can shorten postoperative hospital stay, reduce intraoperative blood loss, and has significant advantages in enhanced recovery after surgery, but SVATS can reduce postoperative pain in patients.

Fecal microbiota transplantation(FMT)is a therapeutic approach that targets intestinal microorganisms by transplanting fecal microorganisms from healthy individuals into the gastrointestinal tract of diseased individuals,thus restoring the recipient's disordered gastrointestinal microbiota by restructuring the intestinal flora.However,the mechanism of action and adverse effects of FMT in different diseases have not yet been clarified,thus limiting its wide clinical application.Its use still relies on in-depth preclinical studies;however,highly inconsistent or incomplete experimental details provided in current reports,coupled with a lack of authoritative standards and recommendations,seriously affect the interpretation of the study findings and replication of the experimental procedures,as well as hindering the clinical translation of the result.We therefore review and discuss the key steps of recipient selection and graft sample collection,storage,graft material preparation,and grafting route,with the aim of improving the utilization of experimental animals,consumables,and labor,and providing method ological recommendations and references to achieve replicability and standardization of preclinical FMT studies.

Objective To explore the changes and significane of USP20 and HIF-α expression in breast cancer.Methods Following transfection of shRNA-USP20 lentivirus into breast cancer MDA-MB-231 cells,the gene and protein expression levels of USP20 were detected using fluorescence quantitative PCR and Western Blot.Subsequently,the overexpression of USP20 was observed to determine its effect on HIF-α expression.Similarly,siRNA-USP20 was used to knock down USP20 in breast cancer MDA-MB-231 cells,followed by detection of gene and protein expression levels using fluorescence quantitative PCR and Western Blot.The subsequent changes in HIF-α expression were then examined.Rusults The positive expression rates of USP20 and HIF-α in breast cancer tissues were 69.6%and 46.83%,respectively,while they were negatively expressed in the adjacent normal tissues,with statistically significant differences(P＜0.01).The positive expressions of USP20 and HIF-α were predomi-nantly observed in the cytoplasm of breast cancer tissue,with a smaller amount present in the nucleus.There was a significant positive correlation between USP20 and HIF-α in breast cancer.Following transfection of shRNA-USP20 lentivirus into MDA-MB-231 cells,both the protein and gene expression levels of USP20 significantly increased(P＜0.01).Over-expression of USP20 did not affect HIF-α mRNA levels but led to a significant increase in HIF-α protein expression(P＜0.01).Conversely,siRNA-USP20 interference resulted in a significant decrease in both the protein and gene expression levels of USP20(P＜0.01),without affecting HIF-α mRNA levels;however,it caused a notable reduction in HIF-α protein expression(P＜0.01).Conclusion The expression of USP20 exhib-ited a significant positive correlation with HIF-α in breast cancer.Overexpression of USP20 led to a substantial increase in HIF-α protein expression,while knock-down of the USP20 gene resulted in a significant decrease in HIF-α protein levels.Therefore,it can be inferred that USP20 may exert its influence on the development of breast cancer through modulation of HIF-α expression,thereby providing crucial experimental evidence for clinical treat-ment,prognosis,and further investigations.

Peripapillary hyperreflective ovoid mass-like structure (PHOMS) is a novel imaging feature newly defined in recent years in the study of optic disc drusen (ODD).In OCT images, it shows an oval hyper-reflective signal on the Bruch membrane around the optic papilla, ring or crescent non-fluorescence in autofluorescence, and light gray peripapillary elevations around the optic disc in non-red infrared images.There is no conclusive evidence as to whether PHOMS is a subtype or precursor of ODD.Histopathologically, PHOMS might correspond to the lateral bulging or herniation of distended axons into the peripapillary retina.Since there may be different pathological properties between PHOMS and ODD in histology, there are different imaging features in multimodal imaging.To make the diagnosis of ODD more accurate, the Optic Disc Drusen Studies Consortium recommends that PHOMS be distinguished from ODD.PHOMS is mainly differentiated from ODD and perioptic vessels on OCT.It can be seen in many diseases such as ODD, optic papilledema, non-arteritis anterior ischemic optic neuropathy, multiple sclerosis, central retinal vein occlusion, pediatric tilted disc syndrome, isolated optic neuritis and optic atrophy, and is an important cause of pseudopapilloedema in children.This article summarizes the origin, imaging features, differential diagnosis and related diseases of PHOMS, to increase domestic researchers' understanding of the structure of PHOMS, enhance the differential diagnosis between PHOMS and ODD, and provide a reference for clinical research and diagnosis.

The variable domain of heavy-chain antibody (VHH) has been developed widely in drug therapy, diagnosis, and research. Escherichia coli is the most popular expression system for VHH production, whereas low bioactivity occurs sometimes. Mammalian cells are one of the most ideal hosts for VHH expression at present. To improve the yield of VHH in Expi293F cells, we optimized the signal peptide (SP) and codons of VHH. Firstly, the fusion protein VHH1-Fc was used to screen SPs. The SP IFN-α2 showed the highest secretion as quantified by enzyme-linked immunosorbent assay (ELISA). Subsequently, codon optimization by improving GC3 and GC content doubled the yield of VHH1 and kept its binding activity to Senecavirus A (SVA). Finally, the mean yields of other 5 VHHs that fused with SP IFN-α2 and codon-optimized were over 191.6 mg/L, and these VHHs had high recovery and high purity in the culture supernatant. This study confirms that SP IFN-α2 and codon optimization could produce VHHs in Expi293F cells efficiently, which provides a reference for the large-scale production of VHHs.
Codon/genetics* ; Protein Sorting Signals/genetics* ; Escherichia coli/metabolism* ; Humans ; Recombinant Fusion Proteins/biosynthesis* ; Interferon-alpha/metabolism* ; Immunoglobulin Heavy Chains/immunology* ; Cell Line ; Immunoglobulin Variable Region/immunology*

To further enhance the immune effect of the foot-and-mouth disease (FMD) virus-like particles (VLPs) vaccine, this study prepared FMDV VLPs-zeolitic imidazolate (framework-8, ZIF-8) complexes with different particle sizes. We used a biomimetic mineralization method with Zn²⁺ and 2-methylimidazole in different concentration ratios to investigate the effect of size on the immunization effect. The results showed that FMDV VLPs-ZIF-8 with three different sizes were successfully prepared, with an approximate size of 70 nm, 100 nm, and 1 000 nm, respectively. Cytotoxicity and animal toxicity tests showed that all three complexes exhibited excellent biological safety. Immunization tests in mice showed that all three complexes enhanced the titers of neutralizing and specific antibodies, and their immune effects improved as the size of the complexes decreased. This study showed that ZIF-8 encapsulation of FMDV VLPs significantly enhanced their immunogenic effect in a size-dependent manner.
Animals ; Mice ; Foot-and-Mouth Disease/prevention & control* ; Foot-and-Mouth Disease Virus ; Antibodies, Neutralizing ; Immunity, Humoral ; Immunization ; Vaccines, Virus-Like Particle ; Antibodies, Viral ; Viral Vaccines

Transient expression is the major method to express foot-and-mouth disease virus (FMDV) capsid proteins in mammalian cells. To achieve stable expression of FMDV capsid proteins and efficient assembly of virus like particles (VLPs) in cells, the plasmids of piggyBac (PB) transposon-constitutive expression and PB transposon-tetracycline (Tet) inducible expression vectors were constructed. The function of the plasmids was tested by fluorescent proteins. By adding antibiotics, the constitutive cell pools (C-WT, C-L127P) expressing P12A3C (WT/L127P) genes and the inducible cell pools (I-WT, I-L127P) expressing P12A3C (WT/L127P) genes were generated. The genes of green fluorescent protein, 3C protease and reverse tetracycline transactivator (rtTA) were integrated into chromosome, which was confirmed by fluorescence observation and PCR testing. The cell pool I-L127P has a stronger production capacity of capsid proteins and VLPs, which was confirmed by Western blotting and enzyme linked immunosorbent assay (ELISA), respectively. In conclusion, inducing the chromosomal expression of FMDV capsid proteins was firstly reported, which may facilitate the technical process of mammalian production of FMDV VLPs vaccine and the construction of mammalian inducible expression systems for other proteins.
Animals ; Foot-and-Mouth Disease Virus/genetics* ; Capsid Proteins ; Viral Proteins/metabolism* ; Foot-and-Mouth Disease/prevention & control* ; Tetracyclines/metabolism* ; Viral Vaccines ; Antibodies, Viral ; Mammals/metabolism*