Species-specific IL-1ß is an inflammatory sensor of Seneca Valley Virus 3C Protease.

Inflammasomes play pivotal roles in inflammation by processing and promoting the secretion of IL-1ß. Caspase-1 is involved in the maturation of IL-1ß and IL-18, while human caspase-4 specifically processes IL-18. Recent structural studies of caspase-4 bound to Pro-IL-18 reveal the molecular basis of Pro-IL-18 activation by caspase-4. However, the mechanism of caspase-1 processing of pro-IL-1ß and other IL-1ß-converting enzymes remains elusive. Here, we observed that swine Pro-IL-1ß (sPro-IL-1ß) exists as an oligomeric precursor unlike monomeric human Pro-IL-1ß (hPro-IL-1ß). Interestingly, Seneca Valley Virus (SVV) 3C protease cleaves sPro-IL-1ß to produce mature IL-1ß, while it cleaves hPro-IL-1ß but does not produce mature IL-1ß in a specific manner. When the inflammasome is blocked, SVV 3C continues to activate IL-1ß through direct cleavage in porcine alveolar macrophages (PAMs). Through molecular modeling and mutagenesis studies, we discovered that the pro-domain of sPro-IL-1ß serves as an 'exosite' with its hydrophobic residues docking into a positively charged 3C protease pocket, thereby directing the substrate to the active site. The cleavage of sPro-IL-1ß generates a monomeric and active form of IL-1ß, initiating the downstream signaling. Thus, these studies provide IL-1ß is an inflammatory sensor that directly detects viral protease through an independent pathway operating in parallel with host inflammasomes.

Lipid A-modified Escherichia coli can produce porcine parvovirus virus-like particles with high immunogenicity and minimal endotoxin activity.

BACKGROUND: A cost-effective Escherichia coli expression system has gained popularity for producing virus-like particle (VLP) vaccines. However, the challenge lies in balancing the endotoxin residue and removal costs, as residual endotoxins can cause inflammatory reactions in the body. RESULTS: In this study, porcine parvovirus virus-like particles (PPV-VLPs) were successfully assembled from Decreased Endotoxic BL21 (BL21-DeE), and the effect of structural changes in the lipid A of BL21 on endotoxin activity, immunogenicity, and safety was investigated. The lipopolysaccharide purified from BL21-DeE produced lower IL-6 and TNF-α than that from wild-type BL21 (BL21-W) in both RAW264.7 cells and BALB/c mice. Additionally, mice immunized with PPV-VLP derived form BL21-DeE (BL21-DeE-VLP) showed significantly lower production of inflammatory factors and a smaller increase in body temperature within 3 h than those immunized with VLP from BL21-W (BL21-W-VLP) and endotoxin-removed VLP (ReE-VLP). Moreover, mice in the BL21-DeE-VLP immunized group had similar levels of serum antibodies as those in the BL21-W-VLP group but significantly higher levels than those in the ReE-VLP group. Furthermore, the liver, lungs, and kidneys showed no pathological damage compared with the BL21-W-VLP group. CONCLUSION: Overall, this study proposes a method for producing VLP with high immunogenicity and minimal endotoxin activity without chemical or physical endotoxin removal methods. This method could address the issue of endotoxin residues in the VLP and provide production benefits.

Generation of porcine circovirus type 4 virus-like particles and their use to detect serum antibodies.

Porcine circovirus type 4 (PCV4), first identified in 2019 as a newly emerging pathogen, has been found in several provinces of China, as well as in Korea and Thailand. Since PCV4 is not included in immunization programs, epidemiological investigations should be conducted for detection of anti-PCV4 antibodies. Virus-like particles (VLPs) are frequently used for serological analysis of pathogen infections. However, there have been no reports on using PCV4 VLPs for serological investigation of PCV4 infection. In this study, we generated self-assembled PCV4 VLPs using an E. coli expression system, purified them using a two-step process, and used them to develop an indirect ELISA. This ELISA method was found to be highly specific, sensitive, and repeatable, making it suitable for PCV4 antibody detection in serum samples. Finally, the ELISA was used to analyze 422 serum samples collected from across several regions in China, 134 of which tested positive. Thus, the PCV4-VLP-based ELISA can effectively detect antibodies against PCV4 in serum samples, making it a useful tool for PCV4 epidemiology.

Transforming growth factor-induced gene TGFBI is correlated with the prognosis and immune infiltrations of breast cancer.

BACKGROUND: Transforming growth factor ß (TGFß) is a critical regulator of lung metastasis of breast cancer and is correlated with the prognosis of breast cancer. However, not all TGFß stimulated genes were functional and prognostic in breast cancer lung metastatic progress. In this study, we tried to determine the prognosis of TGFß stimulated genes in breast cancer. METHODS: TGFß stimulated genes in MDA-MB-231 cells and lung metastasis-associated genes in LM2-4175 cells were identified through gene expression microarray. The prognosis of the induced gene (TGFBI) in breast cancer was determined through bioinformatics analysis and validated using tissue microarray. The immune infiltrations of breast cancer were determined through "ESTIMATE" and "TIMER". RESULTS: TGFBI was up-regulated by TGFß treatment and over-expressed in LM2-4175 cells. Through bioinformatics analysis, we found that higher expression of TGFBI was associated with shorted lung metastasis-free survival, relapse-free survival, disease-free survival, and overall survival of breast cancer. Moreover, the prognosis of TGFBI was validated in 139 Chinese breast cancer patients. Chinese breast cancer patients with higher TGFBI expression had lower overall survival. Correspondingly, breast cancer patients with higher TGFBI methylation had higher overall survival. TGFBI was correlated with the score of the TGFß signaling pathway and multiple immune-related signaling pathways in breast cancer. The stromal score, immune score, and the infiltrations of immune cells were also correlated with TGFBI expression in breast cancer. CONCLUSIONS: TGFß-induced gene TGFBI was correlated with the prognosis and immune infiltrations of breast cancer.

The therapeutic effect of Loranthus parasiticus lignan derivatives on collagen-induced arthritis in rats through the SHBG/NFκB pathway.

Lignan-rich beans, nuts, and various seeds are the main foods with antioxidative and hormone-modulating activities. Although the role of lignans in mediating hormone-dependent cancers and cardiovascular diseases is well characterized, the function of lignans in anti-arthritic activity and its underlying mechanisms remain unknown. Three new lignan derivatives, (-)-nortrachelogenin, trachelogenin, and matairesinol, were extracted from Loranthus parasiticus. After establishing the collagen-induced arthritis (CIA) model by intradermal injection of collagen, rats were treated with three new lignan derivatives ((-)-nortrachelogenin: 37%; trachelogenin: 27%; matairesinol: 25.7%) at a concentration of 50 mg/kg and 100 mg/kg, or methotrexate at 0.3 mg/kg. Mixed lignan derivatives significantly attenuated the immune responses in the joints of CIA rats, leading to lower levels of proinflammatory cytokines (IL-6 and TNF-α) and higher levels of free androgen in the serum compared to the CIA model. The results of molecular docking using AutoDock Vina showed that the lignan derivative (-)-nortrachelogenin was the most effective compound for binding to sex hormone-binding globulin (SHBG), thus inhibiting the activity of NFκB in LPS-stimulated macrophages. In this study, (-)-nortrachelogenin was identified as a novel natural lignan derivative with previously unrecognized anti-inflammatory activity. Its molecular mechanism appears related to the regulation of the NFκB/SHBG pathway. Our findings suggest that further application of sex hormone-like compounds in the treatment of rheumatoid arthritis and the potential clinical applications of (-)-nortrachelogenin are promising.

ASPP2 Upregulation as a Novel Approach to TGF-ß2-Induced Proliferative Vitreoretinopathy In Vivo and In Vitro.

PURPOSE: Proliferative vitreoretinopathy (PVR) can cause blindness and the pathogenesis is unclear. Transforming growth factor (TGF)-ß-induced epithelial-mesenchymal transition (EMT) of RPE cells is vital. P53 protein 2 (ASPP2) was previously reported to inhibit EMT in PVR rats, but the specific mechanism is unveiled. METHODS: TGF-ß was used to induce EMT in ARPE-19 cells, and evaluated by immunofluorescence and western blot. ARPE-19 cells were transfected with scrambled/ASPP2-lentivirus, followed by TGF-ß treatment. After that, alterations of EMT and autophagy were measured by western blot and transmission electron microscopy. Moreover, TGF-ß and ARPE-19 cells treated with scrambled/ASPP2-lentivirus were employed to establish the PVR model via intravitreal injection to SD rats, and retinal changes as well as EMT and autophagy activity were evaluated accordingly. RESULTS: ASPP2 expression was decreased during TGF-ß-induced EMT in ARPE-19 cells. In vitro, EMT and autophagy was activated by TGF-ß, which could be partly reversed by ASPP2 upregulation. In vivo, ASPP2 upregulation protected against structural and functional changes in PVR retinas. Additionally, expressions of EMT and autophagy markers in retinas were inhibited by ASPP2 upregulation. CONCLUSIONS: ASPP2 upregulation inhibited the EMT and autophagy process caused by TGF-ß in ARPE-19 cells. Correspondingly, upregulation of ASPP2 alleviated intraocular fibrosis and protected visual function in PVR rats.

Research trends in functionalized Fe3O4 composites based on affinity recognition systems for targeted extraction of natural products.

In recent years, target-specific affinity recognition systems based on Fe3O4-based composites have proven to be an effective method for screening natural products. Herbal medicines contain a wide range of natural products and are considered to be a major source for the development of novel drugs. However, the process of isolating and obtaining these bioactive components for the production of novel drugs is complex. Meanwhile, the complexity and diversity of herbal constituents have posed a great challenge to the screening studies of herbal active ingredients. Currently, traditional extraction and screening studies of active ingredients in herbal medicine include extraction and chromatographic separation technology development, serum medicinal chemistry, metabolomics and computerized virtual screening. In order to achieve integrated targeting of Fe3O4 for extraction and separation of natural products from herbs, various Fe3O4-based composites need to be synthesized so that the composites can be further functionalized and modified. Composites such as Fe3O4@SiO2, Fe3O4-based magnetic graphene oxide and Fe3O4-based magnetic carbon nanotubes were used to achieve targeted extraction and isolation of natural products from herbal medicines. The main extraction techniques involved based on these Fe3O4-based composites are molecularly imprinted techniques, immobilized ligand fishing techniques, and cell membrane-coated bionanotechnology methods. This article will present recent advances in the synthesis and modification of Fe3O4 composites and their applications for the extraction of natural products in conjunction with molecular imprinting, immobilization-targeted fishing, and cell-membrane-coated biomimetic techniques, as well as the future goals and challenges of functionalized modification of Fe3O4 composites for the targeted extraction of natural products, like protein overexpression modification, doping of fluorescent substances and genetic engineering development. A deeper understanding of the multi-level, multidisciplinary, and applied studies in materials science and phytochemistry will be provided by this article.

Identification of a conserved B-cell epitope on the capsid protein of porcine circovirus type 4.

Porcine circovirus type 4 (PCV4), a recently identified circovirus, is prevalent in numerous provinces in China, as well as in South Korea, Thailand, and Europe. PCV4 virus rescued from an infectious clone showed pathogenicity, suggesting the economic impact of PCV4. However, there remains a lack of understanding regarding the immunogenicity and epitopes of PCV4. This study generated a monoclonal antibody (MAb) 1D8 by immunizing mice with PCV4 virus-like particles (VLPs). Subsequently, the epitope recognized by the MAb 1D8 was identified by truncated protein expression and alanine scanning mutagenesis analysis. Results showed that the 225PKQG228 located at the C-terminus of the PCV4 Cap protein is the minimal motif binding to the MAb. Homology modeling analysis and immunoelectron microscopy revealed that the epitope extends beyond the outer surface of the PCV4 VLP. Moreover, the epitope is highly conserved among PCV4 strains and does not react with other PCVs. Together, the MAb 1D8 recognized epitope shows potential for detecting PCV4. These findings significantly contribute to the design of antigens for PCV4 detection and control strategies. IMPORTANCE: Porcine circovirus type 4 (PCV4) is a novel circovirus. Although PCV4 has been identified in several countries, including China, Korea, Thailand, and Spain, no vaccine is available. Given the potential pathogenic effects of PCV4 on pigs, PCV4 could threaten the global pig farming industry, highlighting the urgency for further investigation. Thus, epitopes of PCV4 remain to be determined. Our finding of a conserved epitope significantly advances vaccine development and pathogen detection.

Effects of Heat Stress and Lipopolysaccharides on Gene Expression in Chicken Immune Cells.

Prolonged exposure to high temperatures and humidity can trigger heat stress in animals, leading to subsequent immune suppression. Lipopolysaccharides (LPSs) act as upstream regulators closely linked to heat stress, contributing to their immunosuppressive effects. After an initial examination of transcriptome sequencing data from individual samples, 48 genes displaying interactions were found to potentially be associated with heat stress. Subsequently, to delve deeper into this association, we gathered chicken bone marrow dendritic cells (BMDCs). We combined heat stress with lipopolysaccharides and utilized a 48 × 48 Fluidigm IFC quantitative microarray to analyze the patterns of gene changes under various treatment conditions. The results of the study revealed that the combination of heat stress and LPSs in a coinfection led to reduced expressions of CRHR1, MEOX1, and MOV10L1. These differentially expressed genes triggered a pro-inflammatory response within cells via the MAPK and IL-17 signaling pathways. This response, in turn, affected the intensity and duration of inflammation when experiencing synergistic stimulation. Therefore, LPSs exacerbate the immunosuppressive effects of heat stress and prolong cellular adaptation to stress. The combination of heat stress and LPS stimulation induced a cellular inflammatory response through pathways involving cAMP, IL-17, MAPK, and others, consequently leading to decreased expression levels of CRHR1, MEOX1, and MOV10L1.

Novel GJB2 mutation c.188delT compound with c.235delC causing non-syndromic hearing loss in a Chinese family: A case report.

RATIONALE: Congenital sensorineural hearing loss is a significant global health issue, primarily driven by genetic factors, such as mutations in the GJB2 gene. This report presents a Chinese girl with congenital deafness and a novel mutation of the GJB2 gene. PATIENT CONCERNS: A newborn Chinese girl exhibited signs of congenital deafness. DIAGNOSIS: Congenital deafness was confirmed through comprehensive newborn hearing screenings that included otologic, audiologic, and physical examinations. Genetic analysis revealed a compound heterozygous mutation involving c.188delT and c.235delC in the GJB2 gene, indicating a genetic basis for her hearing loss. INTERVENTIONS: The patient underwent cochlear implantation, which resulted in stable auditory outcomes. OUTCOMES: Despite follow-up difficulties, stable auditory outcomes were achieved post-cochlear implantation, highlighting the potential efficacy of this intervention in GJB2-related hearing loss. LESSONS: This case study enriches our understanding of GJB2 mutations and underscores the critical role of genetic testing in diagnosing congenital sensorineural hearing loss. It emphasizes the necessity for early intervention and sustained interdisciplinary care to enhance the quality of life for patients with genetic hearing impairment.

Passive exercise is an effective alternative to HRT for restoring OVX induced mitochondrial dysfunction in skeletal muscle.

Background: Postmenopausal women are more prone to develop muscle weakness, which is strongly associated with impairment of mitochondrial function in skeletal muscle. This study aimed to examine the impact of a passive exercise modality, whole-body vibration training (WBVT), on muscle mitochondrial function in ovariectomized (OVX) mice, in comparison with 17ß-estradiol (E2) replacement. Methods: Female C57BL/6J mice were assigned to four groups: sham operation control group (Sham), ovariectomized group (OVX), OVX with E2 supplement group (OVX+E), and OVX with WBVT group (OVX+W). The estrous cycle, body weight, body composition, and muscle strength of the mice were monitored after the operation. Serum E2 level was assessed by enzyme-linked immunosorbent assay (ELISA). The ATP levels were determined using a luciferase-catalyzed bioluminescence assay. The activity of mitochondrial respiration chain complexes was evaluated using high-resolution respirometry (O2K). Expression levels of oxidative phosphorylation (OXPHOS), peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α), and mitochondrial transcription factor A (TFAM) were detected using western blotting. Results: We observed decreased muscle strength and impaired mitochondrial function in the skeletal muscle of OVX mice. The vibration training alleviated these impairments as much as the E2 supplement. In addition, the vibration training was superior to the ovariectomy and the estradiol replacement regarding the protein expression of PGC-1α and TFAM. Conclusion: WBVT improves the OVX-induced decline in muscle strength and impairment of mitochondrial function in the skeletal muscle. This passive exercise strategy may be useful as an alternative to E2 replacement for preventing menopausal muscular weakness. Further studies are needed to understand the effects of WBVT on various physiological systems, and precautions should be taken when implementing it in patient treatment.

Genome-wide characteristics and potential functions of circular RNAs from the embryo muscle development in Chengkou mountain chicken.

The Chengkou mountain chicken, a native Chinese poultry breed, holds significant importance in the country's poultry sector due to its delectable meat and robust stress tolerance. Muscle growth and development are pivotal characteristics in poultry breeding, with muscle fiber development during the embryonic period crucial for determining inherent muscle growth potential. Extensive evidence indicates that non-coding RNAs (ncRNAs) play a regulatory role in muscle growth and development. Among ncRNAs, circular RNAs (circRNAs), characterized by a closed-loop structure, have been shown to modulate biological processes through the regulation of microRNAs (miRNAs). This study seeks to identify and characterize the spatiotemporal-specific expression of circRNAs during embryonic muscle development in Chengkou mountain chicken, and to construct the potential regulatory network of circRNAs-miRNA-mRNAs. The muscle fibers of HE-stained sections became more distinct, and their boundaries were more defined over time. Subsequent RNA sequencing of 12 samples from four periods generated 9,904 novel circRNAs, including 917 differentially expressed circRNAs. The weighted gene co-expression network analysis (WGCNA)-identified circRNA source genes significantly enriched pathways related to cell fraction, cell growth, and muscle fiber growth regulation. Furthermore, a competitive endogenous RNA (ceRNA) network constructed using combined data of present and previous differentially expressed circRNAs, miRNA, and mRNA revealed that several circRNA transcripts regulate MYH1D, MYH1B, CAPZA1, and PERM1 proteins. These findings provide insight into the potential pathways and mechanisms through which circRNAs regulate embryonic muscle development in poultry, a theoretical support for trait improvement in domestic chickens.

VP0 Myristoylation Is Essential for Senecavirus A Replication.

Many picornaviruses require the myristoylation of capsid proteins for viral replication. Myristoylation is a site-specific lipidation to the N-terminal G residue of viral proteins, which is catalyzed by the ubiquitous eukaryotic enzyme N-myristoyltransferase (NMT) by allocating the myristoyl group to the N-terminal G residue. IMP-1088 and DDD85646 are two inhibitors that can deprive NMT biological functions. Whether Senecavirus A (SVA) uses NMT to modify VP0 and regulate viral replication remains unclear. Here, we found that NMT inhibitors could inhibit SVA replication. NMT1 knock-out in BHK-21 cells significantly suppressed viral replication. In contrast, the overexpression of NMT1 in BHK-21 cells benefited viral replication. These results indicated that VP0 is a potential NMT1 substrate. Moreover, we found that the myristoylation of SVA VP0 was correlated to the subcellular distribution of this protein in the cytoplasm. Further, we evaluated which residues at the N-terminus of VP0 are essential for viral replication. The substitution of N-terminal G residue, the myristoylation site of VP0, produced a nonviable virus. The T residue at the fifth position of the substrates facilitates the binding of the substrates to NMT. And our results showed that the T residue at the fifth position of VP0 played a positive role in SVA replication. Taken together, we demonstrated that SVA VP0 myristoylation plays an essential role in SVA replication.

Identification of a linear B-cell epitope on the "puff" loop of the Senecavirus A VP2 protein involved in receptor binding.

Senecavirus A (SVA) is an important emerging swine pathogen that causes vesicular lesions in swine and acute death in newborn piglets. VP2 plays a significant role in the production of antibodies, which can be used in development of diagnostic tools and vaccines. Herein, the aim of the current study was to identify B-cell epitopes (BCEs) of SVA for generation of epitope-based SVA marker vaccine. Three monoclonal antibodies (mAbs), named 2E4, 1B8, and 2C7, against the SVA VP2 protein were obtained, and two novel linear BCEs, 177SLGTYYR183 and 266SPYFNGL272, were identified by peptide scanning. The epitope 177SLGTYYR183 was recognized by the mAb 1B8 and was fully exposed on the VP2 surface, and alanine scanning analysis revealed that it contained a high continuity of key amino acids. Importantly, we confirmed that 177SLGTYYR183 locates on "the puff" region within the VP2 EF loop, and contains three key amino acid residues involved in receptor binding. Moreover, a single mutation, Y182A, blocked the interaction of the mutant virus with the mAb 1B8, indicating that this mutation is the pivotal point for antibody recognition. In summary, the BCEs that identified in this study could be used to develop diagnostic tools and an epitope-based SVA marker vaccine.

Multivalent nanobody-based sandwich enzyme-linked immunosorbent assay for sensitive detection of porcine reproductive and respiratory syndrome virus.

The pandemic of the porcine reproductive and respiratory syndrome virus (PRRSV) has caused huge economic losses and continues to threaten the swine industry worldwide. Nucleocapsid protein (N protein) is the primary antigen of PRRSV for development of sensitive diagnostic assays. Two high affinity nanobodies against N protein, Nb12 and Nb35, were selected and employed to develop a sandwich ELISA. Further we improved the ELISA method to obtain greater sensitivity, a trivalent nanobody (3 × Nb35) and a bivalent nanobody-HRP fusion protein (2 × Nb12-HRP) were expressed and used. This modified ELISA was found to have high sensitivity for detecting PRRSV, with a detection limit of 10 TCID50/ml (median tissue culture infectious dose), which was approximately 200-fold greater than the single-copy nanobody-based sandwich ELISA. The developed assay shows high specificity and can detect almost all circulating lineages of PRRSV-2 in China. This study provides suggestions for reforming nanobodies and for the further development of multivalent nanobody-based ELISAs for other various viruses.