Capping Protein Research Articles

Goose IFIT5 positively regulates goose astrovirus replication in GEF cells

Interferon-induced protein with tetratricopeptide repeats (IFITs), a family of proteins strongly induced by type I interferon (IFN-I), are deeply involved in many cellular and viral processes. IFIT5, the sole protein in this family found in birds, also plays a crucial role in regulating virus infection. In this study, goose IFIT5 (gIFIT5) was first cloned from peripheral blood lymphocyte (PBL) and phylogenetic analysis showed that it was highly homologous with duck IFIT5 (dIFIT5), sharing 94.6% identity in amino acid sequence. Subsequently, the expression kinetics of gIFIT5 during goose astrovirus (GAstV) infection and the regulatory effect of gIFIT5 on GAstV proliferation were evaluated. Results showed that the mRNA and protein expression level of gIFIT5 was greatly induced by GAstV infection, especially at 12 hpi. Importantly, gIFIT5 could conversely promote GAstV replication in GEF cells. Virus titers in gIFIT5 overexpression group were significantly higher than those in control group at 12 and 24 hpi. Western blot and quantitative real-time PCR (qRT-PCR) further demonstrated that the production of viral cap protein was significantly facilitated in gIFIT5-transfected group. Collectively, GAstV facilitates self-replication via promoting gIFIT5 expression.

CRACD loss induces neuroendocrine cell plasticity of lung adenocarcinoma

Tumor cell plasticity contributes to intratumoral heterogeneity and therapy resistance. Through cell plasticity, some lung adenocarcinoma (LUAD) cells transform into neuroendocrine (NE) tumor cells. However, the mechanisms of NE cell plasticity remain unclear. CRACD (capping protein inhibiting regulator of actin dynamics), a capping protein inhibitor, is frequently inactivated in cancers. CRACD knockout (KO) is sufficient to de-repress NE-related gene expression in the pulmonary epithelium and LUAD cells. In LUAD mouse models, Cracd KO increases intratumoral heterogeneity with NE gene expression. Single-cell transcriptomic analysis showed that Cracd KO-induced NE cell plasticity is associated with cell de-differentiation and stemness-related pathway activation. The single-cell transcriptomic analysis of LUAD patient tumors recapitulates that the distinct LUAD NE cell cluster expressing NE genes is co-enriched with impaired actin remodeling. This study reveals the crucial role of CRACD in restricting NE cell plasticity that induces cell de-differentiation of LUAD.

Establishment and preliminary application of duplex fluorescence quantitative PCR for porcine circoviruses type 2 and type 3

Porcine circovirus types 2 (PCV2) and 3 (PCV3) are the two most prevalent porcine circoviruses in China, all of which can infect swine herds and cause serious diseases. To detect coinfection with PCV2 and PCV3, primers and probes for duplex PCV2 and PCV3 real-time PCR were designed to target their cap genes based on the constructed plasmids pUC57-PCV2 and pUC57-PCV3. The established duplex PCV2 and PCV3 real-time PCRs were specific to PCV2 and PCV3 and showed no cross-reactions with other porcine viral pathogens. The limit of detection was 5 and 50 copies for the PCV2 and PCV3 plasmids, respectively. The intra- and interassay repeatability had coefficients of variation below 3 %. The established methods were used to analyze clinical samples from Liaoning and Jilin provinces of China. The coinfection rates of PCV2 and PCV3 in pigs extensively fed in Liaoning and Jilin, large-scale farmed pigs in Liaoning and large-scale farmed pigs in Jilin were 15.0 % (6/40), 36.7 % (11/30) and 35.4 % (62/175), respectively. This study established a useful duplex PCV2 and PCV3 real-time PCR method that can be used for the detection of PCV2 and PCV3 in local clinical samples.

Identification and Evolutionary Analysis of the Widely Distributed CAP Superfamily in Spider Venom.

Venom plays a crucial role in the defense and predation of venomous animals. Spiders (Araneae) are among the most successful predators and have a fascinating venom composition. Their venom mainly contains disulfide-rich peptides and large proteins. Here, we analyzed spider venom protein families, utilizing transcriptomic and genomic data, and highlighted their similarities and differences. We show that spiders have specific combinations of toxins for better predation and defense, typically comprising a core toxin expressed alongside several auxiliary toxins. Among them, the CAP superfamily is widely distributed and highly expressed in web-building Araneoidea spiders. Our analysis of evolutionary relationships revealed four subfamilies (subA-subD) of the CAP superfamily that differ in structure and potential functions. CAP proteins are composed of a conserved CAP domain and diverse C-terminal domains. CAP subC shares similar domains with the snake ion channel regulator svCRISP proteins, while CAP subD possesses a sequence similar to that of insect venom allergen 5 (Ag5). Furthermore, we show that gene duplication and selective expression lead to increased expression of CAP subD, making it a core member of the CAP superfamily. This study sheds light on the functional diversity of CAP subfamilies and their evolutionary history, which has important implications for fully understanding the composition of spider venom proteins and the core toxin components of web-building spiders.

Development a high-sensitivity sandwich ELISA for determining antigen content of porcine circovirus type 2 vaccines

Porcine circovirus type 2 (PCV2) is intensely prevalent in global pig farms. The PCV2 vaccine is an important means of preventing and controlling PCV2. The quality control of PCV2 vaccines is predominantly based on detection techniques such as animal testing and neutralizing antibody titration. Measuring the content of effective proteins in vaccines to measure vaccine efficacy is an excellent alternative to traditional methods, which can greatly accelerate the development speed and testing time of vaccines. In this study, we screened a monoclonal antibody (mAb) that can effectively recognize not only the exogenous expression of PCV2 Cap protein but also PCV2 virus. The double antibody sandwich ELISA (DAS-ELISA) was developed using this mAb that specifically recognize PCV2 Cap. The minimum protein content detected by this method is 3.5 ng/mL. This method can be used for the quality control of PCV2 inactivated vaccine and subunit vaccine, and the detection results are consistent with the results of mice animal experiments. This method has the advantages of simple operation, good sensitivity, high specificity and wide application. It can detect the effective antigen Cap protein content of various types of PCV2 vaccines, which not only shorten the vaccine inspection time but also save costs.

CARMIL1 regulates liver cancer cell proliferation by activating the ERK/mTOR pathway through the TRIM27/p53 axis

Capping protein regulatory factor and myosin 1 linker 1 is termed CARMIL1. CARMIL1 is involved in several physiological processes; it forms an actin filament network and plasma membrane-bound cellular projection tissues and positively regulates the cellular components and tissues. CARMIL1 exhibits important biological functions in cancer; nonetheless, these functions have not been completely explored. We aimed to investigate the novel functions of CARMIL1 in liver cancer, particularly in cell proliferation. The cell counting kit-8, 5-ethynyl-2′-deoxyuridine, Component A experiments, and subcutaneous tumor formation model suggest that CARMIL1 is central to the proliferation of liver cancer cells both in vivo and in vitro. We extracted CARMIL1 samples from The Cancer Genome Atlas Program and analyzed its enrichment. CARMIL1 regulated the pathway activity by affecting the expression of star molecular proteins of the extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin (mTOR). Moreover, it influenced the proliferation ability of liver cancer cells. Western blotting suggested that CARMIL1 downregulation could affect ERK and mTOR phosphorylation. Results of the co-immunoprecipitation demonstrated that CARMIL1 binds to tripartite motif (TRIM)27, which in turn binds to p53. Subsequently, CARMIL1 can regulate p53 stability and promote its degradation through TRIM27. Additionally, CARMIL1 inhibition enhanced the sensitivity of liver cancer cells to sorafenib. Tumor growth was significantly inhibited in the group treated with sorafenib and CARMIL1, compared with the group treated with CARMIL1 alone. Sorafenib is a first-line targeted chemotherapeutic drug for hepatocellular carcinoma treatment. It increases the long-term survival of hepatocellular carcinoma by 44%. In this study, downregulated CARMIL1 combined with sorafenib significantly reduced the tumor volume and weight of the mouse subcutaneous tumor model, indicating the potential possibility of combining CARMIL1 with sorafenib in hepatocellular carcinoma treatment. In summary, CARMIL1 promotes liver cancer cell proliferation by regulating the TRIM27/p53 axis and activating the ERK/mTOR pathway.

NR1I2 as a core biological target in chronic venous ulcer tissues treated with ultrasound therapy.

Ultrasound therapy is a method of applying ultrasonic energy to the stimulation produced by human body to change the function and tissue state of the body in order to achieve the purpose of treating diseases. Chronic venous ulcer is a common chronic skin ulcer. GSE222503 for ultrasound therapy of chronic venous ulcers was downloaded from gene expression omnibus database, which were used to identify differentially expressed genes. Weighted gene co-expression network analysis, functional enrichment analysis, gene set enrichment analysis, immune infiltration analysis and construction and analysis of protein-protein interaction network were performed. Draw gene expression heatmaps. Comparative toxicogenomics database analysis was performed. Two hundred thirty-five differentially expressed genes were obtained. According to gene ontology analysis, in biological process analysis, they were mainly enriched in positive regulation of cellular biosynthetic process, reproductive cell development, vasculogenesis, vascular morphogenesis, and inflammatory response. In cellular component analysis, they were mainly enriched in leading edge of growing cell, extracellular matrix binding organelle, F-actin capping protein complex. In molecular function analysis, they were mainly concentrated in receptor ligand activity, cytokine receptor binding. In Kyoto encyclopedia of genes and genomes analysis, they were mainly enriched in cytokine-cytokine receptor interaction, PI3K-Akt signaling pathway, HIF-1 signaling pathway, heme biosynthesis. In weighted gene co-expression network analysis, the soft threshold power was set to 9. Thirty modules were generated. PF4, NR1I2, TTC16, H3C12, KLRB1, CYP21A2 identified by 4 algorithms (MCC, EPC, closeness, stress). Heatmap of core gene expression showed that H3C12, KLRB1, PF4, NR1I2 were all underexpressed in samples of ultrasound-treated chronic venous ulcers and overexpressed in samples of untreated chronic venous ulcers. Comparative toxicogenomics database analysis showed that H3C12, KLRB1, PF4, NR1I2 are associated with thrombophlebitis, phlebitis, vascular malformations, metabolic syndrome, ulcers, and inflammation. In samples of chronic venous ulcer tissue treated with ultrasound, NR1I2 shows low expression, while in samples of chronic venous ulcer tissue without ultrasound treatment, it shows high expression. This finding suggests a potential role of NR1I2 in the process of ultrasound therapy for chronic venous ulcers, which may be related to the therapeutic effect of ultrasound therapy on chronic venous ulcers.

Adducin Regulates Sarcomere Disassembly During Cardiomyocyte Mitosis.

Recent interest in understanding cardiomyocyte cell cycle has been driven by potential therapeutic applications in cardiomyopathy. However, despite recent advances, cardiomyocyte mitosis remains a poorly understood process. For example, it is unclear how sarcomeres are disassembled during mitosis to allow the abscission of daughter cardiomyocytes. Here, we use a proteomics screen to identify adducin, an actin capping protein previously not studied in cardiomyocytes, as a regulator of sarcomere disassembly. We generated many adeno-associated viruses and cardiomyocyte-specific genetic gain-of-function models to examine the role of adducin in neonatal and adult cardiomyocytes in vitro and in vivo. We identify adducin as a regulator of sarcomere disassembly during mammalian cardiomyocyte mitosis. α/γ-adducins are selectively expressed in neonatal mitotic cardiomyocytes, and their levels decline precipitously thereafter. Cardiomyocyte-specific overexpression of various splice isoforms and phospho-isoforms of α-adducin in vitro and in vivo identified Thr445/Thr480 phosphorylation of a short isoform of α-adducin as a potent inducer of neonatal cardiomyocyte sarcomere disassembly. Concomitant overexpression of this α-adducin variant along with γ-adducin resulted in stabilization of the adducin complex and persistent sarcomere disassembly in adult mice, which is mediated by interaction with α-actinin. These results highlight an important mechanism for coordinating cytoskeletal morphological changes during cardiomyocyte mitosis.

Silver nanoparticles biosynthesis using mixture of Lactobacillus sp. and Bacillus sp. growth and their antibacterial activity

The biosynthesis of nanoparticles offers numerous advantages, including ease of production, cost-effectiveness, and environmental friendliness. In our research, we focused on the bioformation of silver nanoparticles (AgNPs) using a combination of Lactobacillus sp. and Bacillus sp. growth. These AgNPs were then evaluated for their biological activities against multidrug-resistant bacteria. Our study involved the isolation of Bacillus sp. from soil samples and Lactobacillus sp. from raw milk in Dhamar Governorate, Yemen. The synthesized AgNPs were characterized using various techniques such as UV–visible spectroscopy, X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), and transmission electron microscopy (TEM). The antibacterial properties of the AgNPs were assessed using the modified Kirby Bauer disk diffusion method against multidrug-resistant strains of Staphylococcus aureus and Pseudomonas aeruginosa. Our results demonstrated that the use of a bacterial mixture for biosynthesis led to faster and more effective production of AgNPs compared to using a single bacterium. The UV–visible spectra showed characteristic peaks indicative of silver nanoparticles, while XRD analysis confirmed the crystalline nature of the synthesized particles. FTIR results suggested the presence of capping proteins that contribute to the synthesis and stability of AgNPs. Furthermore, TEM images revealed the size and morphology of the AgNPs, which exhibited spherical shapes with sizes ranging from 4.65 to 22.8 nm. Notably, the antibacterial activity of the AgNPs was found to be more pronounced against Staphylococcus aureus than Pseudomonas aeruginosa, indicating the potential of these nanoparticles as effective antimicrobial agents. Overall, our study highlights the promising antibacterial properties of AgNPs synthesized by a mixture of Lactobacillus sp. and Bacillus sp. growth. Further research is warranted to explore the potential of utilizing different bacterial combinations for enhanced nanoparticle synthesis.

Development and application of a TaqMan-probe-based multiplex real-time PCR assay for simultaneous detection of porcine circovirus 2, 3, and 4 in Guangdong province of China.

Porcine circoviruses disease (PCVD), caused by porcine circovirus (PCVs), is an important swine disease characterized by porcine dermatitis, nephrotic syndrome and reproductive disorders in sows. However, diseases caused by PCV2, PCV3, or PCV4 are difficult to distinguish, so a simple, rapid, accurate and high-throughput diagnostic and identification method is urgently needed to differentiate these three types. In this study, specific primers and probes were designed based on the conserved region sequences of the Rep gene of PCV2, and the Cap gene of PCV3 and PCV4. A multiplex qPCR assay was developed and optimized that the limit of detection concentration could reach as low as 3.8 copies/μL, with all correlation coefficients (R2) exceeding 0.999. Furthermore, the method showed no cross-reaction with other crucial porcine viral pathogens, and both intra-repeatability and inter-reproducibility coefficients of variation were below 2%. The assay was applied to the detection of 738 pig samples collected from 2020 to 2021 in Guangdong Province, China. This revealed positive infection rates of 65.18% for PCV2, 29.27% for PCV3, and 0% for PCV4, with a PCV2/PCV3 co-infection rate of 23.17%. Subsequently, complete genome sequences of 17 PCV2 and 4 PCV3 strains were obtained from the above positive samples and pre-preserved positive circovirus samples. Nucleotide sequence analysis revealed that the 17 PCV2 strains shared 96.7-100% complete nucleotide identity, with 6 strains being PCV2b and 11 strains being PCV2d; the 4 PCV3 strains shared 98.9-99.4% complete nucleotide identity, with 2 strains being PCV3a-1 and 2 strains being PCV3b. This research provides a reliable tool for rapid PCVs identification and detection. Molecular epidemiological investigation of PCVs in pigs in Guangdong Province will help us to understand PCV2 and PCV3 epidemiological characteristics and evolutionary trends.

Stability and integrity of self-assembled bovine parvovirus virus‑like particles (BPV‑VLPs) of VP2 and combination of VP1VP2 assisted by baculovirus-insect cell expression: a potential logistical platform for vaccine deployment

BackgroundBovine parvovirus (BPV) is an autonomous DNA virus with a smaller molecular size and subtle differences in its structural proteins, unlike other animal parvoviruses. More importantly, this virus has the potential to produce visible to silent economic catastrophes in the livestock business, despite receiving very little attention. Parvoviral virus-like particles (VLPs) as vaccines and as logistical platforms for vaccine deployment are well studied. However, no single experimental report on the role of VP1 in the assembly and stability of BPV-VLPs is available. Furthermore, the self-assembly, integrity and stability of the VLPs of recombinant BPV VP2 in comparison to VP1 VP2 Cap proteins using any expression method has not been studied previously. In this study, we experimentally evaluated the self-assembling ability with which BPV virus-like particles (VLPs) could be synthesized from a single structural protein (VP2) and by integrating both VP2 and VP1 amino acid sequences.MethodsIn silico and experimental cloning methods were carried out. His-tagged and without-His-tag VP2 and V1VP2-encoding amino acid sequences were cloned and inserted into pFastbacdual, and insect cell-generated recombinant protein was evaluated by SDS‒PAGE and western blot. Period of infectivity and expression level were determined by IFA. The integrity and stability of the BPV VLPs were evaluated by transmission electron microscopy. The secondary structure of the BPV VLPs from both VP2 and V1VP2 was analyzed by circular dichroism.ResultsOur findings show that VP2 alone was equally expressed and purified into detectable proteins, and the stability at different temperatures and pH values was not appreciably different between the two kinds of VLPs. Furthermore, BPV-VP2 VLPs were praised for their greater purity and integrity than BPV-VP1VP2 VLPs, as indicated by SDS‒PAGE. Therefore, our research demonstrates that the function of VP1 has no bearing on the stability or integrity of BPV-VLPs.ConclusionsIn summary, incredible physiochemically stable BPV VP2-derived VLPs have been found to be promising candidates for the development of multivalent vaccines and immunodiagnostic kits against enteric viruses and to carry heterogeneous epitopes for various economically important livestock diseases.

Assessment of the anti-inflammatory and biological properties of Bioroot Flow: A novel bioceramic sealer.

IntroductionBioRoot Flow (BRF) is a novel premixed bioceramic sealer indicated for endodontic treatments, but the biological and immunomodulatory effects of this endodontic sealer on human periodontal ligament stem cells (hPDLSCs) have not been elucidated. MethodsTo ascertain the biological impact of BRF, TotalFill BC Sealer (TFbc), and AH Plus (AHP) on human Periodontal Ligament Stem Cells (hPDLSCs), assessments were conducted to evaluate the cytocompatibility, cellular proliferation, migratory capacity, osteo/cementogenic differentiation potential, the ability to form mineralized nodules, and the immunomodulatory characteristics of hPDLSCs following treatment with these endodontic sealers. ResultsBiological assays showed adequate cell metabolic activity and cell migration in BRF, while SEM assay evidenced that TFbc and BRF groups demonstrated a superior cell adhesion process, including substrate adhesion, cytoskeleton development, and spreading on the niche-like structures of the cement as compared to the AHP group. TFbc and BRF-treated groups exhibited a significantly lower IL6 and IL8 production than AHP (* p <.05). The bioceramic sealers stimulated heightened expression of BSP, CEMP-1, and CAP genes within a 7–14 day period. Notably, BRF and TFbc demonstrated a significant enhancement in the mineralization of hPDLSCs when compared to the negative control. Among these, cells treated with BRF showed a more substantial accumulation of calcium (*** p < .001). ConclusionsTaken together, these findings indicate that BRF can potentially enhance cell differentiation by promoting the expression of essential genes related to bone and cement formation. In addition, BRF and TFbc displayed anti-inflammatory effects.

Investigating pigeon circovirus infection in a pigeon farm: molecular detection, phylogenetic analysis and complete genome analysis

BackgroundPigeon circovirus infections in pigeons (Columba livia domestica) have been reported worldwide. Pigeons should be PiCV-free when utilized as qualified experimental animals. However, pigeons can be freely purchased as experimental animals without any clear guidelines to follow. Herein, we investigated the status quo of PiCV infections on a pigeon farm in Beijing, China, which provides pigeons for experimental use.ResultsPiCV infection was verified in at least three types of tissues in all forty pigeons tested. A total of 29 full-length genomes were obtained and deposited in GenBank. The whole genome sequence comparison among the 29 identified PiCV strains revealed nucleotide homologies of 85.8–100%, and these sequences exhibited nucleotide homologies of 82.7–98.9% as compared with those of the reference sequences. The cap gene displayed genetic diversity, with a wide range of amino acid homologies ranging from 64.5% to 100%. Phylogenetic analysis of the 29 full-genome sequences revealed that the PiCV strains in this study could be further divided into four clades: A (17.2%), B (10.4%), C (37.9%) and D (34.5%). Thirteen recombination events were also detected in 18 out of the 29 PiCV genomes obtained in this study. Phylogenetic research using the rep and cap genes verified the recombination events, which occurred between clades A/F, A/B, C/D, and B/D among the 18 PiCV strains studied.ConclusionsIn conclusion, PiCV infection, which is highly genetically varied, is extremely widespread on pigeon farms in Beijing. These findings indicate that if pigeons are to be used as experimental animals, it is necessary to evaluate the impact of PiCV infection on the results.

Mtc6/Ehg2 is a novel endoplasmic reticulum-resident glycoprotein essential for high-pressure tolerance.

Hydrostatic pressure is a common mechanical stressor that modulates metabolism and reduces cell viability. Eukaryotic cells have genetic programs to cope with hydrostatic pressure stress and maintain intracellular homeostasis. However, the mechanism underlying hydrostatic pressure tolerance remains largely unknown. We have recently demonstrated that maintenance of telomere capping protein 6 (Mtc6) plays a protective role in the survival of the budding yeast Saccharomyces cerevisiae under hydrostatic pressure stress by supporting the integrity of nutrient permeases. The current study demonstrates that Mtc6 acts as an endoplasmic reticulum (ER) membrane protein. Mtc6 comprises two transmembrane domains, a C-terminal cytoplasmic domain and a luminal region with 12 Asn (N)-linked glycans attached to it. Serial mutational analyses showed that the cytoplasmic C-terminal amino acid residues GVPS Mtc6 activity. Multiple N-linked glycans in the luminal region are involved in the structural conformation of Mtc6. Moreover, deletion of MTC6 led to increased degradation of the leucine permease Bap2 under hydrostatic pressure, suggesting that Mtc6 facilitates the proper folding of nutrient permeases in the ER under stress conditions. We propose a novel model of molecular function in which the glycosylated luminal domain and cytoplasmic GVPS sequences of Mtc6 cooperatively support the nutrient permease activity.

Recombinant expression and characterization of Canine circovirus capsid protein for diagnosis.

Canine circovirus (CanineCV) is a contagious virus that causes severe gastroenteritis, diarrhea, respiratory disease, and vasculitis, often resulting in fatality among infected dogs. In this study, a recombinant Capsid protein (rCap) of CanineCV was expressed in the Escherichia coli (E. coli) Rosetta (DE3) pLysS host cell, followed by affinity purification, and then analyzed by SDS-PAGE, revealing a molecular weight of approximately 31 kDa. The antigenicity of the CanineCV rCap protein was confirmed through recognition by a rabbit anti-CanineCV rCap protein polyclonal antibody (PoAb). Additionally, the reactivity and specificity of this PoAb were assessed using indirect enzyme-linked immunosorbent assay (ELISA) and Western blot analysis before applying in an immunohistochemistry (IHC), namely, immunoperoxidase detection. The immunoperoxidase assay using rabbit anti-CanineCV rCap protein PoAb demonstrated that the CanineCV Cap protein was predominantly located in immune cells, especially lymphocytes and macrophages, within the spleen, lung, tracheobronchial lymph nodes, small intestine, and kidney. Similarly, the Cap protein was also found in pneumocytes in the lung and renal tubular epithelial cells in the kidney. These findings reflected the biological activity and cell tropism of the virus. Therefore, the recombinant Cap protein and its PoAb could be used for the development of a valuable diagnostic tool for CanineCV detection.

Prevalence, genotypes, and infection risk factors of psittacine beak and feather disease virus and budgerigar fledgling disease virus in captive birds in Hong Kong

Psittacine beak and feather disease virus (PBFDV) and budgerigar fledgling disease virus (BFDV) are significant avian pathogens that threaten both captive and wild birds, particularly parrots, which are common hosts. This study involved sampling and testing of 516 captive birds from households, pet shops, and an animal clinic in Hong Kong for PBFDV and BFDV. The results showed that PBFDV and BFDV were present in 7.17% and 0.58% of the samples, respectively. These rates were lower than those reported in most parts of Asia. Notably, the infection rates of PBFDV in pet shops were significantly higher compared to other sources, while no BFDV-positive samples were found in pet shops. Most of the positive samples came from parrots, but PBFDV was also detected in two non-parrot species, including Swinhoe’s white-eyes (Zosterops simplex), which had not been reported previously. The ability of PBFDV to infect both psittacine and passerine birds is concerning, especially in densely populated urban areas such as Hong Kong, where captive flocks come into close contact with wildlife. Phylogenetic analysis of the Cap and Rep genes of PBFDV revealed that the strains found in Hong Kong were closely related to those in Europe and other parts of Asia, including mainland China, Thailand, Taiwan, and Saudi Arabia. These findings indicate the presence of both viruses among captive birds in Hong Kong. We recommend implementing regular surveillance for both viruses and adopting measures to prevent contact between captive and wild birds, thereby reducing the transmission of introduced diseases to native species.

Tumor vascular disrupting agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA): Suppresses macrophage capping protein beyond STING activation

The vascular disrupting agent (VDA) 5,6-dimethylxanthenone-4-acetic acid (DMXAA) induces apoptosis in vascular endothelial cells and leads to tumor hemorrhagic necrosis. While DMXAA has been proven to be a potent agonist of murine stimulator of interferon genes (mSTING), it has little effect on human-STING (hSTING). This species selectivity of DMXAA may explain its effectiveness against solid tumors in mice and its failure in clinical trials. However, DMXAA did reduce tumor volume in some patients during clinical trials. These paradoxical results have prompted us to investigate the anti-tumor mechanism of DMXAA beyond STING in the destruction of tumor vasculature in humans. In this study, we demonstrated that DMXAA binds to both human and mouse macrophage capping protein (CapG), with a KD of 5.839 μM for hCapG and a KD of 2.867 μM for mCapG, as determined by surface plasmon resonance (SPR) analysis. Homology modeling and molecular docking analysis of hCapG indicated that the critical residues involved in the hydrogen bond interaction of DMXAA with hCapG were Arg153, Thr151, and GLN141, Asn234. In addition, electrostatic pi-cation interaction occurred between DMXAA and hCapG. Further functional studies revealed that CapG protein plays a crucial role in the effects of DMXAA on human umbilical endothelial vein cell (HUEVC) angiogenesis and migration, as well as the expression of cytoskeletal proteins actin and tubulin, and the invasion of A549 lung adenocarcinoma cells. Our study has originally uncovered a novel cross-species pathway underlying the antitumor vascular disruption of DMXAA extends beyond STING activation. This finding deepens our understanding of the multifaceted actions of flavonoid VDAs in animal models and in clinical settings, and may provide insights for the precise therapy of DMXAA based on the biomarker CapG protein.

Optimized production of full-length PCV2d virus-like particles in Escherichia coli: A cost-effective and high-yield approach for potential vaccine antigen development

Porcine circovirus type 2 (PCV2) is a globally prevalent infectious pathogen affecting swine, with its capsid protein (Cap) being the sole structural protein critical for vaccine development. Prior research has demonstrated that PCV2 Cap proteins produced in Escherichia coli (E. coli) can form virus-like particles (VLPs) in vitro, and nuclear localization signal peptides (NLS) play a pivotal role in stabilizing PCV2 VLPs. Recently, PCV2d has emerged as an important strain within the PCV2 epidemic. In this study, we systematically optimized the PCV2d Cap protein and successfully produced intact PCV2d VLPs containing NLS using E. coli. The recombinant PCV2d Cap protein was purified through affinity chromatography, yielding 7.5 mg of recombinant protein per 100 ml of bacterial culture. We augmented the conventional buffer system with various substances such as arginine, β-mercaptoethanol, glycerol, polyethylene glycol, and glutathione to promote VLP assembly. The recombinant PCV2d Cap self-assembled into VLPs approximately 20 nm in diameter, featuring uniform distribution and exceptional stability in the optimized buffer. We developed the vaccine and immunized pigs and mice, evaluating the immunogenicity of the PCV2d VLPs vaccine by measuring PCV2-IgG, IL-4, TNF-α, and IFN-γ levels, comparing them to commercial vaccines utilizing truncated PCV2 Cap antigens. The HE staining and immunohistochemical tests confirmed that the PCV2 VLPs vaccine offered robust protection. The results revealed that animals vaccinated with the PCV2d VLPs vaccine exhibited high levels of PCV2 antibodies, with TNF-α and IFN-γ levels rapidly increasing at 14 days post-immunization, which were higher than those observed in commercially available vaccines, particularly in the mouse trial. This could be due to the fact that full-length Cap proteins can assemble into more stable PCV2d VLPs in the assembling buffer. In conclusion, our produced PCV2d VLPs vaccine elicited stronger immune responses in pigs and mice compared to commercial vaccines. The PCV2d VLPs from this study serve as an excellent candidate vaccine antigen, providing insights for PCV2d vaccine research.

Comparison of immune effects of porcine circovirus type 2d (PCV2d) capsid protein expressed by Escherichia coli and baculovirus-insect cells

Porcine circovirus type 2 (PCV2) is an important pathogen harmful to global pig production, which causes immunosuppression and serious economic losses. PCV2 capsid (Cap) protein expressed by E. coli or baculovirus-insect cells are often used in preparation of PCV2 subunit vaccines, but the latter is expensive to produce. It is therefore crucial to comparison of the immune effects of Cap protein expressed by the above two expression systems for reducing the production cost and guaranteeing PCV2 vaccine quality. In this study, the PCV2d-Cap protein lacking nuclear localization signal (NLS), designated as E. coli-Cap and Bac-Cap, was expressed by E. coli and baculovirus-Spodoptera frugiperda Sf9 (Bac-Sf9) cells, respectively. The expressed Cap proteins could self-assemble into virus-like particles (VLPs), but the Bac-Cap-assembled VLPs were more regular. The two system-expressed Cap proteins induced similar specific IgG responses in mice, but the neutralizing antibody levels of Bac-Cap-immunized mice was higher than those of E. coli-Cap. After PCV2 challenge, IL-10 in Bac-Cap immunized mice decreased significantly than that in E. coli-Cap. The lesions and PCV2 antigen positive cells in tissues of mice immunized with E. coli-Cap and Bac-Cap were significantly reduced, and Bac-Cap appeared mild lesions and fewer PCV2 antigen-positive cells compared with E. coli-Cap immunized mice. The study indicated that Cap proteins expressed by E. coli and Bac-Sf9 cells could induce specific protective immunity, but the latter induced more effective immunity, which provides valuable information for the research and development of PCV2 vaccine.

Investigation of porcine circovirus type 2 and porcine circovirus type 3 infections based on dual TaqMan fluorescent quantitative PCR method and genetic evolutionary analysis of these two viruses.

Porcine circovirus type 2 (PCV2) is the pathogen of Porcine Circovirus Associated Diseases. Porcine circovirus type 3 (PCV3) is a novel porcine circovirus associated with porcine dermatitis and nephropathy syndrome (PDNS) and reproductive failure. PCV2 is clearly pathogenic, while the pathogenicity of PCV3 remains controversial, so it is crucial to monitor the prevalence of PCV2 and PCV3 in healthy and diseased pigs to investigate the effects of PCV3 and PCV2 on the health status of pigs. Here, we developed a PCV2 and PCV3 dual TaqMan quantitative PCR (qPCR) method to test samples from healthy and diseased pigs, to clarify the differences in the positive rates and viral copy numbers of PCV2 and PCV3, and to analyze the genetic evolution and molecular characterization of the viral genomes obtained with sequence alignment and phylogenetic analysis, homology and structural analysis of Cap proteins, and selection pressure analysis. We successfully established a dual TaqMan qPCR method for PCV2 and PCV3 with good repeatability, specificity and sensitivity. In total, 1,385 samples from 15 Chinese provinces were tested with the established qPCR. The total positive rates were 37.47% for PCV3 and 57.95% for PCV2, and the coinfection rate for was 25.49%. The positive rates of PCV3 and PCV2 in 372 healthy pigs were 15.05 and 69.89%, respectively, and the coinfection rate was 12.90%. The positive rates of PCV3 and PCV2 in 246 diseased pigs were 55.69 and 83.33%, respectively, and the coinfection rate was 47.97%. Eighteen PCV3 genomes and 64 PCV2 genomes were identified, including nine each of the PCV3a-1 and PCV3b genotypes, eight of PCV2a, 16 of PCV2b, and 40 of PCV2d. The amino acid identity within the PCV3 Cap proteins was 94.00-100.0%, whereas the PCV2 Cap proteins showed an identity of 81.30-100.0%. PCV3 Cap was most variable at amino acid sites 24, 27, 77, 104 and 150, whereas PCV2 Cap had 10-13 unique sites of variation between genotypes. These results clarify the prevalence and variations of PCV2 and PCV3 in healthy and diseased pigs, which will provide a basis for the prevention and control of the two viral infections.