P65 Protein Research Articles

Fault-tolerant quantum chemical calculations with improved machine-learning models.

Easy and effective usage of computational resources is crucial for scientific calculations. Following our recent work of machine-learning (ML) assisted scheduling optimization [J. Comput. Chem. 2023, 44, 1174], we further propose (1) the improved ML models for the better predictions of computational loads, and as such, more elaborate load-balancing calculations can be expected; (2) the idea of coded computation, that is, the integration of gradient coding, in order to introduce fault tolerance during the distributed calculations; and (3) their applications together with re-normalized exciton model with time-dependent density functional theory (REM-TDDFT) for calculating the excited states. Illustrated benchmark calculations include P38 protein, and solvent model with one or several excitable centers. The results show that the improved ML-assisted coded calculations can further improve the load-balancing and cluster utilization, owing primarily profit in fault tolerance that aims at the automated quantum chemical calculations for both ground and excited states.

Casticin inhibits the hedgehog signaling and leads to apoptosis in AML stem-like KG1a and mature KG1 cells

Abstract Objectives Acute myeloid leukemia (AML) is a severe blood cancer with less than 50 % long-term survival. Despite advancements in treatment options, relapse is still the major obstacle. The main reason of this problem is ineffective targeting of leukemic stem cells (LSCs), which play an important role in tumor development and relapse. In our previous studies, we found that casticin, the major polyphenolic component of Vitex trifolia’s fruit, targets both leukemic cells and LSCs without affecting healthy tissues. Therefore, in this study, we aimed to investigate the effect of casticin-mediated cell death in relation to the LSCs-favored survival pathways at gene and protein expression levels using in vitro LSC-like and parental leukemic cell models. Methods We validated the LSC character of KG1a and KG1 cells (84.55 % CD34+, CD38- and 93.55 % CD34+, CD38+, respectively) by flow cytometry. For the investigation of casticin’s mechanism of action, we employed real time-PCR, western blotting and bioinformatics analyses. Results Our results showed an increase in cleaved PARP/β-actin ratio but no change in LC3BI/II and SQSTM/β-actin ratios. Our gene expression, bioinformatics and immunoblotting analyses represented significant decrease in Shh, Gli and Wnt levels. We also elucidated a possible crosstalk between Hedgehog and other oncogenic cascades via the Gli, Notch, YAP, p38, Mcl-1, and Myc proteins in casticin mediated anti-leukemic effect. Conclusions In conclusion, we found that casticin induces apoptosis in both LSC-like and parental leukemia cells mainly by suppressing Shh signaling, which is crucial for LSC survival and AML relapse.

Evaluation of the effect of phellodendrin application on rats creating an experimental model of non-compression lumbar disc herniation on the NF-κB-related inflammatory signaling pathway

ObjectiveTo explore the therapeutic effects of phellodendrine on non-compression lumbar disc herniation (NCLDH).MethodsThe Sprague Dawley rat model of NCLDH was established via autologous caudal nucleus pulposus transplantation. Behavioral observations and neurological function scoring were conducted in Sprague Dawley rats, and the serum levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were measured via enzyme-linked immunosorbent assay (ELISA). Real-time quantitative polymerase chain reaction (RT‒qPCR) was used to detect the expression of nuclear factor kappa-B (NF-κB) p65 mRNA in L5 nerve roots and surrounding tissues. Western blotting was used to assess the protein expression of NF-κB p65 and TNF-α. Immunofluorescence and immunohistochemical analyses were performed to investigate the distribution and expression of the NF-κB p65 protein in the L5 nerve and its surrounding tissues.ResultsIn this animal study, phellodendrine was found to downregulate the expression of p65 mRNA, decrease the release of inflammatory factors, and alleviate motor dysfunction caused by lumbar disc herniation(LDH). Therefore, the phellodendrine technique has potential value for the treatment of NCLDH.ConclusionIn this animal experiment, phellodendrine was found to significantly reduce the expression level of p65 mRNA, decrease the release of inflammatory cytokines, and alleviate lumbar disc pain.Clinical trial numberNot applicable.

Resolving the differential distribution of structural proteins in baculovirus using single-molecule localization microscopy

Baculovirus is one of the most complex viruses found in nature. Proteomic analysis of budded viruses (BVs) indicates that they are formed by at least 50 different structural proteins. The function of most of these structural proteins and their specific localization in individual virions remain unknown. In the present study, we have conducted single-molecule localization microscopy analysis of the spatial distribution of the nucleocapsid protein P24 and the envelope proteins GP64 and E25. Our results show that P24 and GP64 are polarized to one end of the baculovirus, while E25 distributes more homogenously along the viral envelope. This is the first study using optical microscopy to demonstrate the polarized distribution of structural proteins in individual baculoviruses.

Identification of a novel conserved B-cell epitope in p15 of the African swine fever virus

African Swine Fever Virus (ASFV), a highly contagious DNA virus, causes severe economic losses to the global swine industry. The ASFV p15 protein, which is found in the core shell, is essential to the assembly of viral particles. In addition, protein p15 is a candidate target for the development of diagnostic reagents for African Swine Fever (ASF) because of its excellent immunogenicity. In this research, we prepared the p15 protein using eukaryotic expression system and validated it with sera from ASFV-infected pigs. The p15 protein could be well identified by the sera from ASFV-infected pigs, suggesting that some linear epitopes are located in the p15 protein. Furthermore, we successfully prepared two lgG1 subclass monoclonal antibodies (1E6-A7 and 3D7C9) specific against p15 using hybridoma technology. Using the peptide scanning method, we discovered the two mAbs well recognized the same linear epitope23LEIINNLCML32. The23LEIINNLCML32 epitope in the ASFV p15 N-terminus was identified and characterized for the first time, and it reacted well with the ASFV-positive serum, implying that it was a natural B cell linear epitope. These findings may help in the development of novel serologic diagnosis tools and the improvement of antiviral drug designs for ASF.

High cell throughput, programmable fixation reveals the RNA and protein co-regulation with spatially resolved NFκB pseudo-signaling.

RNA translation to protein is paramount to creating life, yet RNA and protein correlations vary widely across tissues, cells, and species. To investigate these perplexing results, we utilize a time-series fixation method that combines static stimulation and a programmable formaldehyde perfusion to map pseudo-Signaling with Omics signatures (pSigOmics) of single-cell data from hundreds of thousands of cells. Using the widely studied nuclear factor kappa B (NFκB) mammalian signaling pathway in mouse fibroblasts, we discovered a novel asynchronous pseudotime regulation (APR) between RNA and protein levels in the quintessential NFκB p65 protein using single molecule spatial imaging. Prototypical NFκB dynamics are successfully confirmed by the rise and fall of NFκB response as well as A20 negative inhibitor activity by 90 min. The observed p65 translational APR is evident in both statically sampled timepoints and dynamic response gradients from programmable formaldehyde fixation, which successfully creates continuous response measurements. Finally, we implement a graph neural network model capable of predicting APR cell subpopulations from GAPDH RNA spatial expression, which is strongly correlated with p65 RNA signatures. Successful decision tree classifiers on Potential of Heat-diffusion for Affinity-based Trajectory Embedding embeddings of our data, which illustrate partitions of APR cell subpopulations in latent space, further confirm the APR patterns. Together, our data suggest an RNA-protein regulatory framework in which translation adapts to signaling events and illuminates how immune signaling is timed across various cell subpopulations.

EP300-mediated H3 acetylation elevates MTHFD2 expression to reduce mitochondrial dysfunction in lipopolysaccharide-induced tubular epithelial cells.

Sepsis represents an organ dysfunction resulting from the host's maladjusted response to infection, and can give rise to acute kidney injury (AKI), which significantly increase the morbidity and mortality of septic patients. This study strived for identifying a novel therapeutic strategy for patients with sepsis-induced AKI (SI-AKI). Rat tubular epithelial NRK-52E cells were subjected to lipopolysaccharide (LPS) exposure for induction of in-vitro SI-AKI. The expressions of E1A binding protein p300 (EP300) and methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) in NRK-52E cells were assessed by western blot and qRT-PCR, and their interaction was explored by chromatin immunoprecipitation performed with antibody for H3K27 acetylation (H3K27ac). The effect of them on SI-AKI-associated mitochondrial dysfunction of tubular epithelial cells was investigated using transfection, MTT assay, TUNEL staining, 2',7'-Dichlorodihydrofluorescein diacetate probe assay, Mitosox assay, and JC-1 staining. MTHFD2 and EP300 were upregulated by LPS exposure in NRK-52E cells. LPS increased the acetylation of H3 histone in the MTHFD2 promoter region, and EP300 suppressed the effect of LPS. EP300 ablation inhibited the expression of MTHFD2. MTHFD2 overexpression antagonized LPS-induced viability reduction, apoptosis promotion, reactive oxygen species overproduction, and mitochondrial membrane potential collapse of NRK-52E cells. By contrast, MTHFD2 knockdown and EP300 ablation brought about opposite consequences. Furthermore, MTHFD2 overexpress and EP300 ablation counteracted each other's effect in LPS-exposed NRK-52E cells. EP300-mediated H3 acetylation elevates MTHFD2 expression to reduce mitochondrial dysfunction of tubular epithelial cells in SI-AKI.

Deubiquitinase inhibitor bAP-15 suppresses renal epithelial to mesenchymal transition via inhibition of p300 stability

Renal fibrosis is an irreversible disease that is common in patients with chronic kidney disease. Elevated levels of the histone acetyltransferase p300 have been reported in various fibrotic diseases, including renal fibrosis, suggesting that p300 may be a promising therapeutic target. To investigate the specific deubiquitinase (DUB) involved in the regulation of p300 protein stability in renal epithelial cells, we tested 13 DUB inhibitors using a kidney tubular epithelial cell line. We found that the p300-specific DUB inhibitor, bAP-15 reduces p300 protein stability by targeting ubiquitin-specific protease 14 (USP14) and ubiquitin C-terminal hydrolase L5 (UCHL5). The mRNA levels of USP14 and UCHL5 were increased in patients with chronic kidney disease, and increased protein levels of USP14 and UCHL5 during fibrosis progression were validated using a mouse renal fibrosis model. Both USP14 and UCHL5 interacted with p300 in kidney tubular epithelial cells, with increased binding affinity in response to TGF-β. Moreover, bAP-15-induced p300 degradation inhibited epithelial-to-mesenchymal transition and reduced the expression of pro-fibrotic target genes. Our findings demonstrate an anti-fibrotic effect of bAP-15 through the regulation of p300 stability and suggest that bAP-15 may be a potential therapeutic agent for renal fibrosis.

Expression of Concern for Galdiero et al., "Role of Surface-Exposed Loops of Haemophilus influenzae Protein P2 in the Mitogen-Activated Protein Kinase Cascade".

Expression of Concern for Galdiero et al., "Role of Surface-Exposed Loops of Haemophilus influenzae Protein P2 in the Mitogen-Activated Protein Kinase Cascade".

Host protein PRPS2 interact with the non-structural protein p17 of Avian Reovirus and promote viral replication

Avian reovirus (ARV) is highly prevalent in healthy poultry flocks and has been linked to viral arthritis/tendonitis, dwarf syndrome, chronic respiratory disease, and immunosuppression in avian species, resulting in significant economic losses within the poultry industry. The non-structural protein p17 encoded by ARV induces cellular autophagy and facilitates viral proliferation, playing a pivotal role in viral pathogenesis. To further elucidate the pathogenic mechanism basis of ARV p17 protein function, we employed a yeast two-hybrid system to identify Phosphoribosyl pyrophosphate synthetase 2 (PRPS2) as an interacting host protein with p17. In this study, we validated the interaction between PRPS2 and p17 using laser confocal microscopy, coimmunoprecipitation, and GST-Pulldown assays. Moreover, our findings demonstrate that the C-terminal region of PRPS2 is responsible for its binding to the p17 protein. Intriguingly, ARV infection significantly upregulated PRPS2 expression levels. Additionally, PRPS2 was shown to have a substantial impact on ARV replication; overexpression of PRPS2 increased ARV replication while knockdown of PRPS2 resulted in decreased quantities of ARV particles. Furthermore, our findings suggest that this process involves cellular apoptosis as a potential mechanism underlying these observations. Overall, this research provides valuable insights into elucidating the function of the p17 protein and sheds light on the pathogenic mechanism involving ARV-induced cellular apoptosis while offering novel perspectives for exploring therapeutic targets against ARV.

FOXN3 Downregulation in Colorectal Cancer Enhances Tumor Cell Stemness by Promoting EP300-Mediated Epigenetic Upregulation of SOX12.

Cancer stemness plays a crucial role in promoting the progression of colorectal cancer (CRC). Forkhead box N3 (FOXN3) is a tumor suppressor protein. Herein, we investigated the role of FOXN3 in the regulation of CRC cell stemness. Cell viability, proliferation, migration, and invasion were assessed utilizing cell counting kit-8 assay, 5-ethynyl-20-deoxyuridine assay, and Transwell assay, respectively. Cell-sphere formation was assessed using a sphere-forming assay. The enrichment of H3K27ac modifications at the SRY-related HMG-box 12 (SOX12) promoter, interactions among FOXN3, SOX12, and E1A binding protein p300 (EP300) were analyzed using chromatin immunoprecipitation or dual luciferase reporter assays. We found that FOXN3 overexpression inhibited CRC cell proliferation, migration, invasion, stemness, and tumor formation in mice by inactivating the Wnt/β-catenin signaling, while these effects of FOXN3 overexpression were reversed by the overexpression of SOX12. Mechanistically, EP300 increased SOX12 expression in CRC cells by promoting H3K27ac enrichment in the SOX12 promoter. In addition, FOXN3 transcriptionally inhibited EP300 expression in CRC cells by binding to the EP300 promoter. As expected, EP300 overexpression weakened the inhibitory effect of FOXN3 overexpression on CRC cell stemness. Collectively, FOXN3 upregulation inhibited CRC cell stemness by suppressing EP300-mediated epigenetic upregulation of SOX12.

Ginsenoside Rb1 ameliorates hippocampal neuroinflammation in rats after intracerebral hemorrhage by inactivating the TLR4/NF-kB pathway.

This work elucidated the therapeutic effect and mechanism of ginsenoside Rb1 on intracerebral hemorrhage (ICH). ICH rat models were treated by ginsenoside Rb1. Modified neurological deficit score, and Y-maze and Morris water-maze tests were performed on rats. Hippocampal neuronal damage was observed by Nissl staining. Rat primary astrocytes were exposed to ginsenoside Rb1, Hemin, and lipopolysaccharide (LPS). TNF-α, IL-1β, and IL-6 levels were assessed via enzyme-linked immunosorbent assay. TLR4/NF-kB pathway activity was appraised by Western blot. Immunofluorescence staining was for hippocampal glial fibrillary acidic protein (GFAP) expression and P65 protein location in hippocampus and astrocytes. In rats after ICH, ginsenoside Rb1 ameliorated neurological impairment and hippocampal neuronal damage; improved learning and memory ability; reduced brain water content; decreasedhippocampal TNF-α, IL-1β, and IL-6; inactivated TLR4/NF-kB pathway; and declined hippocampal GFAP expression. In rat primary astrocytes exposed to Hemin, ginsenoside Rb1 declined TNF-α, IL-1β, and IL-6; inactivated TLR4/NF-kB pathway; and hindered P65 protein entry into nucleus. However, these functions of ginsenoside Rb1 on the Hemin-induced astrocytes were abolished by LPS. Ginsenoside Rb1 has promising future for clinical ICH treatment, which exerts therapeutic effect on ICH by ameliorating hippocampal neuroinflammation via inactivating the TLR4/NF-kB pathway.

Knockout of RIG-I in HEK293 cells by CRISPR/Cas9

We knocked out the retinoic acid-inducible gene I (RIG-I) in HEK293 cells via CRISPR/Cas9 to reveal the effects of RIG-I knockout on the key factors in the type I interferon signaling pathway. Three single guide RNAs (sgRNAs) targeting RIG-I were designed, and the recombination vectors were constructed on the basis of the pX459 vector and used to transfect HEK293 cells, which were screened by puromycin subsequently. Furthermore, a mimic of virus, poly I: C, was used to transfect the cells screened out. RIG-I knockout was checked by sequencing, real-time quantitative PCR, Western blotting, and immunofluorescence assay. Meanwhile, the expression levels of key factors of type I interferon signaling pathway such as melanoma differentiation-associated gene 5 (MDA5), interferonβ1 (IFNβ1), and nuclear factor-kappa B p65 [NF-κB(p65)], as well as cell viability, were determined. The results showed that two HEK293 cell lines (S1 and S3) with RIG-I knockout were obtained, which exhibited lower mRNA and protein levels of RIG-I than the wild type HEK293 cells (P < 0.05). The mRNA levels of MDA5 and IFNβ1 in S1 and S3 cells and the protein level of NF-κB(p65) in S3 cells were lower than those in the wild type (P < 0.05). More extranuclear NF-κB(p65) protein was detected in S1 cells than in the wild type after transfection with poly I: C. Plus, the wild-type and S1 cells transfected with poly I: C for 48 h showcased reduced viability (P < 0.05), while S3 cells did not display the reduction in cell viability. In summary, the present study obtained two HEK293 cell lines with RIG-I knockout via CRISPR/Cas9, which provided a stable cell model for exploring the mechanism of type I interferon signaling pathway.

A peptide encoded by LINC00944 suppresses the growth of melanoma cells by diminishing EP400-MYC interaction

The peptides encoded by long noncoding RNAs (lncRNAs) have been shown to participate in cancer pathogenesis. In this study, lncRNA LINC00944 was validated to encode an endogenous 102-amino acid (aa) small peptide (named LINC00944 peptide). Functionally, LINC00944 peptide exerted an anti-growth effect in melanoma cells in vitro. Mechanistically, LINC00944 peptide interacted with the E1A binding protein p400 (EP400)/c-MYC complex. LINC00944 peptide also inhibited c-MYC protein expression. Furthermore, LINC00944 peptide repressed the transcriptional activity of MYC by reducing the EP400-MYC interaction, thereby reducing the levels of fatty acid metabolism- and glucose metabolism-related proteins. Our findings uncovered that LINC00944 peptide might be a promising adjuvant therapeutic agent for melanoma.Implications: This study provided the first evidence that LINC00944-encoded peptide played a critical role in the growth of melanoma cells.

The RNA Silencing Suppressor P8 From High Plains Wheat Mosaic Virus is a Functional Tetramer

In plants, RNA interference (RNAi) serves as a critical defense mechanism against viral infections by regulating gene expression. However, viruses have developed RNA silencing suppressor (RSS) proteins to evade this defense mechanism. The High Plains wheat mosaic virus (HPWMoV) is responsible for the High Plains disease in wheat and produces P7 and P8 proteins, which act as RNA silencing suppressors. P8, in particular, lacks sequence similarity to known suppressors, prompting inquiries into its structure and function.Here, we present a comprehensive analysis of P8, elucidating its structure and function. Using X-ray crystallography, we resolved the full-length P8 structure at 1.9 Å resolution, revealing a tetrameric arrangement formed by two identical dimers. Through structure-based mutagenesis, biochemical assays, and functional studies in plants, we demonstrate that HPWMoV P8’s RNA silencing suppression activity relies on its oligomeric state.Contrary to previous report, our findings indicate that while a P8 fused to maltose-binding protein (MBP-P8) was hypothesized to bind short double-stranded RNA, the native P8 tetramer does not interact with small interfering RNA (siRNA). This suggests an alternative mechanism for its function, yet to be determined.Our study sheds light on the structural and functional characteristics of HPWMoV P8, providing valuable insights into the complex interplay between viral suppressors and host defense mechanisms. Significance statementEffective action to address malnutrition in all its forms requires an understanding of the mechanisms affecting it. Wheat, crucial for human and animal consumption, faces threats from biotic and abiotic stresses. RNA silencing is a key defense against viral infections in plants. Plant viruses employ various mechanisms, including encoding viral RNA silencing suppression (VRS) proteins, to evade host immune responses. Despite the conservation of RNA-silencing pathways, viral RSS proteins exhibit diverse sequences, structures, and mechanisms. Our study focuses on P8, an RSS protein from HPWMoV. Understanding its structure and assembly is a crucial step toward comprehending how these viruses counteract host defenses, aiding in combatting malnutrition.

A high titer antibody response against P22 protein immunocomplex is not correlated with protection in naturally tuberculosis-infected goats

Caprine livestock are significant reservoirs of the Mycobacterium tuberculosis complex (MTBC), contributing to tuberculosis (TB) transmission among animals and humans. The P22 protein immunocomplex (P22PI), derived from bovine tuberculin, shows immunostimulating capacity and is used for TB diagnosis. This study assessed the immunogenicity and protective efficacy of P22PI in two groups of goats: 24 naïve goats (12 immunised, 12 controls) from a TB-free herd, and 24 infected goats (12 immunised, 12 controls), referred to as pre-infected animals, from a M. bovis-infected herd. Both were exposed for 5 months to M. bovis-naturally infected goats. Reactors to single and comparative intradermal tuberculin (SIT and SCIT, respectively) tests and interferon-gamma release assay (IGRA) significantly increased (p < 0.05) in both groups 5 months’ post-exposure, with no significant differences between immunised and control animals. However, immunised animals exhibited a significantly higher (p < 0.05) antibody response against P22PI. Most naïve animals (83.3%) and all pre-infected animals developed TB-compatible lesions, with extensive necrosis in the lungs and associated lymph nodes, compared to 50% and 83.3% of control animals, respectively. These findings suggest that while P22PI stimulates an intense antibody response under the conditions of the present study, it does not confer protection against TB and may exacerbate disease severity.

Role of the CTCF/p300 axis in osteochondrogenic-like differentiation of polyploid giant cancer cells with daughter cells

BackgroundPolyploid giant cancer cells (PGCCs) have properties of cancer stem cells (CSCs). PGCCs with daughter cells (PDCs) undergo epithelial–mesenchymal transition and show enhanced cellular plasticity. This study aimed to elucidate the mechanisms underlying the osteo/chondrogenic-like differentiation of PDCs, which may be exploited therapeutically by transdifferentiation into post-mitotic and functional cells.MethodsCobalt chloride was used to induce PGCC formation in MDA-MB-231 and HEY cells, and PDCs were cultured in osteo/chondrogenic differentiation media. Alcian blue staining was used to confirm osteo/chondrogenic differentiation, and the cell cycle was detected using flow cytometry. The expression of osteo/chondrogenic differentiation-related proteins was compared, and a co-immunoprecipitation assay was used to demonstrate the interactions between proteins. Bioinformatic analysis was used to explore the regulatory mechanism of osteo/chondrogenic differentiation, and a dual-luciferase reporter assay was performed to validate the interaction between transcriptional factors and target genes. Animal xenograft models were used to confirm the osteo/chondrogenic differentiation of PDCs.ResultsWhen cultured in osteo/chondrogenic medium, the stemness of PDCs decreased, and the expression of osteo/chondrogenic-related markers increased. This osteo/chondrogenic-like process was regulated by the transforming growth factor-β pathway in a time-dependent manner. A concurrent increase in the expression of histone acetyltransferase p300 and the transcription factor CCCTC-binding factor (CTCF) was observed. Co-immunoprecipitation assays revealed that p300 acetylated the osteo/chondrogenic marker RUNT-related transcription factor 2 (RUNX2). Analysis of chromatin immunoprecipitation sequencing datasets revealed that both CTCF and histone H3 lysine 27 acetylation (H3K27ac) were enriched in the promoter region of E1A-associated protein p300 (P300). The four predicted binding sites for CTCF and P300 were validated using dual-luciferase reporter assays. We examined the interaction between CTCF and H3K27ac and found that these two proteins had a combined effect on the transactivation of P300.ConclusionCTCF, in synergy with H3K27ac, amplified the expression of P300, facilitating acetyl group transfer to RUNX2. This acetylation stabilized RUNX2 and promoted osteo/chondrogenic differentiation, thereby reducing the incidence of PDC malignancies.

A subunit vaccine based on P97R1, P46, P42, and P65 from Mycoplasma hyopneumoniae can induce significant immune response in piglets.

Mycoplasma pneumonia (MPS), caused by Mycoplasma hyopneumoniae (Mhp), is a chronic, airborne respiratory disease that poses a significant threat to the global swine industry. The P97 and P46 proteins are major antigens of Mhp, with the R1 region of P97 possessing full adhesive capability. Studies have shown that the main antigenic regions of Mhp P42 and P65 proteins exhibit strong immunogenicity. In this study, we first linked the genes encoding P97R1 and P46 proteins to form the P97R1P65 gene and subsequently constructed three shuttle plasmids: pFBD-P97R1P46, pFBD-P97R1P46-p65, and pFBD-P65-P42. These proteins were expressed using the Bac to Bac system and formulated into subunit vaccines for mouse immunization. Mouse experiments indicated that the P97R1P46 + P65-P42 protein combination elicited higher levels of specific antibodies, IL-2, IL-4, and CD8+ T cells compared to other subunit vaccine groups, a finding further validated in subsequent mouse challenge protection experiments. Therefore, we utilized the MultiBac expression system to co-express P97R1P46, P65, and P42 proteins in the pFastMultibacDual vector for immunization experiments in piglets. The piglet immunization experiments demonstrated that the Mhp subunit vaccine prepared in this study could induce specific antibodies against Mhp, with the combination of P97R1P46, P65, and P42 proteins inducing the highest level of humoral immunity. This study provides valuable insights for the development of Mhp subunit vaccines.

TRIM28 regulates the coagulation cascade inhibited by p72 of African swine fever virus

In 2018, African swine fever virus (ASFV) emerged in China, causing extremely serious economic losses to the domestic pig industry. Infection with ASFV can cause disseminated coagulation, leading to the consumption of platelets and coagulation factors and severe bleeding. However, the mechanism of virus-induced coagulation has yet to be established. In our study, ASFV downregulated the coagulation process, as detected by D-dimer (D2D) and Factor X (F10) expression in pigs challenged with ASFV HLJ/18. In vitro, ASFV infection increased Factor IX (F9) and Factor XII (F12) expression while downregulating F10 expression in porcine alveolar macrophages (PAMs). African swine fever virus induced both intrinsic and extrinsic coagulation cascades. In addition, several encoded proteins affect the expression of the crucial coagulation protein F10, and among the encoded proteins, p72 inhibits the activity and expression of F10. Proteomic analysis also revealed that p72 is involved in the coagulation cascade. p72 can interact with F10, and its inhibitory functional domains include amino acids 423–432 and amino acids 443–452. Finally, we found that F10 and p72 interact with tripartite motif-containing protein 28 (TRIM28). TRIM28 knockdown resulted in a decrease in F10 expression. Importantly, TRIM28 contributes to the reduction in F10 protein expression regulated by p72. Our findings revealed an inhibitory effect of the viral protein p72 on the ASFV infection-induced coagulation cascade and revealed a role of TRIM28 in reducing F10 expression, revealing a molecular mechanism of ASFV-associated coagulation.

Molecular detection and sequencing of beet necrotic yellow vein virus and beet cryptic virus 2 in sugar beet from Kazakhstan.

Beet necrotic yellow vein virus (BNYVV) is a common viral pathogen that causes considerable economic loss globally. In the present study, a commercial realtime PCR test system and custom loop mediated amplification primers were used to detect the virus in asymptomatic sugar beet samples. A total of 107 of 124 samples tested positive for the presence of the A type BNYVV coat protein gene. Near complete sequences of RNA-3 and RNA-4 were obtained using reverse transcription, followed by nanopore sequencing of 14 samples. A comparison with available sequences, including previously published isolates Kas2 and Kas3 from Kazakhstan, identified RNA-3 as similar to such of the P-type isolates Puthiviers and Kas3. RNA-5 was not detected using real-time PCR or cDNA amplification. Unique variable sites were identified in the p25 protein sequence translated from RNA-3. Another virus, beet cryptic virus 2 (BCV2), was identified and sequenced in samples infected with BNYVV. With 85.28% genome coverage, the identified BCV2 samples were very similar to the previously reported isolates from Hungary and Germany.