Screening optimal DC-targeting peptide to enhance the immune efficacy of recombinant Lactobacillus expressing RHDV VP60.

Dendritic cells (DCs) present an ideal target for delivering immunogenic cargo due to their potent antigen-presenting capabilities. This targeting approach holds promise in vaccine development by enhancing the efficiency of antigen recognition and capture by DCs. To identify a high-affinity targeting peptide binding to rabbit DCs, rabbit monocyte-derived DCs (raMoDCs) were isolated and cultured, and a novel peptide, HS (HSLRHDYGYPGH), was identified using a phage-displayed peptide library. Alongside HS, two other DC-targeting peptides, KC1 and MY, previously validated in our laboratory, were employed to construct recombinant Lactgobacillus reuteri fusion-expressed rabbit hemorrhagic disease virus (RHDV) capsid protein VP60. These recombinant Lactobacillus strains were named HS-VP60/L. reuteri, KC1-VP60/L. reuteri, and MY-VP60/L. reuteri. The ability of these recombinant Lactobacillus to bind rabbit DCs was evaluated both in vivo and in vitro. Results demonstrated that the DC-targeting peptide KC1 significantly enhanced the capture efficiency of recombinant Lactobacillus by raMoDCs, promoted DC maturation, and increased cytokine secretion. Furthermore, oral administration of KC1-VP60/L. reuteri effectively induced SIgA and IgG production in rabbits, prolonged rabbit survival post-challenge, and reduced RHDV copies in organs. In summary, the DC-targeting peptide KC1 exhibited robust binding to raMoDCs, and recombinant Lactobacillus expressing KC1-VP60 protein antigens efficiently induced systemic and mucosal immune responses in rabbits, conferring protective efficacy against RHDV. This study offers valuable insights for the development of novel RHDV vaccines.

N6-methyladenosine-modified SENP1, identified by IGF2BP3, is a novel molecular marker in acute myeloid leukemia and aggravates progression by activating AKT signal via de-SUMOylating HDAC2.

BACKGROUND: Elevated evidence suggests that the SENPs family plays an important role in tumor progression. However, the role of SENPs in AML remains unclear. METHODS: We evaluated the expression pattern of SENP1 based on RNA sequencing data obtained from OHSU, TCGA, TARGET, and MILE datasets. Clinical samples were used to verify the expression of SENP1 in the AML cells. Lentiviral vectors shRNA and sgRNA were used to intervene in SENP1 expression in AML cells, and the effects of SENP1 on AML proliferation and anti-apoptosis were detected using in vitro and in vivo models. Chip-qPCR, MERIP-qPCR, CO-IP, RNA pulldown, and dual-luciferase reporter gene assays were used to explore the regulatory mechanisms of SNEP1 in AML. RESULTS: SENP1 was significantly upregulated in high-risk AML patients and closely related to poor prognosis. The AKT/mTOR signaling pathway is a key downstream pathway that mediates SENP1's regulation of AML proliferation and anti-apoptosis. Mechanistically, the CO-IP assay revealed binding between SENP1 and HDAC2. SUMO and Chip-qPCR assays suggested that SENP1 can desumoylate HDAC2, which enhances EGFR transcription and activates the AKT pathway. In addition, we found that IGF2BP3 expression was upregulated in high-risk AML patients and was positively correlated with SENP1 expression. MERIP-qPCR and RIP-qPCR showed that IGF2BP3 binds SENP1 3-UTR in an m6A manner, enhances SENP1 expression, and promotes AKT pathway conduction. CONCLUSIONS: Our findings reveal a distinct mechanism of SENP1-mediated HDAC2-AKT activation and establish the critical role of the IGF2BP3/SENP1signaling axis in AML development.

Porcine epidemic diarrhea virus strain CH/HLJ/18 isolated in China: characterization and phylogenetic analysis.

BACKGROUND: Porcine epidemic diarrhea (PED) is an infectious disease of the digestive tract caused by the porcine epidemic diarrhea virus (PEDV), characterized by vomiting, severe diarrhea, and high mortality rates in piglets. In recent years, the distribution of this disease in China has remarkably increased, and its pathogenicity has also increased. PEDV has been identified as the main cause of viral diarrhea in piglets. This study aimed to understand the genetic evolution and diversity of PEDV to provide a theoretical basis for the development of new vaccines and the prevention and treatment of PED. METHODS: A PEDV strain was isolated from the small intestine of a diarrheal piglet using Vero cells. The virus was identified using reverse transcription-polymerase chain reaction (RT-PCR), indirect immunofluorescence assay (IFA), and transmission electron microscopy. The whole genome sequence was sequenced, phylogenetic analysis was conducted using MEGA (version 7.0), and recombination analysis was performed using RDP4 and SimPlot. The S protein amino acid sequence was aligned using Cluster X (version 2.0), and the S protein was modeled using SWISS-MODEL to compare differences in structure and antigenicity. Finally, the piglets were inoculated with PEDV to evaluate its pathogenicity in newborn piglets. RESULT: PEDV strain CH/HLJ/18 was isolated. CH/HLJ/18 shared 89.4-99.2% homology with 52 reference strains of PEDV belonging to the GII-a subgroup. It was a recombinant strain of PEDV BJ-2011-1 and PEDV CH_hubei_2016 with a breakpoint located in ORF1b. Unique amino acid deletions and mutations were observed in the CH/HLJ/18 S protein. The piglets then developed severe watery diarrhea and died within 7 d of inoculation with CH/HLJ/18, suggesting that CH/HLJ/18 was highly pathogenic to newborn piglets. CONCLUSION: A highly pathogenic recombinant PEDV GII-a strain, CH/HLJ/18, was identified in China, with unique deletion and mutation of amino acids in the S protein that may lead to changes in protein structure and antigenicity. These results will be crucial for understanding the prevalence and variation of PEDV and for preventing and controlling PED.

Delivery of antigen to porcine dendritic cells by fusing antigen with porcine dendritic cells targeting peptide.

Dendritic cells (DCs) are professional antigen-presenting cells that can recognize, capture, and process antigens. Fusing molecules targeting DCs with antigens can effectively improve the efficiency with which antigens are recognized and captured by DCs. This targeting strategy can be used for vaccine development to effectively improve the efficiency of antigen recognition and capture by DCs. The targeting sequence of porcine cytotoxic T-lymphocyte associated protein 4 (CTLA4), which binds porcine DCs, was identified in this study. Recombinant Lactobacillus reuteri (L. reuteri) expressing CTLA4-6aa (LYPPPY) and CTLA4-87aa fused to the porcine epidemic diarrhea virus (PEDV) protective antigen core neutralizing epitope (COE) were used to evaluate the ability of the two targeting motifs to bind the B7 molecule on DCs. Our results demonstrate that CTLA4-6aa could bind porcine DCs, and recombinant Lactobacillus expressing the CTLA4-6aa captured by porcine DCs was more efficient than those expressing CTLA4-87aa. In addition, the expression of DC markers, toll-like receptors, and cytokines was significantly higher in the 6aa-COE/L. reuteri-stimulated porcine DCs compared to DCs treated with 87aa-COE/L. reuteri (p<0.01) and recombinant Lactobacillus expressing CTLA4-6aa enhanced the ability of porcine DCs to activate T-cell proliferation. Our analysis of the protein structure revealed that CTLA4-87aa contains intramolecular hydrogen bonds, which may have weakened the intermolecular force between the residues on porcine CTLA4 and that on B7. In conclusion, recombinant Lactobacillus expressing CTLA4-6aa were more efficiently captured by porcine DCs and had a stronger ability to promote DC maturation and enhance T-cell proliferation. The LYPPPY motif is the optimal sequence for binding to porcine DCs. Piglets immunized with recombinant Lactobacillus showed that recombinant Lactobacillus expressing CTLA4-6aa induced significant levels of anti-PEDV-specific IgG and IgA antibody responses. Our study may promote research on DC-targeting strategies to enhance the effectiveness of porcine vaccines.

System Analysis of Adaptor-Related Protein Complex 1 Subunit Mu 2 (AP1M2) on Malignant Tumors: A Pan-Cancer Analysis.

OBJECTIVE: To identify new tumor marker genes available for early tumor screening, differentially expressed gene profiles of multiple tumors were compared using Genotype-Tissue Expression (GTEx), Cancer Cell Line Encyclopedia (CCLE), and The Cancer Genome Atlas (TCGA) databases. As AP1M2 was highly and differentially expressed in invasive breast carcinoma, the purpose of this study was to explore the association of AP1M2 gene with the survival, immune invasion, and tumor neoantigens of patients on a pan-cancer basis. METHODS: The expression and distribution of AP1M2 gene in tumor tissues and the corresponding normal control tissues were analyzed using the pan-cancer databases GTEx, CCLE, and TCGA. Kaplan-Meyer survival plots and proportional hazards model (COX) were employed to evaluate actions of AP1M2 on the clinical prognosis of tumor patients. Subsequently, the association of AP1M2 expression with immune invasion in different tumor types was explored. Simultaneously, the investigation of the interrelationship of AP1M2 and tumor neoantigens of the immune system, unstable microsatellite, DNA repair genes, and DNA methyltransferases were explored, and the mutation frequency of AP1M2 gene in diverse tumors was studied. Several tumor types were analyzed using gene-set enrichment analysis (GSEA). RESULTS: AP1M2 was abundantly expressed in a wide range of cancers, and its expression level was positively correlated with the outcome of tumor victims. Through a study on AP1M2 action on clinical prognosis and immune infiltration in tumor patients, AP1M2 expression in breast-infiltrating carcinoma was found to be highly associated with patients' overall survival and infiltration levels of macrophages, dendritic cells, T cells (CD4+ and CD8+), and B cells. Also, AP1M2 expression was positively correlated with tumor immune neoantigens and microsatellite instability in breast invasive carcinoma. The effect of AP1M2 on tumors was analyzed by GSEA, and findings demonstrated that AP1M2 expression levels in most tumors influenced the activation of tumor-associated pathways and immune-associated pathways. CONCLUSIONS: These findings suggest that AP1M2 expression levels are significantly correlated to patients' outcomes and levels of immune infiltration in most cancer types, including T cells (CD8+ and CD4+), macrophages, neutrophils, and dendritic cells (DCs), particularly in breast cancer. The results indicate that AP1M2 may influence the tumor environment of invasive breast cancer patients and it may be a target contributing to early screening and treatment for breast cancer, helping improve the efficiency of early screening and overall survival rate in invasive breast cancer patients.

Porcine epidemic diarrhea virus S1 protein is the critical inducer of apoptosis.

BACKGROUND: Porcine Epidemic Diarrhea (PED) is an acute and highly contagious enteric disease caused by PED virus (PEDV), characterized by vomitting, watery diarrhea and fatal dehydration with high mortality in sucking piglets of one week of age. Although PEDV induced cell apoptosis has been established in vitro and in vivo, the functional protein that contributes to this event remains unclear. METHODS: The activation or cleavage of main apoptosis-associated molecular such as AIFM1, caspase-3, caspase-8, caspase-9 and PARP in PEDV infected host cells were analyzed by western blotting. The nuclear change of infected cell was monitored by confocal immunofluorescence assay. The overexpressing plasmids of 16 non-structural proteins (Nsp1-16) and 6 structural proteins (M, N, E, ORF3, S1 and S2) were constructed by cloning. Cell apoptosis induced by PEDV or overexpression non-structural or structural proteins was measured by the flow cytometry assay. RESULTS: PEDV could infect various host cells including Vero, Vero-E6 and Marc-145 and cause obvious cytopathic effects, including roundup, cell fusion, cell membrane vacuolation, syncytium formation and cause apparent apoptosis. In infected cells, PEDV-induced apoptosis is accompanied by nuclear concentration and fragmentation as a result of caspase-3 and caspase-8 activation and AIFM1 and PARP cleavage. Overexpression of S1 Spike protein of PEDV SM98 strain effectively induced host cell apoptosis, while the expression of the other non-structure proteins (Nsp1-16) and structural proteins (M, N, E, S2 and ORF3) has no or less effect on cell apoptosis. Similarly, expression of S1 protein from wild-type strain BJ2011 or cell-adapted strain CV777, also induce apoptosis in transfected cells. Finally, we demonstrated that the S1 proteins from various coronavirus family members such as TGEV, IBV, CCoV, SARS and MERS could also induce Vero-E6 cells apoptosis. CONCLUSION: S1 Spike protein is one of the most critical functional proteins that contribute to cell apoptosis. Expression of S1 proteins of the coronavirus tested in this study could all induce cell apoptosis suggesting S1 maybe is an effective inducer in Coronavirus-induced cell apoptosis and targeting S1 protein expression probably is a promising strategy to inhibit coronavirus infection and thus mediated apoptosis on host cells.

Development of the full-length cDNA clones of two porcine epidemic diarrhea disease virus isolates with different virulence.

The recently emerged highly virulent variants of porcine epidemic and diarrhea virus (PEDV) remain a huge threat to the worldwide swine industry. Here, we describe the development of a bacterial artificial chromosome (BAC) reverse genetics system for PEDV based on two recent Chinese field isolates, namely CHM2013 and BJ2011C. Phylogenetically, CHM2013 is closely related to the vaccine strain SM98 whereas the isolate BJ2011C belongs to the GIIb group, a cluster that contains many recent pandemic strains. The full-length cDNA clones of the two isolates were constructed into BAC under the control of CMV promoter. The rescued viruses rBJ2011C and rCHM2013 were found to replicate at the kinetics similar to their respective parental viruses in cell culture. When tested in the 2-day-old pig model, rBJ2011C caused severe diarrhea of piglets with extensive damages to the intestinal epithelium, leading to 100% fatality within 48 hours. In contrast, the rCHM2013-inoculated piglets all survived with only very minor tissue damage observed. Thus, we have successfully established a convenient platform for PEDV genome manipulation. This study also represents the first description of a DNA-launched reverse genetics system for the highly virulent PEDV.

Phylogenetic analysis of porcine epidemic diarrhea virus field strains prevailing recently in China.

Porcine epidemic diarrhea virus (PEDV) is the causative agent of porcine epidemic diarrhea (PED), which is characterized by severe diarrhea, dehydration and high mortality in the affected pigs. Recently, clinical outbreaks of diarrhea in suckling piglets emerged in pig-producing areas of China. In this study, molecular detection of PEDV was conducted using RT-PCR (targeting the M gene) on samples collected from piglets with watery diarrhea from 15 pig farms, and phylogenetic analysis of PEDV field strains was carried out based on their M and S genes. In addition, the complete genome sequence of a PEDV field strain was determined. PEDV was detected in 92.7 % of the samples (267/288). The 15 M genes that were amplified shared 99.6-100 % nucleotide identity and 99.1-100 % amino acid similarity with each other. The 15 S genes exhibited 98.6-99.9 % homology, both at the nucleotide level and at the deduced amino acid level. Phylogenetic analysis showed that all of the amplified M genes grouped in cluster 3, together with some Chinese, Korean and Thai strains, while all of the amplified S genes were in cluster 3 and were closely related to Korean strains. Compared with previous PEDV strains, all of the S genes have common characteristics, namely, a 4-aa (GENQ) insertion between positions 55 and 56, a 1-aa (N) insertion between positions 135 and 136, and a 2-aa (DG) deletion between positions 155 and 156, similar or identical to Korean KNU-serial strains reported in recent years. The genome of the sequenced PEDV field strain is 28,038 nucleotides in length, excluding the poly (A) tail. Our findings suggest that a novel PEDV with a characteristic variant S gene is responsible for recent outbreaks of clinical diarrhea in piglets in China.

Interaction of cellular poly(C)-binding protein 2 with nonstructural protein 1ß is beneficial to Chinese highly pathogenic porcine reproductive and respiratory syndrome virus replication.

Non-structural protein1ß (Nsp1ß) of porcine reproductive and respiratory syndrome virus (PRRSV) has been recognized to be involved in suppressing the host innate immune response and mediating viral subgenomic mRNA transcription. In the present study, we have analyzed the interaction of Nsp1ß of Chinese highly pathogenic PRRSV (HP-PRRSV) with cellular poly(C)-binding 2 (PCBP2) by means of the yeast two-hybrid screening in a pulmonary alveolar macrophages (PAMs) cDNA library and co-immunoprecipitation (Co-IP) assay. Our results indicated that the Nsp1ß of the HP-PRRSV is able to bind and interact with cellular PCBP2 strongly in both the infected cells and plasmid transfected cells. Their minimal binding regions were identified to be the residues 85-203 aa (PCPß and CTE domains) for the Nsp1ß and the residues 96-168 aa (KH2 domain) for PCBP2, respectively. Next, we used confocal immunofluorescence analysis and discovered that, during PRRSV infection in MARC-145 cells and/or plasmid-transfected cells, the Nsp1ß and PCBP2 mainly colocalized in the cytoplasm and perinuclear pattern. Moreover, the siRNA-mediated silencing of PCBP2 gene in the MARC-145 cells resulted in significant reduction of the virus titer in supernatants as well as viral proteins, while no significant effects on the expression of the type I interferon α and interferon ß, suggesting that the interaction of the Nsp1ß with cellular PCBP2 is beneficial to Chinese HP-PRRSV replication in MARC-145 cells.