Construction of an IFNAR1 knockout MDBK cell line using CRISPR/Cas9 and its effect on bovine virus replication.

The type I interferon (IFN) pathway is important for eukaryotic cells to resist viral infection, as well as an impediment to efficient virus replication. Therefore, this study aims to create an IFNAR1 knockout (KO) Madin-Darby bovine kidney (MDBK) cell line using CRISPR/Cas9 and investigate its application and potential mechanism in increasing viral replication of bovines. The IFNAR1 KO cells showed increased titers of bovine viral diarrhea virus (BVDV) (1.5 log10), with bovine enterovirus and bovine parainfluenza virus type 3 (0.5-0.8 log10). RNA-seq revealed reduced expression of the genes related IFN-I pathways including IFNAR1, STAT3, IRF9, and SOCS3 in IFNAR1 KO cells compared with WT cells. In WT cells, 306 differentially expressed genes (DEGs) were identified between BVDV-infected and -uninfected cells. Of these, 128 up- and 178 down-regulated genes were mainly associated with growth cycle and biosynthesis, respectively. In IFNAR1 KO cells, 286 DEGs were identified, with 82 up-regulated genes were associated with signaling pathways, and 204 down-regulated genes. Further, 92 DEGs were overlapped between WT and IFNAR1 KO cells including ESM1, IL13RA2, and SLC25A34. Unique DEGs in WT cells were related to inflammation and immune regulation, whereas those unique in IFNAR1 KO cells involved in cell cycle regulation through pathways such as MAPK. Knocking down SLC25A34 and IL13RA2 in IFNAR1 KO cells increased BVDV replication by 0.3 log10 and 0.4 log10, respectively. Additionally, we constructed an IFNAR1/IFNAR2 double-knockout MDBK cell line, which further increased BVDV viral titers compared with IFNAR1 KO cells (0.6 log10). Overall, the IFNAR1 KO MDBK cell line can support better replication of bovine viruses and therefore provides a valuable tool for bovine virus research on viral pathogenesis and host innate immune response.

US-based radiomics analysis of different machine learning models for differentiating benign and malignant BI-RADS 4A breast lesions.

RATIONALE AND OBJECTIVES: To investigate and authenticate the effectiveness of various radiomics models in distinguishing between benign and malignant BI-RADS 4A lesions. METHODS: A total of 936 patients with pathologically confirmed 4A lesions were included in the study (training cohort: n = 655; test cohort: n = 281). Radiomic features were derived from greyscale US images. Following dimensionality reduction and feature selection, radiomics models were developed using logistic regression (LR), support vector machine (SVM), random forest (RF), eXtreme gradient boosting (XGBoost) and multilayer perceptron (MLP) algorithms. Univariate and multivariable logistic regression analyses were employed to investigate clinical-radiological characteristics and determine variables for creating a clinical model. Five combined models integrating radiomic and clinical parameters were constructed by using each algorithm, and comparison with radiologists' performance was performed. SHapley Additive exPlanations (SHAP) approach was used to elucidate the radiomic model by ranking the significance of features based on their contribution to the evaluation. RESULTS: A total of 1561 radiomic features were extracted. Thirty-six features were deemed significant by dimensionality reduction and selection. The radiomic models showed good performance with AUCs of 0.829-0.945 in training cohort; and 0.805-0.857 in test cohort. The combined model developed by using LR showed the best performance (AUC, training cohort: 0.909; test cohort: 0.905), which is superior to radiologists' performance. Decision curve analysis (DCA) of this combined model indicated better clinical efficacy than clinical and radiomic models. CONCLUSIONS: The combined model integrating radiomic and clinical features demonstrated excellent performance in differentiating between benign and malignant 4A lesions. It may offer a non-invasive and efficient approach to aid in clinical decision-making.

Evaluating clinically translatable conditioning for platelet gene therapy in murine hemophilia A with inhibitors.

BACKGROUND: Platelet gene therapy is effective in hemophilia A (HA) mice even with inhibitors. Fludarabine (Flu), along with busulfan (Bu) or melphalan (Mel), preconditioning has been shown to be highly effective for hematopoietic stem cell transplantation in the clinic. OBJECTIVES: To evaluate the efficacy of Bu-Flu and Mel-Flu preconditioning in platelet gene therapy of HA with inhibitors. METHODS: Bu-Flu and Mel-Flu were used to condition HA mice preimmunized with recombinant human factor (F)VIII. An optimal 660 centigray total body irradiation was used as a control regimen in parallel. Platelet-FVIII expression was introduced by transplantation of 2bF8 lentivirus (LV)-transduced hematopoietic stem cells. Animals were analyzed by fluorescence-activated cell sorting, quantitative polymerase chain reaction, FVIII assays, and tail bleeding tests. RESULTS: Bu-Flu, but not Mel-Flu, enabled successful 2bF8 gene therapy. All recipients achieved >55% chimerism post hematopoietic stem cell transplantation in both Bu-Flu and 660 centigray groups, with comparable copy numbers of 2bF8 cassette and the platelet-FVIII levels. The bleeding phenotype was rescued in 2bF8LV-transduced recipients. FVIII inhibitor titers declined with time, with comparable disappearance time of inhibitors between the 2 groups. When animals were rechallenged with recombinant human FVIII after the titers dropped to undetectable levels, no inhibitors were detected in 2bF8LV-transduced recipients. In contrast, all untransduced transplanted control mice produced inhibitors. These data demonstrate that immune tolerance was established in 2bF8LV-transduced primed HA mice under Bu-Flu conditioning. CONCLUSION: Bu-Flu preconditioning allows for successfully introducing platelet-FVIII expression to restore hemostasis and induce immune tolerance in primed HA mice, suggesting that this approach is a promising clinically translatable strategy for gene therapy of HA with inhibitors.

Ultrasound and clinical features for differential diagnosis of low-grade appendiceal mucinous neoplasm and acute suppurative appendicitis.

AIM: To investigate the application of ultrasound along with clinical features for the differential diagnosis of low-grade appendiceal mucinous neoplasm (LAMN) and acute suppurative appendicitis (ASA). MATERIAL AND METHODS: The ultrasound and clinical data of 76 patients with histopathologically confirmed LAMN (31 patients) and ASA (45 patients) were retrospectively analyzed. Univariate analysis and binary logistic regression analysis of the influencing factors were conducted to identify LAMN and ASA. The AUROC was calculated to analyze the diagnostic efficacy of these independent factors. A four-grid table was established to determine the diagnostic efficacy of the ultrasound marks for diagnosing LAMN. RESULTS: Patient age and appendix short diameter in the LAMN group were found to be significantly higher than those in the ASA group. The neutrophil ratio and thickness of the appendix wall in the LAMN group were significantly lower than they were in the ASA group. Patient age (OR=1.112, p=0.015) and appendix short diameter (OR=1.476, p=0.008) were independent risk factors for LAMN. The AUROCs for age and short diameter were 0.898 [95% CI: 0.807, 0.956] and 0.953 [95% CI: 0.879, 0.988], respectively. The LAMN group tumors were characterized by the appearance of an "onion skin" sign or a purely cystic mark on sonograms, with specificities of 100% for both. Neutrophil ratio (OR<0.001, p=0.064) and thickness of the appendix wall (OR=0.776, p=0.414) were not independent risk factors for ASA. CONCLUSION: Employing ultrasonography with clinical features is useful for distinguishing LAMN from ASA. Patient age, short diameter of the appendix, and sonographic appearance of "onion skin" or purely cystic mark could be key factors in diagnosing LAMN.

Identification of potential nucleomodulins of Mycoplasma bovis by direct biotinylation and proximity-based biotinylation approaches.

Mycoplasma bovis (M. bovis) is a significant bovine pathogen associated with various diseases, including bovine bronchopneumonia and mastitis resulting in substantial economic losses within the livestock industry. However, the development of effective control measures for M. bovis is hindered by a limited understanding of its virulence factors and pathogenesis. Nucleomodulins are newly identified secreted proteins of bacteria that internalize the host nuclei to regulate host cell gene expression and serve as critical virulence factors. Although recent reports have initiated exploration of mycoplasma nucleomodulins, the efficiency of conventional techniques for identification is very limited. Therefore, this study aimed to establish high-throughput methods to identify novel nucleomodulins of M. bovis. Using a direct biotinylation (DB) approach, a total of 289 proteins were identified including 66 high abundant proteins. In parallel, the use of proximity-based biotinylation (PBB), identified 28 proteins. Finally, seven nucleomodulins were verified to be nuclear by transfecting the bovine macrophage cell line BoMac with the plasmids encoding EGFP-fused proteins and observed with Opera Phenix, including the known nucleomodulin MbovP475 and six novel nucleomodulins. The novel nucleomodulins were four ribosomal proteins (MbovP599, MbovP678, MbovP710, and MbovP712), one transposase (MbovP790), and one conserved hypothetical protein (MbovP513). Among them, one unique nucleomodulin MbovP475 was identified with DB, two unique nucleomodulins (MbovP513 and MbovP710) with PBB, and four nucleomodulins by both. Overall, these findings established a foundation for further research on M. bovis nucleomodulin-host interactions for identification of new virulence factors.

Anti-mCD20 in combination with α-mCXCL13 monoclonal antibody inhibits anti-FVIII antibody development in hemophilia A mice.

Anti-factor VIII (FVIII) antibody development poses a significant challenge in hemophilia A (HA) patients receiving FVIII protein replacement therapy. There is an urgent need for novel therapeutic strategies to inhibit the production of anti-FVIII inhibitory antibodies (inhibitors) in HA. This study aimed to investigate a combination monoclonal antibody (mAb) therapy targeting CXCL13 and CD20 on the development of anti-FVIII antibodies in a HA murine model, along with the underlying mechanisms involved. Specifically, mAbs targeting mouse CD20 (18B12) with an IgG2a backbone and mouse CXCL13 (2C4) with an IgG1 backbone were synthesized. HA mice with FVIII inhibitors were established, and the results revealed that the combination therapy of anti-mCD20 with α-mCXCL13 significantly suppressed anti-FVIII antibody development and induced FVIII tolerance. Furthermore, this combination therapy led to a marked reduction of peripheral and splenic follicular helper T cells and an enhancement of regulatory T cell induction, along with sustained depletion of bone marrow and splenic plasma cells in HA mice with preexisting FVIII immunity. Thus, the concurrence of blockage of CD20 and neutralization of CXCL13 hold promise as a therapeutic strategy for HA patients with inhibitors.

The crosstalk between metabolic reprogramming and epithelial-mesenchymal transition and their synergistic roles in distant metastasis in breast cancer.

BACKGROUND: Metabolic reprogramming (MR) and epithelial-mesenchymal transition (EMT) are crucial phenomena involved in the distant metastasis of breast cancer (BRCA). This study aims to assess the risk of distant metastasis in BRCA patients based on MR and EMT processes and investigate their underlying mechanisms. METHODS: Gene sets related to EMT and MR were downloaded. MR-related genes (MRG) and EMT-related genes (ERG) were obtained. Principal Component Analysis method was used to define the EMT Potential Index (EPI) and MR Potential Index (MPI) to quantify the EMT and MR levels in each tumor tissue. A linear scoring model, the Metastasis Score, was derived using the union of MRGs and ERGs to evaluate the risk of distant metastasis/recurrence in BRCA. The Metastasis Score was then validated in multiple datasets. Additionally, our study explored the underlying mechanism of the Metastasis Score and its association with tumor immunity, focusing on HPRT1 gene expression in breast cancer tissues of transfer and untransferred groups using experimental methods. RESULTS: A total of 59 MRGs and 30 ERGs were identified in the present study. Stratifying the dataset based on EPI and MPI revealed significantly lower survival rates (P < .05) in the MPI_high and EPI_high groups. Kaplan-Meier analysis indicated the lowest survival rate in the EPI-high + MPI-high group. The Metastasis Score demonstrated its ability to distinguish prognoses in GSE2034, GSE17705, and TCGA-BRCA datasets. Additionally, differences in mutated genes were found between the high- and the low-Metastasis Score groups, displaying significant associations with immune cell infiltration and anti-tumor immune status. Notably, the 13 genes included in the Metastasis Score showed a strong association with prognosis and tumor immunity. Immunohistochemistry and western blot results revealed high expression of the HPRT1 gene in the transfer group. CONCLUSION: This study established the Metastasis Score as a reliable tool for evaluating the risk of distant metastasis/recurrence in BRCA patients. Additionally, we identified key genes involved in MR and EMT crosstalk, offering valuable insights into their roles in tumor immunity and other relevant aspects.

Mycobacterium tuberculosis FadD18 Promotes Proinflammatory Cytokine Secretion to Inhibit the Intracellular Survival of Bacillus Calmette-Guérin.

Mycobacterium tuberculosis causes 6.4 million cases of tuberculosis and claims 1.6 million lives annually. Mycobacterial adhesion, invasion of host cells, and subsequent intracellular survival are crucial for the infection and dissemination process, yet the cellular mechanisms underlying these phenomena remain poorly understood. This study created a Bacillus Calmette-Guérin (BCG) transposon library using a MycomarT7 phage carrying a Himar1 Mariner transposon to identify genes related to mycobacteria adhesion and invasion. Using adhesion and invasion model screening, we found that the mutant strain B2909 lacked adhesion and invasion abilities because of an inactive fadD18 gene, which encodes a fatty-acyl CoA ligase, although the specific function of this gene remains unclear. To investigate the role of FadD18, we constructed a complementary strain and observed that fadD18 expression enhanced the colony size and promoted the formation of a stronger cord-like structure; FadD18 expression also inhibited BCG growth and reduced BCG intracellular survival in macrophages. Furthermore, FadD18 expression elevated levels of the proinflammatory cytokines IL-6, IL-1ß, and TNF-α in infected macrophages by stimulating the NF-κB and MAPK signaling pathways. Overall, the FadD18 plays a key role in the adhesion and invasion abilities of mycobacteria while modulating the intracellular survival of BCG by influencing the production of proinflammatory cytokines.

Superwetting Nanofluids of NiOx-Nanocrystals/CsBr Solution for Fabricating Quality NiOx-CsPbBr3 Gradient Hybrid Film in Carbon-Based Perovskite Solar Cells.

The wettability of precursor solution on substrates is the critical factor for fabricating quality film. In this work, superwetting nanofluids (NFs) of non-stoichiometric nickel oxide (NiOx) nanocrystals (NCs)-CsBr solution are first utilized to fabricate quality NiOx-CsPbBr3 hybrid film with gradient-distributed NiOx NCs in the upper part for constructing hole transport ladder in carbon-based perovskite solar cells (C-PSCs). As anticipated, the crystalline properties (improved crystalline grain diameters and reduced impurity phase) and hole extraction/transport of the NiOx-CsPbBr3 hybrid film are improved after incorporating NiOx NCs into CsPbBr3. This originates from the superb wettability of NiOx-CsBr NFs on substrates and the excellent hole-transport properties of NiOx. Consequently, the C-PSCs with the structure of FTO/SnO2/NiOx-CsPbBr3/C displays a power conversion efficiency of 10.07%, resulting in a 23.6% improvement as compared with the pristine CsPbBr3 cell. This work opens up a promising strategy to improve the absorber layer in PSCs by incorporating NCs into perovskite layers through the use of the superwettability of NFs and by composition gradient engineering.

A Novel Multiepitope Fusion Antigen as a Vaccine Candidate for the Prevention of Enterotoxigenic E. coli-Induced Calf Diarrhea.

Calf diarrhea caused by enterotoxigenic E. coli (ETEC) poses an enormous economic challenge in the cattle industry. Fimbriae and enterotoxin are crucial virulence factors and vaccine targets of ETEC. Since these proteins have complicated components with large molecular masses, the development of vaccines by directly expressing these potential targets is cumbersome Therefore, this study aimed to develop a multiepitope fusion antigen designated as MEFA by integrating major epitopes of FanC and Fim41a subunits and a toxoid epitope of STa into the F17G framework. The 3D modeling predicted that the MEFA protein displayed the epitopes from these four antigens on its surface, demonstrating the desired structural characteristics. Then, the MEFA protein was subsequently expressed and purified for mouse immunization. Following that, our homemade ELISA showed that the mouse antiserum had a consistent increase in polyclonal antibody levels with the highest titer of 1:217 to MEFA. Furthermore, the western blot assay demonstrated that this anti-MEFA serum could react with all four antigens. Further, this antiserum exhibited inhibition on ETEC adhesion to HCT-8 cells with inhibitory rates of 92.8%, 84.3%, and 87.9% against F17+, F5+, and F41+ ETEC strains, respectively. Additionally, the stimulatory effect of STa toxin on HCT-8 cells was decreased by approximately 75.3% by anti-MEFA serum. This study demonstrates that the MEFA protein would be an antigen candidate for novel subunit vaccines for preventing ETEC-induced diarrhea in cattle.

The Establishment of a Novel γ-Interferon In Vitro Release Assay for the Differentiation of Mycobacterial Bovis-Infected and BCG-Vaccinated Cattle.

BCG vaccination is increasingly reconsidered in the effective prevention of bovine tuberculosis (bTB). However, the primary challenge in BCG vaccination for cattle is the lack of a technique for differentiating between infected and vaccinated animals (DIVA). This study aimed to establish a novel DIVA diagnostic test based on an interferon-gamma in vitro release assay (IGRA). The plasmid encoding three differential antigens (Rv3872, CFP-10, and ESAT-6) absent in BCG genes but present in virulent M. bovis was previously constructed. Thus, a recombinant protein called RCE (Rv3872, CFP-10, and ESAT-6) was expressed, and an RCE-based DIVA IGRA (RCE-IGRA) was established. The RCE concentration was optimized at 4 µg/mL by evaluating 97 cattle (74 of which were bTB-positive, and 23 were negative) using a commercial IGRA bTB diagnostic kit. Further, 84 cattle were tested in parallel with the RCE-IGRA and commercial PPD-based IGRA (PPD-IGRA), and the results showed a high correlation with a kappa value of 0.83. The study included BCG-vaccinated calves (n = 6), bTB-positive cattle (n = 6), and bTB-negative non-vaccinated calves (n = 6). After 3 months post-vaccination, PPD-IGRA generated positive results in both vaccinated and infected calves. However, RCE-IGRA developed positive results in infected calves but negative results in vaccinated calves. In conclusion, this DIVA method has broad prospects in differentiating BCG vaccination from natural infection to prevent bTB.

Individual- and Herd-Level Milk ELISA Test Status and Incidence for Paratuberculosis in Hubei Province, China.

Mycobacterium avium subsp. paratuberculosis (MAP) is responsible for the persistent infectious illness known as bovine paratuberculosis, which is one of the most easily overlooked diseases in China amid a lack of epidemiological data. In this study, we evaluated the agreement of milk and blood antibody tests for paratuberculosis and showed an overall agreement of 92.0%, with a 95.0% negative coincidence rate and a 78.6% positive coincidence rate. The milk test was then used to examine the prevalence and incidence of dairy cows in Hubei Province, China. We found that, at the individual level, the highest lacto-prevalence reached up to 22.9%; the farm-level prevalence was as high as 92.3% (12/13) and 84.6% (11/13) in January and April 2018, respectively. The total incidence risk of all farms was 6% per three months. We also found that large-scale farms had a significantly lower prevalence and incidence than small-scale farms. Finally, the correlation between paratuberculosis and milk quality was evaluated, and we confirmed that MAP can significantly alter milk quality and raise somatic cell counts in the milk. This study provides valuable information for assessing the prevalence and incidence risk of paratuberculosis in China. It further provides an essential basis for calling for the prevention and control of paratuberculosis in China.

Soil Microbial Community Characteristics and Their Effect on Tea Quality under Different Fertilization Treatments in Two Tea Plantations.

Fertilization is an essential aspect of tea plantation management that supports a sustainable tea production and drastically influences soil microbial communities. However, few research studies have focused on the differences of microbial communities and the variation in tea quality in response to different fertilization treatments. In this work, the soil fertility, tea quality, and soil microbial communities were investigated in two domestic tea plantations following the application of chemical and organic fertilizers. We determined the content of mineral elements in the soil, including nitrogen, phosphorus, and potassium, and found that the supplementation of chemical fertilizer directly increased the content of mineral elements. However, the application of organic fertilizer significantly improved the accumulation of tea polyphenols and reduced the content of caffeine. Furthermore, amplicon sequencing results showed that the different ways of applying fertilizer have limited effect on the alpha diversity of the microbial community in the soil while the beta diversity was remarkably influenced. This work also suggests that the bacterial community structure and abundance were also relatively constant while the fungal community structure and abundance were dramatically influenced; for example, Chaetomiaceae at the family level, Hypocreaceae at the order level, Trichoderma at the genus level, and Fusarium oxysporum at the species level were predominantly enriched in the tea plantation applying organic fertilizer. Moreover, the bacterial and fungal biomarkers were also analyzed and it was found that Proteobacteria and Gammaproteobacteria (bacteria) and Tremellomycetes (fungi) were potentially characterized as biomarkers in the plantation under organic fertilization. These results provide a valuable basis for the application of organic fertilizer to improve the soil of tea plantations in the future.

Deletion of Tmem268 in mice suppresses anti-infectious immune responses by downregulating CD11b signaling.

Transmembrane protein 268 (TMEM268) is a novel, tumor growth-related protein first reported by our laboratory. It interacts with the integrin subunit ß4 (ITGB4) and plays a positive role in the regulation of the ITGB4/PLEC signaling pathway. Here, we investigated the effects and mechanism of TMEM268 in anti-infectious immune response in mice. Tmem268 knockout in mice aggravated cecal ligation and puncture-induced sepsis, as evidenced by higher bacterial burden in various tissues and organs, congestion, and apoptosis. Moreover, Tmem268 deficiency in mice inhibited phagocyte adhesion and migration, thus decreasing phagocyte infiltration at the site of infection and complement-dependent phagocytosis. Further findings indicated that TMEM268 interacts with CD11b and inhibits its degradation via the endosome-lysosome pathway. Our results reveal a positive regulatory role of TMEM268 in ß2 integrin-associated anti-infectious immune responses and signify the potential value of targeting the TMEM268-CD11b signaling axis for the maintenance of immune homeostasis and immunotherapy for sepsis and related immune disorders.

Investigation of heat stress responses and adaptation mechanisms by integrative metabolome and transcriptome analysis in tea plants (Camellia sinensis).

Extreme high temperature has deleterious impact on the yield and quality of tea production, which has aroused the attention of growers and breeders. However, the mechanisms by which tea plant varieties respond to extreme environmental heat is not clear. In this study, we analyzed physiological indices, metabolites and transcriptome differences in three different heat-tolerant tea plant F1 hybrid progenies. Results showed that the antioxidant enzyme activity, proline, and malondialdehyde were significantly decreased in heat-sensitive 'FWS' variety, and the accumulation of reactive oxygen molecules such as H2O2 and O2- was remarkably increased during heat stress. Metabolomic analysis was used to investigate the metabolite accumulation pattern of different varieties in response to heat stress. The result showed that a total of 810 metabolites were identified and more than 300 metabolites were differentially accumulated. Transcriptional profiling of three tea varieties found that such genes encoding proteins with chaperon domains were preferentially expressed in heat-tolerant varieties under heat stress, including universal stress protein (USP32, USP-like), chaperonin-like protein 2 (CLP2), small heat shock protein (HSP18.1), and late embryogenesis abundant protein (LEA5). Combining metabolomic with transcriptomic analyses discovered that the flavonoids biosynthesis pathway was affected by heat stress and most flavonols were up-regulated in heat-tolerant varieties, which owe to the preferential expression of key FLS genes controlling flavonol biosynthesis. Take together, molecular chaperons, or chaperon-like proteins, flavonols accumulation collaboratively contributed to the heat stress adaptation in tea plant. The present study elucidated the differences in metabolite accumulation and gene expression patterns among three different heat-tolerant tea varieties under extreme ambient high temperatures, which helps to reveal the regulatory mechanisms of tea plant adaptation to heat stress, and provides a reference for the breeding of heat-tolerant tea plant varieties.

Identification of KDM4C as a gene conferring drug resistance in multiple myeloma.

Bortezomib (BTZ), a proteasome inhibitor, is a promising therapeutic option for multiple myeloma (MM) patients. However, drug resistance often occurs, leading to disease relapse and poor prognosis. In this study, we aimed to identify novel genes associated with drug resistance and investigate their roles in BTZ resistance. Through the screening of 26 genes frequently associated with chemosensitivity or drug resistance, we discovered that KDM4C, a histone demethylase, exhibited increased expression in BTZ-resistant MM cells compared to their sensitive counterparts. Overexpression of KDM4C enhanced the tolerance of a MM cell line to the drug, whereas the knockdown of KDM4C, using shRNA, increased the sensitivity of resistant cells to BTZ treatment. This suggests that KDM4C plays a pivotal role in conferring BTZ resistance. Our study offers fresh insights into BTZ resistance in MM and highlights KDM4C as a potential target for overcoming drug resistance.

The Identification of a Novel Nucleomodulin MbovP467 of Mycoplasmopsis bovis and Its Potential Contribution in Pathogenesis.

Mycoplasmopsis bovis is a causative agent of crucial diseases in both dairy and beef cattle leading to substantial economic losses. However, limited control measures for M. bovis-related diseases exist due to a lack of understanding about the virulence factors of this pathogen, a common challenge in mycoplasma research. Consequently, this study aimed to characterize a novel nucleomodulin as a virulence-related factor of M. bovis. Employing bioinformatic tools, we initially predicted MbovP467 to be a secreted protein with a nuclear localization signal based on SignalP scores and the cNLS (Nuclear Localization Signal) Mapper, respectively. Subsequently, the MbovP467 gene was synthesized and cloned into a pEGFP plasmid with EGFP labeling to obtain a recombinant plasmid (rpEGFP-MbovP467) and then was also cloned in pET-30a with a consideration for an Escherichia coli codon bias and expressed and purified for the production of polyclonal antibodies against the recombinant MbovP467 protein. Confocal microscopy and a Western blotting assay confirmed the nuclear location of MbovP467 in bovine macrophages (BoMacs). RNA-seq data revealed 220 up-regulated and 20 down-regulated genes in the rpEGFP-MbovP467-treated BoMac group compared to the control group (pEGFP). A GO- and KEGG-enrichment analysis identified associations with inflammatory responses, G protein-coupled receptor signaling pathways, nuclear receptor activity, sequence-specific DNA binding, the regulation of cell proliferation, IL-8, apoptotic processes, cell growth and death, the TNF signaling pathway, the NF-κB signaling pathway, pathways in cancer, and protein families of signaling and cellular processes among the differentially expressed up-regulated mRNAs. Further experiments, investigating cell viability and the inflammatory response, demonstrated that MbovP467 reduces BoMac cell viability and induces the mRNA expression of IL-1ß, IL-6, IL-8, TNF-α, and apoptosis in BoMac cells. Further, MbovP467 increased the promoter activity of TNF-α. In conclusion, this study identified a new nucleomodulin, MbovP467, for M. bovis, which might have an important role in M. bovis pathogenesis.

Investigation of the safety and protective efficacy of an attenuated and marker M. bovis-BoHV-1 combined vaccine in bovines.

Bovine respiratory disease (BRD) is one of the most common diseases in the cattle industry worldwide; it is caused by multiple bacterial or viral coinfections, of which Mycoplasma bovis (M. bovis) and bovine herpesvirus type 1 (BoHV-1) are the most notable pathogens. Although live vaccines have demonstrated better efficacy against BRD induced by both pathogens, there are no combined live and marker vaccines. Therefore, we developed an attenuated and marker M. bovis-BoHV-1 combined vaccine based on the M. bovis HB150 and BoHV-1 gG-/tk- strain previously constructed in our lab and evaluated in rabbits. This study aimed to further evaluate its safety and protective efficacy in cattle using different antigen ratios. After immunization, all vaccinated cattle had a normal rectal temperature and mental status without respiratory symptoms. CD4+, CD8+, and CD19+ cells significantly increased in immunized cattle and induced higher humoral and cellular immune responses, and the expression of key cytokines such as IL-4, IL-12, TNF-α, and IFN-γ can be promoted after vaccination. The 1.0 × 108 CFU of M. bovis HB150 and 1.0 × 106 TCID50 BoHV-1 gG-/tk- combined strain elicited the most antibodies while significantly increasing IgG and cellular immunity after challenge. In conclusion, the M. bovis HB150 and BoHV-1 gG-/tk- combined strain was clinically safe and protective in calves; the mix of 1.0 × 108 CFU of M. bovis HB150 and 1.0 × 106 TCID50 BoHV-1 gG-/tk- strain was most promising due to its low amount of shedding and highest humoral and cellular immune responses compared with others. This study introduces an M. bovis-BoHV-1 combined vaccine for application in the cattle industry.

MbovP0725, a secreted serine/threonine phosphatase, inhibits the host inflammatory response and affects metabolism in Mycoplasma bovis.

Mycoplasma species are able to produce and release secreted proteins, such as toxins, adhesins, and virulence-related enzymes, involved in bacteria adhesion, invasion, and immune evasion between the pathogen and host. Here, we investigated a novel secreted protein, MbovP0725, from Mycoplasma bovis encoding a putative haloacid dehalogenase (HAD) hydrolase function of a key serine/threonine phosphatase depending on Mg2+ for the dephosphorylation of its substrate pNPP, and it was most active at pH 8 to 9 and temperatures around 40°C. A transposon insertion mutant strain of M. bovis HB0801 that lacked the protein MbovP0725 induced a stronger inflammatory response but with a partial reduction of adhesion ability. Using transcriptome sequencing and quantitative reverse transcription polymerase chain reaction analysis, we found that the mutant was upregulated by the mRNA expression of genes from the glycolysis pathway, while downregulated by the genes enriched in ABC transporters and acetate kinase-phosphate acetyltransferase pathway. Untargeted metabolomics showed that the disruption of the Mbov_0725 gene caused the accumulation of 9-hydroxyoctadecadienoic acids and the consumption of cytidine 5'-monophosphate, uridine monophosphate, and adenosine monophosphate. Both the exogenous and endogenous MbvoP0725 protein created by purification and transfection inhibited lipopolysaccharide (LPS)-induced IL-1ß, IL-6, and TNF-α mRNA production and could also attenuate the activation of MAPK-associated pathways after LPS treatment. A pull-down assay identified MAPK p38 and ERK as potential substrates for MbovP0725. These findings define metabolism- and virulence-related roles for a HAD family phosphatase and reveal its ability to inhibit the host pro-inflammatory response. IMPORTANCE: Mycoplasma bovis (M. bovis) infection is characterized by chronic pneumonia, otitis, arthritis, and mastitis, among others, and tends to involve the suppression of the immune response via multiple strategies to avoid host cell immune clearance. This study found that MbovP0725, a haloacid dehalogenase (HAD) family phosphatase secreted by M. bovis, had the ability to inhibit the host pro-inflammatory response induced by lipopolysaccharide. Transcriptomic and metabolomic analyses were used to identify MbovP0725 as an important phosphatase involved in glycolysis and nucleotide metabolism. The M. bovis transposon mutant strain T8.66 lacking MbovP0725 induced a higher inflammatory response and exhibited weaker adhesion to host cells. Additionally, T8.66 attenuated the phosphorylation of MAPK P38 and ERK and interacted with the two targets. These results suggested that MbovP0725 had the virulence- and metabolism-related role of a HAD family phosphatase, performing an anti-inflammatory response during M. bovis infection.