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Abstract Lumpy skin disease (LSD) is a highly contagious disease caused by lumpy skin disease virus (LSDV) in bovines. Rapid and accurate diagnosis is very important to controll it. However, current commercial detection kits need to be improved in terms of sensitivity or specificity. This study aimed to develop a novel diagnostic competitive enzyme-linked immunosorbent assay (cELISA) based on the newly identified antigen gene LSDV034. The rLSDV034 protein was identified as a potential diagnostic antigen, and it was expressed, purified, and used to immunize BALB/c mice. Using laboratory-prepared indirect ELISA (iELISA), the positive cell lines were screened, and their blocking activity was further verified by competitive ELISA (cELISA). The cell line, 1H7, was chosen to produce mouse ascites, which were purified for a monoclonal antibody (mAb, 5.395 mg/mL). The heavy chain type of the 1H7 mAb was identified as IgG1a, and its light chain subtype was identified as κ. Furthermore, cELISA was developed to detect bovine serum antibodies, with rLSDV034 (4 μg/mL) as the coating antigen and HRP-1H7 mAb (1:300) as the competitive antibody. The cutoff value of cELISA was 55%, based on 32 negative bovine serum samples. The analytical sensitivity was 1:8, and no cross-reaction was detected with bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheitis virus (IBRV), Pasteurella multocida (P. multocida), or Mycoplasma bovis (M. bovis) from the serum samples. The diagnostic sensitivity and specificity of cELISA were 98.46% (95% confidence interval, CI: 91.7–100) and 100% (95% CI: 89.1–100), respectively, based on the analysis of 30 LSDV-infected bovine serum samples, 35 GTPV-vaccinated samples, and 32 negative samples. The overall coincidence of the cELISA with the virus neutralization test (VNT) reached 98.97% (95% CI: 94.4–100). Furthermore, we used cELISA to analyze 230 clinical bovine serum samples (including 59 infected and 171 vaccinated samples) and found that the serum positivity rates of the immunized samples (on d 60 postimmunization) and infected samples were 77.78% (95% CI: 70.8–83.8%) and 71.19% (95% CI: 57.9–82.2), respectively. These results indicate that the developed cELISA is promising for detecting serum antibodies in naturally infected or vaccinated cattle.

Abstract 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.

The Mycobacterium tuberculosis variant bovis (M. bovis) is a highly pathogenic environmental microorganism that causes bovine tuberculosis (bTB), a significant zoonotic disease. Currently, “test and culling” is the primary measure for controlling bTB, but it has been proven to be inadequate in animals due to their high susceptibility to the pathogen. Selective breeding for increased host resistance to bTB to reduce its prevalence is feasible. In this study, we found a vital host-dependent factor, RBMX2, that can potentially promote M. bovis infection. By knocking RBMX2 out, we investigated its function during M. bovis infection. Through transcriptome sequencing and alternative splicing transcriptome sequencing, we concluded that after M. bovis infection, embryo bovine lung (EBL) cells were significantly enriched in RNA splicing associated with apoptosis compared with wild-type EBL cells. Through protein/molecular docking, molecular dynamics simulations, and real-time quantitative PCR, we demonstrated that RBMX2 promotes the apoptosis of epithelial cells by upregulating and binding to apoptotic peptidase activating factor 1 (APAF-1), resulting in the alternative splicing of APAF-1 as a retention intron. To our knowledge, this is the first report of M. bovis affecting host epithelial cell apoptosis by hijacking RBMX2 to promote the intron splicing of downstream APAF-1. These findings may represent a significant contribution to the development of novel TB prevention and control strategies.

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.

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.

ABSTRACTMycoplasma 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-1β, 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.IMPORTANCEMycoplasma 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.

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.

Abstract The E3 ubiquitin ligase RING finger protein 115 (RNF115), also known as breast cancer-associated gene 2 (BCA2), has been linked with the growth of some cancers and immune regulation, which is negatively correlated with prognosis. Here, it is demonstrated that the RNF115 deletion can protect mice from acute liver injury (ALI) induced by the treatment of lipopolysaccharide (LPS)/D-galactosamine (D-GalN), as evidenced by decreased levels of alanine aminotransaminase, aspartate transaminase, inflammatory cytokines (e.g., tumor necrosis factor α and interleukin-6), chemokines (e.g., MCP1/CCL2) and inflammatory cell (e.g., monocytes and neutrophils) infiltration. Moreover, it was found that the autophagy activity in Rnf115 −/− livers was increased, which resulted in the removal of damaged mitochondria and hepatocyte apoptosis. However, the administration of adeno-associated virus Rnf115 or autophagy inhibitor 3-MA impaired autophagy and aggravated liver injury in Rnf115 −/− mice with ALI. Further experiments proved that RNF115 interacts with LC3B, downregulates LC3B protein levels and cell autophagy. Additionally, Rnf115 deletion inhibited M1 type macrophage activation via NF-κB and Jnk signaling pathways. Elimination of macrophages narrowed the difference in liver damage between Rnf115 +/+ and Rnf115 −/− mice, indicating that macrophages were linked in the ALI induced by LPS/D-GalN. Collectively, for the first time, we have proved that Rnf115 inactivation ameliorated LPS/D-GalN-induced ALI in mice by promoting autophagy and attenuating inflammatory responses. This study provides new evidence for the involvement of autophagy mechanisms in the protection against acute liver injury.

Abstract Background Gene expression profiles have important significance for gene expression characteristics and further functional studies. More attention has been given to the expression databases in humans and mice, but less attention has been given to rats, while rat models also play an irreplaceable role in biomedical experiments. Results To depict the rat gene expression profiles in mRNA expression levels, we analyzed over 2,700 RNA sequencing (RNA-Seq) samples from 48 tissues, 40 primary cell types and 25 cell lines; and then mapped them to the latest version of the rat genome reference, mRatBN7.2. Based on these datasets and reanalysis, we constructed a new database, the Omic Horizon Expression Database ( http://immudb.bjmu.edu.cn/expression.html ), which allows expressional profile query of over 25,000 rat genes based on non-redundant gene symbols. The database supports requests using gene symbols (or alias), Ensemble and Entrez gene IDs. Gene expression profiles can be queried in three categories: tissues, primary cells and cell lines. Application examples including expression profiling and comparison, as well as identification of novel rat genes, were illustrated to show the utility of the database. Conclusions As an omic resource, the Omic Horizon Expression Database provides horizons of gene expression profiles across various tissues and cells, which greatly facilitates the identification of rat genes as well as functional clues.

Abstract A cross-sectional study was conducted to evaluate the risk factors associated with the Salmonella infection status of dairy herds in Henan and Hubei provinces, China. Herds were assigned a Salmonella status based on the isolation of Salmonella from fecal samples obtained from adult lactating dairy cows. Information on potential biosecurity risk factors was collected using a questionnaire template via a 15-min face-to-face interview with dairy cattle producers from May 2020 to March 2021. The questionnaire consisted of 23 questions across two broad categories of potential biosecurity risk factors: farm and biosecurity managemental factors. A total of 48 farms were surveyed. In all (100%, 48/48) of the surveyed farms, although sick animals were separated from healthy animals using fencing, there were no strict quarantine protocols for newly introduced animals. Mixed species rearing was practiced in 35.4% (17/48) of the farms. Feces were removed more than once a day in 45.8% (22/48) of the farms, whereas the remainder (54.2%, 26/48) only removed animal feces once a day. A total of 29.2% (14/48) of the farms were located

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-1β, 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.

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.

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.

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.

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.

Nucleotide second messengers play an important role in bacterial adaptation to environmental changes. Recent evidence suggests that some of these regulatory molecular pathways were conserved upon the degenerative evolution of the wall-less mycoplasmas. We have recently reported the occurrence of a phosphodiesterase (PDE) in the ruminant pathogen Mycoplasma bovis, which was involved in c-di-AMP metabolism. In the present study, we demonstrate that the genome of this mycoplasma species encodes a PDE of the GdpP family with atypical DHH domains. Characterization of M. bovis GdpP (MbovGdpP) revealed a multifunctional PDE with unusual nanoRNase and single-stranded DNase activities. The alarmone ppGpp was found unable to inhibit c-di-NMP degradation by MbovGdpP but efficiently blocked its nanoRNase activity. Remarkably, MbovGdpP was found critical for the osmotic tolerance of M. bovis under K+ and Na+ conditions. Transcriptomic analyses further revealed the biological importance of MbovGdpP in tRNA biosynthesis, pyruvate metabolism, and several steps in genetic information processing. This study is an important step in understanding the role of PDE and nucleotide second messengers in the biology of a minimal bacterial pathogen.

Background Pyroptosis, a lytic form of programmed cell death initiated by inflammasomes, has been reported to be closely associated with tumor proliferation, invasion and metastasis. However, the roles of pyroptosis genes (PGs) in low-grade glioma (LGG) remain unclear. Methods We obtained information for 1,681 samples, including the mRNA expression profiles of LGGs and normal brain tissues and the relevant corresponding clinical information from two public datasets, TCGA and GTEx, and identified 45 differentially expressed pyroptosis genes (DEPGs). Among these DEPGs, nine hub pyroptosis genes (HPGs) were identified and used to construct a genetic risk scoring model. A total of 476 patients, selected as the training group, were divided into low-risk and high-risk groups according to the risk score. The area under the curve (AUC) values of the receiver operating characteristic (ROC) curves verified the accuracy of the model, and a nomogram combining the risk score and clinicopathological characteristics was used to predict the overall survival (OS) of LGG patients. In addition, a cohort from the Gene Expression Omnibus (GEO) database was selected as a validation group to verify the stability of the model. qRT-PCR was used to analyze the gene expression levels of nine HPGs in paracancerous and tumor tissues from 10 LGG patients. Results Survival analysis showed that, compared with patients in the low-risk group, patients in the high-risk group had a poorer prognosis. A risk score model combining PG expression levels with clinical features was considered an independent risk factor. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated that immune-related genes were enriched among the DEPGs and that immune activity was increased in the high-risk group. Conclusion In summary, we successfully constructed a model to predict the prognosis of LGG patients, which will help to promote individualized treatment and provide potential new targets for immunotherapy.

Abstract Bovine tuberculosis (bTB) is a chronic zoonotic disease that is endemic in China. Current in-vitro tests for bTB are mainly based on blood assays. Collection of samples results in some stress to the sampled cattle and associated economic losses for the herd owner. This study was designed to investigate the relationship between milk and serum antibody tests for bTB in dairy cows using 85 cows with milk and corresponding blood samples. Totally 4,395 milk samples were used to assesse the apparent (test) prevalence and incidence of bTB using the milk antibody ELISA. The association between levels of bTB milk antibody and milk quality was also evaluated. Milk and serum antibody tests showed a good correlation with a 87.5% (95% CI: 61.7%, 98.4) positive agreement and 98.7% (95% CI: 95.4, 99.8) negative agreement. The animal level lactoprevalence ranged from 0.3% (95% CI: 0, 1.2) to 33.3% (95% CI: 26.6, 40.6) in different farms and the incidence rate ranged from 0 head/cow-month (95% CI: 0, 0.02) to 0.04 head/cow-month (95% CI: 0.02, 0.07). Twenty percent of sampled farms met the criteria for bTB control in China. The prevalence on large-scale farms was lower (p

Abstract Background Tuberculosis (TB) is a zoonotic disease that affects humans and domesticated and wild animals. Animals in zoos are potentially an important source of TB for humans; however they are often neglected in routine disease surveillance programs. This investigation reports an outbreak of TB in milu deer and northern pig‐tailed macaques in a zoo in Wuhan, China, which highlighted the need for improved prevention and control of TB in China. Methods Between 24 November and 9 December 2020 two milu deer and a northern pig‐tailed macaque that were displaying signs of wasting died. Post‐mortem, histopathological diagnosis and acid fast staining were used for the dead animals. Multiple PCR for Mycobacterium tuberculosis complex (MTBC) was performed to identify the bacterial in both milu deer and northern pig‐tailed macaque. The serum antibody iELISA for MTBC was then performed for all the surviving milu deer and northern pig‐tailed macaques. Six seropositive milu deer and a seropositive northern pig‐tailed macaque were subsequently euthanised and, along with two other dead milu deer, necropsied. DNA from these tissue samples was extracted and detected MTBC using PCR and Real‐time PCR. Subsequently bacterial isolation was used to confirm the infection. Results The lungs of the dead animals displayed gross and histological TB‐like lesions and changes, and red staining bacilli were detected in smears of the lesions by microscopy after acid fast staining. Mycobacterium bovis (M. bovis) was detected in the two milu deer and Mycobacterium tuberculosis (M. tb) in the northern pig‐tailed macaque using multiple PCR for MTBC. 35.3% surviving milu deer and 50% surviving northern pig‐tailed macaques MTBC serologically positive. Six of the euthanised milu deer were also positive on a DNA test for M. bovis and the euthanised northern pig‐tailed macaque was positive to M. tb. Conclusions This is the first report of tuberculosis in the endangered species, milu deer and northern pig‐tailed macaques, in China, and warrants urgent attention by researchers and conservation authorities. These cases highlight the need for expanding surveillance for MTBC to zoos in China.

Abstract Background The snub-nosed monkey (Rhinopithecus roxellanae) is an endangered animal species mainly distributed in China and needs to be protected. Gut microbiome is an important determinant of animal health and population survival as it affects the adaptation of the animals to different foods and environments under kinetic changes of intrinsic and extrinsic factors. Therefore, this study aimed to elucidate gut fecal microbiome profiles of snub-nosed monkeys affected by several extrinsic and intrinsic factors, including raising patterns (captive vs. wild), age, sex, and diarrheal status to provide a reference for making protection strategies. Results The 16S rRNA gene sequencing was firstly used to pre-check clustering of 38 fecal samples from the monkeys including 30 wild and 8 captive (5 healthy and 3 diarrheal) from three Regions of Shennongjia Nature Reserve, Hubei Province, China. Then the 24 samples with high-quality DNA from 18 wild and 6 captive (4 healthy and 2 diarrheal) monkeys were subjected to shotgun metagenomic sequencing to characterize bacterial gut microbial communities. We discovered that the raising pattern (captive and wild) rather than age and sex was the predominant factor attributed to gut microbiome structure and proportionality. Wild monkeys had significantly higher bacterial diversity and lower Bacteroidetes/Firmicutes ratios than captive animals. Moreover, the gut microbiomes in wild healthy monkeys were enriched for the genes involved in fatty acid production, while in captive animals, genes were enriched for vitamin biosynthesis and metabolism and amino acid biosynthesis from carbohydrate intermediates. Additionally, a total of 37 antibiotic resistant genes (ARG) types were detected. Unlike the microbiome diversity, the captive monkeys have a higher diversity of ARG than the wild animals. Conclusion Taken together, we highlight the importance of self-reprogramed metabolism in the snub-nosed monkey gut microbiome to help captive and wild monkeys adapt to different intrinsic and extrinsic environmental change.

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-1β, 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.

Bovine parainfluenza virus type 3 (BPIV-3) is one of the major pathogens of the bovine respiratory disease complex (BRDC). BPIV-3 surveillance in China has been quite limited. In this study, we used PCR to test 302 cattle in China, and found that the positive rate was 4.64% and the herd-level positive rate was 13.16%. Six BPIV-3C strains were isolated and confirmed by electron microscopy, and their titers were determined. Three were sequenced by next-generation sequencing (NGS). Phylogenetic analyses showed that all isolates were most closely related to strain NX49 from Ningxia; the genetic diversity of genotype C strains was lower than strains of genotypes A and B; the HN, P, and N genes were more suitable for genotyping and evolutionary analyses of BPIV-3. Protein variation analyses showed that all isolates had mutations at amino acid sites in the proteins HN, M, F, and L. Genetic recombination analyses provided evidence for homologous recombination of BPIV-3 of bovine origin. The virulence experiment indicated that strain Hubei-03 had the highest pathogenicity and could be used as a vaccine candidate. These findings apply an important basis for the precise control of BPIV-3 in China.

Bovine respiratory disease (BRD) is one of the most common diseases in the cattle industry; it is a globally prevalent multifactorial infection primarily caused by viral and bacterial coinfections. In China, Mycoplasma bovis (M. bovis) and bovine herpesvirus type 1 (BoHV-1) are the most notable pathogens associated with BRD. Our previous study attempted to combine the two vaccines and conducted a preliminary investigation of their optimal antigenic ratios. Based on this premise, the research extended its investigation by administering varying vaccine doses in a rabbit model to identify the most effective immunization dosage. After immunization, all rabbits in other immunization dose groups had a normal rectal temperature without obvious clinical symptoms. Furthermore, assays performed on the samples collected from immunized rabbits indicated that there were increased humoral and cellular immunological reactions. Moreover, the histological analysis of the lungs showed that immunized rabbits had more intact lung tissue than their unimmunized counterparts after the challenge. Additionally, there appears to be a positive correlation between the protective efficacy and the immunization dose. In conclusion, the different immunization doses of the attenuated and marker M. bovis HB150 and BoHV-1 gG-/tk- combined vaccine were clinically safe in rabbits; the mix of 2.0 × 108 CFU of M. bovis HB150 and 2.0 × 106 TCID50 BoHV-1 gG-/tk- strain was most promising due to its highest humoral and cellular immune responses and a more complete morphology of the lung tissue compared with others. These findings determined the optimal immunization dose of the attenuated and marker M. bovis HB150 and BoHV-1 gG-/tk- combined vaccine, laying a foundation for its clinical application.

Nucleomodulins are secreted bacterial proteins whose molecular targets are located in host cell nuclei. They are gaining attention as critical virulence factors that either modify the epigenetics of host cells or directly regulate host gene expression. Mycoplasma bovis is a major veterinary pathogen that secretes several potential virulence factors. The aim of this study was to determine whether any of their secreted proteins might function as nucleomodulins. After an initial in silico screening, the nuclear localization of the secreted putative lipoprotein MbovP475 of M. bovis was demonstrated in bovine macrophage cell line (BoMac) experimentally infected with M. bovis. Through combined application of ChIP-seq, Electrophoretic mobility shift assay (EMSA) and surface plasmon resonance (SPR) analysis, MbovP475 was determined to bind the promoter regions of the cell cycle central regulatory genes CRYAB and MCF2L2. MbovP475 has similar secondary structures with the transcription activator-like effectors (TALEs). Screening of various mutants affecting the potential DNA binding sites indicated that the residues 242NI243 within MbovP475 loop region of the helix-loop-helix domain were essential to its DNA binding activity, thereby contributing to decrease in BoMac cell viability. In conclusion, this is the first report to confirm M. bovis secretes a conserved TALE-like nucleomodulin that binds the promoters of CRYAB and MCF2L2 genes, and subsequently down-regulates their expression and decreases BoMac cell viability. Therefore, this study offers a new understanding of mycoplasma pathogenesis.

Abstract Brucellosis is an important zoonosis that results in substantial economic losses to the livestock industry through abortions and reduced milk yield. This study investigated an abortion outbreak in a dairy herd and then explored the effects of emergency vaccination with Brucella abortus A19 vaccine on the incidence of abortion and milk yield. A full dose of vaccine (6 × 1010—12 × 1010 colony forming units, CFU) was administered subcutaneously to calves and non-pregnant heifers, and a reduced dose (6 × 108—12 × 108 CFU) to adult cows and pregnant replacement heifers. Rose Bengal Test was used to screen Brucella infection status and then positive samples were tested with a C-ELISA. Animals that tested positive for both tests were considered positive to Brucella spp. The animal-level seroprevalence of brucellosis was 23.1% (95% CI: 17.0, 30.2), and the attributable fraction of abortions in seropositive animals was 89.1% (95% CI: 64.3, 96.7). The odds of seropositivity were significantly higher in cows that aborted compared to cows that calved normally (OR = 21.4, 95% CI: 4.4, 168.4). Cows in sheds A2 and C1 were 10.2 (95% CI: 1.4, 128.0) and 17.0 (95% CI: 2.8, 190.3) times more likely to be seropositive than cows in shed B1. Antibodies were not detectable in most heifers 12 months post-vaccination. The effectiveness of the vaccine in preventing abortions was estimated to be 56.8% (95% CI: 15.8, 77.8) for the entire herd, but increased to 86.7% (95% CI: 4.4, 98.1) when only primiparous heifers were considered. Furthermore, a significant increase in the average herd 305-day milk yield one-year after vaccination was also observed relative to that in the previous three years. It is concluded that emergency vaccination of a dairy herd undergoing an abortion outbreak with the A19 vaccine effectively reduced the incidence of abortion and indirectly increased milk yield one-year after vaccination.

Abstract Elevated extrachromosomal circular DNA (eccDNA) has been reported to accelerate tumor pathogenesis. Although the eccDNA profiles of other tumors have been established, the landscape of the eccDNA of acute myeloid leukemia (AML) has not been revealed. Our study first depicted the eccDNA profile of normal hematopoiesis and AML evolution by exploiting the ATAC-seq and RNA-seq data from nine healthy donors and 12 AML patients, which contained a total of 137 cell samples and 96 RNA-seq samples (including 16 blood cell types of the normal hematopoietic and AML hierarchies). We found the number of eccDNAs generally increased with the evolution of normal hematopoiesis and AML. The ecDNAs and ring chromosomes were found to reappear both in normal hematopoiesis and AML cells. Furthermore, we compared the eccDNAs of AML with normal cells. There were almost 300 AML-specific genes, including the known oncogenes NRAS, MCL1, EVI1, GATA2, WT1, and PAK1. And the ecDNA (chr11: 58668376-58826008) occurred in five out of 17 AML evolution-related cells, which was associated with the high expression of the GLYATL1 gene and the high expressed GLYATL1 was a poor prognostic factor. In conclusion, the eccDNA profiles of normal hematopoiesis and AML evolution were depicted and the recurrent eccDNAs we revealed might be utilized in the treatment of AML as biomarkers.

Circular RNAs (circRNAs) are noncoding RNAs with diverse functions. However, most Mycobacterium tuberculosis (M.tb)-related circRNAs remain undiscovered. In this study, we infected THP-1 cells with virulent and avirulent M.tb strains and then sequenced the cellular circRNAs. Bioinformatic analysis predicted 58,009 circRNAs in all the cells. In total, 2035 differentially expressed circRNAs were identified between the M.tb-infected and uninfected THP-1 cells and 1258 circRNAs were identified in the virulent and avirulent M.tb strains. Further, the top 10 circRNAs were confirmed by Sanger sequencing, among which four circRNAs, namely circSOD2, circCHSY1, circTNFRSF21, and circDHTKD1, which were highly differentially expressed in infected cells compared with those in uninfected cells, were further confirmed by ring formation, specific primers, and RNase R digestion. Next, circRNA-miRNA-mRNA subnetworks were constructed, such as circDHTKD1/miR-660-3p/IL-12B axis. Some of the individual downstream genes, such as miR-660-3p and IL-12B, were previously reported to be associated with cellular defense against pathological processes induced by M.tb infection. Because macrophages are important immune cells and the major host cells of M.tb, these findings provide novel ideas for exploring the M.tb pathogenesis and host defense by focusing on the regulation of circRNAs during M.tb infection.

The most important pathogenic Mycoplasma species in bovines are Mycoplasma bovis (M. bovis) and Mycoplasma mycoides subsp. mycoides (Mmm). Mmm causes contagious bovine pleuropneumonia (CBPP), which is a severe respiratory disease widespread in sub-Saharan Africa but eradicated in several countries, including China. M. bovis is an important cause of the bovine respiratory disease complex (BRD), characterized worldwide by pneumonia, arthritis, and mastitis. Secreted proteins of bacteria are generally considered virulence factors because they can act as toxins, adhesins, and virulent enzymes in infection. Therefore, this study performed a comparative proteomic analysis of the secreted proteins of M. bovis and Mmm in order to find some virulence-related factors as well as discover differential diagnostic biomarkers for these bovine mycoplasmas. The secretome was extracted from both species, and liquid chromatography-tandem mass spectrometry was used, which revealed 55 unique secreted proteins of M. bovis, 44 unique secreted proteins of Mmm, and 4 homologous proteins. In the M. bovis secretome, 19 proteins were predicted to be virulence factors, while 4 putative virulence factors were identified in the Mmm secretome. In addition, five unique secreted proteins of Mmm were expressed and purified, and their antigenicity was confirmed by Western blotting assay and indirect ELISA. Among them, Ts1133 and Ts0085 were verified as potential candidates for distinguishing Mmm infection from M. bovis infection.

Tuberculosis, caused by Mycobacterium tuberculosis (M. tb), remains a significant global health challenge. The survival of M. tb in hostile extracellular and intracellular microenvironments is crucial for its pathogenicity. In this study, we discovered a Bacillus Calmette–Guérin (BCG) mutant B1033 that potentially affected mycobacterium pathogenicity. This mutant contained an insertion mutation gene, fadD33, which is involved in lipid metabolism; however, its direct role in regulating M. tb infection is not well understood. Here, we found that the absence of fadD33 reduced BCG adhesion and invasion into human pulmonary alveolar epithelial cells and increased the permeability of the mycobacterial cell wall, allowing M. tb to survive in the low pH and membrane pressure extracellular microenvironment of the host cells. The absence of fadD33 also inhibited the survival of BCG in macrophages by promoting the release of proinflammatory cytokines, such as interleukin (IL)-1β, IL-6, and tumors necrosis factor-α, through the mitogen-activated protein kinase p38 signaling pathway. Overall, these findings provide new insights into M. tb mechanisms to evade host defenses and might contribute to identifying potential therapeutic and vaccine targets for tuberculosis prevention.

E. coli is a ubiquitous pathogen that is responsible for over one million fatalities worldwide on an annual basis. In animals, E. coli can cause a variety of diseases, including mastitis in dairy cattle, which represents a potential public health hazard. However, the pathophysiology of E. coli remains unclear. We found that E. coli could induce global upregulation of m6A methylation and cause serious apoptosis in bovine mammary epithelial cells (MAC-T cells). Furthermore, numerous m6A-modified lncRNAs were identified through MeRIP-seq. Interestingly, we found that the expression of LOC4191 with hypomethylation increased in MAC-T cells upon E. coli-induced apoptosis. Knocking down LOC4191 promoted E. coli-induced apoptosis and ROS levels through the caspase 3–PARP pathway. Meanwhile, knocking down ALKBH5 resulted in the promotion of apoptosis through upregulated ROS and arrested the cell cycle in MAC-T cells. ALKBH5 silencing accelerated LOC4191 decay by upregulating its m6A modification level, and the process was recognized by hnRNP A1. Therefore, this indicates that ALKBH5 stabilizes m6A-modified LOC4191 to suppress E. coli-induced apoptosis. This report discusses an initial investigation into the mechanism of m6A-modified lncRNA in cells under E. coli-induced apoptosis and provides novel insights into infectious diseases.

Bovine respiratory disease (BRD) is a global prevalent multifactorial infection primarily caused by viral and bacterial coinfections. In China, Mycoplasma bovis (M. bovis) and bovine herpesvirus type 1 (BoHV-1) are the predominant pathogens associated with BRD. Our previous study involved the development of attenuated M. bovis HB150 and BoHV-1 gG-/tk- vaccine strains, which were thoroughly assessed for their safety profiles and protective efficacy in cattle. In this study, we applied a combination of vaccines in varying ratios and used a rabbit model to determine the safety and protective efficacy. We used PCR/RT-PCR to detect the postimmunization and challenge shedding of M. bovis and BoHV-1. Additionally, we measured antibody titers and the expression of IFN-β and TNF-α to evaluate the humoral and cellular immune responses, respectively. Furthermore, we performed a histopathological analysis to assess lung damage. Our study provides evidence of the safety and effectiveness of the bivalent M. bovis–BoHV-1 vaccine in rabbits, particularly when applying a combination of 1.0 × 108 CFU of M. bovis HB150 and 1.0 × 106 TCID50 of the BoHV-1 gG-/tk- strain. The bivalent vaccine significantly enhanced both the long-term antibody immune response and cellular protection against the M. bovis and BoHV-1 challenge. These findings provide a valuable model for the potential application in cattle.

Infectious bovine rhinotracheitis (IBR) caused by bovine herpes virus 1 (BoHV-1) can lead to enormous economic losses in the cattle industry. Vaccine immunization is preferentially used to decrease its transmission speed and resultant clinical signs, rather than to completely stop viral infection. Therefore, a drug effective in treating IBR is urgently needed. Our previous work demonstrated that ivermectin significantly inhibited viral replication in a cell infection model. This study aimed to investigate its antiviral effects in vivo by using a rabbit infection model. The viral inhibition assay was first used to confirm that ivermectin at low concentrations (6–25 nM) could reduce viral titers (TCID50) significantly (p < 0.001) at 24 h post-infection. In rabbits, ivermectin was administrated with one to three doses, based on the recommended anti-parasite treatment dosage (0.2 mg/kg bodyweight) through subcutaneous injection at different days post-infection in the treated IBRV infection groups, while non-treated infection group was used as the control. The infected rabbits showed hyperthermia and other clinical signs, but the number of high-fever rabbits in the ivermectin treatment groups was significantly lower than that in the non-treated infection group. Furthermore, in ivermectin treatment groups, the cumulative clinical scores correlated negatively with drug doses and positively with delay of administration time post-infection. The overall nasal shedding time in ivermectin-treated groups was two days shorter than the non-treated challenge group. At the same time point, the titer of neutralizing antibodies in the treatment group with triple doses was higher than the other two-dose groups, but the difference between the treatment groups decreased with the delay of drug administration. Correspondingly, the serious extent of lung lesions was negatively related to the dosage, but positively related to the delay of drug administration. The qPCR with tissue homogenates showed that the virus was present in both the lung tissues and trigeminals of the infected rabbits. In conclusion, ivermectin treatment had therapeutic effect by decreasing clinical signs and viral shedding, but could not stop virus proliferation in lung tissues and trigeminals.

Abstract ULK1 is crucial for initiating autophagosome formation and its activity is tightly regulated by post-translational modifications and protein-protein interactions. In the present study, we demonstrate that TMEM189 (Transmembrane protein 189), also known as plasmanylethanolamine desaturase 1 (PEDS1), negatively regulates the proteostasis of ULK1 and autophagy activity. In TMEM189-overexpressed cells, the formation of autophagesome is impaired, while TMEM189 knockdown increases cell autophagy. Further investigation reveals that TMEM189 interacts with and increases the instability of ULK1, as well as decreases its kinase activities. The TMEM189 N-terminal domain is required for the interaction with ULK1. Additionally, TMEM189 overexpression can disrupt the interaction between ULK1 and TRAF6, profoundly impairs K63-linked polyubiquitination of ULK1 and self-association, leading to the decrease of ULK1 stability. Moreover, in vitro and in vivo experiments suggest that TMEM189 deficiency results in the inhibition of tumorigenicity of gastric cancer. Our findings provide a new insight into the molecular regulation of autophagy and laboratory evidence for investigating the physiological and pathological roles of TMEM189.

Platelets are small, versatile blood cells that are critical for hemostasis/thrombosis. Local platelet accumulation is a known contributor to proinflammation in various disease states. However, the anti-inflammatory/immunosuppressive potential of platelets has been poorly explored. Here, we uncovered, unexpectedly, desialylated platelets (dPLTs) down-regulated immune responses against both platelet-associated and -independent antigen challenges. Utilizing multispectral photoacoustic tomography, we tracked dPLT trafficking to gut vasculature and an exclusive Kupffer cell-mediated dPLT clearance in the liver, a process that we identified to be synergistically dependent on platelet glycoprotein Ibα and hepatic Ashwell–Morell receptor. Mechanistically, Kupffer cell clearance of dPLT potentiated a systemic immunosuppressive state with increased anti-inflammatory cytokines and circulating CD4+ regulatory T cells, abolishable by Kupffer cell depletion. Last, in a clinically relevant model of hemophilia A, presensitization with dPLT attenuated anti-factor VIII antibody production after factor VIII ( infusion. As platelet desialylation commonly occurs in daily-aged and activated platelets, these findings open new avenues toward understanding immune homeostasis and potentiate the therapeutic potential of dPLT and engineered dPLT transfusions in controlling autoimmune and alloimmune diseases.

ContextThe malignant potential and molecular signature of atypical parathyroid adenoma (APA) remain elusive. Data from Asia are still lacking.Design and settingThis was a retrospective study on a large APA cohort in a single center from mainland China.MethodsA total of 320 patients with primary hyperparathyroidism (PHPT), containing 79 APA, 79 Parathyroid cancer (PC) and 162 benign lesions cases, were enrolled after surgery for collection of clinical data and genetic analysis.ResultsAPA patients showed earlier mean onset age than benign group (46.9 ± 17.1 vs. 52.0 ± 14.3 yrs). Less bone involvement and gastrointestinal symptoms were presented in APA compared to PC (35.4% vs. 62.0%, and 17.7% vs. 41.8%), while more urolithiasis was seen in APA than in benign lesions (57.0% vs. 29.6%). The APA group had moderate hypercalcemia (mean 3.02 ± 0.44mmol/L) with elevated serum PTH (median 593.0pg/ml) and proportion of hypercalcemic crisis as 22.8%, all higher than those of benign lesions but lower than those of PC group. The recurrence/no remission rate of the APA group was significantly lower than that of the PC and similar to the benign group (5.1% vs. 31.6% vs. 3.1%). Germline CDC73 mutation was the most common molecular abnormality in both PC and APA subjects. APA patients with nonsynonymous germline variants showed earlier onset age (28.5 ± 16.9 vs. 48.1 ± 17.7 yrs) and more cases developing no remission/recurrence (25.0% vs. 0.0%).ConclusionsPatients with APA presented clinical and biochemical characteristics much less severe than PC and resembling the benign neoplasms, with a relatively good prognosis. Germline gene variations were associated with earlier onset and probably more recurrence of PHPT in APA.

Bovine herpes virus -1 (BoHV-1) infection leads to upper respiratory tract infection, conjunctivitis and genital disorders in cattle. To control BoHV-1, it is important to understand the role of viral proteins in viral infection. BoHV-1 has several gene products to help in viral replication in infected cell. One such gene is deoxyuridine triphosphate nucleotidohydrolase (dUTPase) also known as UL50. In this study, we analyzed the amino acid sequence of UL50 (dUTPase) using bioinformatics tools and found that it was highly conserved among herpesvirus family. Then, it was cloned and expressed in Escherichia coli Rosetta (DE3), induced by isopropy1-b-D-thiogalactopyranoside (IPTG) and the recombinant UL50 protein was purified to immunize rabbits for the preparation of polyclonal antiserum. The results indicated that the UL50 gene of BoHV-1 was composed of 978 nucleotides, which encoded 323 amino acids. Western blot analysis revealed that polyclonal sera against UL50 reacted with a band of 34 kDa. Furthermore, immunofluorescence assay showed that UL50 localized in the cytoplasmic area. Taken together, UL50 was successfully cloned, expressed and detected in BoHV-1-infected cells and was localized in the cytoplasm to help in the replication of BoHV-1 in infected cells.

The development of coagulation factor VIII (FVIII) inhibitory antibodies is a serious complication in hemophilia A (HA) patients after FVIII replacement therapy. Inhibitors render regular prophylaxis ineffective and increase the risk of morbidity and mortality. Immune tolerance induction (ITI) regimens have become the only clinically proven therapy for eradicating these inhibitors. However, this is a lengthy and costly strategy. For HA patients with high titer inhibitors, bypassing or new hemostatic agents must be used in clinical prophylaxis due to the ineffective ITI regimens. Since multiple genetic and environmental factors are involved in the pathogenesis of inhibitor generation, understanding the mechanisms by which inhibitors develop could help identify critical targets that can be exploited to prevent or eradicate inhibitors. In this review, we provide a comprehensive overview of the recent advances related to mechanistic insights into anti-FVIII antibody development and discuss novel therapeutic approaches for HA patients with inhibitors.

Bovine viral diarrhea virus (BVDV) is an important livestock viral pathogen responsible for causing significant economic losses. The emerging and novel BVDV isolates are clinically and biologically important, as there are highly antigenic diverse and pathogenic differences among BVDV genotypes. However, no study has yet compared the virulence of predominant genotype isolates (BVDV-1a, 1b, and 1m) in China and the emerging genotype isolate BVDV-1v. The serological relationship among these genotypes has not yet been described. In this study, we isolated three BVDV isolates from calves with severe diarrhea, characterized as BVDV-1a, 1m, and novel 1v, based on multiple genomic regions [including 5-untranslated region (5′-UTR), Npro, and E2] and the phylogenetic analysis of nearly complete genomes. For the novel genotype, genetic variation analysis of the E2 protein of the BVDV-1v HB-03 strain indicates multiple amino acid mutation sites, including potential host cell-binding sites and neutralizing epitopes. Recombination analysis of the BVDV-1v HB-03 strain hinted at the possible occurrence of cross-genotypes (among 1m, 1o, and 1q) and cross-geographical region transmission events. To compare the pathogenic characters and virulence among these BVDV-1 genotypes, newborn calves uninfected with common pathogens were infected intranasally with BVDV isolates. The calves infected with the three genotype isolates show different symptom severities (diarrhea, fever, slowing weight gain, virus shedding, leukopenia, viremia, and immune-related tissue damage). In addition, these infected calves also showed bovine respiratory disease complexes (BRDCs), such as nasal discharge, coughing, abnormal breathing, and lung damage. Based on assessing different parameters, BVDV-1m HB-01 is identified as a highly virulent strain, and BVDV-1a HN-03 and BVDV-1v HB-03 are both identified as moderately virulent strains. Furthermore, the cross-neutralization test demonstrated the antigenic diversity among these Chinese genotypes (1a, 1m, and 1v). Our findings illustrated the genetic evolution characteristics of the emerging genotype and the pathogenic mechanism and antigenic diversity of different genotype strains, These findings also provided an excellent vaccine candidate strain and a suitable BVDV challenge strain for the comprehensive prevention and control of BVDV.

‘Zijuan’ (Camellia sinensis var. assamica), a somatic mutant with purple foliage and stem selected from the Yunnan Daye cultivar, has been well developed owing to abnormal pattern of anthocyanin accumulation. However, the genetic basis for the specific accumulation of phloem glycosides is not clear. Tea plants are self-incompatible, so parents with large differences in foliage color were used for crosses to investigate the genetic mechanism of anthocyanins. In this study, ‘Zijuan’ and green foliage cultivar ‘Fudingdabaicha’ (C. sinensis var. sinensis) were used as female and male parents, respectively, to generated F1 hybrid progenies with various anthocyanin contents. In order to decipher the genetic rules of anthocyanins accumulation, we performed widely targeted metabolic and transcriptomic profiling. The results showed that cyanidin-3-O-galactoside, delphinidin-3-O-galactoside and petunidin-3-O-galactoside were the major types of anthocyanins and factors directly led to the color variation between parents and F1 plants. Transcriptomic analyses suggested the significant up-regulation of anthocyanidin synthase gene (CsANS1) and CsAN1, a MYB family gene positively regulated the expression of CsANS1, in anthocyanin-rich tea plants. Furthermore, the deletion mutation of CsAN1 was found by cloning and alignment in anthocyanin-lacking cultivars. Taken together, the function deficiency of CsAN1 is predominantly responsible for the inability of anthocyanins accumulation, and this trait is heritable in progenies through hybridization. The present study elucidated the molecular basis of leaf purple trait formation in ‘zijuan’ and ‘Fudingdabaicha’ and their F1 plants, which helps to elucidate the genetic mechanism of leaf anthocyanin accumulation regulation in tea plants, and the results provide a research reference for the selection and breeding of high anthocyanin type tea varieties.

Mycoplasma bovis (M. bovis) is an important pathogen of the bovine respiratory disease complex, invading lower respiratory tracts and causing severe pneumonia. However, its molecular mechanism largely remains unknown. Host annexin A2 (ANXA2) is a calcium-dependent phospholipid-binding protein. The current study sought to determine whether ANXA2 could mediate M. bovis adhesion and invasion thereby affecting its induction of inflammatory response. ANXA2 expression was upregulated in M. bovis-infected bovine lung epithelial cells (EBL), and blocking ANXA2 with an anti-ANXA2 antibody reduced M. bovis adhesion to EBL. Compared with uninfected cells, more ANXA2 was translocated from the cytoplasm to the cell surface after M. bovis infection. Furthermore, RNA interference knockdown of ANXA2 expression in EBL cells resulted in a significant decrease in M. bovis invasion and F-actin polymerization. Next, the transcriptomic study of M. bovis-infected EBL cells with and without ANXA2 knockdown were performed. The data exhibited that ANXA2 knockdown EBL cells had 2487 differentially expressed genes (DEGs), with 1175 upregulated and 1312 downregulated compared to control. According to GO and KEGG analyses, 50 genes potentially linked to inflammatory responses, 23 involved in extracellular matrix (ECM) receptor interaction, and 48 associated with PI3K-AKT signal pathways were upregulated, while 38 mRNA binding genes, 16 mRNA 3′-UTR binding genes, and 34 RNA transport genes were downregulated. Furthermore, 19 genes with various change-folds were selected for qPCR verification, and the results agreed with the RNA-seq findings. Above all, the transcription of two chemokines (IL-8 and CXCL5) and a key bovine β-defensin TAP in IL-17 signaling pathway were significantly increased in ANXA2 knockdown cells. Moreover, ANXA2 knockdown or knockout could increase NF-κB and MAPK phosphorylation activity in response to M. bovis infection. Additionally, ANXA2 knockdown also significantly decreased the CD44 transcripts via exon V3 and V7 skipping after M. bovis infection. We concluded that M. bovis borrowed host ANXA2 to mediate its adhesion and invasion thereby negatively regulating molecular expression essential to IL-17 signal pathway. Furthermore, CD44 V3 and V7 isoforms might contribute to this ANXA2 meditated processes in M. bovis infected EBL cells. These findings revealed a new understanding of pathogenesis for M. bovis infection.

Mycoplasma bovis (M. bovis) is one of the major pathogens in the bovine respiratory disease complex, which includes pneumonia, mastitis, and arthritis and causes a great economic loss in the cattle industry. In China, a live-attenuated vaccine strain M. bovis P150 was obtained by a continuous culture of the wild-type strain M. bovis HB0801 (P1) in vitro for 150 passages. Using the infected bovine macrophage cell line BoMac, this work attempted to investigate the mechanism of P150 attenuation and protective immune response. To begin, we show that M. bovis P150 effectively triggered cytotoxicity and apoptosis in BoMac, although with lower intracellular survival than P1. The transcriptomes of BoMac after infection with M. bovis strains P1 and P150 were sequenced, and bioinformatic analysis identified 233 differentially expressed genes (DEGs), with 185 upregulated and 48 downregulated. Further Gene Ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analyses revealed that the majority of the DEGs were linked to CHOP complex, MAP kinase phosphatase activity and were involved in the IL-17 signaling pathway in immune response, MAPK signaling pathway in signal transduction, and p53 signaling pathway in cell growth and death. Among them, the level of C/EBP homologous protein (CHOP) was significantly upregulated in P150-infected BoMac compared to P1-infected cells at different time points, along with its upstream and downstream genes phosphorylated-PERK, phosphorylated-EIF2α, ATF4, and GADD45A increased in the PERK-dependent ER stress response. The role of CHOP in apoptosis was further verified by M. bovis-induced siCHOP knockdown in BoMac cells. The results showed that CHOP knockdown enhanced P150-induced apoptosis and dramatically increased the M. bovis P1 and P150 intracellular survival, particularly for P150. These data suggest that P150 infection upregulates CHOP expression, which can increase apoptosis and mediate a crosstalk between ER stress and apoptosis during infection, and hence, contribute to high cytotoxicity and low intracellular survival.

Thrombocytopenia is a multifactorial condition that frequently involves concomitant defects in platelet production and clearance. The physiopathology of low platelet count in thrombocytopenia remains unclear. Sialylation on platelet membrane glycoprotein and follicular helper T cells (TFHs) are thought to be the novel platelet clearance pathways. The aim of this study was to clarify the roles of platelet desialylation and circulating TFHs in patients with immune thrombocytopenia (ITP) and non-ITP thrombocytopenia. We enrolled 190 patients with ITP and 94 patients with non-ITP related thrombocytopenia including case of aplastic anemia (AA) and myelodysplastic syndromes (MDS). One hundred and ten healthy volunteers were included as controls. We found significantly increased desialylated platelets in patients with ITP or thrombocytopenia in the context of AA and MDS. Platelet desialylation was negatively correlated with platelet count. Meanwhile, the circulating TFH levels in patients with thrombocytopenia were significantly higher than those of normal controls, and were positively correlated with desialylated platelet levels. Moreover, TFHs-related chemokine CXCL13 and apoptotic platelet levels were abnormally high in ITP patients. The upregulation of pro-apoptotic proteins and the activation of the MAPK/mTOR pathway were observed in the same cohort. These findings suggested that platelet desialylation and circulating TFHs may become the potential biomarkers for evaluating the disease process associated with thrombocytopenia in patients with ITP and non-ITP.

Bovine rotavirus (BRV) causes massive economic losses in the livestock industry worldwide. Elucidating the pathogenesis of BRV would help in the development of more effective measures to control BRV infection. The MA-104 cell line is sensitive to BRV and is thereby a convenient tool for determining BRV–host interactions. Thus far, the role of the microRNAs (miRNAs) of MA-104 cells during BRV infection is still ambiguous. We performed Illumina RNA sequencing analysis of the miRNA libraries of BRV-infected and mock-infected MA-104 cells at different time points: at 0 h post-infection (hpi) (just after 90 min of adsorption) and at 6, 12, 24, 36, and 48 hpi. The total clean reads obtained from BRV-infected and uninfected cells were 74,701,041 and 74,184,124, respectively. Based on these, 579 were categorized as known miRNAs and 144 as novel miRNAs. One hundred and sixty differentially expressed (DE) miRNAs in BRV-infected cells in comparison with uninfected MA-104 cells were successfully investigated, 95 of which were upregulated and 65 were downregulated. The target messenger RNAs (mRNAs) of the DE miRNAs were examined by bioinformatics analysis. Functional annotation of the target genes with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) suggested that these genes mainly contributed to biological pathways, endocytosis, apoptotic process, trans-Golgi membrane, and lysosome. Pathways such as the mammalian target of rapamycin (mTOR) (mml-miR-486-3p and mml-miR-197-3p), nuclear factor kappa B (NF-κB) (mml-miR-204-3p and novel_366), Rap1 (mml-miR-127-3p), cAMP (mml-miR-106b-3p), mitogen-activated protein kinase (MAPK) (mml-miR-342-5p), T-cell receptor signaling (mml-miR-369-5p), RIG-I-like receptor signaling (mml-miR-504-5p), AMP-activated protein kinase (AMPK) (mml-miR-365-1-5p), and phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) signaling (mml-miR-299-3p) were enriched. Moreover, real-time quantitative PCR (qPCR) verified the expression profiles of 23 selected DE miRNAs, which were consistent with the results of deep sequencing, and the 28 corresponding target mRNAs were mainly of regulatory pathways of the cellular machinery and immune importance, according to the bioinformatics analysis. Our study is the first to report a novel approach that uncovers the impact of BRV infection on the miRNA expressions of MA-104 cells, and it offers clues for identifying potential candidates for antiviral or vaccine strategies.

Mastitis is one of the most common and significant infectious diseases in dairy cattle and is responsible for significant financial losses for the dairy industry globally. An important pathogen of bovine mastitis, Mycoplasma bovis (M. bovis) has a high infection rate, requires a long course of treatment, and is difficult to cure. Bovine mammary epithelial cells (BMECs) are the first line of defense of the mammary gland, and their natural immune system plays a critical role in resisting M. bovis infection. This study aimed to explore and demonstrate the regularity of Toll-like receptors (TLRs) activation during M. bovis infection and their function during M. bovis mastitis. An in vitro model of M. bovis-induced mastitis showed that the expression of IL-6, IL-8, and TNF-α increased significantly following infection. M. bovis infection also upregulated the expression of TLR1/2/6 on the cell membrane and TLR3/9 in the cytoplasm. There is a crosstalk effect between TLR1–TLR2 and TLR2–TLR6. Furthermore, M. bovis infection was found to activate the TLR1/2/6/9/MyD88/NF-κB and TLR3/TRIF/IRF signal transduction pathways, which in turn activate inflammatory factors. These findings lay the theoretical foundation for understanding the pathogenesis of M. bovis, permitting the development of effective measures for preventing and controlling M. bovis mastitis.

Mastitis is a common disease that hinders the development of dairy industry and animal husbandry. It leads to the abuse of antibiotics and the emergence of super drug-resistant bacteria, and poses a great threat to human food health and safety. Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) are the most common pathogens of mastitis in dairy cows and usually cause subclinical or clinical mastitis. CircRNAs and N6-methyladenosine (m6A) play important roles in immunological diseases. However, the mechanisms by which m6A modifies circRNA in bovine mammary epithelial cells remain poorly understood. The aim of our study was to investigate m6A-modified circRNAs in bovine mammary epithelial cells (MAC-T cells) injured by S. aureus and E. coli. The profile of m6A-modified circRNA showed a total of 1,599 m6A peaks within 1,035 circRNAs in the control group, 35 peaks within 32 circRNAs in the S. aureus group, and 1,016 peaks within 728 circRNAs in the E. coli group. Compared with the control group, 67 peaks within 63 circRNAs were significantly different in the S. aureus group, and 192 peaks within 137 circRNAs were significantly different in the E. coli group. Furthermore, we found the source genes of these differentially m6A-modified circRNAs in the S. aureus and E. coli groups with similar functions according to GO and KEGG analyses, which were mainly associated with cell injury, such as inflammation, apoptosis, and autophagy. CircRNA–miRNA–mRNA interaction networks predicted the potential circRNA regulation mechanism in S. aureus- and E. coli-induced cell injury. We found that the mRNAs in the networks, such as BCL2, MIF, and TNFAIP8L2, greatly participated in the MAPK, WNT, and inflammation pathways. This is the first report on m6A-modified circRNA regulation of cells under S. aureus and E. coli treatment, and sheds new light on potential mechanisms and targets from the perspective of epigenetic modification in mastitis and other inflammatory diseases.

Abstract Immune thrombocytopenia (ITP) is an autoimmune disease characterized by immune-mediated destruction of one’s own platelets. The progression of thrombocytopenia involves an imbalance of platelet production and clearance. B cells can induce autoantibodies, and T cells contribute to the pathological progression as well. Some patients with ITP have a poor response to common first-line therapies. Recent studies have shown that a novel Fc-independent platelet clearance pathway is associated with poor prognosis in these patients. By this pathway, desialylated platelets can be cleared by Ashwell-Morell receptor (AMR) on hepatocytes. Research has demonstrated that patients with refractory ITP usually have a high level of desialylation, indicating the important role of sialylation on platelet membrane glycoprotein (GP) in patients with primary immune thrombocytopenia, and neuraminidase 1(NEU1) translocation might be involved in this process. Patients with ITP who are positive for anti-GPIbα antibodies have a poor prognosis, which indicates that anti-GPIbα antibodies are associated with this Fc-independent platelet clearance pathway. Experiments have proven that these antibodies could lead to the desialylation of GPs on platelets. The T follicular helper (TFH) cell level is related to the expression of the anti-GPIbα antibody, which indicates its role in the progression of desialylation. This review will discuss platelet clearance and production, especially the role of the anti-GPIbα antibody and desialylation in the pathophysiology of ITP and therapy for this disease.

Golden snub-nosed monkeys (Rhinopithecus roxellanae) belong to Class A, the highest level of endangered primate species. Exploring the infection status of potential pathogens in golden snub-nosed monkeys is important for controlling associated diseases and protecting this species. The objective of this study was to investigate the seroprevalence for a number of potential pathogens and the prevalence of fecal adenovirus and rotavirus. A total of 283 fecal samples were collected from 100 golden snub-nosed monkeys in December 2014, June 2015, and January 2016; 26 blood samples were collected from 26 monkeys in June 2014, June 2015, January 2016 and November 2016 at Shennongjia National Reserve in Hubei, China. The infection of 11 potential viral diseases was examined serologically using an Indirect Enzyme-linked Immunosorbent Assay (iELISA) and Dot Immunobinding Assays (DIA), while the whole blood IFN-γ in vitro release assay was used to test tuberculosis (TB). In addition, fecal Adenovirus and Rotavirus were detected using Polymerase Chain Reaction (PCR). As a result, the Macacine herpesvirus-1 (MaHV-1), Golden snub-nosed monkey cytomegalovirus (GsmCMV), Simian foamy virus (SFV) and Hepatitis A virus (HAV) were detected with the seroprevalence of 57.7% (95% CI: 36.9, 76.6), 38.5% (95% CI: 20.2, 59.4), 26.9% (95% CI: 11.6, 47.8), and 7.7% (95% CI: 0.0, 84.2), respectively. Two fecal samples tested positive for Adenovirus (ADV) by PCR, with a prevalence of 0.7% (95% CI: 0.2, 2.5), and further, the amplification products were sequenced. Phylogenetic analysis revealed that they belonged to the HADV-G group. However, other pathogens, such as Coxsackievirus (CV), Measles virus (MeV), Rotavirus (RV), Simian immunodeficiency virus (SIV), Simian type D retroviruses (SRV), Simian-T-cell lymphotropic virus type 1 (STLV-1), Simian varicella virus (SVV), Simian virus 40 (SV40) and Mycobacterium tuberculosis complex (TB) were negative in all samples. In addition, a risk factor analysis indicated that the seroprevalence of MaHV-1 infection was significantly associated with old age (≥4 years). These results have important implications for understanding the health status and conservation of the endangered golden snub-nosed monkey population at Shennongjia Nature Reserve.

Mycobacterium tuberculosis (M. tb) is the causative agent of tuberculosis (TB) that leads to millions of deaths each year. Extensive evidence has explored the involvement of microRNAs (miRNAs) in M. tb infection. Limitedly, the concrete function of microRNA-100-5p (miR-100-5p) in M. tb remains unexplored and largely elusive. In this study, using Bacillus Calmette–Guérin (BCG) as the model strain, we validated that miR-100-5p was significantly decreased in BCG-infected THP-1 cells. miR-100-5p inhibition effectively facilitated the apoptosis of infected THP-1 cells and reduced BCG survival by regulating the phosphatidylinositol 3-kinase/AKT pathway. Further, SMARCA5 was the target of miR-100-5p and reduced after miR-100-5p overexpression. Since BCG infection down-regulated miR-100-5p in THP-1 cells, the SMARCA5 expression was up-regulated, which in turn increased apoptosis through caspase-3 and Bcl-2 and, thereby, reducing BCG intracellular survival. Collectively, the study uncovered a new molecular mechanism of macrophage to suppress mycobacterial infection through miR-100-5p and SMARCA5 pathway.

Diabetes and its related metabolic disorders have been reported as the leading comorbidities in patients with coronavirus disease 2019 (COVID-19). This clinical study aims to investigate the clinical features, radiographic and laboratory tests, complications, treatments, and clinical outcomes in COVID-19 patients with or without diabetes. This retrospective study included 208 hospitalized patients (≥ 45 years old) with laboratory-confirmed COVID-19 during the period between 12 January and 25 March 2020. Information from the medical record, including clinical features, radiographic and laboratory tests, complications, treatments, and clinical outcomes, were extracted for the analysis. 96 (46.2%) patients had comorbidity with type 2 diabetes. In COVID-19 patients with type 2 diabetes, the coexistence of hypertension (58.3% vs 31.2%), coronary heart disease (17.1% vs 8.0%), and chronic kidney diseases (6.2% vs 0%) was significantly higher than in COVID-19 patients without type 2 diabetes. The frequency and degree of abnormalities in computed tomography (CT) chest scans in COVID-19 patients with type 2 diabetes were markedly increased, including ground-glass opacity (85.6% vs 64.9%, P

Tuberculosis is the second cause in infectious diseases leading to human death. Understanding the virulence mechanism is inevitable if the disease needs to be fully cured. Therefore, this study aimed to reveal this mechanism by comparing proteomic profiles of intracellular and extracellular virulent strain M.tb and bacille Calmette–Guérin (BCG) from infected THP-1cells. First, M.tb and BCG infected THP-1 at MOI 10:1. Twelve hours postinfection, intracellular bacteria of M.tb and BCG were collected, whereas the two bacilli cultured in 7H9 broth media were used as the control. Then four groups of bacilli were subjected to proteomic analysis, and differential proteomic profiles between M.tb and BCG were comparatively analyzed with bioinformatics tools. As a result, we identified a total of 1,557 proteins. Further, they were divided into four groups for comparison of M.tb versus BCG under 7H9 culture (shorten as out), M.tb in (intracellular) versus M.tb out, BCG in versus BCG out and M.tb in versus BCG in. Between M.tb in versus BCG in, a total of 211 differentially expressed proteins were found. Eight proteins like ESAT-6 distributed in six RDs and some known proteins related to virulence. Besides, five uncharacterized proteins were differentially expressed. Further analysis revealed enriched pathways were associated with glyoxylate and dicarboxylate metabolism pathways. In M.tb out versus BCG out, a total of 144 differential proteins were identified and mainly involved in metabolism pathways. Then, 121 differential proteins in the group of M.tb in versus M.tb out were enriched in ribosome and oxidative phosphorylation related to adaptation to the host environment. The group of BCG in versus BCG out shared the same trend of different pathways to the M.tb in versus M.tb out. Finally, 42 proteins were identified to be up-regulated only in intracellular M.tb including eight RD proteins, whereas 22 up-regulated uniquely in intracellular BCG. Besides, only two proteins (Pks13 and Rv1405c) were commonly up-regulated in intracellular M.tb and BCG. Further, some unknown proteins were uniquely up-regulated in the intracellular M.tb and BCG. These findings provide valuable data for further exploration of molecular mechanism for M.tb virulence and BCG immune response.