Search

Abstract Background Cystic echinococcosis (CE) is a life-threatening zoonotic disease caused by the larval stage of Echinococcus granulosus sensu lato, which employs various strategies to evade the host immune system for survival. Recent advances have revealed the role of annexins as excretory/secretory products, providing new insights into the immune regulation by these proteins in the pathogenesis of CE. Methods Echinococcus granulosus annexin B proteins EgANXB2, EgANXB18, EgANXB20, and EgANXB23 were cloned, expressed, and analyzed using bioinformatic tools. Membrane binding analysis was used to assess their bioactivity, while their immunoreactivity and tissue distribution characteristics were determined experimentally using western blotting and immunofluorescence staining, respectively. Furthermore, quantitative real-time reverse transcription PCR (qRT-PCR) was used to analyze the mRNA expression profiles of EgANXBs in different developmental stages of E. granulosus. Finally, immunofluorescence staining, cell counting kit 8 assays, flow cytometry, transwell migration assays, and qRT-PCR were used to evaluate the functional effects of rEgANXB18 and rEgANXB20 on mouse peripheral blood mononuclear cells (PBMCs). Results In this study, we identified four EgANXBs with conserved protein structures and calcium-dependent phospholipid binding activities. rEgANXBs were recognized by serum from sheep infected with E. granulosus and distributed in the germinal layer of fertile cysts. Interestingly, transcription levels of the four EgANXBs were significantly higher in protoscoleces than in 28-day strobilated worms. Moreover, we demonstrated that rEgANXB18 and rEgANXB20 were secretory proteins that could bind to PBMCs and regulate their function. Specifically, rEgANXB18 inhibited cell proliferation and migration while promoting cell apoptosis, NO production, and cytokine profile shifting. In contrast, rEgANXB20 showed limited effects on apoptosis but inhibited NO production. Conclusions Our findings suggested that among the four identified EgANXBs, EgANXB2 and EgANXB23 might play a pivotal role for the development of protoscoleces, while EgANXB18 and EgANXB20, as secretory proteins, appeared to participate in the host-parasite interaction by regulating the function of immune cells. Graphical Abstract

BackgroundCystic echinococcosis (CE) is caused by the infection of Echinococcus granulosus sensu lato (E. granulosus s.l.), one of the most harmful zoonotic helminths worldwide. Infected dogs are the major source of CE transmission. While praziquantel-based deworming is a main measure employed to control dog infections, its efficacy is at times compromised by the persistent high rate of dog re-infection and the copious discharge of E. granulosus eggs into the environment. Therefore, the dog vaccine is a welcome development, as it offers a substantial reduction in the biomass of E. granulosus. This study aimed to use previous insights into E. granulosus functional genes to further assess the protective efficacy of six recombinant proteins in dogs using a two-time injection vaccination strategy.MethodsWe expressed and combined recombinant E. granulosus triosephosphate isomerase (rEgTIM) with annexin B3 (rEgANXB3), adenylate kinase 1 (rEgADK1) with Echinococcus protoscolex calcium binding protein 1 (rEgEPC1), and fatty acid-binding protein (rEgFABP) with paramyosin (rEgA31). Beagle dogs received two subcutaneous vaccinations mixed with Quil-A adjuvant, and subsequently orally challenged with protoscoleces two weeks after booster vaccination. All dogs were sacrificed for counting and measuring E. granulosus tapeworms at 28 days post-infection, and the level of serum IgG was detected by ELISA.ResultsDogs vaccinated with rEgTIM&rEgANXB3, rEgADK1&rEgEPC1, and rEgFABP-EgA31 protein groups exhibited significant protectiveness, with a worm reduction rate of 71%, 57%, and 67%, respectively, compared to the control group (P < 0.05). Additionally, the vaccinated groups exhibited an inhibition of worm growth, as evidenced by a reduction in body length and width (P < 0.05). Furthermore, the level of IgG in the vaccinated dogs was significantly higher than that of the control dogs (P < 0.05).ConclusionThese verified candidates may be promising vaccines for the prevention of E. granulosus infection in dogs following two injections. The rEgTIM&rEgANXB3 co-administrated vaccine underscored the potential for the highest protective efficacy and superior protection stability for controlling E. granulosus infections in dogs.

Objective: To propose a nerve stereoscopic reconstruction technique based on ultrasound imaging for site diagnosis, intuitive reflection of disease severity, and classification of neuralgic amyotrophy (NA).Methods: We enrolled 44 patients with NA who underwent high-frequency ultrasonography examination. Multiple sites on the normal side and the affected side were scanned to calculate the ratio of the cross-section area (CSA) of the affected side to the normal side at each location measured, i.e., the cross-section area swelling ratio (CSASR). The CSASR of 44 patients and 30 normal controls was analyzed to determine their threshold value for the diagnosis of NA. Then, ultrasound images of the cross-section were used to reconstruct the stereoscopic model of the nerve on the affected side and the normal side. Using the CSASR values in each measurement location, a CSASR stereoscopic model was developed.Results: The threshold value of CSASR for ultrasound diagnosis of NA was 1.55. The average diseased segments per patient was 2.49 ± 1.97, with an average overall length of 10.03 ± 7.95 cm. Nerve stereoscopic reconstruction could be conducted for swelling, torsion, incomplete constriction, and complete constriction.Conclusion: The ultrasound image reconstruction method proposed in this study can accurately determine the site, range, and type of neuropathies in patients with NA, and simultaneously provide complete and accurate data information and intuitive morphological information.

African swine fever (ASF) is an infectious disease caused by African swine fever virus (ASFV) that is highly contagious and has an extremely high mortality rate (infected by virulent strains) among domestic and wild pigs, causing huge economic losses to the pig industry globally. In this study, SDS−PAGE gel bands hybridized with ASFV whole virus protein combined with ASFV-convalescent and ASFV-positive pig serum were identified by mass spectrometry. Six antigens were detected by positive serum reaction bands, and eight antigens were detected in ASFV-convalescent serum. In combination with previous literature reports and proteins corresponding to MHC-II presenting peptides screened from ASFV-positive pig urine conducted in our lab, seven candidate antigens, including KP177R (p22), K78R (p10), CP204L (p30), E183L (p54), B602L (B602L), EP402R-N (CD2V-N) and F317L (F317L), were selected. Subunit-Group 1 was prepared by mixing above-mentioned seven ASFV recombinant proteins with MONTANIDETM1313 VG N mucosal adjuvant and immunizing pigs intranasally and intramuscularly. Subunit-Group 2 was prepared by mixing four ASFV recombinant proteins (p22, p54, CD2V-N1, B602L) with Montanide ISA 51 VG adjuvant and immunizing pigs by intramuscular injection. Anticoagulated whole blood, serum, and oral fluid were collected during immunization for flow cytometry, serum IgG as well as secretory sIgA antibody secretion, and cytokine expression testing to conduct a comprehensive immunogenicity assessment. Both immunogen groups can effectively stimulate the host to produce ideal humoral, mucosal, and cellular immune responses, providing a theoretical basis for subsequent functional studies, such as immunogens challenge protection and elucidation of the pathogenic mechanism of ASFV.

Abstract Mycoplasma hyopneumoniae, the causative agent of swine respiratory disease, demonstrates differences in virulence. However, factors associated with this variation remain unknown. We herein evaluated the association between differences in virulence and genotypes as well as phenotype (i.e., biofilm formation ability). Strains 168 L, RM48, XLW-2, and J show low virulence and strains 232, 7448, 7422, 168, NJ, and LH show high virulence, as determined through animal challenge experiments, complemented with in vitro tracheal mucosa infection tests. These 10 strains with known virulence were then subjected to classification via multilocus sequence typing (MLST) with three housekeeping genes, P146-based genotyping, and multilocus variable-number tandem-repeat analysis (MLVA) of 13 loci. MLST and P146-based genotyping identified 168, 168 L, NJ, and RM48 as the same type and clustered them in a single branch. MLVA assigned a different sequence type to each strain. Simpson’s index of diversity indicates a higher discriminatory ability for MLVA. However, no statistically significant correlation was found between genotypes and virulence. Furthermore, we investigated the correlation between virulence and biofilm formation ability. The strains showing high virulence demonstrate strong biofilm formation ability, while attenuated strains show low biofilm formation ability. Pearson correlation analysis revealed a significant positive correlation between biofilm formation ability and virulence. To conclude, there was no association between virulence and our genotyping data, but virulence was found to be significantly associated with the biofilm formation ability of M. hyopneumoniae.

Abstract Background Mycoplasma hyopneumoniae (M. hyopneumoniae) is the etiological agent of enzootic pneumonia, a highly infectious swine respiratory disease that distributed worldwide. The pathogenesis and virulence factors of M. hyopneumoniae are not fully clarified. As an important virulence factor of bacteria, nicotinamide adenine dinucleotide (NADH) oxidase (NOX) participates in host-pathogen interaction, however, the function of NOX involved in the pathogenesis of M. hyopneumoniae is not clear. Results In this study, significant differences in NOX transcription expression levels among different strains of M. hyopneumoniae differed in virulence were identified, suggesting that NOX may be correlated with M. hyopneumoniae virulence. The nox gene of M. hyopneumoniae was cloned and expressed in Escherichia coli, and polyclonal antibodies against recombinant NOX (rNOX) were prepared. We confirmed the enzymatic activity of rNOX based on its capacity to oxidize NADH to NAD+. Flow cytometry analysis demonstrated the surface localization of NOX, and subcellular localization analysis further demonstrated that NOX exists in both the cytoplasm and cell membrane. rNOX was depicted to mediate adhesion to immortalized porcine bronchial epithelial cells (hTERT-PBECs). Pre-neutralizing M. hyopneumoniae with anti-rNOX antibody resulted in a more than 55% reduction in the adhesion rate of high- and low-virulence M. hyopneumoniae strains to hTERT-PBECs. Moreover, a significant difference appeared in the decline in CCU50 titer between virulent (168) and virulence-attenuated (168L) strains. NOX not only recognized and interacted with host fibronectin but also induced cellular oxidative stress and apoptosis in hTERT-PBECs. The release of lactate dehydrogenase by NOX in hTERT-PBECs was positively correlated with the virulence of M. hyopneumoniae strains. Conclusions NOX is considered to be a potential virulence factor of M. hyopneumoniae and may play a significant role in mediating its pathogenesis.

Abstract Mycoplasma hyorhinis may cause systemic inflammation of pigs, typically polyserositis and arthritis, and is also associated with several types of human cancer. However, the pathogenesis of M. hyorhinis colonizing and breaching the respiratory barrier to establish systemic infection is poorly understood. Glycolytic enzymes are important moonlighting proteins and virulence-related factors in various bacteria. In this study, we investigated the functions of a glycolytic critical enzyme, enolase in the infection and systemic spread of M. hyorhinis. Bacterial surface localization of enolase was confirmed by flow cytometry and colony hybridization assay. Recombinant M. hyorhinis enolase (rEno) was found to adhere to pig kidney (PK-15) cells, and anti-rEno serum significantly decreased adherence. The enzyme was also found to bind host plasminogen and fibronectin, and interactions were specific and strong, with dissociation constant (K D) values of 1.4 nM and 14.3 nM, respectively, from surface plasmon resonance analysis. Activation of rEno-bound plasminogen was confirmed by its ability to hydrolyze plasmin-specific substrates and to degrade a reconstituted extracellular matrix. To explore key sites during these interactions, C-terminal lysine residues of enolase were replaced with leucine, and the resulting single-site and double-site mutants show significantly reduced interaction with plasminogen in far-Western blotting and surface plasmon resonance tests. The binding affinities of all mutants to fibronectin were reduced as well. Collectively, these results imply that enolase moonlights as an important adhesin of M. hyorhinis, and interacts with plasminogen and fibronectin. The two lysine residues in the C-terminus are important binding sites for its multiple binding activities.

Mycoplasma hyorhinis (M. hyorhinis) is responsible for infections in the swine population. Such infections are usually cured by using antimicrobials and lead to develop resistance. Until now, there has been no effective vaccine to eradicate the disease. This study used conserved domains found in seven members of the variable lipoprotein (VlpA-G) family in order to design a multi-epitope candidate vaccine (MEV) against M. hyorhinis. The immunoinformatics approach was followed to predict epitopes, and a vaccine construct consisting of an adjuvant, two B cell epitopes, two HTL epitopes, and one CTL epitope was designed. The suitability of the vaccine construct was identified by its non-allergen, non-toxic, and antigenic nature. A molecular dynamic simulation was executed to assess the stability of the TLR2 docked structure. An immune simulation showed a high immune response toward the antigen. The protein sequence was reverse-translated, and codons were optimized to gain a high expression level in E. coli. The proposed vaccine construct may be a candidate for a multi-epitope vaccine. Experimental validation is required in future to test the safety and efficacy of the hypothetical candidate vaccine.

Proteomics is playing an increasingly important role in identifying pathogens, emerging and re-emerging infectious agents, understanding pathogenesis, and diagnosis of diseases. Recently, more advanced and sophisticated proteomics technologies have transformed disease diagnostics and vaccines development. The detection of pathogens is made possible by more accurate and time-constrained technologies, resulting in an early diagnosis. More detailed and comprehensive information regarding the proteome of any noxious agent is made possible by combining mass spectrometry with various gel-based or short-gun proteomics approaches recently. MALDI-ToF has been proved quite useful in identifying and distinguishing bacterial pathogens. Other quantitative approaches are doing their best to investigate bacterial virulent factors, diagnostic markers and vaccine candidates. Proteomics is also helping in the identification of secreted proteins and their virulence-related functions. This review aims to highlight the role of cutting-edge proteomics approaches in better understanding the functional genomics of pathogens. This also underlines the limitations of proteomics in bacterial secretome research.

PurposesThis study aimed to establish a predictive model of microvascular invasion (MVI) in hepatocellular carcinoma (HCC) by contrast-enhanced computed tomography (CT), which relied on a combination of machine learning approach and imaging features covering Liver Imaging and Reporting and Data System (LI-RADS) features.MethodsThe retrospective study included 279 patients with surgery who underwent preoperative enhanced CT. They were randomly allocated to training set, validation set, and test set (167 patients vs. 56 patients vs. 56 patients, respectively). Significant imaging findings for predicting MVI were identified through the Least Absolute Shrinkage and Selection Operator (LASSO) logistic regression method. Predictive models were performed by machine learning algorithm, support vector machine (SVM), in the training set and validation set, and evaluated in the test set. Further, a combined model adding clinical findings to the radiologic model was developed. Based on the LI-RADS category, subgroup analyses were conducted.ResultsWe included 116 patients with MVI which were diagnosed through pathological confirmation. Six imaging features were selected about MVI prediction: four LI-RADS features (corona enhancement, enhancing capsule, non-rim aterial phase hyperehancement, tumor size) and two non-LI-RADS features (internal arteries, non-smooth tumor margin). The radiological feature with the best accuracy was corona enhancement followed by internal arteries and tumor size. The accuracies of the radiological model and combined model were 0.725–0.714 and 0.802–0.732 in the training set, validation set, and test set, respectively. In the LR-4/5 subgroup, a sensitivity of 100% and an NPV of 100% were obtained by the high-sensitivity threshold. A specificity of 100% and a PPV of 100% were acquired through the high specificity threshold in the LR-M subgroup.ConclusionA combination of LI-RADS features and non-LI-RADS features and serum alpha-fetoprotein value could be applied as a preoperative biomarker for predicting MVI by the machine learning approach. Furthermore, its good performance in the subgroup by LI-RADS category may help optimize the management of HCC patients.

The novel variant IBDV (nVarIBDV, especially genotype A2dB1) mainly affects broilers in China. It causes an infection characterized by the atrophy of the bursa, a decrease in the level of lymphocytes, proliferation of fibrous tissue around the follicle, and severe atrophy of the follicle in the bursa. Poultry vaccinated with live IBDV vaccines do not have the challenge present with bursa atrophy, which is misdiagnosed for nVarIBDV because of the lack of other gross clinical symptoms. The present study sought to explore the potential and reliability of the real-time TaqMan analysis method for the detection and discrimination of the nVarIBDV genotype from that of the non-nVarIBDV, especially in live vaccine strains. This method will help monitor vaccinated poultry to control and manage infection with the nVarIBDV IBDVs. The nucleotide polymorphism in the 5′-UTR region and the vp5/vp2 overlapping region of the segment A sequences of IBDV were used to establish a one-step real-time TaqMan reverse transcription polymerase chain reaction (RT-PCR) method in this study. The results showed that the method accurately distinguished the nVarIBDV and non-nVarIBDV strains (especially live vaccine strains), and there were no cross-reactions with the infectious bronchitis virus (IBV), Newcastle disease virus (NDV), avian influenza virus (AIV), infectious laryngotracheitis virus (ILTV), fowlpox virus (FPV), Mycoplasma gallisepticum (M. gallisepticum), Mycoplasma synoviae (M. synoviae), and IBDV-negative field samples. The method showed a linear dynamic range between 102 and 107 DNA copies/reaction, with an average R2 of 0.99 and an efficiency of 93% for nVarIBDV and an average R2 of 1.00 and an efficiency of 94% for non-nVarIBDV. The method was also used for the detection of 84 clinical bursae of chickens vaccinated with the live vaccine. The results showed that this method accurately distinguished the nVarIBDV and non-nVarIBDV strains (vaccine strains), compared with a strategy based on the sequence analysis of HVRs at the vp2 gene or the reverse transcription PCR (RT-PCR) for the vp5 gene. These findings showed that this one-step real-time TaqMan RT-PCR method provides a rapid, sensitive, specific, and simple approach for detection of infections caused by nVarIBDV and is a useful clinical diagnostic tool for identifying and distinguishing nVarIBDV from non-nVarIBDV, especially live vaccine strains.

Abstract Mycoplasma hyorhinis infects pigs causing polyserositis and polyarthritis, and has also been reported in a variety of human tumor tissues. The occurrence of disease is often linked with the systemic invasion of the pathogen. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH), one of the key enzymes of glycolysis, was reported as a surface multifunctional molecule in several bacteria. Here, we investigated whether GAPDH could manifest binary functions; as an adhesin to promote colonization as well as a plasminogen receptor functioning in extracellular matrix (ECM) degradation to promote systemic invasion. The surface localization of GAPDH was observed in M. hyorhinis with flow cytometry and colony blot analysis. Recombinant GAPDH (rGAPDH) was found to be able to bind porcine-derived PK-15 and human-derived NCI-H292 cells. The incubation with anti-GAPDH antibody significantly decreased the adherence of M. hyorhinis to both cell lines. To investigate its function in recruiting plasminogen, firstly, the interaction between rGAPDH and plasminogen was demonstrated by ELISA and Far-Western blot assay. The activation of the rGAPDH-bound plasminogen into plasmin was proved by using a chromogenic substrate, and furtherly confirmed to degrade extracellular matrix by using a reconstituted ECM. Finally, the ability of rGAPDH to bind different ECM components was demonstrated, including fibronectin, laminin, collagen type IV and vitronectin. Collectively, our data imply GAPDH as an important adhesion factor of M. hyrohinis and a receptor for hijacking host plasminogen to degrade ECM. The multifunction of GAPDH to bind both plasminogen and ECM components is believed to increase the targeting of proteolysis and facilitate the dissemination of M. hyorhinis.

Magnetic particle imaging (MPI) is a novel emerging noninvasive and radiation-free imaging modality that can quantify superparamagnetic iron oxide nanoparticles tracers. The zero endogenous tissue background signal and short image scanning times ensure high spatial and temporal resolution of MPI. In the context of precision medicine, the advantages of MPI provide a new strategy for the integration of the diagnosis and treatment of diseases. In this review, after a brief explanation of the simplified theory and imaging system, we focus on recent advances in the biomedical application of MPI, including vascular structure and perfusion imaging, cancer imaging, the MPI guidance of magnetic fluid hyperthermia, the visual monitoring of cell and drug treatments, and intraoperative navigation. We finally optimize MPI in terms of the system and tracers, and present future potential biomedical applications of MPI.

ABSTRACT Mycoplasma hyorhinis is a widespread pathogen in pig farms worldwide. Although the majority of M. hyorhinis-colonized pigs have no apparent clinical disease, the pathogen can induce diseases such as polyserositis, arthritis, and eustachitis in some cases. To explore the mechanisms for the occurrence of these diseases, we challenged 4 groups of Bama miniature pigs with M. hyorhinis isolated from pigs without clinical symptoms (non-clinical origin [NCO] strain) or with typical clinical symptoms (clinical origin [CO] strain) and investigated the impacts of different strains and inoculation routes (intranasal [IN], intravenous [IV] + intraperitoneal [IP], and IV+IP+IN) on disease induction. Another group of pigs was set as a negative control. Pigs inoculated with the CO strain through a combined intravenous and intraperitoneal (IV+IP) route showed a significant decrease in average daily weight gain (ADWG), serious joint swelling, and lameness compared with the pigs in the negative-control group. Furthermore, this group developed moderate-to-severe pericarditis, pleuritis, peritonitis, and arthritis, as well as high levels of IgG and IgM antibodies. Pigs inoculated IV+IP with the NCO strain developed less marked clinical, pathological changes and a weaker specific antibody response compared with the pigs inoculated with the CO strain. The challenging results of the NCO strain via different routes (IV+IP, IV+IP+IN, and IN) indicated that the combined route (IV+IP) induced the most serious disease compared to the other inoculation routes. Intranasal inoculation induced a smaller decrease in ADWG without obvious polyserositis or arthritis. These data suggest that differences in both strain virulence and inoculation route affect the consequences of M. hyorhinis infection. IMPORTANCE Mycoplasma hyorhinis is a widespread pathogen in pig farms worldwide. The mechanisms or conditions that lead to the occurrence of disease in M. hyorhinis-infected pigs are still unknown. The objective of this study was to evaluate the impact of differences in the virulence of strain and the inoculation route on the consequences of M. hyorhinis infection.

Infrared dim small target detection is one of the important contents in the research of military applications such as remote sensing intelligence reconnaissance, long-range precision strike, aerospace offense–defense confrontation, etc. In this article, we focus on the detection of low-flying fixed-wing unmanned aerial vehicle target based on infrared imaging. To this end, we propose a simple and effective detection model, which can be viewed as a combination of low-rank approximation and multiple sparse constraints. We first model the infrared image that to be detected as a sum of three patch matrices called background, target, and noise. Then, we put a nonconvex low-rank approximation on the background patch matrix to suppress the background edges and put a reweighted $l_{1,1}$-norm constraint on the target matrix to better preserve the dim small target. Moreover, in order to eliminate the strong residual edges left in the target image under complex background, both the $l_{1,1}$ matrix norm and the $l_{2,1}$ matrix norm are used to constrain the noise patch. Finally, we develop an alternating optimization algorithm to solve the associated minimization problem. Extensive experiments carried out on a recently released real low-flying UAV database show that the proposed approach works well in detecting infrared dim small target measured by qualitative analysis and quantitative analysis.

Mycoplasmas persist in the host for a long time, suggesting that they possess mechanisms for immune evasion. Factor H is a negative regulator of the complement system, which binds to host cells to avoid unexpected complement activation. In this study, we revealed that many mycoplasmas, such as Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, Mycoplasma hyosynoviae, Mycoplasma gallisepticum, Mycoplasma pneumoniae, Mycoplasma genitalium, Mycoplasma flocculare, and Mycoplasma bovis could hijack factor H such that they present themselves as a host tissue and thus escape from complement attack. Furthermore, the mechanism of recruiting factor H was identified in M. hyopneumoniae. M. hyopneumoniae binds factor H via factor H binding proteins, such as elongation factor thermo unstable (EF-Tu), P146, pyruvate dehydrogenase (acetyl-transferring) E1 component subunit alpha (PdhA), P46, Pyruvate dehydrogenase E1 component subunit beta (PdhB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and three different hypothetical proteins. The binding of factor H by EF-Tu further contributes to decreased C3 deposition on the M. hyopneumoniae surface and ultimately blocks further complement activation. In fact, binding of factor H occurs in a multifactorial manner; factor H is not only exploited by M. hyopneumoniae via its regulator activity to help mycoplasmas escape from complement killing, but also increases M. hyopneumoniae adhesion to swine tracheal epithelial cells, partially through EF-Tu. Meanwhile, the high sequence identity among EF-Tu proteins in the above-mentioned mycoplasmas implied the universality of the mechanism. This is the first report that mycoplasmas can escape complement killing by binding to factor H.

Abstract Esophageal cancer is one of the leading causes of cancer‐related deaths worldwide. FAM225A is a novel lncRNA, only has been explored in nasopharyngeal carcinoma tumorigenesis. This study aims to investigate the regulatory mechanism of FAM225A in esophageal squamous cell carcinoma (ESCC). We discovered that FAM225A exhibited higher expression in ESCC. The silence of FAM225A attenuated cell viability, migration, and invasion, but facilitated cell apoptosis in ESCC. Exosome‐mediated transfer of lncRNA FAM225A could participate in ESCC progression. In addition, we found that miR‐206 bound to FAM225A. Moreover, we further demonstrated that FAM225A absorbed miR‐206 to upregulate NETO2 and FOXP1 expression, and FOXP1 acted as a transcription factor to enhance FAM225A expression. Eventually, it was revealed that the overexpression of NETO2 or FOXP1 rescued the effects of FAM225A repression on ESCC progression. Our results suggested that FAM225A upregulated NETO2 and FOXP1 expression by sponging miR‐206 to accelerate ESCC progression and angiogenesis. These results determined the biological role of lncRNA FAM225A in ESCC tumorigenesis, and FAM225A may be a promising biomarker for ESCC treatment.

Mycoplasma hyorhinis is a common pathogen of swine and is also associated with various human tumors. It causes systemic inflammation, typically polyserositis and polyarthritis, in some infected pigs. However, the pathogenic mechanism of M. hyorhinis remains unclear. DnaK is a highly conserved protein belonging to the heat-shock protein 70 family of molecular chaperones, which plays important roles as a moonlighting protein in various bacteria. In the present study, we identified the surface exposure of M. hyorhinis DnaK. Two virulent strains expressed more DnaK on their surface than the avirulent strain. Thereafter, the potential moonlighting functions of DnaK were investigated. Recombinant M. hyorhinis DnaK (rMhr-DnaK) was found to be able to adhere to swine PK-15 cells and human NCI-H292 cells. It also bound to four extracellular matrix components—fibronectin, laminin, type IV collagen, and vitronectin—in a dose-dependent manner. ELISA demonstrated an interaction between rMhr-DnaK and plasminogen, which was significantly inhibited by a lysine analog, ε-aminocaproic acid. rMhr-DnaK-bound plasminogen was activated by tissue-type plasminogen activator (tPA), and the addition of rMhr-DnaK significantly enhanced the activation. Finally, a DnaK-specific antibody was detected in the serum of pigs immunized with inactivated vaccines, which indicated good immunogenicity of it. In summary, our findings imply that DnaK is an important multifunctional moonlighting protein in M. hyorhinis and likely participates extensively in the infection and pathogenesis processes of M. hyorhinis.

Abstract Mycoplasma hyopneumoniae (M. hyopneumoniae), is the primary aetiological agent of enzootic pneumonia leading to chronic respiratory disease prevalent worldwide. Conventional pigs are the only animals used for pathogenicity studies and vaccine evaluations of M. hyopneumoniae. Considering that the challenge animals have better genetic stability and a smaller body size to operate with, an alternative experimental animal model of M. hyopneumoniae infection with Bama miniature pigs was established. Nine seven-week-old snatch-farrowed, porcine colostrum-deprived (SF-pCD) Bama miniature pigs and nine conventional pigs were randomly divided into two infected groups (Bama miniature-infected (BI) and conventional-infected groups (CI), BI and CI, n = 6) and two control groups (Bama miniature control (BC) and conventional control (CC) groups, BC and CC, n = 3). Every piglet was tracheally inoculated with 5 × 108 CCU/mL containing 10% suspension of a stock of frozen lung homogenate from SF-pCD pigs infected with virulent strain JS or sterilized KM2 medium. Typical lung lesions appeared in all infected pigs after necropsy, and the mean gross lung lesions was 17.3 and 13.7 in groups of BI and CI. Serum IgG and nasal sIgA antibody titres were increased significantly. Cilia shedding and mucus staining increased greatly in JS-infected bronchi. Obvious reddish gross lesions and M. hyopneumoniae antigen were detected, especially apparently observed in group of BI. Moreover, DNA copies of M. hyopneumoniae from bronchoalveolar lavage fluid (BALF) of each JS-infected piglet reached more than 108, and M. hyopneumoniae could be re-isolated from each infected BALF. These results indicate that Bama miniature pigs could be used as an alternative and more maneuverable experimental infection model for M. hyopneumoniae and display typical clinical and pathological features consistent with those in conventional pigs.

Mycoplasmas are a group of prokaryotes without cell walls that have evolved through several rounds of degenerative evolution. With a low cell DNA G + C content and definitively long genetic lineages, mycoplasmas are thought to be in a state of rapid evolution. However, little associated evidence has been provided. Enolase is a key enzyme in glycolysis that is widely found in all species from the three domains, and it is evolutionarily conserved. In our previous studies, enolase acted as a virulence factor and participated in cell-surface adhesion in Mycoplasma hyopneumoniae. Furthermore, unique loop regions were first found in the crystal structure of Mhp Eno. Here, enolase structures from Mycoplasma pneumoniae and Mycoplasma bovis were determined. An extra helix 7 is specific and conservatively found in almost all mycoplasma enolases, as confirmed by crystal structures and sequence alignment. Particular motifs for helix 7, which is composed of F-K/G-K-L/F-K-X-A-I, have been proposed and could be regarded as molecular markers. To our surprise, the genetic distances between any two mycoplasma enolases were obviously longer than those between the two corresponding species themselves, indicating divergent evolution of mycoplasma enolases, whereas no horizontal gene transfer was detected in mycoplasma enolase genens. Furthermore, different evolutionary patterns were adopted by different loop regions of mycoplasma enolase. Enolases from different Mycoplasma species also showed different affinities for PLG and fibronectin. Our results indicate the rapid and divergent evolution of mycoplasma enolase and mycoplasmas. This study will also aid understanding the independent evolution of Mycoplasma species after separation from their common ancestor.

Mycoplasma hyopneumoniae (Mhp) is the main pathogen that causes enzootic pneumonia, a disease that has a significant impact on the pig industry worldwide. The pathogenesis of enzootic pneumonia, especially possible virulence factors of Mhp, has still not been fully elucidated. The transcriptomic and proteomic analyses of different Mhp strains reported in the literature have revealed differences in virulence, and differences in RNA transcription levels between high- and low-virulence strains initially indicated that nicotinamide adenine dinucleotide (NADH)-dependent flavin oxidoreductase (NFOR) was related to Mhp pathogenicity. Prokaryotic expression and purification of the NFOR protein from Mhp were performed, a rabbit-derived polyclonal antibody against NFOR was prepared, and multiple sequence alignment and evolutionary analyses of Mhp NFOR were performed. For the first time, it was found that the NFOR protein was conserved among all Mhp strains, and NFOR was localized to the cell surface and could adhere to immortalized porcine bronchial epithelial cells (hTERT-PBECs). Adhesion to hTERT-PBECs could be specifically inhibited by an anti-NFOR polyclonal antibody, and the rates of adhesion to both high- and low-virulence strains, 168 and 168L, significantly decreased by more than 40%. Moreover, Mhp NFOR not only recognized and interacted with host fibronectin and plasminogen but also induced cellular oxidative stress and apoptosis in hTERT-PBECs. The release of lactate dehydrogenase by hTERT-PBECs incubated with Mhp NFOR was significantly positively correlated with the virulence of Mhp. Overall, in addition to being a metabolic enzyme related to oxidative stress, NFOR may also function as a potential novel virulence factor of Mhp, thus contributing to the pathogenesis of Mhp; these findings provide new ideas and theoretical support for studying the pathogenic mechanisms of other mycoplasmas.

The objective of this study was to develop a TaqMan probe-based, sensitive, specific duplex real-time PCR assay for simultaneous detection of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis. The specific primers and probes, labeled with FAM and Texas Red, respectively, were designed to amplify the p97 gene of M. hyopneumoniae and p37gene of M. hyorhinis. The duplex real-time PCR reaction mixtures were established and optimized and the sensitivity, specificity and reproducibility of the assay were assessed. The sensitivity of the duplex real-time PCR was found to be 10 copies/μL for both M. hyopneumoniae and M. hyorhinis, respectively. There was no cross reaction with other common viral and bacterial pathogens. The concentration of standard coefficient of variation of Ct values was less than 5%, indicating a good reproducibility. Clinical samples (n = 937) were tested by the duplex real-time PCR assay, including broncho-alveolar lavage fluids, nasal swabs, tissues and cell culture supernatant. Duplex real-time PCR for simultaneous detection of M. hyopneumoniae and M. hyorhinis was highly sensitive and can be utilized for diagnosing clinical samples. It is timeefficient and economic, thereby providing a new approach to control both M. hyopneumoniae and M. hyorhinis.

Abstract Mycoplasma hyopneumoniae is an important respiratory pathogen that causes great economic losses to the pig industry worldwide. Although some putative virulence factors have been reported, pathogenesis remains poorly understood. Herein, we evaluated the relative abundance of proteins in virulent 168 (F107) and attenuated 168L (F380) M. hyopneumoniae strains to identify virulence-associated factors by two-dimensional electrophoresis (2-DE). Seven proteins were found to be ≥ 1.5-fold more abundant in 168, and protein–protein interaction network analysis revealed that all seven interact with putative virulence factors. Unexpectedly, six of these virulence-associated proteins are encoded by core rather than accessory genomic elements. The most differentially abundant of the seven, fructose-1,6-bisphosphate aldolase (FBA), was successfully cloned, expressed and purified. Flow cytometry demonstrated the surface localisation of FBA, recombinant FBA (rFBA) mediated adhesion to swine tracheal epithelial cells (STEC), and anti-rFBA sera decreased adherence to STEC. Surface plasmon resonance showed that rFBA bound to fibronectin with a moderately strong K D of 469 nM. The results demonstrate that core gene expression contributes to adhesion and virulence in M. hyopneumoniae, and FBA moonlights as an important adhesin, mediating binding to host cells via fibronectin.

Mycoplasma hyorhinis is ubiquitous in swine, and it is a common pathogen of swine that causes polyserositis, arthritis, and maybe pneumonia. Fluoroquinolones are effective antimicrobials used for the treatment of mycoplasmal infection. However, a decrease in fluoroquinolones susceptibility in mycoplasma was observed. The molecular mechanisms have been studied in many mycoplasma species, while the mechanism in M. hyorhinis is still unknown. This study aimed to illustrate the in vitro development of fluoroquinolone resistance in M. hyorhinis and unveil the resistance mechanisms in both in vitro selected mutants and field strains. Seven ciprofloxacin-sensitive M. hyorhinis isolates were chosen to induce the fluoroquinolone resistance in vitro, and the point mutations in the quinolone resistance-determining regions (QRDRs) were characterized. The substitutions first occurred in ParC, resulting in a 2- to 8-fold increase in resistance, followed by additional mutations in GyrA and/or ParE to achieve a 32-fold increase. The mutations occurred in hot spots of QRDRs, and they were diverse and variable, including five in ParC (Ser80Phe, Ser80Tyr, Phe80Tyr, Glu84Gly, and Glu84Lys), four in GyrA (Ala83Val, Ser84Pro, Asp87Tyr, and Asp87Asn) and one in ParE (Glu470Lys). Target mutations in field strains were observed in the ParC (Ser80Phe, Ser81Pro, and Glu84Gln) of isolates with MICCIP = 2 μg/mL. This study characterized the point mutations in the QRDRs of M. hyorhinis and could be useful for the rapid detection of fluoroquinolone resistance in M. hyorhinis field isolates.

Homoepitaxial growth of step-flow single crystal diamond was performed by microwave plasma chemical vapor deposition system on high-pressure high-temperature diamond substrate. A coarse surface morphology with isolated particles was firstly deposited on diamond substrate as an interlayer under hillock growth model. Then, the growth model was changed to step-flow growth model for growing step-flow single crystal diamond layer on this hillock interlayer. Furthermore, the surface morphology evolution, cross-section and surface microstructure, and crystal quality of grown diamond were evaluated by scanning electron microscopy, high-resolution transmission electron microcopy, and Raman and photoluminescence spectroscopy. It was found that the surface morphology varied with deposition time under step-flow growth parameters. The cross-section topography exhibited obvious inhomogeneity in crystal structure. Additionally, the diamond growth mechanism from the microscopic point of view was revealed to illustrate the morphological and structural evolution.

Enolase is an evolutionarily conserved enzyme involved in the processes of glycolysis and gluconeogenesis. Mycoplasma hyopneumoniae belongs to Mycoplasma, whose species are wall-less and among the smallest self-replicating bacteria, and is an important colonizing respiratory pathogen in the pig industry worldwide. Mycoplasma hyopneumoniae enolase (Mhp Eno) expression is significantly increased after infection and was previously found to be a virulence factor candidate. Our studies show that Mhp Eno is a cell surface-localized protein that can adhere to swine tracheal epithelial cells (STECs). Adhesion to STECs can be specifically inhibited by an Mhp Eno antibody. Mhp Eno can recognize and interact with plasminogen with high affinity. Here, the first crystal structure of the mycoplasmal enolase from Mycoplasma hyopneumoniae was determined. The structure showed unique features of Mhp Eno in the S3/H1, H6/S6, H7/H8, and H13 regions. All of these regions were longer than those of other enolases and were exposed on the Mhp Eno surface, making them accessible to host molecules. These results show that Mhp Eno has specific structural characteristics and acts as a multifunctional adhesin on the Mycoplasma hyopneumoniae cell surface.

Mycoplasma hyopneumoniae is a colonizing respiratory pathogen that can cause great economic losses to the pig industry worldwide. Although putative virulence factors have been reported, the pathogenesis of this species remains unclear. Here, we used the virulent M. hyopneumoniae strain 168 to infect swine tracheal epithelial cells (STEC) to identify the infection-associated factors by two-dimensional electrophoresis (2-DE). Whole proteins of M. hyopneumoniae were obtained and compared with samples cultured in broth. Six differentially expressed proteins with an increase in abundance of ≥1.5 in the cell infection group were successfully identified. A String network of virulence-associated proteins showed that all the six differential abundance proteins were involved in virulence of M. hyopneumoniae. One of the most important upregulated hubs in this network, elongation factor thermo unstable (EF-Tu), which showed a relatively higher expression in M. hyopneumoniae-infected STEC and obtained a higher score on mass spectrometry was successfully recombined. In addition to its canonical enzymatic activities in protein synthesis, EF-Tu was also reported to be located on the cell surface as an important adhesin in many other pathogens. The cell surface location of EF-Tu was then observed in M. hyopneumoniae with flow cytometry. Recombinant EF-Tu (rEF-Tu) was found to be able to adhere to STEC and anti-rEF-Tu antibody enclosed M. hyopneumoniae decreased adherence to STEC. In addition, surface plasmon resonance (SPR) analysis showed that rEF-Tu could bind to fibronectin with a specific and moderately strong interaction, a dissociation constant (KD) of 605 nM. Furthermore, the block of fibronectin in STEC also decreased the binding of M. hyopneumoniae to the cell surface. Collectively, these data imply EF-Tu as an important adhesin of M. hyopneumoniae and fibronectin as an indispensable receptor on STEC. The binding between EF-Tu with fibronectin contributes to the adhesion of M. hyopneumoniae to STEC.HIGHLIGHTSElongation factor thermo unstable (EF-Tu) exists on the cell surface of M. hyopneumoniae.EF-Tu moonlights as an adhesin of M. hyopneumoniae.The adhesive effect of EF-Tu is partly meditated by fibronectin.

Mycoplasma, the smallest self-replicating organism with a minimal metabolism and little genomic redundancy, is expected to be a close approximation to the minimal set of genes needed to sustain bacterial life. This study employs comparative evolutionary analysis of twenty Mycoplasma genomes to gain an improved understanding of essential genes. By analyzing the core genome of mycoplasmas, we finally revealed the conserved essential genes set for mycoplasma survival. Further analysis showed that the core genome set has many characteristics in common with experimentally identified essential genes. Several key genes, which are related to DNA replication and repair and can be disrupted in transposon mutagenesis studies, may be critical for bacteria survival especially over long period natural selection. Phylogenomic reconstructions based on 3,355 homologous groups allowed robust estimation of phylogenetic relatedness among mycoplasma strains. To obtain deeper insight into the relative roles of molecular evolution in pathogen adaptation to their hosts, we also analyzed the positive selection pressures on particular sites and lineages. There appears to be an approximate correlation between the divergence of species and the level of positive selection detected in corresponding lineages.