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1.
Int J Biol Macromol ; 187: 683-689, 2021 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-34333004

RESUMO

Glycoprotein 3 (GP3), a highly glycosylated membrane protein, is a protective antigen and minor structural protein of porcine reproductive and respiratory syndrome virus (PRRSV), and plays a crucial role in virus assembly and infection. In the present study, we synthesized 23 overlapping peptides span GP3 protein sequence and used pig anti-PRRSV serums to identify immunodominant peptides by indirect ELISA. Five immunodominant peptides GP3-P3, P4, P5, P6 and P7 were identified and GP3-P4 (P55LCPTRQAAAEILEPGKS72) was conjugated to carrier protein BSA. One mAb 1E5 against GP3 was generated from BALB/c mice immunized with the conjugates BSA-P4. The Characterization of mAb was identified by Western blot, Dot-ELISA, IPMA and IFA. We found that mAb 1E5 can specifically react with HP-PRRSV strains but not C-PRRSV or NADC30-like PRRSV strains tested in this study. Site-directed alanine substitution analysis revealed that 8 amino acid residues were involved in antibody binding, among them E65, L67 and P69 were critical residue recognized by mAb 1E5. Taken together, this study provided a novel strategy for generating specific mAbs against virus proteins by using immunodominant peptides as targets, and the mAb 1E5 may be useful for development of rapid differential detection method differentiating HP-PRRSV from C-PRRSV and NADC30-like PRRSV.

2.
Bioelectrochemistry ; 141: 107877, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34171508

RESUMO

Tiamulin (TML) is a pleuromutilin antibiotic and mainly used to treat pulmonary and gastrointestinal infections. However, excessive use of TML can bring health threats to consumers. In this work, a label-free electrochemical immunosensor was proposed for sensitive detection of TML in pork and pork liver. Silver nanoparticles (AgNPs) were synthesized in situ on graphene oxide (GO), in which GO acted as a carrier for loading more AgNPs and AgNPs exhibited both strong conductivity and good redox property. In addition, staphylococcal protein A (SPA) was applied to oriented immobilization of fragment crystallizable (Fc) region of the TML monoclonal antibody. Under the optimal condition, the developed electrochemical immunosensor exhibited a good linear response with a concentration of TML ranging from 0.05 ng mL-1 to 100 ng mL-1 and the limit of detection (LOD) was 0.04 ng mL-1. Furthermore, the designed immunosensor was applied to detect TML in real samples with a good accuracy. Therefore, the label-free electrochemical immunosensor could be used as a potential method to detect TML and other antibiotic residues in animal derived foods.

3.
Front Immunol ; 12: 619362, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33659004

RESUMO

Mycoplasma bovis causes important diseases and great losses on feedlots and dairy farms. However, there are only a few measures to control M. bovis-related diseases. As in other mycoplasma species, this is predominantly because the virulence related factors of this pathogen are largely unknown. Therefore, in this study, we aimed to identify novel virulence-related factors among the secreted proteins of M. bovis. Using bioinformatic tools to analyze its secreted proteins, we preliminarily predicted 39 secreted lipoproteins, and then selected 11 of them for confirmation based on SignalP scores >0.6 or SceP scores >0.8 and conserved domains. These 11 genes were cloned after gene modification based on the codon bias of Escherichia coli and expressed. Mouse antiserum to each recombinant protein was developed. A western blotting assay with these antisera confirmed that MbovP280 and MbovP475 are strongly expressed and secreted proteins, but only MbovP280 significantly reduced the viability of bovine macrophages (BoMac). In further experiments, MbovP280 induced the apoptosis of BoMac treated with both live M. bovis and MbovP280 protein. The conserved coiled-coil domain of MbovP280 at amino acids 210-269 is essential for its induction of apoptosis. Further, immunoprecipitation, mass spectrometry, and coimmunoprecipitation assays identified the anti-apoptosis regulator αB-crystallin (CRYAB) as an MbovP280-binding ligand. An αß-crystallin knockout cell line BoMac-cryab-, Mbov0280-knockout M. bovis strain T9.297, and its complemented M. bovis strain CT9.297 were constructed and the apoptosis of BoMac-cryab- induced by these strains was compared. The results confirmed that CRYAB is critical for MbovP280 function as an apoptosis inducer in BoMac. In conclusion, in this study, we identified MbovP280 as a novel secreted protein of M. bovis that induces the apoptosis of BoMac via its coiled-coil domain and cellular ligand CRYAB. These findings extend our understanding of the virulence mechanism of mycoplasmal species.


Assuntos
Apoptose , Proteínas de Bactérias/metabolismo , Macrófagos/metabolismo , Infecções por Mycoplasma/genética , Infecções por Mycoplasma/microbiologia , Mycoplasma bovis/fisiologia , Animais , Apoptose/imunologia , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/farmacologia , Bovinos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Genoma Bacteriano , Humanos , Ligantes , Macrófagos/imunologia , Camundongos , Modelos Biológicos , Infecções por Mycoplasma/imunologia , Infecções por Mycoplasma/metabolismo
4.
PLoS Pathog ; 16(6): e1008661, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32598377

RESUMO

Mycoplasmas are host-restricted prokaryotes with a nearly minimal genome. To overcome their metabolic limitations, these wall-less bacteria establish intimate interactions with epithelial cells at mucosal surfaces. The alarming rate of antimicrobial resistance among pathogenic species is of particular concern in the medical and veterinary fields. Taking advantage of the reduced mycoplasma genome, random transposon mutagenesis was combined with high-throughput screening in order to identify key determinants of mycoplasma survival in the host-cell environment and potential targets for drug development. With the use of the ruminant pathogen Mycoplasma bovis as a model, three phosphodiesterases of the DHH superfamily were identified as essential for the proliferation of this species under cell culture conditions, while dispensable for axenic growth. Despite a similar domain architecture, recombinant Mbov_0327 and Mbov_0328 products displayed different substrate specificities. While rMbovP328 protein exhibited activity towards cyclic dinucleotides and nanoRNAs, rMbovP327 protein was only able to degrade nanoRNAs. The Mbov_0276 product was identified as a member of the membrane-associated GdpP family of phosphodiesterases that was found to participate in cyclic dinucleotide and nanoRNA degradation, an activity which might therefore be redundant in the genome-reduced M. bovis. Remarkably, all these enzymes were able to convert their substrates into mononucleotides, and medium supplementation with nucleoside monophosphates or nucleosides fully restored the capacity of a Mbov_0328/0327 knock-out mutant to grow under cell culture conditions. Since mycoplasmas are unable to synthesize DNA/RNA precursors de novo, cyclic dinucleotide and nanoRNA degradation are likely contributing to the survival of M. bovis by securing the recycling of purines and pyrimidines. These results point toward proteins of the DHH superfamily as promising targets for the development of new antimicrobials against multidrug-resistant pathogenic mycoplasma species.


Assuntos
Proteínas de Bactérias/metabolismo , Mycoplasma bovis/enzimologia , Pirofosfatases/metabolismo , Ribonucleases/metabolismo , Animais , Proteínas de Bactérias/genética , Linhagem Celular , Camundongos , Camundongos Endogâmicos BALB C , Mycoplasma bovis/genética , Pirofosfatases/genética , Ribonucleases/genética
5.
Microb Pathog ; 143: 104135, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32165330

RESUMO

Mycoplasma bovis is a risky pathogen mainly responsible for pneumonia and mastitis in cattle. Up to date, its pathogenesis is not clear. Since secreted proteins have a tricky role in M. bovis pathogenesis, this study was designed to systematically reveal M. bovis secretome and potential role in virulence of the pathogen. By using bioinformatics tools, a total of 246 secreted proteins were predicted based on M. bovis genome. Among them, 14 were classical, 154 non-classical and 78 both pathways. Then by using 2-dimensional gel electrophoresis (2-DE) and Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF- MS), 169 proteins were revealed. Of them, 60 were predicted to be secreted including 3 classical, 43 non-classical, and 14 both classical and non-classical. Further 8 proteins (MbovP0038, MbovP0338, MbovP0341, MbovP0520, MbovP0581, MbovP0674, MbovP0693, MbovP0845) were predicted to be virulence-related factors with VFDB. In addition, MbovP0581 (ABC transporter protein) was validated experimentally as secreted in nature and highly immunogenic reacting with sera of cattle experimentally infected with M. bovis. In conclusion, this study might be a crucial step towards a better understanding of pathogenesis and leading to the development of novel diagnostic marker and potent vaccine against M. bovis.


Assuntos
Proteínas de Bactérias/metabolismo , Mycoplasma bovis/metabolismo , Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Sequência Conservada/genética , Eletroforese em Gel Bidimensional , Genoma Bacteriano/genética , Genômica , Espectrometria de Massas , Mycoplasma bovis/genética , Mycoplasma bovis/patogenicidade , Proteômica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Virulência
6.
Microorganisms ; 8(2)2020 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-31979335

RESUMO

Molecules contributing to microbial cytoadhesion are important virulence factors. In Mycoplasma bovis, a minimal bacterium but an important cattle pathogen, binding to host cells is emerging as a complex process involving a broad range of surface-exposed structures. Here, a new cytoadhesin of M. bovis was identified by producing a collection of individual knock-out mutants and evaluating their binding to embryonic bovine lung cells. The cytoadhesive-properties of this surface-exposed protein, which is encoded by Mbov_0503 in strain HB0801, were demonstrated at both the mycoplasma cell and protein levels using confocal microscopy and ELISA. Although Mbov_0503 disruption was only associated in M. bovis with a partial reduction of its binding capacity, this moderate effect was sufficient to affect M. bovis interaction with the host-cell tight junctions, and to reduce the translocation of this mycoplasma across epithelial cell monolayers. Besides demonstrating the capacity of M. bovis to disrupt tight junctions, these results identified novel properties associated with cytoadhesin that might contribute to virulence and host colonization. These findings provide new insights into the complex interplay taking place between wall-less mycoplasmas and the host-cell surface.

7.
Front Microbiol ; 10: 2753, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31849895

RESUMO

Microbial access to host nutrients is a key factor of the host-pathogen interplay. With their nearly minimal genome, wall-less bacteria of the class Mollicutes have limited metabolic capacities and largely depend on host nutrients for their survival. Despite these limitations, host-restricted mycoplasmas are widely distributed in nature and many species are pathogenic for humans and animals. Yet, only partial information is available regarding the mechanisms evolved by these minimal pathogens to meet their nutrients and the contribution of these mechanisms to virulence. By using the ruminant pathogen Mycoplasma bovis as a model system, extracellular DNA (eDNA) was identified as a limiting nutrient for mycoplasma proliferation under cell culture conditions. Remarkably, the growth-promoting effect induced by supplementation with eDNA was associated with important cytotoxicity for actively dividing host cells, but not confluent monolayers. To identify biological functions mediating M. bovis cytotoxicity, we produced a library of transposon knockout mutants and identified three critical genomic regions whose disruption was associated with a non-cytopathic phenotype. The coding sequences (CDS) disrupted in these regions pointed towards pyruvate metabolism as contributing to M. bovis cytotoxicity. Hydrogen peroxide was found responsible for eDNA-mediated M. bovis cytotoxicity, and non-cytopathic mutants were unable to produce this toxic metabolic compound. In our experimental conditions, no contact between M. bovis and host cells was required for cytotoxicity. Further analyses revealed important intra-species differences in eDNA-mediated cytotoxicity and H2O2 production, with some strains displaying a cytopathic phenotype despite no H2O2 production. Interestingly, the genome of strains PG45 and HB0801 were characterized by the occurrence of insertion sequences (IS) at close proximity to several CDSs found disrupted in non-cytopathic mutants. Since PG45 and HB0801 produced no or limited amount of H2O2, IS-elements might influence H2O2 production in M. bovis. These results confirm the multifaceted role of eDNA in microbial communities and further identify this ubiquitous material as a nutritional trigger of M. bovis cytotoxicity. M. bovis may thus take advantage of the multiple sources of eDNA in vivo to modulate its interaction with host cells, a way for this minimal pathogen to overcome its limited coding capacity.

8.
Genes (Basel) ; 10(9)2019 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-31466385

RESUMO

Mycoplasma bovis is a critical bovine pathogen, but its pathogenesis remains poorly understood. Here, the virulent HB0801 (P1) and attenuated HB0801-P150 (P150) strains of M. bovis were used to explore the potential pathogenesis and effect of induced immunity from calves' differential transcriptomes post infection. Nine one-month-old male calves were infected with P1, P150, or mock-infected with medium and euthanized at 60 days post-infection. Calves in P1 group exhibited other clinical signs and pathological changes compared to the other two groups. Transcriptome profiles of peripheral blood mononuclear cells revealed seven and 10 hub differentially expressed genes (DEGs) in P1 and P150 groups compared with mock-infected group, respectively. Then, P1-induced pathogenesis was predicted to be associated with enhanced Th17, and P150-induced immunity with Th1 response and expression of ubiquitination-associated enzymes. Association analysis showed that 14 and 11 DEGs were positively and negatively correlated with pathological changes, respectively. Furthermore, up-regulated expression in molecules critical to differentiation of pathogenic Th17 cells in lung and peripheral blood mononuclear cells in P1 group was validated at RNA and protein levels. The results confirmed virulent and attenuated strains might be associated with biased differentiation of pro-inflammatory pathogenic Th17 and Th1 subsets respectively.


Assuntos
Infecções por Mycoplasma/imunologia , Mycoplasma bovis/patogenicidade , Células Th1/imunologia , Células Th17/imunologia , Transcriptoma , Animais , Bovinos , Células Cultivadas , Masculino , Infecções por Mycoplasma/genética , Infecções por Mycoplasma/microbiologia , Infecções por Mycoplasma/veterinária
9.
Nanoscale Res Lett ; 14(1): 19, 2019 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-30635803

RESUMO

A general one-pot approach is developed to synthesize a series of binary metal sulfide nanocrystals (NCs) including PbS, Cu2S, ZnS, CdS, Ag2S, and ternary CuInS2 and CdS:Cu(I) NCs. This synthetic approach involves thermal decomposition of the mixture of inorganic metal salts and n-dodecanethiol (DDT) without pre-synthesis of any organometallic precursors. In this method, layered metal-thiolate compound is formed at the beginning of the reaction and then this intermediate compound is decomposed into small particles, leading to further growth as the reaction time increases. The as-obtained CdS NCs exhibits a broad but weak surface-state emission, and the Cu(I) doping leads to a red-shift of the emission band due to the Cu(I)-related emission. It is expected that this one-pot approach can be extended to prepare multinary metal sulfide NCs.

10.
Microb Pathog ; 111: 108-117, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28826770

RESUMO

This study was undertaken to determine the genotypic distribution of Chinese M. bovis strains and their similarity to isolates from other countries. Two multilocus sequence typing (MLST) schemes (MLST-1 and MLST-2) and pulsed field gel electrophoresis (PFGE) were used to compare 44 Chinese strains and the M. bovis type strain PG45. The results showed a high genetic homogeneity of Chinese isolates; 43 of 44 (97.7%) Chinese isolates were identified as ST-10 and as ST-34 by MLST-1, while for MLST-2 42 of 44 (95.5%) were identified as ST-10 with the two remaining isolates of ST-32 and ST43. PFGE clustered 42 of 44 (95.5%) of the Chinese isolates into PT-I. The overall agreement rate between the three typing methods was 97.8% (95% CI:86.8-99.9%). The type strain PG45 was identified as a unique type by all three methods. When the MLST-2 scheme was further used to analyze 16 isolates of Australian and Israeli origin ST-10 was more dominant among Australian isolates (7/8), compared with those from Israel (3/8). The evolutionary relationship of the 60 isolates typed in this study assessed together with 206 additional isolates retrieved from pubmlst/mbovis database analyzed by geoBURST Minimum spanning tree (MST) confirmed that the Chinese, Israeli and Australian M. bovis isolates typed in this study that were predominantly ST-10, were clustered in CC3 with isolates originating from the USA. Our results suggest that ST-10 is an emerging clone of M. bovis population. We hypothesized that the widespread distribution of this type is a result of global livestock movements. These findings will help further the understanding of the global evolution of M. bovis and development of novel vaccines against M. bovis.


Assuntos
Evolução Molecular , Genótipo , Mycoplasma bovis/classificação , Mycoplasma bovis/genética , Mycoplasma bovis/isolamento & purificação , Análise de Variância , Animais , Austrália , Bovinos , Doenças dos Bovinos/microbiologia , China , DNA Bacteriano , Eletroforese em Gel de Campo Pulsado/métodos , Genes Bacterianos/genética , Variação Genética , Israel , Epidemiologia Molecular , Tipagem de Sequências Multilocus/métodos , Análise de Sequência de DNA , Estados Unidos , Sequenciamento Completo do Genoma
11.
Vision Res ; 137: 24-28, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28688906

RESUMO

The conventional view was that cones are responsible for pupil constriction in photopic lighting conditions. With the discovery of intrinsically photosensitive retinal ganglion cells (ipRGC), it was found that signals from ipRGCs along with cones mediated the pupil light reflex in photopic lighting conditions. Although both signals contributed, it was unclear how these signals were summed. In the work reported here, steady-state pupil size was measured with an infrared camera under LED lighting conditions with different color temperatures and luminance. A formula was then derived for pupil size according to the linear summation of cirtopic and photopic luminance. This formula allowed direct calculations to predict pupil size well when LED photopic luminance ranged from about 50cd/m2 to 300cd/m2, which is the general luminance level range for computer and smartphone screens.


Assuntos
Visão de Cores/fisiologia , Estimulação Luminosa , Pupila/fisiologia , Reflexo Pupilar/fisiologia , Células Fotorreceptoras Retinianas Cones/efeitos da radiação , Células Ganglionares da Retina/efeitos da radiação , Células Fotorreceptoras Retinianas Bastonetes/efeitos da radiação , Adolescente , Feminino , Humanos , Masculino , Adulto Jovem
12.
Artigo em Inglês | MEDLINE | ID: mdl-28553620

RESUMO

Mycoplasma bovis is an important cause of bovine respiratory disease worldwide. To understand its virulence mechanisms, we sequenced three attenuated M. bovis strains, P115, P150, and P180, which were passaged in vitro 115, 150, and 180 times, respectively, and exhibited progressively decreasing virulence. Comparative genomics was performed among the wild-type M. bovis HB0801 (P1) strain and the P115, P150, and P180 strains, and one 14.2-kb deleted region covering 14 genes was detected in the passaged strains. Additionally, 46 non-sense single-nucleotide polymorphisms and indels were detected, which confirmed that more passages result in more mutations. A subsequent collective bioinformatics analysis of paralogs, metabolic pathways, protein-protein interactions, secretory proteins, functionally conserved domains, and virulence-related factors identified 11 genes that likely contributed to the increased attenuation in the passaged strains. These genes encode ascorbate-specific phosphotransferase system enzyme IIB and IIA components, enolase, L-lactate dehydrogenase, pyruvate kinase, glycerol, and multiple sugar ATP-binding cassette transporters, ATP binding proteins, NADH dehydrogenase, phosphate acetyltransferase, transketolase, and a variable surface protein. Fifteen genes were shown to be enriched in 15 metabolic pathways, and they included the aforementioned genes encoding pyruvate kinase, transketolase, enolase, and L-lactate dehydrogenase. Hydrogen peroxide (H2O2) production in M. bovis strains representing seven passages from P1 to P180 decreased progressively with increasing numbers of passages and increased attenuation. However, eight mutants specific to eight individual genes within the 14.2-kb deleted region did not exhibit altered H2O2 production. These results enrich the M. bovis genomics database, and they increase our understanding of the mechanisms underlying M. bovis virulence.


Assuntos
Genes Bacterianos/genética , Genoma Bacteriano/genética , Mycoplasma bovis/genética , Fatores de Virulência/genética , Virulência/genética , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bovinos , Biologia Computacional , Peróxido de Hidrogênio/metabolismo , Redes e Vias Metabólicas/genética , Mycoplasma bovis/enzimologia , Mycoplasma bovis/metabolismo , Polimorfismo de Nucleotídeo Único , Mapas de Interação de Proteínas , Análise de Sequência de DNA , Deleção de Sequência , Virulência/imunologia
13.
J Sep Sci ; 40(9): 2054-2061, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28252250

RESUMO

A novel method named effective length calibration method has been developed to process the fluorescence signal detected by charge-coupled device during capillary electrophoresis. The new method treated each pixel as an individual point detector, and effectively binned a large number of pixels into a final electropherogram without losing the narrow detection window defined by a single pixel. Capillary electrophoresis separations of DNA were carried out and detected by charge-coupled device and conventional detector (photomultiplier tube). Detection properties including signal-to-noise ratio, peak width, detection frequency, and tilt of detector were investigated. It was found that the new method achieved much higher signal-to-noise ratio and smaller peak width than the conventional detector did. A Detection width of 0.5 µm was easily achieved.


Assuntos
DNA/análise , Eletroforese Capilar , Fluorescência , Calibragem
14.
Sci Rep ; 7(1): 44, 2017 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-28246386

RESUMO

Mycoplasma bovis causes considerable economic losses in the cattle industry worldwide. In mycoplasmal infections, adhesion to the host cell is of the utmost importance. In this study, the amino acid sequence of NOX was predicted to have enzymatic domains. The nox gene was then cloned and expressed in Escherichia coli. The enzymatic activity of recombinant NOX (rNOX) was confirmed based on its capacity to oxidize NADH to NAD+ and reduce O2 to H2O2. The adherence of rNOX to embryonic bovine lung (EBL) cells was confirmed with confocal laser scanning microscopy, enzyme-linked immunosorbent assay, and flow cytometry. Both preblocking EBL cells with purified rNOX and preneutralizing M. bovis with polyclonal antiserum to rNOX significantly reduced the adherence of M. bovis to EBL cells. Mycoplasma bovis NOX-expressed a truncated NOX protein at a level 10-fold less than that of the wild type. The capacities of M. bovis NOX- for cell adhesion and H2O2 production were also significantly reduced. The rNOX was further used to pan phage displaying lung cDNA library and fibronectin was determined to be potential ligand. In conclusion, M. bovis NOX functions as both an active NADH oxidase and adhesin, and is therefore a potential virulence factor.


Assuntos
Adesinas Bacterianas/metabolismo , Complexos Multienzimáticos/metabolismo , Mycoplasma bovis/enzimologia , NADH NADPH Oxirredutases/metabolismo , Oxigênio/metabolismo , Adesinas Bacterianas/genética , Sequência de Aminoácidos , Animais , Aderência Bacteriana , Bovinos , Células Cultivadas , Escherichia coli/genética , Fibronectinas/metabolismo , Peróxido de Hidrogênio/metabolismo , Camundongos Endogâmicos BALB C , Complexos Multienzimáticos/genética , Mycoplasma bovis/genética , Mycoplasma bovis/metabolismo , NAD/metabolismo , NADH NADPH Oxirredutases/genética , Oxirredução
15.
Front Microbiol ; 8: 2500, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29312206

RESUMO

The Mycobacterium tuberculosis complex causes tuberculosis (TB) in humans and other animal species, but Mycobacterium tuberculosis has a distinct host preference to humans. The present study aimed to determine whether a bovine M. tb strain 1458 has evolved some genetic properties in their genome that might be associated with their bovine adaptation. The genome of the M. tb strain 1458 was sequenced and subjected to an extensive comparative genomic analysis. A phylogenetic analysis showed that strain 1458 is most closely related to a Chinese M. tb strain, CCDC5079, of the same Beijing family. Compared with three human M. tb Beijing family strains, the strain 1458 has the fewest unique genes. However, there are most (21) IS6110 insertion sequences in the strain 1458 genome at either intragenic or intergenic sites, resulting in the interruption of 11 genes including three PPE family-encoding genes (PPE16, PPE38, and PPE59). Only the strain 1458 genome has the upstream insertion in esxS and phoP genes. PCR confirmed four upstream insertions and qPCR determined that transcription of esxS, phoP, dnaN, and ctpD genes differed significantly between M. tb strain 1458 and H37Rv or M. bovis. A Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed that the genes affected by non-synonymous SNPs are enriched in RNA polymerase. Moreover, 127 of the 133 unique SNPs in strain 1458 are either different to those in the M. bovis genome. In conclusion, some critical genes responsible for bacterial virulence and immunogenicity were interrupted in the genome of bovine M. tb strain 1458 by IS insertions and non-synonymous SNPs, which might contribute to its bovine adaptation, and the modification of its virulence and immunogenicity in cattle.

16.
Inflammation ; 40(1): 1-12, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27718095

RESUMO

Indirubin plays an important role in the treatment of many chronic diseases and exhibits strong anti-inflammatory activity. However, the molecular mode of action during mastitis prophylaxis remains poorly understood. In this study, a lipopolysaccharide (LPS)-induced mastitis mouse model showed that indirubin attenuated histopathological changes in the mammary gland, local tissue necrosis, and neutrophil infiltration. Moreover, indirubin significantly downregulated the production of interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α (TNF-α). We explored the mechanism whereby indirubin exerts protective effects against LPS-induced inflammation of mouse mammary epithelial cells (MMECs). The addition of different concentrations of indirubin before exposure of cells to LPS for 1 h significantly attenuated inflammation and reduced the concentrations of the three inflammatory cytokines in a dose-dependent manner. Indirubin downregulated LPS-induced cyclooxygenase-2 (COX-2) and Toll-like receptor 4 (TLR4) expression, inhibited phosphorylation of the LPS-induced nuclear transcription factor-kappa B (NF-kB) P65 protein and its inhibitor IkBα of the NF-kB signaling pathway. Furthermore, indirubin suppressed phosphorylation of P38, extracellular signal-regulated kinase (ERK), and c-Jun NH2-terminal kinase (JNK) of the mitogen-activated protein kinase (MAPK) signal pathways. Thus, indirubin effectively suppressed LPS-induced inflammation via TLR4 abrogation mediated by the NF-kB and MAPK signaling pathways and may be useful for mastitis prophylaxis.


Assuntos
Inflamação/tratamento farmacológico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , NF-kappa B/metabolismo , Receptor 4 Toll-Like/fisiologia , Animais , Citocinas/efeitos dos fármacos , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Feminino , Indóis/farmacologia , Indóis/uso terapêutico , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos , Mastite/tratamento farmacológico , Camundongos , Transdução de Sinais/efeitos dos fármacos
17.
Talanta ; 160: 425-430, 2016 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-27591633

RESUMO

High-speed capillary electrophoresis (HSCE) is a promising technology applied in ultra-rapid and high-performance analysis of biomolecules (such as nucleic acids, protein). In present study, the short-end capillary electrophoresis coupled with one novel space domain internal standard method (SDIS) was employed for the rapid and simultaneous analysis of specific genes from three oral bacteria (Porphyromonas gingivalis (P.g), Treponema denticola (T.d) and Tannerela forsythia (T.f)). The reliability, reproducibility and accuracy properties of above mentioned SDIS method were investigated in detail. The results showed the target gene fragments of P.g, T.d and T.f could be precisely, fast identified and quantitated within 95s via present short-end CE system. The analyte concentration and the ratio of space domain signals (between target sample and internal standard sample) featured a well linear relationship calculated via SDIS method. And the correlation coefficients R(2) and detection limits for P.g, T.d, T.f genes were 0.9855, 0.9896, 0.9969 and 0.077, 0.114 and 0.098ng/µl, respectively.


Assuntos
DNA Bacteriano/análise , Boca/microbiologia , Porphyromonas gingivalis/genética , Tannerella forsythia/genética , Treponema denticola/genética , Eletroforese Capilar , Genes Bacterianos , Humanos , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes
18.
Oncotarget ; 7(26): 39376-39395, 2016 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-27281618

RESUMO

A lack of knowledge regarding the antigenic properties of Mycoplasma bovis proteins prevents the effective control of bovine infections using immunological approaches. In this study, we detected and characterized a specific and sensitive M. bovis diagnostic biomarker. After M. bovis total proteins and membrane fractions were separated with two dimensional gel electrophoresis, proteins reacting with antiserawere detected using MALDI-TOF MS. Thirty-nine proteins were identified, 32 of which were previously unreported. Among them, immunoinformatics predicted eight antigens, encoded by Mbov_0106, 0116, 0126, 0212, 0275, 0579, 0739, and 0789, to have high immunological value. These genes were expressed in E. coli after mutagenesis of UGA to UGG using overlap extension PCR. A lipoprotein, MbovP579, encoded by a functionally unknown gene, was a sensitive and specific antigen for detection of antibodies in sera from both M. bovis-infected and vaccinated cattle. The specificity of MbovP579 was confirmed by its lack of cross-reactivity with other mycoplasmas, including Mycoplasma agalactiae. An iELISA based on rMbovP579 detected seroconversion 7 days post-infection (dpi). The ELISA had sensitivity of 90.2% (95% CI: 83.7%, 94.3%) and a specificity of 97.8% (95% CI: 88.7%, 99.6%) with clinical samples. Additional comparative studies showed that both diagnostic and analytic sensitivities of the ELISA were higher than those of a commercially available kit (p<0.01). We have thus detected and characterized the novel antigen, MbovP579, and established an rMbovP579-based ELISA as a highly sensitive and specific method for the early diagnosis of M. bovis infection.


Assuntos
Biomarcadores/metabolismo , Infecções por Mycoplasma/diagnóstico , Infecções por Mycoplasma/microbiologia , Proteômica/métodos , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Monoclonais/química , Antígenos de Bactérias/imunologia , Bovinos , Biologia Computacional , Eletroforese em Gel Bidimensional , Ensaio de Imunoadsorção Enzimática , Feminino , Lipoproteínas/química , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Mycoplasma/sangue , Mycoplasma agalactiae , Mycoplasma bovis , Curva ROC , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
19.
Cytotechnology ; 68(4): 1473-87, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27006302

RESUMO

Endothelial cell activation, injury and dysfunction have been regarded as one of the initial key events in the pathogenesis of atherosclerosis. Lipopolysaccharide (LPS), an important mediator of inflammation, can cause endothelial cell damage and apoptosis. Naringin (Nar), one major flavanone glycoside from citrus fruits, shows various pharmacological actions, but the effect of Nar on LPS-induced damage in human umbilical vein endothelial cells (HUVECs) remains unknown. The present results showed that Nar significantly improved the survival rate of HUVECs, and decreased reactive oxygen species and intracellular Ca(2+) levels caused by LPS compared with model group. In addition, Nar obviously decreased cytochrome c release from mitochondria into cytosol. Moreover, Nar significantly down-regulated the protein or mRNA levels of IL-1, IL-6, TNF-α, VCAM-1, ICAM-1, NF-κB, AP-1, cleaved-3,-7,-9, p53, Bak and Bax, and up-regulated the expressions of Bcl-xl, Bcl-2 to suppress inflammation and apoptosis. Furthermore, Nar obviously inhibited phosphorylation levels of JNK, ERK and p38 MAPK. In conclusion, Nar exhibited potent effects against LPS-induced damage in HUVECs through the modulation of oxidative stress, inflammation, apoptosis and MAPK pathways, which should be developed as a potent candidate for the treatment of atherosclerosis in the future.

20.
J Sep Sci ; 39(5): 986-92, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26648455

RESUMO

Capillary polymer electrophoresis is identified as a promising technology for the analysis of DNA from bacteria, virus and cell samples. In this paper, we propose an innovative capillary polymer electrophoresis protocol for the quantification of polymerase chain reaction products. The internal standard method was modified and applied to capillary polymer electrophoresis. The precision of our modified internal standard protocol was evaluated by measuring the relative standard deviation of intermediate capillary polymer electrophoresis experiments. Results showed that the relative standard deviation was reduced from 12.4-15.1 to 0.6-2.3%. Linear regression tests were also implemented to validate our protocol. The modified internal standard method showed good linearity and robust properties. Finally, the ease of our method was illustrated by analyzing a real clinical oral sample using a one-run capillary polymer electrophoresis experiment.


Assuntos
Bactérias/genética , Proteínas de Bactérias/genética , Eletroforese Capilar/métodos , Boca/microbiologia , Reação em Cadeia da Polimerase/métodos , Bactérias/química , Bactérias/isolamento & purificação , Proteínas de Bactérias/análise , Eletroforese Capilar/instrumentação , Humanos , Polímeros/química
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