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Toxoplasmosis, while often asymptomatic and prevalent as a foodborne disease, poses a considerable mortality risk for immunocompromised individuals during pregnancy. Point-of-care serological tests that detect specific IgG and IgM in patient sera are critical for disease management under limited resources. Despite many efforts to replace the T. gondii total lysate antigens (TLAs) by recombinant antigens (rAgs) in commercial kits, while IgG detection provides significant specificity and sensitivity, IgM detection remains comparatively low in sensitivity. In this study, we attempted to identify novel antigens targeting IgM in early infection, thereby establishing an IgM on-site detection kit. Using two-dimensional gel electrophoresis (2DE) and mouse serum immunoblotting, three novel antigens, including EF1γ, PGKI, and GAP50, were indicated to target T. gondii IgM. However, rAg EF1γ was undetectable by IgM of mice sera in Western blotting verification experiments, and ELISA coated with PGKI did not eliminate cross-reactivity, in contrast to GAP50. Subsequently, the lateral flow reaction employing a strip coated with 0.3 mg/mL purified rAg GAP50 and exhibited remarkable sensitivity compared with the conventional ELISA based on tachyzoite TLA, which successfully identified IgM in mouse sera infected with tachyzoites, ranging from 10³ to 104 at 5 dpi and 104 at 7 dpi, respectively. Furthermore, by using standard T. gondii-infected human sera from WHO, the limit of detection (LOD) for the rapid fluorescence immunochromatographic test (FICT) using GAP50 was observed at 0.65 IU (international unit). These findings underline the particular immunoreactivity of GAP50, suggesting its potential as a specific biomarker for increasing the sensitivity of the FICT in IgM detection. [ABSTRACT FROM AUTHOR]

Vibrio cholerae can cause pandemic cholera in humans. The waterborne bacterium is frequently isolated from aquatic products worldwide. However, current literature on the impact of aquatic product matrices on the survival and pathogenicity of cholerae is rare. In this study, the growth of eleven non-O1/0O139 V. cholerae isolates recovered from eight species of commonly consumed fish and shellfish was for the first time determined in the eight aquatic animal matrices, most of which highly increased the bacterial biomass when compared with routine trypsin soybean broth (TSB) medium. Secretomes of the V. cholerae isolates (draft genome size: 3,852,021–4,144,013 bp) were determined using two-dimensional gel electrophoresis (2DE-GE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques. Comparative secretomic analyses revealed 74 differential extracellular proteins, including several virulence- and resistance-associated proteins secreted by the V. cholerae isolates when grown in the eight matrices. Meanwhile, a total of 8,119 intracellular proteins were identified, including 83 virulence- and 8 resistance-associated proteins, of which 61 virulence-associated proteins were absent from proteomes of these isolates when grown in the TSB medium. Additionally, comparative genomic and proteomic analyses also revealed several strain-specific proteins with unknown functions in the V. cholerae isolates. Taken, the results in this study demonstrate that distinct secretomes and proteomes induced by the aquatic animal matrices facilitate V. cholerae resistance in the edible aquatic animals and enhance the pathogenicity of the leading waterborne pathogen worldwide. [ABSTRACT FROM AUTHOR]

Plantaricin E/F (PlnEF) is a pair of two-component class IIb bacteriocin produced by lactic acid bacteria. PlnEF commonly displays potent antimicrobial activity against certain Gram-positive organisms. In this study, we investigated the synergistic activity of PlnEF combined with lactic acid against Gram-negative food and aquaculture potential pathogen Aeromonas hydrophila LPL-1, which is naturally resistant to PlnEF. We applied SDS-PAGE, wavelength-scanning, laser confocal microscopy, flow cytometer, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and two-dimensional electrophoresis to investigate their synergistic inhibitory activities. The results showed that L-lactic acid drove the release of LPS from A. hydrophila , making it possible for PlnEF to contact the inner cell membrane of A. hydrophila. Besides, co-treatment of lactic acid and PlnEF caused severe morphological and intracellular changes of A. hydrophila , including blebs on the cell surface, abnormal cell elongation, inner membrane disruption, pore-forming through the outer and inner membrane, coagulation of the cytoplasm, and structural transformation of DNA. Protein profile analysis revealed that combined treatment of lactic acid and PlnEF inhibited the energy metabolism, protein synthesis, protein folding, and DNA replication in A. hydrophila. These findings proved that PlnEF combined with lactic acid was efficient against A. hydrophila and shed light on bacteriocin's potential and a new inhibition mechanism against A. hydrophila. Importance: Bacteriocins and their producing strains are increasingly used to substitute artificial preservatives and antibiotics in the food and aquaculture industries. However, the bacteriocins produced by lactic acid bacteria are efficient to mainly Gram-positive bacteria. Our paper had demonstrated the antimicrobial activity of class IIb bacteriocin against potential Gram-negative pathogen, A. hydrophila LPL-1, when combined with lactic acid. The results could refresh our knowledge about the potential of class IIb bacteriocins produced by lactic acid bacteria. [ABSTRACT FROM AUTHOR]

Vibrio parahaemolyticus uses bacterial secretion systems and integrative and conjugative elements (ICEs) to induce various diseases and to adapt to harsh environments, respectively. Information pertaining to the identity of secreted proteins and functional characterization of ICEs has been previously reported, but the relationship between these elements remains unclear. Herein we investigated secreted proteins of V. parahaemolyticus strains JHY20 and JHY20△ICE using two-dimensional gel electrophoresis and LC-MS/MS, which led to the identification of an ICE-associated secreted protein – dihydrolipoamide dehydrogenase (DLDH). Considering the data related to its physical and biochemical characterization, we predicted that DLDH is a novel immunogenic protein and associated with virulence in JHY20. Our findings indicate a potential relationship between ICE-associated transport and secreted proteins and shed light on the function of such transport mechanisms. We believe that our data should enhance our understanding of mobile genetic elements. [ABSTRACT FROM AUTHOR]

Bombyx mori nucleopolyhedrovirus (BmNPV) is a major pathogen causing severe economic loss. However, the molecular mechanism of silkworm resistance to BmNPV and the interactions of this virus with the host during infection remain largely unclear. To explore the virus-binding proteins of silkworms, the midgut subcellular component proteins that may interact with BmNPV were analyzed in vitro based on one- and two-dimensional electrophoresis and far-western blotting combined with mass spectrometry (MS). A total of 24 proteins were determined to be specifically bound to budded viruses (BVs) in two subcellular fractions (mitochondria and microsomes). These proteins were involved in viral transportation, energy metabolism, apoptosis and viral propagation, and they responded to BmNPV infection with different expression profiles in different resistant strains. In particular, almost all the identified proteins were downregulated in the A35 strain following BmNPV infection. Interestingly, there were no virus-binding proteins identified in the cytosolic fraction of the silkworm midgut. Two candidate proteins, RACK1 and VDAC2, interacted with BVs, as determined with far-western blotting and reverse far-western blotting. We speculated that the proteins interacting with the virus could either enhance or inhibit the infection of the virus. The data provide comprehensive useful information for further research on the interaction of the host with BmNPV.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a major pathogen causing severe economic loss. However, the molecular mechanism of silkworm resistance to BmNPV and the interactions of this virus with the host during infection remain largely unclear. To explore the virus-binding proteins of silkworms, the midgut subcellular component proteins that may interact with BmNPV were analyzed in vitro based on one- and two-dimensional electrophoresis and far-western blotting combined with mass spectrometry (MS). A total of 24 proteins were determined to be specifically bound to budded viruses (BVs) in two subcellular fractions (mitochondria and microsomes). These proteins were involved in viral transportation, energy metabolism, apoptosis and viral propagation, and they responded to BmNPV infection with different expression profiles in different resistant strains. In particular, almost all the identified proteins were downregulated in the A35 strain following BmNPV infection. Interestingly, there were no virus-binding proteins identified in the cytosolic fraction of the silkworm midgut. Two candidate proteins, RACK1 and VDAC2, interacted with BVs, as determined with far-western blotting and reverse far-western blotting. We speculated that the proteins interacting with the virus could either enhance or inhibit the infection of the virus. The data provide comprehensive useful information for further research on the interaction of the host with BmNPV. [ABSTRACT FROM AUTHOR]

Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli strain C999 was isolated of a Spanish patient with urinary tract infection. Previous genotyping indicated that this strain presented a multidrug-resistance phenotype and carried beta-lactamase genes encoding CTX-M-15, TEM-1, and OXA-1 enzymes. The whole-cell proteome, and the membrane, cytoplasmic, periplasmic and extracellular sub-proteomes of C999 were obtained in this work by two-dimensional gel electrophoresis (2DE) followed by fingerprint sequencing through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). A total of 602 proteins were identified in the different cell fractions, several of which are related to stress response systems, cellular responses, and antibiotic and drug responses, consistent with the multidrug-resistance phenotype. In parallel, whole genome sequencing (WGS) and RNA sequencing (RNA-Seq) was done to identify and quantify the genes present and expressing. The in silico prediction following WGS confirmed our strain as being serotype O25:H4 and sequence type ST131. The presence of proteins related to antibiotic resistance and virulence in an O25:H4-ST131 E. coli clone are serious indicators of the continued threat of antibiotic resistance spread amongst healthcare institutions. On a positive note, a multiomics approach can facilitate surveillance and more detailed characterization of virulent bacterial clones from hospital environments. [ABSTRACT FROM AUTHOR]

In this study, differentially expressed proteins in A549 cells (human lung adenocarcinoma epithelial cell line) infected with H9N2 avian influenza virus (AIV) were investigated by two-dimensional electrophoresis (2-DE). Sixteen different spots between the groups (ratio>2, p

In vitro mimicking conditions are thought to reflect the environment experienced by M. tuberculosis inside the host granuloma. The majority of the in vitro dormancy experimental models used laboratory adapted strain H37Rv or Erdman strain over the prevalent clinical strains involved during disease outbreaks. Thus, we included the most prevalent clinical strains (S7 and S10) of M. tuberculosis from south India in addition to H37Rv for our in vitro oxygen depletion (hypoxia) experimental model. Cytosolic proteins were prepared from the hypoxic cultures, resolved by two-dimensional electrophoresis (2-DE) and protein spots were characterized by mass spectrometry. Totally 49 spots were characterized as over-expressed or newly appeared between the 3 strains. Two antigens (ESAT-6, Lpd) out of the 49 characterized spots were readily available in recombinant form in our lab. Hence, these 2 genes were overexpressed, purified and used for in vitro stimulation of whole blood collected from healthy household contacts (HHC) and active pulmonary tuberculosis patients (PTB). Multicolour flow cytometry analysis showed high levels of antigen specific CD4+ central memory T cells in circulation of HHC when compared to PTB (p

The present study investigated the hypolipidaemic effects of Grifola frondosa and its regulation mechanism involved in lipid metabolism in liver of rats fed a high-cholesterol diet. The body weights and serum lipid levels of control rats, of hyperlipidaemic rats and of hyperlipidaemic rats treated with oral Grifola frondosa were determined. mRNA expression and concentration of key lipid metabolism enzymes were investigated. Serum cholesterol, triacylglycerol and low-density lipoprotein cholesterol levels were markedly decreased in hyperlipidaemic rats treated with Grifola frondosa compared with untreated hyperlipidaemic rats. mRNA expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), acyl-coenzyme A: cholesterol acyltransferase (ACAT2), apolipoprotein B (ApoB), fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC1) were significantly down-regulated, while expression of cholesterol 7-alpha-hydroxylase (CYP7A1) was significantly up-regulated in the livers of treated rats compared with untreated hyperlipidaemic rats. The concentrations of these enzymes also paralleled the observed changes in mRNA expression. Two-dimensional polyacrylamide gel electrophoresis (2-DE) and Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) were used to identify twenty proteins differentially expressed in livers of rats treated with Grifola frondosa compared with untreated hyperlipidemic rats. Of these twenty proteins, seven proteins were down-regulated and thirteen proteins were up-regulated. These findings indicate that the hypolipidaemic effects of Grifola frondosa reflected its modulation of key enzymes involved in cholesterol and triacylglycerol biosynthesis, absorption and catabolic pathways. Grifola frondosa may exert anti-atherosclerotic effects by inhibiting LDL oxidation through down-regulation and up-regulating proteins expression in the liver of rats. Therefore, Grifola frondosa may produce both hypolipidaemic and anti-atherosclerotic effects, and potentially be of use as a functional food for the treatment or prevention of hyperlipidaemia and atherosclerosis.

In vitro mimicking conditions are thought to reflect the environment experienced by Mycobacterium tuberculosis inside the host granuloma. The majority of in vitro dormancy experimental models use laboratory-adapted strains H37Rv or Erdman instead of prevalent clinical strains involved during disease outbreaks. Thus, we included the most prevalent clinical strains (S7 and S10) of M. tuberculosis from south India in addition to H37Rv for our in vitro oxygen depletion (hypoxia) experimental model. Cytosolic proteins were prepared from hypoxic cultures, resolved by two-dimensional electrophoresis and protein spots were characterized by mass spectrometry. In total, 49 spots were characterized as over-expressed or newly emergent between the three strains. Two antigens (ESAT-6, Lpd) out of the 49 characterized spots were readily available in recombinant form in our lab. Hence, these two genes were overexpressed, purified and used for in vitro stimulation of whole blood collected from healthy household contacts (HHC) and active pulmonary tuberculosis patients (PTB). Multicolor flow cytometry analysis showed high levels of antigen specific CD4+ central memory T cells in the circulation of HHC compared to PTB (p < 0.005 for ESAT-6 and p < 0.0005 for Lpd). This shows proteins that are predicted to be up regulated during in vitro hypoxia in most prevalent clinical strains would indicate possible potential immunogens. In vitro hypoxia experiments with most prevalent clinical strains would also elucidate the probable true representative antigens involved in adaptive mechanisms. [ABSTRACT FROM AUTHOR]

In this study, differentially expressed proteins in A549 cells (human lung adenocarcinoma epithelial cell line) infected with H9N2 avian influenza virus (AIV) were investigated by two-dimensional electrophoresis (2-DE). Sixteen different spots between the groups (ratio > 2, p < 0.05) were identified with mass spectrometry identification. Proteins located in the downstream of the NF-κB and IFN transcription factor pathways were identified, e.g., ISG15. Actin and keratin were also identified, suggesting that the cytoskeleton may plays an important role in the AIV infection of mammalian cells. These findings could provide insights into the interaction between host and influenza viruses and might provide valuable information for clarifying the pathogenesis of viral infections as well.

The present study investigated the hypolipidaemic effects of Grifola frondosa and its regulation mechanism involved in lipid metabolism in liver of rats fed a high-cholesterol diet. The body weights and serum lipid levels of control rats, of hyperlipidaemic rats, and of hyperlipidaemic rats treated with oral G. frondosa were determined. mRNA expression and concentration of key lipid metabolism enzymes were investigated. Serum cholesterol, triacylglycerol, and low-density lipoprotein cholesterol levels were markedly decreased in hyperlipidaemic rats treated with G. frondosa compared with untreated hyperlipidaemic rats. mRNA expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), acyl-coenzyme A: cholesterol acyltransferase (ACAT2), apolipoprotein B (ApoB), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC1) were significantly down-regulated, while expression of cholesterol 7-alpha-hydroxylase (CYP7A1) was significantly up-regulated in the livers of treated rats compared with untreated hyperlipidaemic rats. The concentrations of these enzymes also paralleled the observed changes in mRNA expression. Two-dimensional polyacrylamide gel electrophoresis (2-DE) and Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) were used to identify 20 proteins differentially expressed in livers of rats treated with G. frondosa compared with untreated hyperlipidemic rats. Of these 20 proteins, seven proteins were down-regulated, and 13 proteins were up-regulated. These findings indicate that the hypolipidaemic effects of G. frondosa reflected its modulation of key enzymes involved in cholesterol and triacylglycerol biosynthesis, absorption, and catabolic pathways. G. frondosa may exert anti-atherosclerotic effects by inhibiting LDL oxidation through down-regulation and up-regulating proteins expression in the liver of rats. Therefore, G. frondosa may produce both hypolipidaemic and anti-atherosclerotic effects, and potentially be of use as a functional food for the treatment or prevention of hyperlipidaemia and atherosclerosis.