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Electron beam welding of TC4 titanium alloy and 4J29 Kovar alloy was performed by using different thicknesses of silver interlayers. The microstructure and the composition of welded joints were characterized by scanning electron microscopy, X-ray diffraction, and energy-dipersive spectrometry. The mechanical properties of welded joints were evaluated by tensile strength tests. The results indicated that Ag thickness has great effects on the weld appearance, microstructure, and mechanical properties of electron beam-welded joints. In case of 0.3 and 0.4 mm thickness of Ag interlayers, a considerable part of 4J29 and TC4 melts and a large amount of Fe, Ti, and Ni elements diffuse into the molten pool forming intermetallic compounds (IMCs) such as TiFe2, NiTi, and FeTi. In case of 0.6 mm thickness of Ag interlayer, the welded joint exhibits brazing characteristics with little IMCs near the fusion line of 4J29 Kovar alloy side. When the thickness of the Ag interlayer increases to 0.8 mm, the diffusion of Ti and Fe elements is completely inhibited by Ag. As the thickness of the Ag interlayer increases from 0.3 to 0.8 mm, the tensile strength of the welded sample shows a phenomenon that first rises and then falls, and the largest tensile strength is 243 MPa.

Introduction Staphylococcus aureus (S. aureus) is a gram‐positive opportunistic pathogen, there are currently no high effective vaccine against S. aureus in humans and animals, the development of an efficient vaccine remains an important challenge to prevent S. aureus infection. Here, we prepared Als3‐Th‐cell‐epitope‐Target of RNAIII Activating Protein (TRAP) (ATT) proteins plus the novel combined adjuvants to develop a promising vaccine candidate against S. aureus. Methods The recombinant pET‐28a (+)‐att plasmids were constructed, and the ATT proteins were expressed and obtained, then, ATT plus Freund's adjuvant or the novel combined adjuvants of cytosine‐phosphate‐guanosine oligodeoxynucleotides (CpG), muramyl dipeptides (MDP), and FIA were immunized in mice. After booster immunization, the levels of interferon‐γ (IFN‐γ), interleukin‐4 (IL‐4), IL‐10 and IL‐17A cytokine were evaluated, the humoral immune responses against TRAP were detected in mice, and the survival rate of mice was confirmed by challenge assay. Results The mice immunized with ATT plus Freund's adjuvant exhibited significantly higher level of IFN‐γ, IL‐4, IL‐10, and IL‐17A, and displayed the stronger humoral immune response against TRAP than control groups, importantly, the survival rate of these mice was significantly higher than control groups. In addition, compared with the control groups, ATT + CpG + MDP + FIA group was elicited significantly higher level of IFN‐γ, IL‐4, IL‐10, and IL‐17A and was triggered the stronger humoral immune responses against TRAP, moreover, generated the higher survival rate of mice. Conclusion Als3 epitopes significantly enhanced TRAP immunogenicity. ATT plus the novel combined adjuvants of CpG, MDP, and FIA induced the strong immune response and protection against S. aureus, revealing the combination of CpG, MDP, and FIA adjuvant acts the synergistic effect., The recombinant ATT proteins were expressed and prepared. ATT plus the novel combined adjuvants of MDP, CpG, and FIA induced the strong immune response and protection against S. aureus in mice, revealing the novel combined adjuvant acts the synergistic effect.

Here, Als3-Th-cell-epitope （Als3 epitope） was connected to the N-terminal of TRAP by flexible linker, and the Als3-Th-cell-epitope-TRAP (ATT) proteins were prepared, then, the ATT proteins plus freund’s adjuvant were inoculated in mice to evaluate Als3 epitope to increase the immunogenicity of TRAP. To strengthen the immunogenicity of ATT protein, the proteins plus the novel combined adjuvants of MDP, CpG and FIA were immunized in mice. After the booster immunization, the results showed that the mice immunized with ATT protein plus Freund’s adjuvant exhibited significantly higher level for IFN-γ, IL-4, IL-10 and IL-17A, and displayed the stronger humoral immune response against TRAP than the control groups, importantly, the survival rate of these mice was significantly higher than the control groups. In addition, the mice immunized with ATT protein plus CpG+MDP+FIA adjuvants exhibited significantly higher level for IFN-γ and IL-17A than other groups, and the level of IgG antibody against TRAP was higher than other groups, moreover, the survival rate of these mice was obviously higher than other groups. These data suggested that the immune protection triggered with ATT was significantly stronger than TRAP or TRAP+Als3 epitope did, which indicted Als3 epitope significantly enhanced the immune responses triggered with TRAP through their fused forms of expression. Additionally, these data manifested that ATT plus the novel combined adjuvant, MDP, CpG and FIA, induced the strongest immune response and protection against S.aureus among all the groups, revealing the synergistic effect on different adjuvant. This study provides an important reference for the further development of a new effective vaccine against S.aureus.

Here, we prepared the novel combined adjuvants, CTB as intra-molecular adjuvant, CpG and aluminum hydroxide (Alum) to strengthen the immunogenicity of clumping factor A221-550 of Staphylococcus aureus (S. aureus). The protein-immunoactive results showed CTB-ClfA221-550 elicited the strong immune responses to serum from mice immunized with CTB and ClfA221-550, respectively. The mice immunized with CTB-ClfA221-550 plus CpG and Alum adjuvant exhibited significantly stronger CD4+ T cell responses for IFN-γ, IL-2, IL-4, and IL-17 and displayed the higher proliferation response of splenic lymphocytes than the control groups, in addition, these mice generated the strongest humoral immune response against ClfA221-550 among all groups. Our results also showed CTB-ClfA221-550 plus CpG and Alum adjuvant obviously increased the survival percentage of the mice challenged by S. aureus. These data suggested that the novel combined adjuvants, CTB, CpG, and Alum, significantly enhance the immune responses triggered with ClfA221-550, and could provide a new approach against infection of S. aureus. Abbreviations CTB: Cholera Toxin B; CpG: Cytosine preceding Guanosine; ODN: Oligodeoxynucleotides; Alum: Aluminum hydroxide; TRAP: Target of RNAIII-activating Protein; TLR9: Toll-like Receptor 9; TMB: 3, 3ʹ, 5, 5ʹ-tetramethylbenzidine; mAbs: Monoclonal Antibodies; OD: Optical Densities; S. aureus: Staphylococcus aureus; ClfA: Clumping factor A; FnBPA: Fibronection-binding protein A; IsdB: Iron-regulated surface determinant B; SasA: Staphylococcus aureus Surface Protein A; GapC: Glycer-aldehyde-3-phosphate dehydrogenase-C

This study is to analyze the potential contribution of Syndecan 1 (SDC1) to cisplatin resistance in hepatic carcinoma. Cell proliferation and viability were determined by direct counting and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, respectively. The protein levels of SDC1, p-AKT, AKT and β-actin were quantified by western blotting. The SDC1 transcript abundance was measured by real-time polymerase chain reaction. The relative expression of SDC1 in clinical liver tumor samples was analyzed with immunohistochemistry. SDC1 was up-regulated in cisplatin-resistant HepG2 cells (denoted as HepG2 CR hereafter). SDC1-knockdown re-sensitized HepG2 CR cells to cisplatin treatment. Ectopic over-expression of SDC1 conferred drug resistance to naïve HepG2 cells. PI3K/AKT pathway was over-activated in HepG2 CR cells, and simultaneous administration with PI3K inhibitor greatly surmounted the resistance. We also demonstrated that SDC1 was aberrantly up-regulated in clinical hepatocellular carcinoma samples. Our study highlighted the importance of SDC1-PI3K/AKT signaling in the cisplatin resistance in hepatocellular carcinoma.

This study investigates two multiplexing schemes for enabling multiple-input multiple-output (MIMO) operation of orthogonal chirp-division multiplexing (OCDM)-based Radar-Communication (RadCom) systems, namely code-division multiplexing (CDM) via outer coding and frequency-division multiplexing (FDM) via frequency shift precoding (FSP). After detailed system model description for both multiplexing strategies, radar and communication aspects of the studied MIMO-OCDM RadCom system are analyzed having the conventional MIMO-OFDM RadCom as a baseline. Finally, it is concluded that CDM based on outer coding may yield better radar and communication performance than its FDM counterpart, while still outperforming MIMO-OFDM in terms of communication at the cost of higher range sidelobe level.

Here, we prepared the novel combined adjuvants, CTB as intra-molecular adjuvant, CpG and aluminum hydroxide (Alum) to strengthen the immunogenicity of clumping factor ACTB: Cholera Toxin B; CpG: Cytosine preceding Guanosine; ODN: Oligodeoxynucleotides; Alum: Aluminum hydroxide; TRAP: Target of RNAIII-activating Protein; TLR9: Toll-like Receptor 9; TMB: 3, 3', 5, 5'-tetramethylbenzidine; mAbs: Monoclonal Antibodies; OD: Optical Densities

Glyceraldehyde-3-phosphate dehydrogenase C (GapC) of Streptococcus dysgalactiae (S. dysgalactiae) is a highly conserved surface protein that can induce a protective immune response against S. dysgalactiae infection. To investigate the immune response and protective efficacy induced by epitope-vaccines against S. dysgalactiae infection, we constructed epitope-vaccines GTB1, GB1B2, and GTB1B2 using a T cell epitope (GapC63-77, abbreviated as GT) and two B cell epitopes (GapC30-36, abbreviated as GB1, and GapC97-103, abbreviated as GB2), which were identified in GapC1-150 of S. dysgalactiae in tandem by a GSGSGS linker. BALB/c mice were immunized via an intramuscular injection with the epitope vaccines. The levels of the cytokines, IFN-γ, IL-4, and IL-17, secreted by splenic lymphocytes and the antibody levels in the sera of the immunized mice were detected by ELISA. The immunized mice were subsequently challenged with S. dysgalactiae, and the bacterial colonization in the immunized-mouse organs was examined using the plate counting method. The results showed that the level of the cytokines induced by GTB1B2 was lower than that induced by GapC1-150, but higher than that induced by other epitope vaccines. The level of IgG induced by GTB1B2 was lower than that induced by GapC1-150, but higher than the levels induced by other epitope vaccines. The bacterial colonization numbers in the organs of the mice immunized with GTB1B2 were higher those of the mice immunized with GapC1-150, but significantly lower than those from the mice immunized with other epitope-vaccines. Our results demonstrated that the T cell and B cell epitopes in the epitope-vaccines worked synergistically against bacterial challenge. The multi-epitope vaccine, GTB1B2, could induce stronger cellular and humoral immune responses, and provide a better protective effect against S. dysgalactiae infection.

The GapC protein of Staphylococcus aureus (S. aureus) is a surface protein that is highly conserved among Staphylococcus strains, and it can induce protective humoral immune responses. However, B-cell epitopes in S. aureus GapC have not been reported. In this study, we generated a monoclonal antibody (mAb2A9) targeting S. aureus GapC. Through a passive immunity test, mAb2A9 was shown to partially protect mice against S. aureus infection. We screened the motif 236PVATGSLTE243 that is recognized by mAb2A9 using a phage-display system. The motif sequence exactly matched amino acids 236-243 of the S. aureus GapC protein. Then, we identified the key amino acids in the motif using site-directed mutagenesis. Site-directed mutagenesis revealed that residues P236, G240, L242, and T243 formed the core of the 236PVATGSLT243 motif. In addition, this epitope was proven to be located on the surface of S. aureus, and it induced a protective humoral immune response against S. aureus infection in immunized mice. Overall, our results characterized a conserved B-cell epitope, which will be an attractive target for designing effective epitope-based vaccines against S. aureus infection.

Purpose. In this study, we prepared GapC1-150-IsdB126-361-TRAP (GIT) proteins plus heat-labile enterotoxin B (LTB) as an intra-molecular adjuvant, together with CpG to further enhance its immunogenicity. Methodology. Initially, the target genes were acquired and inserted into pET-32a (+) vectors to express LTB–GIT protein. LTB–GIT expression was confirmed by Western blotting and its immunocompetence was estimated through ELISA. Further, we immunized BALB/c mice with the LTB–GIT plus CpG adjuvant. After the second immunization, the antigen-specific CD4+ cell responses for IFN-γ, IL-2, IL-4 and IL-10 were monitored by intracellular cytokine staining (ICS) assay. After the third immunization, the level of IgG antibodies in the serum from immunized groups was assessed by ELISA, and the protective immune response was appraised by Staphylococcus aureus and Streptococcus dysgalactiae challenge. Results. The ELISA results showed that the OD450nm value of the LTB–GIT group was significantly higher than that of the BSA group. The group immunized with LTB–GIT plus CpG exhibited significantly stronger CD4+ T cell responses for IFN-γ, IL-2, IL-4 and IL-10 compared to the group immunized with LTB–GIT, GIT alone orLTB–GIT plus CpG. In addition, the group immunized with LTB–GIT plus CpG generated the highest level of IgG antibodies against GIT among all of the groups, and our results also showed that LTB–GIT plus CpG markedly improved the survival percentage of mice compared to other groups. Conclusion. We confirmed that the novel double adjuvants, LTB and CpG, are able to significantly improve GIT-induced immune responses. This formula could be a promising strategy for enhancing the immune efficacy of multi-subunit vaccines against Staphylococcus aureus and streptococcal infection.

Purpose. To explore an epitope-based vaccine against Staphylococcus aureus, we screened the epitopes in the N2N3 subdomain of fibronectin-binding protein A (FnBPA) as a surface component of S. aureus. Methodology. We expressed N2N3 proteins and prepared monoclonal antibodies (mAbs) against N2N3 by the hybridoma technique, before screening the B-cell epitopes in N2N3 using a phage-displayed random 12-mer peptide library with these mAbs against N2N3. Finally, we analysed the characters of the screened epitopes using immunofluorescence and an S. aureus infection assay. Results. In this paper, we identified a linear B-cell epitope in N2N3 through screening a phage-displayed peptide library with a 3C3 mAb against the N2N3. The 3C3 mAb recognized the 159IETFNKANNRFSH171 sequence of the N2N3 subdomain. Subsequently, site-directed mutagenic analysis demonstrated that residues F162, K164, N167, R168 and F169 formed the core of 159IETFNKANNRFSH171, and this core motif was the minimal determinant of the B-cell epitope recognized by the 3C3 mAb. The epitope 159IETFNKANNRFSH171 showed high homology among different S. aureus strains. Moreover, this epitope was exposed on the surface of the S. aureus by using an enzyme-linked immunosorbent assay (ELISA) assay and an indirect immunofluorescence assay. As expected, the epitope peptide evoked a protective immune response against S. aureus infection in immunized mice. Conclusion. We identified a novel linear B-cell epitope, 159IETFNKANNRFSH171, in the N2N3 subdomain of S. aureus fibronectin-binding protein A that is recognized by 3C3 mAb, which will contribute to the further study of an epitope-based vaccine candidate against S. aureus.

Staphylococcus aureus can cause different types of diseases from mild skin infections to life-threatening sepsis worldwide. Owing to the emergence and transmission of multidrug-resistant strains, developing an impactful immunotherapy especially vaccine control approach against S. aureus infections is increasingly encouraged and supported. S. aureus manganese transport protein C (MntC), which is a highly-conserved cell surface protein, can elicit protective immunity against S. aureus and Staphylococcus epidermidis. In this study, we evaluated the humoral immune response and CD4+ T cell-mediated immune responses in a mouse peritonitis model. The results showed that MntC-specific antibodies conferred an essential protection for mice to reduce invasion of S. aureus, which was corroborated via the opsonophagocytic killing assay and passive immunization experiment in mice, and moreover MntC-induced Th17 played a remarkable part in preventing S. aureus infection since the MntC-induced protective immunity decreased after neutralization of IL-17 by antibody in vivo and the Th17 adoptive transferred-mice could partly resist S. aureus challenge. In conclusion, we considered that the MntC-specific antibodies and MntC-specific Th17 cells play cooperative roles in the prevention of S. aureus infection.

Manganese transport protein C (MntC) of Staphylococcus aureus represents an excellent vaccine-candidate antigen. The important role of CD4+ T cells in effective immunity against S. aureus infection was shown; however, CD4+ T cell-specific epitopes on S. aureus MntC have not been well identified. Here, we used bioinformatics prediction algorithms to evaluate and identify nine candidate epitopes within MntC. Our results showed that peptide M8 emulsified in Freund's adjuvant induced a much higher cell-proliferation rate as compared with controls. Additionally, CD4+ T cells stimulated with peptide M8 secreted significantly higher levels of interferon-γ and interleukin-17A. These results suggested that peptide M8 represented an H-2d (I-E)-restricted Th17-specific epitope.

Streptococcus is one of the main pathogens that cause bovine mastitis. They includes into S.agalactiae, S.dysgalactiae, and S.uberis. The GapC protein is a virulence factor that is expressed on the surface of Streptococcus species. GapC is highly antigenic and immunization with GapC confers cross-protection against all three species. Our previous data showed that amino acids 1-150 of GapC (GapC1-150) of S. dysgalactiae conferred similar immunoprotection compared to full-length GapC. Thus, the present study aimed to construct a recombinant Escherichia coli XL1-Blue strain that displayed GapC1-150 on its surface, and to investigate the immunogenicity of the surface-localized GapC1-150. To do so, the ompA gene of the E. coli XL1-Blue strain was replaced with the lpp'-ompA-gapC11-150 or lpp'-ompA genes by λ Red recombination, the former of which fused GapC1-150 to an Lpp lipoprotein signal peptide and amino acids 1-159 of OmpA; the recombinant strains were named XL1-Blue/LOG76 and XL1-Blue/LO11, respectively. GapC1-150 was confirmed to localize to the surface of the XL1-Blue/LOG76 strain by an indirect enzyme-linked immunosorbent assay (ELISA), a fluorescence-activated cell sorter analysis, and laser-scanning confocal microscopy. Then, ICR mice were immunized intramuscularly with the XL1-Blue/LOG76 or XL1-Blue/LO11 strains, or recombinant GapC1-150. The sera of the immunized mice were collected and the anti-GapC1-150 antibody levels were detected by ELISA. Lymphocytes secreting interleukin (IL)-4 and interferon-γ were detected by an enzyme-linked ImmunoSpot assay, as was the level of IL-17A level in the supernatant of cultured splenic lymphocytes. The mice immunized with the XL1-Blue/LOG76 strain or GapC1-150 exhibited better cellular and humoral immunity. Lastly, the immunized mice were challenged with S. uberis, S. dysgalactiae, and S. agalactiae strains, and mice that were immunized with the XL1-Blue/LOG76 strain were better protected than those that were immunized with the XL1-Blue/LO11 strain. These results indicate that it is feasible to display GapC1-150 on the E. coli surface as a vaccine against Streptococcus species.

Interferon-γ (IFN-γ), a cytokine produced by activated natural killer cells and T lymphocytes, is an important regulator of innate and adaptive immunity. Interleukin (IL)-18, also known as IFN-γ-inducing factor, is a cytokine that induces T and natural killer cells to produce IFN-γ. In this study, the chicken IL-18 (ChIL-18) and chicken IFN-γ (ChIFN-γ) genes were inserted into the pET28a prokaryotic expression vector, resulting in pET28a-IL-18 and pET28a-IFN-γ, respectively. These plasmids were transformed into Escherichia coli strain BL21, and the ChIL-18 and ChIFN-γ proteins were expressed and purified. To determine their antiviral activities, 200 ng/mL of ChIL-18 and/or ChIFN-γ were inoculated into chicken embryonic fibroblast cells. After 24 h, one 50% tissue culture infective dose (TCID50) of infectious bursal disease virus (IBDV) was inoculated into the chicken embryonic fibroblast cells. The results showed that the antiviral effect of ChIL-18 and ChIFN-γ in combination was better than that of ChIL-18 or ChIFN-γ alone. Next, 14-day-old chicken were injected with 200 µg of ChIL-18 and/or ChIFN-γ and then were challenged with 103 TCID50 of IBDV via intraperitoneal injection. The results showed that the proliferation of IBDV was inhibited by the injection of the recombinant proteins, especially the combination of ChIL-18 and ChIFN-γ, as evidenced by cytokine detection, quantitative PCR, and pathology analyses. These results indicate that ChIL-18 and ChIFN-γ could inhibit IBDV infection and the combination of ChIL-18 and ChIFN-γ has a better inhibitory effect than either cytokine alone.

Glyceraldehyde-3-phosphate dehydrogenase-C (GapC) is a highly conserved surface protein of Staphylococcus aureus, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, which represents an excellent vaccine candidate antigen. It can induce protective immune responses to S. aureus infections. However, CD4

The purpose of this investigation was to construct a recombinant Escherichia coli strain displaying the Staphylococcus aureus target of RNAIII activating protein (TRAP) on its surface, and to investigate the strain for its immunogenicity. The lpp'ompA and lpp'ompA-TRAP genes were fused by the overlap polymerase chain reaction and then ligated into expression plasmid pQE30 producing pLO and pLO-TRAP. These two recombinant plasmids were transformed into E. coli XL1-Blue, resulting in XL1-Blue/pLO and XL1-Blue/pLO-TRAP, which were induced to express protein. The expressed TRAP protein was displayed on the surface of XL1-Blue as judged by whole cell ELISA, flow cytometric analysis, and laser scanning confocal microscopy using the lpp'ompA surface display system. ICR mice were intramuscularly immunized with recombinant strains XL1-Blue/pLO and XL1-Blue/pLO-TRAP as well as recombinant protein TRAP. Immunized mice were assessed for anti-TRAP antibody and lymphocytes for secreted IL-4 and IFN-γ by ELISPOT and secreted IL-17A by indirect ELISA. Immunized mice were challenged with S. aureus Newman and HLJ23-1 strains. The results showed both XL1-Blue/pLO-TRAP and TRAP protein immunized mice to produce better cellular and humoral immunity than XL1-Blue/pLO and PBS injected mice.

This study optimized spray drying process of Lactobacillus reuteri ( L. reuteri ) and increased the survival rate of bacteria using the optimized method. Experiments were designed with quadratic orthogonal rotation combination design. Effects of independent variables on response value were analyzed by response surface methodology. The results showed that the spray drying process was optimized as follows: outlet temperature was 90 °C, inlet temperature was 140 °C, skim milk concentration was 20% (w/v), feeding speed was 1200 mL/h. The effect of optimized process was verified by showing that the mean survival rate was 72.50%, which was basically consistent with the predicted value (74.91%). This study showed that quadratic orthogonal rotation combination design and response surface methodology were useful tools for optimizing the spray drying process for L. reuteri DYF5. In addition, L. reuteri preparations produced by optimized spray drying process yielded higher numbers of viable bacteria. Thus, our finding provided a technical reference for industrial production.

Glyceraldehyde-3-phosphate dehydrogenase-C (GapC) is a highly conserved surface protein of Staphylococcus aureus, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, which represents an excellent vaccine candidate antigen. It can induce protective immune responses to S. aureus infections. However, CD4+ T cell epitopes of GapC that induce CD4+ T cell immune responses are currently unclear. In this study, we used bioinformatics prediction algorithms to predict CD4+ T cell epitopes of GapC. Ten peptides were synthesized to investigate the candidate epitopes. Our results showed that the peptides, G4 (GapC 104-123) and G10 (GapC 314-333) were able to induce proliferation of CD4+ T cells and secrete high levels of interferon (IFN)-γ, respectively. In addition, they significantly reduced bacterial loads in tissue and induced immunoprotective effects. It is suggested that G4 and G10 are Th1-type epitopes of S. aureus GapC. This study provides the potential development of the design of epitope-based vaccine against S. aureus.

Staphylococcal and streptococcal species are the most common pathogens that cause bovine mastitis. Induction of a broad-spectrum protective immunity against staphylococci and streptococci by combining multiple antigens into a single vaccine is highlighted. To develop a universal vaccine candidate, a GapC1-tIsdB-TRAP (GIT) construct was generated. The GIT contained the truncated GapC from Streptococcus dysgalactiae, and truncated IsdB and full-length TRAP from Staphylococcus aureus. The humoral and cellular immune responses elicited by GIT were evaluated in mice. Antibody levels against GIT displayed a consistent tendency with antibody levels against GapC, IsdB and TRAP. The level of IFN-γ was higher in the GIT group than in the IsdB group (PPStreptococcus in comparison with GapC group. A significant difference in S. aureus challenge test was detected between the GIT group and the IsdB or TRAP groups (PS. aureus or S. dysgalactiae was produced in the GIT group. These results suggested that the GIT would be a promising common vaccine candidate against S. aureus and Streptococcus.

Manganese transport protein C (MntC) of Staphylococcus aureus represents an excellent vaccine-candidate antigen. The important role of CD4

Staphylococcus aureus can cause different types of diseases from mild skin infections to life-threatening sepsis worldwide. Owing to the emergence and transmission of multidrug-resistant strains, developing an impactful immunotherapy especially vaccine control approach against S. aureus infections is increasingly encouraged and supported. S. aureus manganese transport protein C (MntC), which is a highly-conserved cell surface protein, can elicit protective immunity against S. aureus and Staphylococcus epidermidis. In this study, we evaluated the humoral immune response and CD4+ T cell-mediated immune responses in a mouse peritonitis model. The results showed that MntC-specific antibodies conferred an essential protection for mice to reduce invasion of S. aureus, which was corroborated via the opsonophagocytic killing assay and passive immunization experiment in mice, and moreover MntC-induced Th17 played a remarkable part in preventing S. aureus infection since the MntC-induced protective immunity decreased after neutralization of IL-17 by antibody in vivo and the Th17 adoptive transferred-mice could partly resist S. aureus challenge. In conclusion, we considered that the MntC-specific antibodies and MntC-specific Th17 cells play cooperative roles in the prevention of S. aureus infection.

Interferon-γ (IFN-γ), a cytokine produced by activated natural killer cells and T lymphocytes, is an important regulator of innate and adaptive immunity. Interleukin (IL)-18, also known as IFN-γ-inducing factor, is a cytokine that induces T and natural killer cells to produce IFN-γ. In this study, the chicken IL-18 (ChIL-18) and chicken IFN-γ (ChIFN-γ) genes were inserted into the pET28a prokaryotic expression vector, resulting in pET28a-IL-18 and pET28a-IFN-γ, respectively. These plasmids were transformed into Escherichia coli strain BL21, and the ChIL-18 and ChIFN-γ proteins were expressed and purified. To determine their antiviral activities, 200 ng/mL of ChIL-18 and/or ChIFN-γ were inoculated into chicken embryonic fibroblast cells. After 24 h, one 50% tissue culture infective dose (TCID50) of infectious bursal disease virus (IBDV) was inoculated into the chicken embryonic fibroblast cells. The results showed that the antiviral effect of ChIL-18 and ChIFN-γ in combination was better than that of ChIL-18 or ChIFN-γ alone. Next, 14-day-old chicken were injected with 200 µg of ChIL-18 and/or ChIFN-γ and then were challenged with 103 TCID50 of IBDV via intraperitoneal injection. The results showed that the proliferation of IBDV was inhibited by the injection of the recombinant proteins, especially the combination of ChIL-18 and ChIFN-γ, as evidenced by cytokine detection, quantitative PCR, and pathology analyses. These results indicate that ChIL-18 and ChIFN-γ could inhibit IBDV infection and the combination of ChIL-18 and ChIFN-γ has a better inhibitory effect than either cytokine alone.

The aim of this article was to develop an indirect enzyme-linked immunosorbent assay (ELISA) for efficient detection of the infection of E. coli in cattle. OmpT, a highly conserved protease in all E. coli strains, was successfully expressed in E. coli XL-1-Blue strain with PET32a vector. Molecular weight of recombinant protein was identified by analyzing SDS-PAGE and the immunogenicity of OmpT was confirmed by Western Blotting. The recombinant OmpT was then employed as capture antigen in the ELISA. The antigen concentration and serum dilution were determined using a checkerboard titration. Results showed that the optimal concentration of coated antigen was 1 μg/ml at a serum dilution of 1:640 and the cut-off value of the assay was 0.335. In addition, the cross-reactivity assay showed that the OmpT was E. coli specific and the reproducibility experiments displayed good repeatability of the assay. Three hundred and forty cattle serum samples were tested by rOmpT-ELISA and sera coagulation tests. The ELISA has showed relative sensitivity of 100% and specificity of 96.47%. Results of these experiments indicated that the rOmpT-ELISA is a simple, rapid, and convenient method for detection the infection of E. coli with different serotype strains.

The pathogen Staphylococcus aureus causes a wide range of serious infections, necessitating urgent development of a vaccine against this organism. However, currently developed vaccines are relatively ineffective because of the limited antigenic component that is contained in the vaccine formulations. To develop an effective S. aureus candidate vaccine, overlapping PCR was used to add the truncated immunodominant antigen iron-regulated surface determinant B (IsdB)(N126–P361) (tIsdB) to the N-terminal of intact antigen target of RNAIII activating protein (TRAP) and thus construct a tIsdB-TRAP chimera. The humoral and cellular immune responses against tIsdB-TRAP were compared with those against single or combined formulations. tIsdB-TRAP elicited significantly stronger humoral responses in mice (P

The impact of epidemic Staphylococcus aureus (S. aureus) on public health is increasing. Because of the abuse of antibiotics, the antibiotic resistance of S. aureus is increasing. Thus, there is an urgent need to develop new immunotherapies and immunoprophylaxes. Previous studies showed that the GapC protein of S. aureus, which is a surface protein with high glyceraldehyde 3-phosphate dehydrogenase activity, transferrin binding activity, and other biological activities, is highly conserved. GapC induces an effective humoral immune response in vivo. However, the B-cell epitopes of S. aureus GapC have not been well identified. Here we used the bioinformatics tools to analyze the sequence of GapC, and we generated protective anti-GapC monoclonal antibodies (mAbs). A protective mAb (1F4) showed strong specificity to GapC and the ability to induce macrophages to phagocytose S. aureus. We screened the motif 272GYTEDEIVSSD282, which was recognized by mAb 1F4, using a phage display system. Then, we used site-directed mutagenesis to identify key amino acids in the motif. Residues G272 D276 E277 I278 and V279 formed the core of the 272GYTEDEIVSSD282 motif. In addition, we showed that this epitope peptide induced a protective humoral immune response against S. aureus infection in immunized mice. Our results will be useful for the further study of epitope-based vaccines against S. aureus infection.

The GapC protein is highly conserved surface dehydrogenase among Streptococcus dysgalactiae (S. dysgalactiae) and is shown to be involved in bacterial virulence. Immunization of GapC protein can induce specific CD4(+) T-cell immune responses and protect against S. dysgalactiae infection. However, there are no studies to identify immunodominant CD4(+) T-cell epitopes on GapC protein. In this study, in silico MHC affinity measurement method was firstly used to predict potential CD4(+) T-cell epitopes on GapC protein. Six predictive 15-mer peptides were synthesized and two novel GapC CD4(+) T-cell epitopes, GapC63-77 and GapC96-110, were for the first time identified using CD4(+) T-cells obtained from GapC-immunized BALB/c (H-2(d)) and C57BL/6 (H-2(b)) mice spleen based on cell proliferation and cytokines response. The results showed that peptides containing 63-77 and 96-110 induced significant antigen-specific CD4(+) T-cells proliferation response in vivo. At the same time, high levels of IFN-γ and IL-17A, as well as moderate levels of IL-10 and IL-4 were detected in CD4(+) T-cells isolated from both GapC and peptide-immunized mice in vivo, suggesting that GapC63-77 and GapC96-110 preferentially elicited polarized Th1/Th17-type responses. The characterization of GapC CD4(+) T-cell epitopes not only helps us understand its protective immunity, but also contributes to design effective T-cell epitope-based vaccine against S. dysgalactiae infection.

Iron-regulated surface determinant B (IsdB) of Staphylococcus aureus (S. aureus) is a highly conserved surface protein that can induce protective CD4(+) T-cell immune response. A pivotal role of CD4(+) T-cells in effective immunity against S. aureus infection has been proved, but CD4(+) T-cell epitopes on the S. aureus IsdB have not been well identified. In this study, MHC binding assay was firstly used to predict CD4(+) T-cell epitopes on S. aureus IsdB protein, and six peptides were synthesized to validate the probable epitopes. Two novel IsdB CD4(+) T-cell epitopes, P1 (residues 159-178) and P4 (residues 287-306), were for the first time identified using CD4(+) T-cells obtained from IsdB-immunized C57BL/6 (H-2(b)) and BALB/c (H-2(d)) mice spleen based on cell proliferation and cytokines response. The results showed that P1 and P4 emulsified in Freund's adjuvant (FA) induced much higher cell proliferation compared with PBS emulsified in FA. CD4(+) T-cells stimulated with peptides P1 and P4 secreted significantly higher levels of IFN-γ and IL-17A. However, the level of the cytokine IL-4 almost remained unchanged, suggesting that P1 and P4 preferentially elicited polarized Th1-type responses. In addition, BALB/c mice just respond to P4 not P1, while C57BL/6 mice respond to P1 not P4, implying that epitope P1 and P4 were determined as H-2(b) and H-2(d) restricted epitope, respectively. Taken together, our data may provide an explanation of the IsdB-induced protection against S. aureus and highlight the possibility of developing the epitope-based vaccine against the S. aureus.

The GapC of Streptococcus dysgalactiae (S. dysgalactiae) is a highly conserved surface protein that can induce protective humoral immune response in animals. However, B-cell epitopes on the S. dysgalactiae GapC have not been well identified. In this study, a monoclonal antibody (mAb5B7) against the GapC1-150 protein was prepared. After passive transfer, mAb5B7 could partially protect mice against S. dysgalactiae infection. Eleven positive phage clones recognized by mAb5B7 were identified by screening phage-displayed random 12-peptide library, most of which matched the consensus motif DTTQGRFD. The motif sequence exactly matches amino acids 48-55 of the S. dysgalactiae GapC protein. In addition, the motif 48DTTQGRFD55 shows high homology among various streptococcus species. Site-directed mutagenic analysis further confirmed that residues D48, T50, Q51, G52 and F54 formed the core motif of 48DTTQGRFD55. This motif was the minimal determinant of the B-cell epitope recognized by the mAb5B7. As expected, epitope-peptide evoked protective immune response against S. dysgalactiae infection in immunized mice. Taken together, this identified conserved B-cell epitope within S. dysgalactiae GapC could provide very valuable insights for vaccine design against S. dysgalactiae infection.

Streptococcus dysgalactiae (S. dysgalactiae) GapC protein is a protective antigen that induces partial immunity against S. dysgalactiae infection in animals. To identify the conserved B-cell epitope of S. dysgalactiae GapC, a mouse monoclonal antibody 1E11 (mAb1E11) against GapC was generated and used to screen a phage-displayed 12-mer random peptide library (Ph.D.-12). Eleven positive clones recognized by mAb1E11 were identified, most of which matched the consensus motif TGFFAKK. Sequence of the motif exactly matched amino acids 97-103 of the S. dysgalactiae GapC. In addition, the epitope (97)TGFFAKK(103) showed high homology among different streptococcus species. Site-directed mutagenic analysis further confirmed that residues G98, F99, F100 and K103 formed the core of (97)TGFFAKK(103), and this core motif was the minimal determinant of the B-cell epitope recognized by the mAb1E11. Collectively, the identification of conserved B-cell epitope within S. dysgalactiae GapC highlights the possibility of developing the epitope-based vaccine.

Streptococcus dysgalactiae (S. dysgalactia) GapC is a highly conserved surface dehydrogenase among the streptococcus spp., which is responsible for inducing protective antibody immune responses in animals. However, the B-cell epitope of S. dysgalactia GapC have not been well characterized. In this study, a monoclonal antibody 1F2 (mAb1F2) against S. dysgalactiae GapC was generated by the hybridoma technique and used to screen a phage-displayed 12-mer random peptide library (Ph.D.-12) for mapping the linear B-cell epitope. The mAb1F2 recognized phages displaying peptides with the consensus motif TRINDLT. Amino acid sequence of the motif exactly matched (30)TRINDLT(36) of the S. dysgalactia GapC. Subsequently, site-directed mutagenic analysis further demonstrated that residues R31, I32, N33, D34 and L35 formed the core of (30)TRINDLT(36), and this core motif was the minimal determinant of the B-cell epitope recognized by the mAb1F2. The epitope (30)TRINDLT(36) showed high homology among different streptococcus species. Overall, our findings characterized a conserved B-cell epitope, which will be useful for the further study of epitope-based vaccines.

This paper explores online learning and batch algorithms for detecting malicious Web sites (those involved in criminal scams) using lexical and host-based features of the associated URLs. A data set has been built including malicious and benign URLs, and data mining system Weka has been used as an aid to classify the existent URLs and new coming URLs and evaluate the classification algorithms. A real-time malicious URL detection system has been constructed successfully. The experiment result shows that this method can help to reduce internet access risk effectively.

Candida albicans is the principal human fungal pathogen that leads to life-threatening mycoses worldwide. To study its pathobiology, we characterized genes for two enzymes involved in the de novo purine biosynthesis pathway: ADE8 (encoding phosphoribosylglycinamide formyl-transferase) and GUA1 (GMP synthase). Heterozygous and homozygous disruption strains were constructed for both genes. We found that ADE8 and GUA1 are conditionally essential; i.e. can be bypassed in the presence of exogenous adenine and guanine, respectively, and that ADE8 plays an additional role in the C1-folate pool. Furthermore, the heterozygotes of ADE8/ade8 and GUA1/gua1 were hypersensitive to methotrexate (an inhibitor of de novo synthesis of tetrahydrofolate) and 6-azauracil (a known inhibitor of the IMP dehydrogenase involved in GMP biosynthesis), respectively. In a murine model of systemic candidiasis, the virulence of both heterozygous strains was marginally attenuated, while the ade8/ade8 and gua1/gua1 strains were completely avirulent. Our results and those of others indicate that many conditional essential genes involved in different biosynthesis pathways are required for systemic candidiasis, likely due to the host nutritional constraints imposed on the pathogen.

Type 2C protein phosphatases (PP2C) are monomeric enzymes and their activities require the presence of magnesium or manganese ions. There are seven PP2C genes, ScPTC1, ScPTC2, ScPTC3, ScPTC4, ScPTC5, ScPTC6 and ScPTC7, in Saccharomyces cerevisiae. PTC6 is highly conserved in pathogenic and nonpathogenic yeasts. In the current study we have demonstrated that the Candida albicans CaPTC6 gene could complement the functions of ScPTC6 in the rapamycin and caffeine sensitivities of S. cerevisiae cells, indicating that they are functional homologues. We have also demonstrated that the CaPTC6-encoded protein is a typical PP2C enzyme and that CaPtc6p is localized in the mitochondrion of yeast-form and hyphal cells. However, deletion of CaPTC6 neither affects cell and hyphal growth nor renders Candida cells sensitive to rapamycin and caffeine. Therefore, possibly with a functional redundancy to other mitochondrial phosphatases, CaPtc6p is likely to be involved in the regulation of a mitochondrial physiology. Copyright © 2009 John Wiley & Sons, Ltd.