A novel chimeric recombinant FliC-Pgp3 vaccine promotes immunoprotection against Chlamydia muridarum infection in mice.

The Pgp3 subunit vaccine elicits immune protection against Chlamydia trachomatis infection, but additional adjuvants are still required to enhance its immunoprotective efficacy. Flagellin can selectively stimulate immunity and act as an adjuvant. In this research, the FliC-Pgp3 recombinant was successfully expressed and purified. Tri-immunization with the FliC-Pgp3 vaccine in Balb/C mice induced rapid and persistent germinal center B-cell response and Tfh differentiation, promoting a significantly higher IgG antibody titer compared to the Pgp3 group. FliC-Pgp3 immunization primarily induced Th1-type cellular immunity, leading to higher levels of IFN-γ, TNF-α, and IL-2 secreted by CD4+ T cells than in Pgp3-vaccinated mice. Chlamydia muridarum challenge results showed that FliC-Pgp3-vaccinated mice exhibited more rapid clearance of Chlamydia muridarum colonization in the lower genital tract, ensuring a lower hydrosalpinx rate and cumulative score. Histological analysis showed reduced dilation and inflammatory infiltration in the oviduct and uterine horn of FliC-Pgp3-vaccinated mice compared to the PBS and Pgp3 control. Importantly, tri-immunization with FliC-Pgp3 effectively activated CD4+ T cells and dendritic cells, as confirmed by the adoptive transfer, resulting in better immune protection in recipient mice. In summary, the novel FliC-Pgp3 chimeric is hoped to be a novel vaccine with improved immunoprotection against Chlamydia muridarum.

Preparation of graphene oxide-stabilized Pickering emulsion adjuvant for Pgp3 recombinant vaccine and enhanced immunoprotection against Chlamydia Trachomatis infection.

Background: Traditional emulsion adjuvants are limited in clinical application because of their surfactant dependence. Graphene oxide (GO) has unique amphiphilic properties and therefore has potential to be used as a surfactant substitute to stabilize Pickering emulsions. Methods: In this study, GO-stabilized Pickering emulsion (GPE) was prepared and used as an adjuvant to facilitate an enhanced immune response to the Chlamydia trachomatis (Ct) Pgp3 recombinant vaccine. Firstly, GPE was prepared by optimizing the sonication conditions, pH, salinity, GO concentration, and water/oil ratio. GPE with small-size droplets was characterized and chosen as the candidate. Subsequently, controlled-release antigen delivery by GPE was explored. Cellular uptake behaviors, M1 polarization, and cytokine stimulation by GPE + Pgp3 was considered in terms of the production of macrophages. Finally, GPE's adjuvant effect was evaluated by vaccination with Pgp3 recombinant in BALB/c mouse models. Results: GPE with the smallest droplet sizes was prepared by sonication under 163 W for 2 min at 1 mg/mL GO in natural salinity with a pH of 2 when the water/oil ratio was 10:1 (w/w). The optimized average GPE droplet size was 1.8 µm and the zeta potential was -25.0 ± 1.3 mv. GPE delivered antigens by adsorption onto the droplet surface, demonstrating the controlled release of antigens both in vitro and in vivo. In addition, GPE promoted antigen uptake, which stimulated proinflammatory tumor necrosis factor alpha (TNF-α), enhancing the M1 polarization of macrophages in vitro. Macrophage recruitment was also significantly promoted by GPE at the injection site. In the GPE + Pgp3 treatment group, higher levels of immunoglobin (IgG), immunoglobin G1 (IgG1), immunoglobin G2a (IgG2a) sera, and immunoglobin A (IgA) were detected in vaginal fluid, and higher levels of IFN-γ and IL-2 secretion were stimulated, than in the Pgp3 group, showing a significant type 1 T helper (Th1)-type cellular immune response. Chlamydia muridarum challenging showed that GPE enhanced Pgp3's immunoprotection through its advanced clearance of bacterial burden and alleviation of chronic pathological damage in the genital tract. Conclusion: This study enabled the rational design of small-size GPE, shedding light on antigen adsorption and control release, macrophage uptake, polarization and recruitment, which enhanced augmented humoral and cellular immunity and ameliorated chlamydial-induced tissue damage in the genital tract.

Novel use of graphene oxide quantum dots in a pickering emulsion as a Chlamydia trachomatis vaccine adjuvant.

Graphene oxide quantum dots (GOQDs), which are graphene-based nanoparticles, are potential surfactant substitutes for stabilizing Pickering emulsions, due to their high surface area, biodegradability, and reasonable biocompatibility. In the present study, GOQDs stabilized Pickering emulsion (GQPE) was prepared by simple sonication and then used as an adjuvant to enhance immune responses to the Chlamydia trachomatis Pgp3 recombinant vaccine. Immunization of mice showed that GQPE robustly activates adaptive immunity by efficiently stimulating IgG, sIgA, IFN-γ, IL-4, and TNF-α production. Controlled release repository of antigens both in vivo and in vitro prolonged the immune response. In addition, GQPE enhanced dendritic cell recruitment at the injection site, ensuring rapid and efficient innate immunity. Safety assessment revealed that GQPE does not cause liver, kidney, and myocardial damage in mice, suggesting its favorable biocompatibility. This study provides evidence for the use of GOPE as a facile, effective, and safe strategy to enhance the immune response to Pgp3 recombinant vaccines.

Chitosan particle stabilized Pickering emulsion/interleukin-12 adjuvant system for Pgp3 subunit vaccine elicits immune protection against genital chlamydial infection in mice.

Considering the shortcomings in current chlamydia infection control strategies, a major challenge in curtailing infection is the implementation of an effective vaccine. The immune response induced by C. trachomatis plasmid encoded Pgp3 was insufficient against C. trachomatis infection, which requires adjuvant applications to achieve the robust immune response induced by Pgp3. There is increasing promising in developing adjuvant systems relying on the delivery potential of Pickering emulsions and the immunomodulatory effects of interleukin (IL)-12. Here, owing to the polycationic nature, chitosan particles tended to absorb on the oil/water interphase to prepare the optimized chitosan particle-stabilized Pickering emulsion (CSPE), which was designed as a delivery system for Pgp3 protein and IL-12. Our results showed that the average droplets size of CSPE was 789.47 ± 44.26 nm after a series of optimizations and about 90% antigens may be absorbed by CSPE owing to the positively charged surface (33.2 ± 3mV), and CSPE promoted FITC-BSA proteins uptake by macrophages. Furthermore, as demonstrated by Pgp3-specific antibody production and cytokine secretion, CSPE/IL-12 system enhanced significantly higher levels of Pgp3-specific IgG, IgG1, IgG2a, sIgA and significant cytokines secretion of IFN-γ, IL-2, TNF-α, IL-4. Similarly, vaginal chlamydial shedding and hydrosalpinx pathologies were markedly reduced in mice immunized with Pgp3/CSPE/IL-12. Collectively, vaccination with Pgp3/CSPE/IL-12 regimen elicited robust cellular and humoral immune response in mice resulting in an obvious reduction of live chlamydia load in the vaginal and inflammatory pathologies in the oviduct, which further propells the development of vaccines against C. trachomatis infection.

Lactobacillus Modulates Chlamydia Infectivity and Genital Tract Pathology in vitro and in vivo.

Since we previously reported that women infected with chlamydia had a significant overall reduction in Lactobacillus in the vagina microbiota as compared to those uninfected individuals; the interactions between the altered Lactobacillus and Chlamydia trachomatis, on the other hand, need to be elucidated. Here, we employed both in vitro and in vivo models to evaluate the effects of this changed Lactobacillus on Chlamydia infection. We found that L. iners, L. crispatus, L. jensenii, L. salivarius, L. gasseri, L. mucosae, and L. reuteri all significantly reduced C. trachomatis infection in a dose- and time-dependent manner. The strongest anti-Chlamydia effects were found in L. crispatus (90 percent reduction), whereas the poorest was found in L. iners (50 percent reduction). D (-) lactic acid was the key component in Lactobacillus cell-free supernatants (CFS) to inactivate Chlamydia EBs, showing a positive correlation with the anti-Chlamydia activity. The effects of D (-) lactic acid were substantially attenuated by neutralizing the pH value to 7.0. In vivo, mice intravaginally inoculated with Lactobacillus mixtures (L. crispatus, L. reuteri, and L. iners at a ratio of 1:1:1), but not single Lactobacillus, after genital Chlamydia infection, significantly attenuated the levels of Chlamydia live organism shedding in both the lower genital tract and the intestinal tract, reduced cytokines production (TNF-α, IFN-γ, and IL-1ß) in the vagina, and lessened upper genital tract inflammation and pathogenicity. Taken together, these data demonstrate that Lactobacillus inhibits Chlamydia infectivity both in vivo and in vitro, providing useful information for the development of Lactobacillus as adjunctive treatment in Chlamydia infection.

LncRNA ZEB1-AS1/miR-1224-5p / MAP4K4 axis regulates mitochondria-mediated HeLa cell apoptosis in persistent Chlamydia trachomatis infection.

Persistent infection of Chlamydia trachomatis is thought to be responsible for the debilitating sequelae of blinding trachoma and infertility. Inhibition of host cell apoptosis is a persistent C. trachomatis infection mechanism. ZEB1-AS1 is a long non-coding RNA (lncRNA), which was up-regulated in persistent C. trachomatis infection in our previous work. In this study, we investigated the role of ZEB1-AS1 in persistent infection and the potential mechanisms. The results showed that ZEB1-AS1 was involved in the regulation of apoptosis, and targeted silencing of ZEB1-AS1 could increase the apoptosis rate of persistently infected cells. Mechanically, interference ZEB1-AS1 caused an apparent down-regulation of the Bcl-2/Bax ratio and the repression of the mitochondrial membrane potential with the remarkable release of cytochrome c, resulting in the significant elevation level of caspase-3 activation. Meanwhile, the luciferase reporter assay confirmed that ZEB1-AS1 acted as a sponge for miR-1224-5p to target MAP4K4. The regulatory effect of miR-1224-5p/MAP4K4 on persistent infection-induced antiapoptosis was regulated by ZEB1-AS1. In addition, ZEB1-AS1 inhibited the apoptosis of Chlamydia-infected cells by activating the MAPK/ERK pathway. In conclusion, we found a new molecular mechanism that the ZEB1-AS1/miR-1224-5p/MAP4K4 axis contributes to apoptosis resistance in persistent C. trachomatis infection. This work may help understand the pathogenic mechanisms of persistent C. trachomatis infection and reveal a potential therapeutic strategy for its treatment.

Insights Into Mitochondrial Dynamics in Chlamydial Infection.

Mitochondria are intracellular organelles that are instrumental in the creation of energy, metabolism, apoptosis, and intrinsic immunity. Mitochondria exhibit an extraordinarily high degree of flexibility, and are constantly undergoing dynamic fusion and fission changes. Chlamydia is an intracellular bacterium that causes serious health problems in both humans and animals. Due to a deficiency of multiple metabolic enzymes, these pathogenic bacteria are highly dependent on their eukaryotic host cells, resulting in a close link between Chlamydia infection and host cell mitochondria. Indeed, Chlamydia increase mitochondrial fusion by inhibiting the activation of dynein-related protein 1 (DRP1), which can regulate host cell metabolism for extra energy. Additionally, Chlamydia can inhibit mitochondrial fission by blocking DRP1 oligomerization, preventing host cell apoptosis. These mechanisms are critical for maintaining a favorable environment for reproduction and growth of Chlamydia. This review discusses the molecular mechanisms of mitochondrial fusion and fission, as well as the mechanisms by which Chlamydia infection alters the mitochondrial dynamics and the prospects of limiting chlamydial development by altering mitochondrial dynamics.

Chlamydia trachomatis induces lncRNA MIAT upregulation to regulate mitochondria-mediated host cell apoptosis and chlamydial development.

Chlamydia trachomatis persistent infection is the leading cause of male prostatitis and female genital tract diseases. Inhibition of host cell apoptosis is the key to maintaining Chlamydia survival in vivo, and long noncoding RNAs (lncRNAs) play important roles in its developmental cycle and pathogenesis. However, it is not clear how lncRNAs regulate persistent Chlamydia infection. Here, using a microarray method, we identified 1718 lncRNAs and 1741 mRNAs differentially expressed in IFN-γ-induced persistent C. trachomatis infection. Subsequently, 10 upregulated and 5 downregulated differentially expressed lncRNAs were verified by qRT-PCR to confirm the reliability of the chip data. The GO and KEGG analyses revealed that differentially regulated transcripts were predominantly involved in various signalling pathways related to host immunity and apoptosis response. Targeted silencing of three lncRNAs (MIAT, ZEB1-AS1 and IRF1) resulted in increased apoptosis rates. Furthermore, interference with lncRNA MIAT caused not only an obvious downregulation of the Bcl-2/Bax ratio but also a marked release of cytochrome c, resulting in a significantly elevated level of caspase-3 activation. Meanwhile, MIAT was involved in the regulation of chlamydial development during the persistent infection. Collectively, these observations shed light on the enormous complex lncRNA regulatory networks involved in mitochondria-mediated host cell apoptosis and the growth and development of C. trachomatis.

Long Non-Coding RNA FGD5-AS1 Induced by Chlamydia trachomatis Infection Inhibits Apoptosis via Wnt/ß-Catenin Signaling Pathway.

Background: Chlamydia trachomatis (Ct) is one of the most common bacterial sexually transmitted infection (STI) pathogens in the world, but the exact pathogenic mechanism still needs to be further elucidated. Long non-coding RNAs (lncRNAs) have become vital regulators in many biological processes. Their role in the interaction between Ct and host cells has not been reported. Methods: Microarrays were used to study the expression profiles of lncRNAs and mRNAs in HeLa cells at 12, 24, and 40 h post-infection (hpi). Differentially expressed lncRNAs and mRNAs were verified by RT-qPCR. Coding-non-coding (CNC) network analysis showed co-expression molecules of selected lncRNA. Western blot, flow cytometry, and indirect immunofluorescence were used to detect the effect of lncRNA FGD5-AS1 on apoptosis during Ct infection. Results: Compared with the uninfected group, the number of differential lncRNAs were 2,130, 1,081, and 1,101 at 12, 24, and 40 hpi, and the number of differential mRNAs was 1,998, 1,129, and 1,330, respectively. Ct induced differential expression of large amounts of lncRNAs and mRNAs in HeLa cells, indicating that lncRNAs may play roles in the pathogenesis of Ct. RT-qPCR verified six differential lncRNAs and six differential mRNAs, confirming the reliability of the microarray. Among these molecules, lncRNA FGD5-AS1 was found to be upregulated at 12 and 24 hpi. Coding-non-coding (CNC) network analysis showed that co-expressed differential molecules of FGD5-AS1 at 12 and 24 hpi were enriched in the DNA replication and Wnt signaling pathway. The downregulation of FGD5-AS1 decreased the expression of ß-catenin and inhibited the translocation of ß-catenin and the DNA replication, while it promoted apoptosis of the host cells. Conclusions: DNA replication and apoptosis of host cells were affected by upregulating FGD5-AS1 via Wnt/ß-catenin pathway during Ct infection. This study provides evidence that lncRNAs are involved in the coaction between Ct and hosts, and provides new insights into the study of lncRNAs that regulate chlamydial infection.

Alterations of Vaginal Microbiota in Women With Infertility and Chlamydia trachomatis Infection.

Chlamydia trachomatis (C. trachomatis) is the most common etiological agent of bacterial sexually transmitted infections (STIs) worldwide and causes serious health sequelae such as cervicitis, pelvic inflammatory disease, and even infertility if ascending from the lower to the upper female genital tract. Previous studies have revealed the pivotal role of vaginal microbiota in susceptibility to STIs. However, alterations in the vaginal microbiota in women who are infertile and infected with C. trachomatis remain unknown. This study used metagenomic analysis of sequenced 16S rRNA gene amplicons to examine the vaginal microbial profiles of women with tubal infertility who were C. trachomatis-negative and those who were C. trachomatis-positive pre- and post-antibiotic treatment. Women who were C. trachomatis-negative and deemed healthy were recruited as references of eubiosis and dysbiosis. Women with tubal infertility and C. trachomatis infection presented a unique Lactobacillus iners-dominated vaginal microbiota rather than one dominated by Lactobacillus crispatus and displayed a decrease in Lactobacillus, Bifidobacterium, Enterobacter, Atopobium, and Streptococcus, accompanied by decreased levels of cytokines such as interferon (IFN)-γ and interleukin (IL)-10. This altered vaginal microbiota could be restored with varying degrees after standard treatment for C. trachomatis. This shift could be a predictive vaginal microbiota signature for C. trachomatis infection among females with tubal infertility, while no significant differences in phylum, class, and operational taxonomic unit (OTU) levels were observed between women with tubal infertility who were C. trachomatis-negative and healthy controls. This is the first study to provide data on the association of vaginal microbiota with C. trachomatis infection among women with tubal infertility and highlights unprecedented potential opportunities to predict C. trachomatis infection.

Chlamydia trachomatis Pgp3 protein regulates oxidative stress via activation of the Nrf2/NQO1 signal pathway.

AIM: Chlamydia trachomatis has evolved various strategies to alleviate oxidative stress of host cells to maintain their intracellular survival. However, the exact mechanism of anti-oxidative stress of C. trachomatis is still unclear. The activation of nuclear factor erythroid 2-related factor 2/quinone oxidoreductase (Nrf2/NQO1) signal pathway has been identified as an efficient antioxidant defensive mechanism used by host cells to counteract oxidative stress. Pgp3 is a pivotal virulence factor of C. trachomatis involved in intracellular survival. The aim of this study is to explore the role of Pgp3 on Nrf2/NQO1 signal pathway against oxidative stress. MAIN METHODS: After HeLa cells were stimulated with Pgp3 protein, Nrf2 location and the inclusion bodies of C. trachomatis were detected by indirect immunofluorescence, western blotting and Oxidative stress assay kits were used to separately determine the protein expression and the content of malondialdehyde (MDA), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) before and after the interference of Nrf-2 and NQO1. KEY FINDINGS: Pgp3 promoted the nuclear translocation of Nrf2 to increase NQO1 expression and reduced oxidative stress induced by LPS to contribute to the survival of C. trachomatis. Inhibition of Nrf2/NQO1 signal pathway with Nrf2 inhibitor and down-regulation of NQO1 with siRNA-NQO1 suppressed oxidative stress resistance induced by Pgp3. SIGNIFICANCE: Here we found that Pgp3 alleviated oxidative stress to promote the infectivity of C. trachomatis through activation of Nrf2/NQO1 signal pathway, which provided a novel understanding of the effects of Pgp3 in the pathogenesis of C. trachomatis.

O/W Nanoemulsion as an Adjuvant for an Inactivated H3N2 Influenza Vaccine: Based on Particle Properties and Mode of Carrying.

BACKGROUND AND PURPOSE: Adjuvant can reduce vaccine dosage and acquire better immune protection to the body, which helps to deal with the frequent outbreaks of influenza. Nanoemulsion adjuvants have been proved efficient, but the relationship between their key properties and the controlled release which greatly affects immune response is still unclear. The present work explores the role of factors such as particle size, the polydispersity index (PDI), stability and the safety of nanoemulsions by optimizing the water concentration, oil phase and modes of carrying, to explain the impact of those key factors above on adjuvant effect. METHODS: Isopropyl myristate (IPM), white oil, soybean oil, and grape-kernel oil were chosen as the oil phase to explore their roles in emulsion characteristics and the adjuvant effect. ICR mice were immunized with an emulsion-inactivated H3N2 split influenza vaccine mixture, to compare the nanoemulsion's adjuvant with traditional aluminium hydroxide or complete Freund's adjuvant. RESULTS: Particle size of all the nanoemulsion formed in our experiment ranged from 20 nm to 200 nm and did not change much when diluted with water, while the PDI decreased obviously, indicating that the particles tended to become more dispersive. Formulas with 80% or 85.6% water concentration showed significant higher HAI titer than aluminium hydroxide or complete Freund's adjuvant, and adsorption rather than capsule mode showed higher antigen delivery efficiency. As mentioned about oil phase, G (IPM), F (white oil), H (soybean oil), and I (grape-kernel oil) showed a decreasing trend in their adjuvant efficiency, and nanoemulsion G was the best adjuvant with smaller and uniform particle size. CONCLUSION: Emulsions with a smaller, uniform particle size had a better adjuvant effect, and the adsorption mode was generally more efficient than the capsule mode. The potential adjuvant order of the different oils was as follows: IPM > white oil > soybean oil > grape-kernel oil.

Chlamydia trachomatis Plasmid Protein pORF5 Up-Regulates ZFAS1 to Promote Host Cell Survival via MAPK/p38 Pathway.

Long non-coding RNAs (lncRNAs) have been demonstrated to play essential roles in many diseases. However, few studies have shown that lncRNAs take part in the pathogenesis of Chlamydia trachomatis (C. trachomatis). Here, we used a lncRNA microarray to detect the global lncRNA expression profiles in HeLa cells transfected with pORF5 plasmid protein, an important virulence factor for C. trachomatis. The differentially expressed lncRNAs and mRNAs screened by microarray were selected for validation by quantitative real-time PCR. The up-regulated lncRNA zinc finger antisense 1 (ZFAS1) was presumed to involved in MAPK pathways by bioinformatics analysis. Inhibition of ZFAS1 decreased the apoptotic rate of pORF5 and reduced the infectivity of C. trachomatis, and MAPK/p38 pathway was involved in anti-apoptotic effect induced by ZFAS1. Therefore, the present study confirmed that pORF5 up-regulates ZFAS1 to promote host cell survival via MAPK/p38 pathway and influences the infectivity of C. trachomatis.

Serological diagnosis of Mycoplasma pneumoniae infection by using the mimic epitopes.

Nowadays, there is lack of effective serological detection method for Mycoplasma pneumoniae (M. pneumoniae) infection in clinic. In this study, the mimic epitopes of M. pneumoniae were screened to evaluate the role in the serodiagnosis of M. pneumoniae infection. The M. pneumoniae-positive serum was used as the target for biopanning to phage display random 7-peptide library. The positive phage clones were selected and the DNA were sequenced and analyzed by BLAST. The representative phages were identified using dot immunoblotting and ELISA. The exogenous heptapeptides were synthesized and their reactions with M. pneumonia-positive serum were tested by indirect ELISA. Two heptapeptides, namely heptapeptide 1: TVNFKLY and heptapeptide 2: LPQRLRT, were screened out from the randomly selected 40 phages after the four bio-panning rounds. They had high homologies to some M. pneumoniae antigens. Besides, the representative bacteriophage containing heptapeptide 1 or 2 could react with the M. pneumonia- positive serum. The sensitivities of heptapeptide 1 and heptapeptide 2 for the diagnosis of M. pneumoniae infection were 90.1 and 80.0%, respectively, and the specificities were 94.3 and 97.1%, respectively. Therefore the two heptapeptides were the mimic epitopes of M. pneumoniae and might have potential serological diagnosis value for M. pneumoniae infection.

Visible-light-driven H2 production and decomposition of 4-nitrophenol over nickel phosphides.

Photocatalytic H2 production and photocatalytic decomposition are efficient and economical methods to obtain hydrogen fuel and dispose of organic pollutants. In this paper, amorphous nickel phosphide (Ni2P) is synthesized by an extremely simple precipitation method under low temperature. The prepared nickel phosphide was not only used to produce H2 in the presence of Eosin Y as sensitizer and triethanolamine (TEOA) as a sacrificial electron donor but also to degrade 4-nitrophenol under visible light illumination. The rate of hydrogen evolution is 34.0 µmol h-1 g-1 and the degradation efficiency is 25.5% within 4 h at initial 5.0 mg L-1 4-nitrophenol. Probable photocatalytic mechanisms were discussed. The present work is expected to contribute toward the hydrogen evolution and disposal of highly toxic pollutants by cost-effective photocatalytic means.

[Preparation and identification of the polyclonal antibody against ATRX-C2193-2492].

Objective To prepare the polyclonal antibody against human alpha thalassemia/mental retardation syndrome X-linked (ATRX) C-terminal and study the distribution and expression of ATRX protein in human cervical cancer tissues. Methods The antiserum was obtained from the BALB/c mice immunized with 6His-ATRX-C2193-2492 protein and then purified by the saturated ammonium sulfate precipitation and affinity chromatography. The titer of anti-ATRX polyclonal antibody was determined by ELISA. Its specificity was identified by SDS-PAGE analysis and Western blotting. The expression and location of ATRX in human cervical tissues were analyzed by immunohistochemistry. Results The titer of the polyclonal antibody against 6His-ATRX-C2193-2492 protein was about 1:12 800. The antibody could recognize 6His-ATRX-C2193-2492 protein specifically. With the polyclonal antibody, the target protein was found mainly in the nucleus of para-carcinoma tissues, and it was also expressed in the nucleus of cervical cancer tissue cells, but the expression in the latter was obviously lower. Conclusion The polyclonal antibody against 6His-ATRX-C2193-2492 protein has been produced successfully and used to detect ATRX protein in human cervical cancer tissues.

Evaluation of Rv0220, Rv2958c, Rv2994 and Rv3347c of Mycobacterium tuberculosis for serodiagnosis of tuberculosis.

Tuberculosis (TB), the leading cause of death among infectious diseases worldwide, is caused by Mycobacterium tuberculosis (M. tuberculosis). Early accurate diagnosis means earlier prevention, treatment and control of TB. To confirm efficient diagnostic antigens for M. tuberculosis, the serodiagnosis value of four recombinant proteins including Rv0220, Rv2958c, Rv2994 and Rv3347c was evaluated in this study. The specificities and sensitivities of four recombinant proteins were determined based on enzyme-linked immunosorbent assay (ELISA) by screening sera from smear-positive pulmonary TB patients (n = 92), uninfected individuals (n = 60) and patients with Mycoplasma pneumoniae (n = 32) that potentially cross-react with M. tuberculosis. The ELISAs showed that Rv0220, Rv2958c, Rv2994 and Rv3347c exhibited high specificities and sensitivities in detecting immunoglobulin G (IgG) antibody, with 98.3/91.3%, 91.7/85.9%, 93.3/89.1% and 93.3/80.4% respectively. According to the receiver-operating characteristic (ROC) analysis, the area under the ROC of the target proteins was 0.988, 0.969, 0.929 and 0.945 respectively. Western blot was established to evaluate the immunoreactivities of target proteins to mice and human sera. Results demonstrated that Rv0220, Rv2958c, Rv2994 and Rv3347c could specifically recognize TB-positive sera and the sera of mice immunized with the corresponding protein. Thus, Rv0220, Rv2958c, Rv2994 and Rv3347c were valuable potential diagnostic antigens for M. tuberculosis.

TNF-α-inducing protein of Helicobacter pylori induces epithelial-mesenchymal transition (EMT) in gastric cancer cells through activation of IL-6/STAT3 signaling pathway.

Tumor necrosis factor (TNF)-α-inducing protein (Tipα) is a newly identified carcinogenic factor secreted by Helicobacter pylori (H. pylori). Although it has been proved that Tipα is a strong inducer of epithelial-mesenchymal transition (EMT), a crucial process of migration, the exact molecular mechanism is unknown. Current evidence indicates that the oncogenic transcription factor signal transducers and activators of transcription 3 (STAT3) is inappropriately activated in multiple malignancies, including gastric cancer. In this study, we showed that Tipα significantly down-regulated the expression of EMT-related markers E-cadherin as well as up-regulated N-cadherin and vimentin in SGC7901 cells, with typical morphological changes of EMT. Tipα also promoted proliferation and migration of SGC7901 cells. Furthermore, Tipα activated interleukin-6 (IL-6)/STAT3 signaling pathway in SGC7901 cells. The effects of Tipα treatment observed was abolished when we block IL-6/STAT3 signaling pathway. Altogether, our data demonstrated that Tipα may accelerate tumor aggressiveness in gastric cancer by promoting EMT through activation of IL-6/STAT3 pathway.

The mimic epitopes of Mycobacterium tuberculosis screened by phage display peptide library have serodiagnostic potential for tuberculosis.

Mycobacterium tuberculosis is an obligate pathogenic bacterial species in the family of Mycobacteriaceae and attracts excessive immune responses which cause pathology of the lungs in active tuberculosis. The lack of more sensitive and effective diagnosis reagents advocates a further recognition for the fast diagnostic and immunological measures for tuberculosis. Here, two 12-mer peptides with core sequences of SVSVGMKPSPRP (CS1) and TMGFTAPRFPHY (CS2) were screened from a phage display random peptide library using the purified mixed tuberculosis-positive serum as a target. Enzyme-linked immunosorbent assay (ELISA) and dot immunobinding assay verified that positive phages exhibited strong binding affinity to mixed tuberculosis-positive serum. BLAST analysis showed that the two sequences may be mimotopes of the Mycobacterium tuberculosis The diagnostic potential for two synthetic mimotope peptides CS1 and CS2 was evaluated using different panels of serum samples (n = 181) by ELISA, and the diagnostic parameters were calculated. CS1 and CS2 achieved sensitivity of 89.41% and 85.88%, and specificities were 90.63% and 87.50%, respectively. We hypothesized that the diagnostic based on CS1 and CS2 may become a promising strategy to enhance the detection of Mycobacterium tuberculosis infection due to higher specificity and sensitivity. Therefore, CS1 and CS2 may possess potentials to provide an experimental basis for the diagnosis of tuberculosis.

Identification of potential therapeutic targets for lung cancer by bioinformatics analysis.

The aim of the present study was to identify potential therapeutic targets for lung cancer and explore underlying molecular mechanisms of its development and progression. The gene expression profile datasets no. GSE3268 and GSE19804, which included five and 60 pairs of tumor and normal lung tissue specimens, respectively, were downloaded from Gene Expression Omnibus. Differentially expressed genes (DEGs) between lung cancer and normal tissues were identified, and gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis of the DEGs was performed. Furthermore, protein­protein interaction (PPI) networks and a transcription factor (TF) regulatory network were constructed and key target genes were screened. A total of 466 DEGs were identified, and the PPI network indicated that IL­6 and MMP9 had key roles in lung cancer. A PPI module containing 34 nodes and 547 edges was obtained, including PTTG1. The TF regulatory network indicated that TFs of FOSB and LMO2 had a key role. Furthermore, MMP9 was indicated to be the target of FOSB, while PTTG1 was the target of LMO2. In conclusion, the bioinformatics analysis of the present study indicated that IL­6, MMP9 and PTTG1 may have key roles in the progression and development of lung cancer and may potentially be used as biomarkers or specific therapeutic targets for lung cancer.