Prostein expression on circulating tumor cells as a prognostic marker in metastatic castration-resistant prostate cancer.

BACKGROUND: Identification of emerging molecular biomarkers on circulating tumor cells (CTCs) represents an attractive feature of liquid biopsy that facilitates precision and tailored medicine in the management of metastatic castration-resistant prostate cancer (mCRPC). Prostein is an androgen-regulated transmembrane protein with high prostate specificity. Prostein-positive circulating tumor cell (CTC) was recently suggested to have diagnostic potential; however, no study has been conducted to evaluate its prognostic value in mCRPC. METHODS: CTCs from mCRPC patients were enumerated using the CellSearch System. Prostein-positive CTCs were identified by immunostaining results. The relationships between prostein expression on CTCs and PSA response rate, PSA progression-free survival (PSA-PFS), radiographic progression-free survival (PFS), and overall survival (OS) were tested by Fisher's exact test or evaluated using Kaplan-Meier and multivariate Cox analyses. RESULTS: Prostein-positive CTCs were identified in 31 of 87 baseline samples from mCRPC patients and 16 of 51 samples collected at the first follow-up visit. PSA response rates were significantly lower in baseline prostein-positive patients (0%, 0/31) than in prostein-negative patients (19.6%, 11/56) (p = 0.007). The 31 prostein-positive patients had significantly shorter PSA-PFS (p < 0.001), radiographic PFS (p < 0.001), and OS (p = 0.018), compared to the 56 prostein-negative patients at baseline. The association with PSA-PFS maintained its significance (p = 0.028) in multivariate analyses. Analyzing prostein expression at the first follow-up as well as the conversion of prostein expression from baseline to follow-up samples not only confirmed the association with PSA-PFS, but also demonstrated prognostic significance with OS. CONCLUSION: Our study provides the first evidence to support the potential of prostein expression on CTCs to serve as a novel prognostic marker in mCRPC patients. Future large-scale prospective studies are needed to validate our findings.

Association between the infections of Trichomonas vaginalis and uterine cervical human papillomavirus: a meta-analysis.

Trichomonas vaginalis (TV) may have an impact on other reproductive tract infections. Studies on the connection between the infection of TV and human papillomavirus (HPV) have been inconsistent. We performed a systematic review of the relevant articles through keywords that satisfy the criteria and filtered the articles according to the inclusion and exclusion criteria. A total of 16 eligible studies were screened for the meta-analysis, involving a total of 150,605 women. RevMan 5.4 software was used for meta-analysis of the selected literatures. The results showed that the papers included in this study had good homogeneity and no significant publication bias was found in the current analysis. The pooled estimates using a fixed-effects model showed that TV was more prevalent in HPV-infected women than in non-infected women [odds ratio (OR): 1.51, 95% confidence interval (CI): 1.29-1.75]; In turn, HPV was more widespread in TV-infected women than in uninfected women (OR: 3.62, 95% CI: 2.71-4.85). Moreover, the interaction between TV and HPV infection was insensitive to the deletion of some studies and correlation coefficients, consequently, the results were robust and reliable. These results suggested that TV is positively associated with HPV infection, and HPV is also a risk factor for TV infection.

The correlation between Trichomonas vaginalis infection and reproductive system cancer: a systematic review and meta-analysis.

BACKGROUND: Trichomonas vaginalis (T. vaginalis) is a microaerophilic protozoan parasite which is responsible for trichomoniasis, the most common non-viral sexually transmitted infection in the world. The infection greatly damages the reproductive system. However, whether T. vaginalis infection can cause reproductive system cancer remains controversial. METHODS: This study systematically searched PubMed, EMBASE, Ovid and Google scholar, and 144 relevant articles were retrieved and classified into three categories: epidemiological investigations (68), reviews (30) and research articles (46). These three types of articles were verified according to their respective inclusion and exclusion criteria. Stata 16 was used to conduct a meta-analysis on the articles of epidemiological investigations for analysing the correlation between T. vaginalis infection and reproductive system cancer. RESULTS: The result of meta-analysis indicated that the rate of T. vaginalis infection in the cancer group was significantly higher than that in the non-cancer group (OR = 1.87, 95% CI 1.29-2.71, I2 = 52%). Moreover, the cancer rate of the population infected with T. vaginalis was significantly higher than that of the population without T. vaginalis infection (OR = 2.77, 95% CI 2.37-3.25, I2 = 31%). The review articles and most research articles stated that the infection of T. vaginalis could lead to cancer and the pathogenic mechanisms were as follows: T. vaginalis promoting inflammatory response, T. vaginalis infection changing the internal environment around parasitic sites and signal transduction pathway, the metabolites secreted by T. vaginalis inducing carcinogenesis and T. vaginalis increasing other pathogenic microbial infection to promote the occurrence of cancer. CONCLUSIONS: Our study confirmed that there was a correlation between the infection of T. vaginalis and reproductive system cancer, and provided some possible research directions for clarifying the carcinogenic mechanisms caused by T. vaginalis infection.

Recombinant Toxoplasma gondii Calreticulin protein provides partial protection against acute and chronic toxoplasmosis.

Toxoplasma gondii, a highly prevalent apicomplexan pathogen, can cause serious or even fatal toxoplasmosis in both animals and humans. Immunoprophylaxis is considered a promising strategy for controlling this disease. Calreticulin (CRT) is known as a pleiotropic protein, which is critical for calcium storage and phagocytosis of apoptotic cells. Our study examined the protective effects of recombinant T. gondii Calreticulin (rTgCRT) as a recombinant subunit vaccine against the T. gondii challenge in mice. Here, rTgCRT was successfully expressed in vitro using prokaryptic expression system. Polyclonal antibody (pAb) has been prepared by immunizing Sprague Dawley rats with rTgCRT. Western blotting showed that rTgCRT and natural TgCRT protein were recognized by serum of T. gondii infected mice and rTgCRT pAb, respectively. T lymphocyte subsets and antibody response were monitored using flow cytometry and enzyme-linked immunosorbent assay (ELISA). The results showed that ISA 201 rTgCRT could stimulate lymphocyte proliferation and induce high levels of total and subclasses of IgG. After the RH strain challenge, a longer survival period was given by the ISA 201 rTgCRT vaccine compared to the control groups; after infection with the PRU strain, we observed a 100% survival rate and a significant reduction in cysts load and size. In the neutralization test, high concentrations of rat-rTgCRT pAb provided 100% protection, while in the passive immunization trial, only weak protection was observed after RH challenge, indicating that rTgCRT pAb needs further modification to improve its activity in vivo. Taken together, these data confirmed that rTgCRT can trigger strong cellular and humoral immune responses against acute and chronic toxoplasmosis.

Dynamic changes in TIGIT expression on the T-cell surface and TIGIT-mediated T-cell dysfunction in the brains of mice with chronic Toxoplasma gondii infection.

The immunosuppressive receptor TIGIT plays a vital role in the regulation of the immune system's response to pathogens. However, the expression pattern of this receptor in mouse brains during infection with Toxoplasma gondii cysts is not known. Here, we provide evidence of immunological changes and TIGIT expression in infected mouse brains through flow cytometry and QPCR. The obtained results show that TIGIT expression on brain T cells rose considerably after infection. T. gondii infection triggered the conversion of TIGIT+ TCM cells to TIGIT+ TEM cells and reduced their cytotoxicity. During the whole period of T. gondii infection, high intensity and persistent expression of IFN-γ and TNF-α in brain and serum of mice. This study shows that chronic T. gondii infection increases TIGIT expression on brain T cells and affects their immune function.

Vaccination with recombinant Toxoplasma gondii bradyzoite-formation deficient 1 (rTgBFD1) antigen provides partial protective immunity against chronic T. gondii infection.

As an apicomplexan pathogen, Toxoplasma gondii still remains a major threat to public health and requires special attention. In fact, positive attempts to identify more effective antigens to provide protection are important to control toxoplasmosis. Latest scientific advances in T. gondii study hint at the probability of the T. gondii bradyzoite-formation deficient 1 (TgBFD1) as an ideal vaccine candidate, since this molecule plays a critical role in regulating the chronic infection of T. gondii. Thus, BALB/c mouse models of acute and chronic T. gondii infections were used to evaluate the TgBFD1 protection efficacy in this study. Before conducting animal trials, antigen analysis of TgBFD1 was performed using DNAstar software and Western blots. The preliminary results suggested that TgBFD1 should be a potent immunogen. Then, this conclusion is confirmed by ELISA assays. After immunization with rTgBFD1, high levels of specific IgG, IgG1, IgG2a, and cytokines (Interferon γ and interleukin 10) were observed, indicating that TgBFD1 could induce strong protective antibody responses. While TgBFD1-specific IgG antibodies were measurable in vaccinated mice, no protection was observed in the acute T. gondii infection (RH strain) assay. However, a noticeable decrease in brain cysts counts of immunized mice compared with negative controls in the latent T. gondii infection (PRU strain) assay was observed. Taken together, these results indicated that rTgBFD1 had the remarkable ability to elicit both humoral and cellular immune responses and could provide partial protective immunity against chronic T. gondii infection.

Genomic Aberrations in Circulating Tumor DNAs from Palbociclib-Treated Metastatic Breast Cancer Patients Reveal a Novel Resistance Mechanism.

Previously undescribed molecular mechanisms of resistance will emerge with the increased use of cyclin-dependent kinase 4/6 inhibitors in clinical settings. To identify genomic aberrations in circulating tumor DNA associated with treatment resistance in palbociclib-treated metastatic breast cancer (MBC) patients, we collected 35 pre- and post-treatment blood samples from 16 patients with estrogen receptor-positive (ER+) MBC, including 9 with inflammatory breast cancer (IBC). Circulating cell-free DNAs (cfDNAs) were isolated for sequencing using a targeted panel of 91 genes. Our data showed that FBXW7 and CDK6 were more frequently altered in IBC than in non-IBC, whereas conversely, PIK3CA was more frequently altered in non-IBC than in IBC. The cfDNA samples collected at follow-up harbored more mutations than baseline samples. By analyzing paired samples, we observed a higher percentage of patients with mutations in RB1, CCNE1, FBXW7, EZH2, and ARID1A, but a lower proportion of patients with mutated TSC2 at the post-treatment stage when they developed progression. Moreover, acquisition of CCNE1 mutations or loss of TSC2 mutations after treatment initiation conferred an unfavorable prognosis. These data provide insights into the relevance of novel genomic alterations in cfDNA to palbociclib resistance in MBC patients. Future large-scale prospective studies are warranted to confirm our findings.

A recombinant protein vaccine encoding Toxoplasma gondii Cyst wall 2 (dense granule protein 47) provides partial protection against acute and chronic T. gondii infection in BALB/c mice.

Toxoplasma gondii poses a major threat to economies and public health, and there are still no available vaccines for human against T. gondii infection. T. gondii cyst wall 2 (TgCST2, also known as dense granule protein-47) is a critical molecule in the establishment of chronic infection, making it a potential vaccine candidate. In this research, the recombinant TgCST2 (rTgCST2) was employed to evaluate the protective efficacy of TgCST2 antigen using BALB/c mice model against T. gondii infections via active immunization trials. First, the strong immunogenicity of TgCST2 was indicated by immunoblotting and immunofluorescence, which mean that TgCST2 might elicit robust immune responses in the organism. Then, after triply subcutaneous immunization with rTgCST2/ISA 201 emulsion, high levels of Toxoplasma-specific IgG, IgG1, IgG2a and cytokines (Interferon γ and interleukin 10) further suggested that TgCST2 was a promising immunogenic antigen. More importantly, this antigen could prolong survival in RH strain infected mice and resulted in the lower brain cysts size and number of PRU strain infected mice. These preliminary results demonstrated the immunoprophylactic effects of TgCST2 antigen and will inform new studies in developing subunit recombinant vaccines against T. gondii.

Characterization of the complete chloroplast genome of Brassica oleracea var. italica and phylogenetic relationships in Brassicaceae.

Broccoli (Brassica oleracea var. italica) is an important B. oleracea cultivar, with high economic and agronomic value. However, comparative genome analyses are still needed to clarify variation among cultivars and phylogenetic relationships within the family Brassicaceae. Herein, the complete chloroplast (cp) genome of broccoli was generated by Illumina sequencing platform to provide basic information for genetic studies and to establish phylogenetic relationships within Brassicaceae. The whole genome was 153,364 bp, including two inverted repeat (IR) regions of 26,197 bp each, separated by a small single copy (SSC) region of 17,834 bp and a large single copy (LSC) region of 83,136 bp. The total GC content of the entire chloroplast genome accounts for 36%, while the GC content in each region of SSC,LSC, and IR accounts for 29.1%, 34.15% and 42.35%, respectively. The genome harbored 133 genes, including 88 protein-coding genes, 37 tRNAs, and 8 rRNAs, with 17 duplicates in IRs. The most abundant amino acid was leucine and the least abundant was cysteine. Codon usage analyses revealed a bias for A/T-ending codons. A total of 35 repeat sequences and 92 simple sequence repeats were detected, and the SC-IR boundary regions were variable between the seven cp genomes. A phylogenetic analysis suggested that broccoli is closely related to Brassica oleracea var. italica MH388764.1, Brassica oleracea var. italica MH388765.1, and Brassica oleracea NC_0441167.1. Our results are expected to be useful for further species identification, population genetics analyses, and biological research on broccoli.

Dynamic Expressions of TIGIT on Splenic T Cells and TIGIT-Mediated Splenic T Cell Dysfunction of Mice With Chronic Toxoplasma gondii Infection.

As an immunosuppressive receptor, T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) play a critical part in cellular immune regulation mediated by pathogen infection. Whereas, TIGIT expression on splenic T cells in hosts infected with Toxoplasma gondii cysts has not been studied. In this study, we detected TIGIT expression and the changes of immune function in the spleen by flow cytometry and real-time PCR (RT-PCR). We found that TIGIT expression on splenic T cells increased significantly post infection. At the same time, splenic TIGIT+TCM cells were activated and transformed into TIGIT+TEM cells during the infection, and the cytotoxicity of TIGIT+ T cells was reduced in the later stage of infection. This study shows that chronic T. gondii infection can upregulate TIGIT expression on the surface of T cells and affect immune cell function.

Integration of circulating tumor cell and neutrophil-lymphocyte ratio to identify high-risk metastatic castration-resistant prostate cancer patients.

BACKGROUND: The neutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR), and circulating tumor cells (CTCs) have been associated with survival in castration-resistant prostate cancer (CRPC). However, no study has examined the prognostic value of NLR and PLR in the context of CTCs. METHODS: Baseline CTCs from mCRPC patients were enumerated using the CellSearch System. Baseline NLR and PLR values were calculated using the data from routine complete blood counts. The associations of CTC, NLR, and PLR values, individually and jointly, with progression-free survival (PFS) and overall survival (OS), were evaluated using Kaplan-Meier analysis, as well as univariate and multivariate Cox models. RESULTS: CTCs were detected in 37 (58.7%) of 63 mCRPC patients, and among them, 16 (25.4%) had ≥5 CTCs. The presence of CTCs was significantly associated with a 4.02-fold increased risk for progression and a 3.72-fold increased risk of death during a median follow-up of 17.6 months. OS was shorter among patients with high levels of NLR or PLR than those with low levels (log-rank P = 0.023 and 0.077). Neither NLR nor PLR was individually associated with PFS. Among the 37 patients with detectable CTCs, those with a high NLR had significantly shorter OS (log-rank P = 0.024); however, among the 26 patients without CTCs, the OS difference between high- and low-NLR groups was not statistically significant. Compared to the patients with CTCs and low NLR, those with CTCs and high levels of NLR had a 3.79-fold risk of death (P = 0.036). This association remained significant after adjusting for covariates (P = 0.031). Combination analyses of CTC and PLR did not yield significant results. CONCLUSION: Among patients with detectable CTCs, the use of NLR could further classify patients into different risk groups, suggesting a complementary role for NLR in CTC-based prognostic stratification in mCRPC.

Whole-exome sequencing identifies somatic mutations and intratumor heterogeneity in inflammatory breast cancer.

Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer. Although it is a rare subtype, IBC is responsible for roughly 10% of breast cancer deaths. In order to obtain a better understanding of the genomic landscape and intratumor heterogeneity (ITH) in IBC, we conducted whole-exome sequencing of 16 tissue samples (12 tumor and four normal samples) from six hormone-receptor-positive IBC patients, analyzed somatic mutations and copy number aberrations, and inferred subclonal structures to demonstrate ITH. Our results showed that KMT2C was the most frequently mutated gene (42%, 5/12 samples), followed by HECTD1, LAMA3, FLG2, UGT2B4, STK33, BRCA2, ACP4, PIK3CA, and DNAH8 (all nine genes tied at 33% frequency, 4/12 samples). Our data indicated that PTEN and FBXW7 mutations may be considered driver gene mutations for IBC. We identified various subclonal structures and different levels of ITH between IBC patients, and mutations in the genes EIF4G3, IL12RB2, and PDE4B may potentially generate ITH in IBC.

Improved Prognostic Stratification Using Circulating Tumor Cell Clusters in Patients with Metastatic Castration-Resistant Prostate Cancer.

Liquid biopsy-based biomarkers have advantages in monitoring the dynamics of metastatic castration-resistant prostate cancer (mCRPC), a bone-predominant metastatic disease. Previous studies have demonstrated associations between circulating tumor cells (CTCs) and clinical outcomes of mCRPC patients, but little is known about the prognostic value of CTC-clusters. In 227 longitudinally collected blood samples from 64 mCRPC patients, CTCs and CTC-clusters were enumerated using the CellSearch platform. The associations of CTC and CTC-cluster counts with progression-free survival (PFS) and overall survival (OS), individually and jointly, were evaluated by Cox models. CTCs and CTC-clusters were detected in 24 (37.5%) and 8 (12.5%) of 64 baseline samples, and in 119 (52.4%) and 27 (11.9%) of 227 longitudinal samples, respectively. CTC counts were associated with both PFS and OS, but CTC-clusters were only independently associated with an increased risk of death. Among patients with unfavorable CTCs (≥5), the presence of CTC-clusters signified a worse survival (log-rank p = 0.0185). mCRPC patients with both unfavorable CTCs and CTC-clusters had the highest risk for death (adjusted hazard ratio 19.84, p = 0.0072), as compared to those with <5 CTCs. Analyses using longitudinal data yielded similar results. In conclusion, CTC-clusters provided additional prognostic information for further stratifying death risk among patients with unfavorable CTCs.

A Novel Vaccine Candidate: Recombinant Toxoplasma gondii Perforin-Like Protein 2 Stimulates Partial Protective Immunity Against Toxoplasmosis.

Toxoplasma gondii is an apicomplexan pathogen infecting 2 billion people and numerous livestock, causing a major threat to economies and human health. Passive-active immunoprophylaxis is an efficient approach to provide protection against toxoplasmosis. T. gondii perforin-like protein 2 (TgPLP2) contains a membrane attack complex/perforin (MACPF) domain, making it a potential vaccine candidate. Here, we aimed to assess the protection efficacy of TgPLP2 using Bagg albino/c (BALB/c) mice model. The Escherichia coli system was used to obtain the recombinant TgPLP2 (rTgPLP2). Mice challenged by anti-rTgPLP2 polyclonal antibodies (PcAb) pretreated tachyzoites showed obviously increased survival outcomes. In addition, mice that passively received anti-rTgPLP2 PcAb following a lethal dose of tachyzoites infection had longer survival time compared with phosphate-buffered saline (PBS) controls. Furthermore, we demonstrated that immunization with rTgPLP2 could prolong survival in RH strain infected mice and resulted in the lowest brain cysts size and number of Prugniaud (PRU) genotype II strain infected mice. High levels of Toxoplasma-specific IgG, IgG1, IgG2a, and cytokines (IFN-γ and IL-10) were produced after two immunizations with rTgPLP2. Together these results indicated that TgPLP2 can induce both humoral and cellular immune responses to protect host against infection and thus is a potential candidate for T. gondii vaccines.

Prognostic value of HER2 status on circulating tumor cells in advanced-stage breast cancer patients with HER2-negative tumors.

PURPOSE: Discordance between HER2 expression in tumor tissue (tHER2) and HER2 status on circulating tumor cells (cHER2) has been reported. It remains largely underexplored whether patients with tHER2-/cHER2+ can benefit from anti-HER2 targeted therapies. METHODS: cHER2 status was determined in 105 advanced-stage patients with tHER2- breast tumors. Association between cHER2 status and progression-free survival (PFS) was analyzed by univariate and multivariate Cox models and survival differences were compared by Kaplan-Meier method. RESULTS: Compared to the patients with low-risk cHER2 (cHER2+ < 2), those with high-risk cHER2 (cHER2+ ≥ 2) had shorter survival time and an increased risk for disease progression (hazard ratio [HR] 2.16, 95% confidence interval [CI] 1.20-3.88, P = 0.010). Among the patients with high-risk cHER2, those who received anti-HER2 targeted therapies had improved PFS compared with those who did not (HR 0.30, 95% CI 0.10-0.92, P = 0.035). In comparison, anti-HER2 targeted therapy did not affect PFS among those with low-risk cHER2 (HR 0.70, 95% CI 0.36-1.38, P = 0.306). Similar results were obtained after adjusting covariates. A longitudinal analysis of 67 patients with cHER2 detected during follow-ups found that those whose cHER2 status changed from high-risk at baseline to low-risk at first follow-up exhibited a significantly improved survival compared to those whose cHER2 remained high-risk (median PFS: 11.7 weeks vs. 2.0 weeks, log-rank P = 0.001). CONCLUSION: In advanced-stage breast cancer patients with tHER2- tumors, cHER2 status has the potential to guide the use of anti-HER2 targeted therapy in patients with high-risk cHER2.

Development of a convenient detection method for Trichomonas vaginalis based on loop-mediated isothermal amplification targeting adhesion protein 65.

BACKGROUND: Trichomoniasis resulting from Trichomonas vaginalis (T. vaginalis) has been considered as a commonly seen disease with the transmission way of sex. At present, the detection methods of T. vaginalis mainly include wet mount microscopy, culture, PCR, immunofluorescence and ELISA. However, all of these detection methods exist shortcomings. METHODS: In this study, a loop-mediated isothermal amplification (LAMP) assay that targeted the species-specific sequence of adhesion protein 65 (AP65) gene had been conducted to detect T. vaginalis. The optimum reaction system and conditions were optimized in this rapid detection method. RESULTS: The results of sensitivity analysis showed that the LAMP assay targeting the AP65 gene was 1000 times more sensitive than the nested PCR targeting the actin gene commonly used for detection of T. vaginalis, and the detecting limitation of the former was 10 trichomonad. Moreover, the amplification of the target gene AP65 by LAMP assay exhibited high specificity and the product was exclusively from T. vaginalis. The detection technique of LAMP did not exhibit cross-reactivity with the common pathogens of Trichinella spiralis, Toxoplasma gondii, Escherichia coli, Candida albicans, Staphylococcus aureus, Haemophilus. CONCLUSIONS: According to the present study, the LAMP assay with the target of AP65 gene, was suitable for the early diagnosis of T. vaginalis infections. Consequently, the LAMP assay was proposed by the current study as a point-of-care examination and an alternative molecular tool which exhibited the potential value in the treatment, control and prevention of trichomoniasis transmission and relevant complication.

Identification of Toxoplasma Gondii Tyrosine Hydroxylase (TH) Activity and Molecular Immunoprotection against Toxoplasmosis.

The neurotropic parasite Toxoplasma gondii (T. gondii) infection can change the behavior of rodents and cause neuropsychological symptoms in humans, which may be related to the change in neurotransmitter dopamine in the host brain caused by T. gondii infection. T. gondii tyrosine hydroxylase (TgTH) is an important factor in increasing the neurotransmitter dopamine in the host brain. In this study, the enzyme activity of TgTH catalytic substrate for dopamine production and the molecular characteristics of TgTH were identified. In order to amplify the open reading frame (ORF), the designing of the specific primers for polymerase chain reaction (PCR) was on the basis of the TgTH sequence (GenBank Accession No. EU481510.1), which was inserted into pET-32a (+) for the expression of recombined TgTH (rTgTH). The sequence analysis indicated that the gene of TgTH directed the encoding of a 62.4-kDa protein consisting of 565 amino acid residues, which was predicted to have a high antigen index. The enzyme activity test showed that rTgTH and the soluble proteins extracted separately from T. gondii RH strain and PRU strain could catalyze the substrate to produce dopamine in a dose-dependent manner, and the optimum catalytic temperature was 37 °C. The result of the Western Blotting assay revealed that the rTgTH and the native TgTH extracted from somatic of T. gondii RH tachyzoite were successfully detected by the sera of mice infected with T. gondii and the rat serum after rTgTH immune, respectively. Immunofluorescence analysis using antibody against rTgTH demonstrated that the protein was expressed and located on the surface of T. gondii RH tachyzoite. Freund's adjuvant was used to emulsify the rTgTH, which was subsequently applied to BALB/c mouse immune thrice on week 0, week 2, and week 4, respectively. The result of the animal challenge experiments showed an integral increase in IgG, IgG2a, IgG1, and IFN-γ, IL-4, and IL17 were as well significantly increased, and that the rTgTH vaccinated animals apparently had a prolonged survival time (14.30 ± 2.41) after infection with the RH strain of T. gondii compared with that of the non-vaccinated control animals, which died within 11 days. Additionally, in the rTgTH vaccination group, the number of brain cysts (1275 ± 224) significantly decreased (p < 0.05) compared to the blank control group (2375 ± 883), and the size of the brain cysts in the animals immunized with rTgTH vaccination was remarkably smaller than that of the control mice. All the findings prove that TgTH played an important role in increasing the neurotransmitter dopamine in the host brain and could be used as a vaccine candidate antigen to mediate cell-mediated and humoral immunity.

Molecular characterization of a potential receptor of Eimeria acervulina microneme protein 3 from chicken duodenal epithelial cells.

Eimeria acervulina is one of seven Eimeria spp. that can infect chicken duodenal epithelial cells. Eimeria microneme protein 3 (MIC3) plays a vital role in the invasion of host epithelial tissue by the parasite. In this study, we found that chicken (Gallus gallus) ubiquitin conjugating enzyme E2F (UBE2F) could bind to the MIC3 protein of E. acervulina (EaMIC3), as screened using the yeast two-hybrid system, and that it might be the putative receptor protein of EaMIC3. The UBE2F gene was cloned from chicken duodenal epithelial cells. The recombinant protein of UBE2F (rUBE2F) was expressed in E. coli and the reactogenicity of rUBE2F was analyzed by Western blot. Gene sequencing revealed that the opening reading frame (ORF) of UBE2F was 558 base pairs and encoded a protein of 186 amino acids with a molecular weight of 20.46 kDa. The predicted UBE2F protein did not contain signal peptides or a transmembrane region, but had multiple O-glycosylation and phosphorylation sites. A phylogenetic analysis showed that the chicken UBE2F protein is closely related to those of quail and pigeon (Coturnix japonica and Columba livia). A sporozoite invasion-blocking assay showed that antisera against rUBE2F significantly inhibited the invasion of E. acervulina sporozoites in vitro. Animal experiments indicated that the antisera could significantly enhance average body weight gains and reduce mean lesion scores following a challenge with E. acervulina. These results therefore imply that the chicken UBE2F protein might be the target receptor molecule of EaMIC3 that is involved in E. acervulina invasion.

TITLE: Caractérisation moléculaire d'un récepteur potentiel de la protéine 3 du micronème d'Eimeria acervulina dans les cellules épithéliales duodénales de poulet. ABSTRACT: Eimeria acervulina est l'une des sept Eimeria spp. qui peuvent infecter les cellules épithéliales duodénales de poulet. La protéine 3 du micronème d'Eimeria (MIC3) joue un rôle vital dans l'invasion du tissu épithélial de l'hôte par le parasite. Dans cette étude, nous avons constaté que l'enzyme de conjugaison de l'ubiquitine de poulet E2F (UBE2F) pouvait se lier à la protéine MIC3 d'E. acervulina (EaMIC3), telle que testé à l'aide du système de levure à deux hybrides, et qu'il pourrait s'agir de la protéine réceptrice putative d'EaMIC3. Le gène UBE2F a été cloné à partir de cellules épithéliales duodénales de poulet. La protéine recombinante d'UBE2F (rUBE2F) a été exprimée dans E. coli et la réactogénicité de rUBE2F a été analysée par Western blot. Le séquençage génétique a révélé que le cadre de lecture d'ouverture (ORF) d'UBE2F était de 558 paires de bases et codait une protéine de 186 acides aminés avec un poids moléculaire de 20,46 kDa. La protéine UBE2F prédite ne contenait pas de peptides signaux ni de région transmembranaire, mais avait plusieurs sites d'O-glycosylation et de phosphorylation. Une analyse phylogénétique a montré que la protéine UBE2F de poulet est étroitement liée à celles de la caille et du pigeon (Coturnix japonica et Columba livia). Un test de blocage des invasions de sporozoïtes a montré que les antisérums dirigés contre rUBE2F inhibaient de manière significative l'invasion des sporozoïtes d'E. acervulina in vitro. Les expériences sur les animaux ont indiqué que les antisérums pourraient améliorer de manière significative les gains de poids corporel moyens et réduire les scores moyens de lésions suite à une infection avec E. acervulina. Ces résultats impliquent donc que la protéine UBE2F de poulet pourrait être la molécule de récepteur cible d'EaMIC3 impliquée dans l'invasion d'E. acervulina.

MicroRNA-216a suppresses the proliferation and migration of human breast cancer cells via the Wnt/ß-catenin signaling pathway.

The aim of the present study was to investigate the potential anticancer effects of microRNA-216a on the growth of human breast cancer and the possible underlying mechanisms. The results demonstrated that serum microRNA-216a was significantly decreased in patients with breast cancer compared with healthy controls. MicroRNA-216a overexpression led to a decrease in cell proliferation and migration, as well as increases in apoptosis, caspase-3/8 activities, Bax expression and p53 protein expression in MCF-7 cells. It was also revealed that microRNA-216a suppressed Wnt and ß-catenin expression in MCF-7 cells. The anticancer effects of microRNA-216a were reversed by anti-microRNA-216a by promoting the Wnt/ß-catenin signaling pathway. Inactivation of the Wnt pathway increased the anticancer effects of microRNA-216a in MCF-7 cells. Collectively, the results of the present study indicated that microRNA-216a suppressed the growth of human breast cancer cells by targeting the Wnt/ß­catenin signaling pathway.

The Molecular Characterization and Immunity Identification of Rhoptry Protein 22 of Toxoplasma gondii as a DNA Vaccine Candidate Against Toxoplasmosis.

Toxoplasma gondii (T. gondii) rhoptry proteins (TgROPs) have been considered main targets and indicator molecules for immune diagnosis and prophylaxis since they initially present during the process of invasion. In this study, the effect of intramuscularly injecting the genetic vaccine pVAX-ROP22 was evaluated, made by inserting the TgROP22 sequence into the eukaryotic expression vector of pVAX I, into BALB/c mice. The levels of IgG, IgG1, and IgG2a in pVAX-ROP22 vaccinated animals were integrally increased. It was uncovered by cytokine profile analyses that the levels of IFN-γ and IL-2 were significantly increased, while no significant changes were detected in IL-4 and IL-10 levels. In addition, we found that immunization with pVAX-ROP22 significantly prolonged the survival time (13.80 ± 1.75 d) of mice after challenge infection with the virulent T. gondii RH strain, in comparison with those of control animals (died within 10 d). Moreover, the number of brain cysts (1,406 ± 277) in the animals subjected to pVAX-TgROP22 vaccination decreased remarkably (P < 0.05) compared with the blank control mice (2,333 ± 473), and the size of brain cysts in pVAX-TgROP22 group was significantly smaller than the groups of blank, PBS and pVAXI. These results suggested that TgROP22 as DNA vaccine could trigger strong humoral and cellular responses and induce partial protection against toxoplasmosis.