RNA modifications are reversible, dynamically regulated, and involved in a variety of diseases such as cancers. Given the lack of efficient and reliable biomarkers for early diagnosis of ovarian cancer (OV), this study was designed to explore the role of RNA modification genes (RMGs) in the diagnosis of OV. Herein, 132 RMGs were retrieved in PubMed, 638 OV and 18 normal ovary samples were retrieved in The Cancer Genome Atlas (TCGA), and GSE18520 cohorts were collected for differential analysis. Finally, PUS7 (Pseudouridine Synthase 7) as differentially expressed RMGs (DEGs-RMGs) was identified as a diagnostic biomarker candidate and evaluated for its specificity and sensitivity using Receiver Operating Characteristic (ROC) analysis in TCGA and GEO data. The protein expression, mutation, protein interaction networks, correlated genes, related pathways, biological processes, cell components, and molecular functions of PUS7 were analyzed as well. The upregulation of PUS7 protein in OV was confirmed by the staining images in HPA and tissue arrays. Collectively, the findings of the present study point towards the potential of PUS7 as a diagnostic marker and therapeutic target for ovarian cancer.

Background: Toxoplasma gondiiis a widespread intracellular parasite, which infects most vertebrate animal hosts and causes zoonotic infection in humans. Vaccine strategy remains a promising method for the prevention and control of toxoplasmosis.T. gondiiGRA4 protein has been identified as a potential candidate for vaccine development. In our study, we evaluated the immune response induced by four different immunization vaccination strategies encoding TgGRA4.Methods: BALB/c mice were intramuscularly (i.m.) immunized four times according to specific immunization schedules. Generally, mice in experimental groups were immunized with polypeptide, pGRA4, peptide/DNA, or DNA/peptide, and mice in the control groups were injected with PBS or pEGFP. After immunization, the levels of IgG antibodies and cytokine productions were determined by enzyme-linked immunosorbent assays (ELISA). The survival time of mice was also evaluated after challenge infection with the highly virulentT. gondiiRH strain.Results: The results showed that mice vaccinated with different immunization regimens (polypeptide, pGRA4, peptide/DNA, or DNA/peptide) elicited specific humoral and cellular responses, with high levels of total IgG, IgG2a isotype and gamma interferon (IFN-γ), which suggested a specific Th1 immunity was activated. After lethal challenge, an increased survival time was observed in immunized mice (11.8 ± 4.8 days) compared to the control groups injected with PBS or pEGFP (P < 0.05). Mice injected with PBS or pEGFP died within 8 days, and there was no significant difference in the protection level in two groups (P > 0.05).Conclusions: These results demonstrated that this DNA prime and peptide boost immunization protocol encoding the TgGRA4 can elicit the highest level of humoral and cellular immune responses compared to other immunized groups, which is a promising approach to increase the efficacy of DNA immunization.

Toxoplasma gondii is an obligate intracellular parasite that can pose a serious threat to human health by causing toxoplasmosis. There are no drugs that target the chronic cyst stage of this infection; therefore, development of an effective vaccine would be an important advance. Aspartic proteases play essential roles in the T. gondii lifecycle. The parasite has four aspartic protease encoding genes, which are called toxomepsin 1, 2, 3 and 5 (TgASP1, 2, 3 and 5, respectively). Bioinformatics approaches have enabled us to identify several promising linear-B cell epitopes and potential Th-cell epitopes on TgASP1, thus supporting its potential as a DNA vaccine against toxoplasmosis. We expressed TgASP1 in Escherichia coli and used the purified protein to immunize BALB/c mice. The antibodies obtained were used to determine where TgASP1 was localized in the parasite. We also made a TgASP1 DNA vaccine construct and evaluated it for the level of protection conferred to mice against infection with the virulent RH strain of T. gondii. TgASP1 appears to be a membrane protein located primarily at the tip of the T. gondii tachyzoite. Investigation of its potential as a DNA vaccine showed that it elicited strong humoral and cellular immune responses in mice, and that these responses were mediated by Th-1 cells. Mice immunized with the vaccine had greater levels of protection against mortality following challenge with T. gondii RH tachyzoites than did those immunized with PBS or the empty vector control. TgASP1 is a novel candidate DNA vaccine that merits further investigation.

A widely prevalent disease, toxoplasmosis poses serious health threats to both humans and animals; therefore, development of an ideal DNA vaccine against Toxoplasma gondii is needed eagerly. The purpose of the present study is to assess the protective efficacy of a DNA vaccine encoding the T. gondii toxofilin gene (pEGFP-toxofilin). In addition, toxofilin DNA vaccine combined with the individual adjuvants, alum or monophosphoryl lipid A (MPLA), or a mixture of alum-MPLA adjuvant were screened for their ability to enhance antibody responses. Using bioinformatics, we analyzed the gene and amino acid sequences of the toxofilin protein, recognizing and identifying several potential linear B and T helper (Th)-1 cell epitopes. BALB/c mice were immunized three times with either toxofilin DNA vaccine alone or in combination with the adjuvants such as alum, MPLA or an alum-MPLA mixture. The systemic immune response was evaluated by cytokine, the percentage of CD4 (+) and CD8 (+) T cells and specific antibody measurement. Two weeks after the last immunization, protective efficacy was evaluated by challenging intraperitoneally with 1 × 104 tachyzoites of T. gondii or intragastrically with 20 cysts of T. gondii PRU strain. All experimentally immunized mice developed strong humoral and cellular immune responses compared with the control groups. Moreover, by comparison with the non-adjuvant toxofilin DNA vaccine group, adding alum adjuvant to toxofilin DNA vaccine resulted in an increase in humoral response and a skewed Th2 response. However, the MPLA adjuvant with toxofilin DNA vaccine induced significantly enhanced humoral and Th1-biased immune responses. Importantly, the co-administration of alum-MPLA adjuvant in combination with the toxofilin DNA vaccine shifted the Th2 immune response to a Th1 response compared with the alum-toxofilin group, and elicited the strongest humoral and Th1 responses among all the groups. At the same time, a longer survival time and less cyst amounts against T. gondii infection were also observed in the alum-MPLA-toxofilin group in comparison with single or no adjuvant groups. Toxoplasma gondii toxofilin is a promising vaccine candidate that warrants further development. Co-administration of the alum-MPLA adjuvant mixture with DNA vaccine could effectively enhance immunogenicity and strongly skew the cellular immune response towards a Th1 phenotype.

Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite with a broad host range including most warm-blooded animals, including humans. T. gondii surface antigen 1 (SAG1) is a well-characterized T. gondii antigen. T. gondii expresses five nonmitochondrial rhomboid intramembrane proteases, TgROM1-5. TgROM4 is uniformly distributed on the surface of T. gondii and involved in regulating MIC2, MIC3, MIC6, and AMA1 during T. gondii invasion of host cells. Bioinformatics have predicted ROM4 B-cell and T-cell epitopes. Immunization strategy is also a key factor in determining the effectiveness of the immune response and has gained increasing attention in T. gondii vaccine research. In this study, we used a DNA prime-peptide boost vaccination regimen to assess the protective efficacy of various vaccination strategies using TgROM4. We identified a polypeptide (YALLGALIPYCVEYWKSIPR) using a bioinformatics approach, and immunized mice using a DNA-prime and polypeptide-boost regimen. BALB/c mice were randomly divided into six groups, including three experimental groups (peptide, pROM4 and pROM4/peptide) and three control groups (PBS, pEGFP-C1 and pSAG1). Mice were then immunized intramuscularly four times. After immunization, IgG and cytokine productions were determined using enzyme-linked immunosorbent assays. The survival time of mice was evaluated after challenge with tachyzoites of T. gondii RH strain. Additionally, the number of cysts in the brain was determined after intragastric challenge with cysts of T. gondii PRU strain. Mice vaccinated with different immunization regimens (peptide, pROM4 and pROM4/peptide) elicited specific humoral and cellular responses, with high levels of IgG, IgG2a, and interferon (IFN)-γ. Moreover, IgG, IgG2a and IFN-γ levels were highest in the pROM4/peptide group. Immunized mice, especially those in the pROM4/peptide group, had prolonged survival times after challenge with tachyzoites and reduced numbers of brain cysts after infection compared with negative controls. A DNA prime-peptide boost regimen based on ROM4 elicited the highest level of humoral and cellular immune responses among immunization regimens, and may be a promising approach to increase the efficacy of DNA immunization.

Toxoplasma gondii, an obligate intracellular apicomplexan parasite, infects a wide range of warm-blooded animals including humans. T. gondii expresses five members of the C1 family of cysteine proteases, including cathepsin B-like (TgCPB) and cathepsin L-like (TgCPL) proteins. TgCPB is involved in ROP protein maturation and parasite invasion, whereas TgCPL contributes to proteolytic maturation of proTgM2AP and proTgMIC3. TgCPL is also associated with the residual body in the parasitophorous vacuole after cell division has occurred. Both of these proteases are potential therapeutic targets in T. gondii. The aim of this study was to investigate TgCPB and TgCPL for their potential as DNA vaccines against T. gondii. Using bioinformatics approaches, we analyzed TgCPB and TgCPL proteins and identified several linear-B cell epitopes and potential Th-cell epitopes in them. Based on these results, we assembled two single-gene constructs (TgCPB and TgCPL) and a multi-gene construct (pTgCPB/TgCPL) with which to immunize BALB/c mice and test their effectiveness as DNA vaccines. TgCPB and TgCPL vaccines elicited strong humoral and cellular immune responses in mice, both of which were Th-1 cell mediated. In addition, all of the vaccines protected the mice against infection with virulent T. gondii RH tachyzoites, with the multi-gene vaccine (pTgCPB/TgCPL) providing the highest level of protection. T. gondii CPB and CPL proteases are strong candidates for development as novel DNA vaccines.