Genome-wide DNA methylation and transcriptomic patterns of precancerous gastric cardia lesions.

BACKGROUND: Intestinal metaplasia (IM) and intraepithelial neoplasia (IEN) are considered precursors of gastric cardia cancer (GCC). Here, we investigated the histopathologic and molecular profiles of precancerous gastric cardia lesions (PGCLs) and biomarkers for risk stratification of gastric cardia IM. METHODS: We conducted a hospital-based evaluation (n = 4578) for PGCL profiles in high-incidence and non-high-incidence regions for GCC in China. We next performed 850K methylation arrays (n = 42) and RNA-seq (n = 44) in tissues with PGCLs. We then examined the protein expression of candidate biomarker using immunohistochemistry. RESULTS: Of the 4578 participants, 791 were diagnosed with PGCLs (600 IM, 62 IM with IEN, and 129 IEN). We found that individuals from high-incidence regions (26.7%) were more likely to develop PGCLs than those from non-high-incidence areas (13.5%). DNA methylation and gene expression alterations, indicated by differentially methylated probes (DMPs) and differentially expressed genes (DEGs), exhibited a progressive increase from type I IM (DMP = 210, DEG = 24), type II IM (DMP = 3402, DEG = 129), to type III IM (DMP = 3735, DEG = 328), peaking in IEN (DMP = 47 373, DEG = 2278). Three DEGs with aberrant promoter methylation were identified, shared exclusively by type III IM and IEN. Of these DEGs, we found that OLFM4 expression appears in IMs and increases remarkably in IENs (P < .001). CONCLUSIONS: We highlight that type III IM and IEN share similar epigenetic and transcriptional features in gastric cardia and propose biomarkers with potential utility in risk prediction.

Recombinant interferon-gamma promotes immunoglobulin G and cytokine memory responses to cathepsin L-like cysteine proteinase of Hyalomma asiaticum and the efficacy of anti-tick.

Among bloodsucking arthropods, hard tick is a vector of transmitting the most diverse human and animal pathogens, leading to an increasing number of manifestations worldwide. The development of the anti-tick vaccine has the potential to be an environmentally friendly and cost-effective option for tick management. We have previously demonstrated the induction of both humoral and cellular response against Hyalomma asiaticum (H. asiaticum) following immunization with recombinant cathepsin L-like cysteine protease from H. asiaticum tick (rHasCPL), and could control tick infestations. Interferon-gamma (IFN-γ), is an immunomodulatory factor that plays an important role in the regulation of adaptive immunity against infection. In the present study, recombinant BALB/c mouse IFN-γ (rMus-IFN-γ) was cloned and expressed using a prokaryotic expression system, and verified by Western blotting and IFN-γ-ELISA kit analysis. Female BALB/c mice (n = 12) were used for immunization using rHasCPL (100 µg) plus IFN-γ as adjuvant (10 µg). In immunized female BALB/c mice, the levels of anti-CPL antibodies as well as cytokines were determined using ELISA analysis. Protective efficacy of immunization was evaluated by larvae H. asiaticum challenge of immunized female BALB/c mice. Using rMus-IFN-γ as an adjuvant to rHasCPL vaccine (CPL + IFN-γ) promoted specific antibody IgG (IgG1 > IgG2a) and increased production of IFN-γ and IL-4 compared to immune rHasCPL group (CPL). The protected rate of immunized mice from tick challenge was significantly higher after immunization with CPL + IFN-γ (85.11 %) than with CPL (63.28 %). Immunization using CPL + IFN-γ promoted the activation of anti-HasCPL humoral and cellular immune responses, and could provide better protection against H. asiaticum infestation. This approach may could help develop a candidate vaccine for control tick infestations.

De novo assembly and analysis of the transcriptome of the Dermacentor marginatus genes differentially expressed after blood-feeding and long-term starvation.

BACKGROUND: The ixodid tick Dermacentor marginatus is a vector of many pathogens wide spread in Eurasia. Studies of gene sequence on many tick species have greatly increased the information on tick protective antigen which might have the potential to function as effective vaccine candidates or drug targets for eco-friendly acaricide development. In the current study, RNA-seq was applied to identify D. marginatus sequences and analyze differentially expressed unigenes. METHODS: To obtain a broader picture of gene sequences and changes in expression level, RNA-seq was performed to obtain the whole-body transcriptome data of D. marginatus adult female ticks after engorgement and long-term starvation. Subsequently, the real-time quantitative PCR (RT-qPCR) was applied to validate the RNA-seq data. RESULTS: RNA-seq produced 30,251 unigenes, of which 32% were annotated. Gene expression was compared among groups that differed by status as newly molted, starved and engorged female adult ticks. Nearly one third of the unigenes in each group were differentially expressed compared to the other two groups, and the most numerous were genes encoding proteins involved in catalytic and binding activities and apoptosis. Selected up-regulated differentially expressed genes in each group were associated to protein, lipids, carbohydrate and chitin metabolism. Blood-feeding and long-term starvation also caused genes differentially expressed in the defense response and antioxidant response. RT-qPCR results indicated 6 differentially expressed transcripts showed similar trends in expression changes with RNA-seq results confirming that the gene expression profiles in transcriptome data is in consistent with RT-qPCR validation. CONCLUSIONS: Obtaining the sequence information of D. marginatus and characterizing the expression pattern of the genes involved in blood-feeding and during starvation would be helpful in understanding molecular physiology of D. marginatus and provides data for anti-tick vaccine and drug development for controlling the tick.

Sequence identification and expression profile of seven Dermacentor marginatus glutathione S-transferase genes.

Dermacentor marginatus is a widespread tick species and a vector of many pathogens in Eurasia. Due to the medical importance of D. marginatus, control measures are needed for this tick species. Currently tick control approaches rely mostly on acaricide application, whereas wrong and irrational acaricide use may result in drug resistance and residue problems. Vaccination as an alternative approach for tick control has been proven to be effective towards some tick species. However, immunization against D. marginatus has not yet reached satisfactory protection. The effort of in silico based analysis could predict antigenicity and identify candidates for anti-tick vaccine development. We carried out an in silico analysis of D. marginatus glutathione S-transferases (DmGSTs) in order to identify blood-feeding induced GSTs as antigens that can be used in anti-tick vaccine development. Phylogenetic analysis, linear B-cell epitope prediction, homology modeling, and conformational B-cell epitope mapping on the GST models were performed to identify highly antigenic DmGSTs. Relative gene expressions of the seven GSTs were profiled through real-time quantitative PCR (RT-qPCR) to outline GSTs up-regulated during blood feeding. The phylogenetic analysis indicated that the seven GSTs belonged to four classes of GST, including one in epsilon-class, one in zeta-class, one in omega-class, and four in mu-class. Linear B-cell epitope prediction revealed mu-class GSTs share similar conserved antigenic regions. The conformational B-cell epitope mapped on the homology model of the GSTs displayed that GSTs of mu-class showed stronger antigenicity than that of other classes. RT-qPCR revealed DmGSTM1 and DmGSTM2 were positively related to blood feeding. In sum, the data suggest that DmGSTM1 and DmGSTM2 could be tested for potential anti-tick vaccine trials.

[Establishment of transgenic mice with visualized neovascularization].

OBJECTIVE: To establish transgenic mice with GFP expression in the vascular endothelium during neovascularization. METHODS: The vector nestin-hsp68-gfp containing nestin second intron was introduced into U251 cells and the expression level of GFP was detected by fluorescence microscopy. Transgenic mice were produced by microinjection. The genome of the offspring mice was screened by PCR, and GFP expression in the vascular endothelium was detected using immunohistochemistry. RESULTS: Thirteen offspring mice were obtained and 2 of them were positive for GFP in the vascular endothelium as detected by PCR. GFP was detected in the offspring mice both at the embryonic stage and after birth. CONCLUSIONS: The transgenic mice with GFP expression in the vascular endothelium during neovascularization have been successfully established.