Identifying Qingkailing ingredients-dependent mesenchymal-epithelial transition factor-axiation "π" structuring module with angiogenesis and neurogenesis effects.

OBJECTIVE: To explore the functional role of the drug-dependent mesenchymal-epithelial transition (Met)-axiation "π" structural module of neurogenesis after processing by three components of Qingkailing injection in neurogenesis and angiogenesis in cerebral ischemia. METHODS: We used a Glutathione S-transferase (GST)-pull down assay, isothermal titration calorimetry assay, and other related methods to identify the relationships among Met, inositol polyphosphate phosphatase like 1 (Inppl1), and death associated protein kinase 3 (Dapk3) in this allosteric module. The biological effects of the modules of neurons generation composed of Met, Inppl1, and Dapk3 were measured through Western blot, apoptosis analysis, and double immunofluorescence labeling. RESULTS: The GST-pull down assay revealed that proline-serine-threonine rich domain of Met binds to the Src homology domain of Inppl1 to form a protein-protein complex; Dapk3 with a C-terminal domain interacts weakly with the protein kinase C domain of Met in the intracellular region. Thus, we obtained a "π" structuring module considered a neural regeneration module. The biological effects of angiogenesis and neurogenesis modules composed of Met, Inppl1, and Dapk3 were also verified. CONCLUSION: The study suggested that understanding the functional modules that contribute to pharmaceutics might provide novel signatures that can be used as endpoints to define disease processes under stroke or cerebral ischemia conditions.

LINC00998-encoded micropeptide SMIM30 promotes the G1/S transition of cell cycle by regulating cytosolic calcium level.

The biological functions of short open reading frame (sORF)-encoded micropeptides remain largely unknown. Here, we report that LINC00998, a previously annotated lncRNA, was upregulated in multiple cancer types and the sORF on LINC00998 encoded a micropeptide named SMIM30. SMIM30 was localized in the membranes of the endoplasmic reticulum (ER) and mitochondria. Silencing SMIM30 inhibited the proliferation of hepatoma cells in vitro and suppressed the growth of tumor xenografts and N-nitrosodiethylamine-induced hepatoma. Overexpression of the 5'UTR-sORF sequence of LINC00998, encoding wild-type SMIM30, enhanced tumor cell growth, but this was abolished when a premature stop codon was introduced into the sORF via single-base deletion. Gain- and loss-of-function studies revealed that SMIM30 peptide but not LINC00998 reduced cytosolic calcium level, increased CDK4, cyclin E2, phosphorylated-Rb and E2F1, and promoted the G1/S phase transition and cell proliferation. The effect of SMIM30 silencing was attenuated by a calcium chelator or the agonist of sarco/endoplasmic reticulum calcium ATPase (SERCA) pump. These findings suggest a novel function of micropeptide SMIM30 in promoting G1/S transition and cell proliferation by enhancing SERCA activity and reducing cytosolic calcium level.

Matrine, a potential c-Myc inhibitor, suppresses ribosome biogenesis and nucleotide metabolism in myeloid leukemia.

Previous studies have shown that matrine, a natural compound extracted from the herb Sophora flavescens, has a good anti-leukemia effect, but its key target and mechanism remains unclear. Here, we found that only c-Myc could respond rapidly to matrine treatment in three myeloid leukemia cell lines, and matrine inhibited both transcription and translation of c-Myc. Ribosome biogenesis and nucleotide metabolism, the key downstream of c-Myc, were significantly suppressed after matrine treatment. Therefore, our results confirmed that matrine is a special c-Myc inhibitor which suppresses ribosome biogenesis and nucleotide metabolism by inhibiting c-Myc in myeloid leukemia. This study provides scientific basis for the development of matrine derivatives to c-Myc-driven cancers.

[Epigenetic regulation of microglia-mediated innate immune memory and neurological diseases].

Microglia have the ability to mediate innate immune memory and can be reprogrammed by primary stimuli to enhance or inhibit the immune response of microglia to secondary stimuli. Inflammatory stimulation is an important factor for microglia to mediate innate immune memory. Single or repeated stimulation can induce microglia to form different phenotypes. Microglia-mediated innate immune response is involved in the regulation of immune memory. Enhancer modification is a key pathway of microglia epigenetic regulation, and the H3K27ac enhancer marker is closely related to immune training. TGF-ß1 mediates the interaction between IL-10 and IL-1ß, thereby influencing the microglial phenotype. Microglia glycolysis activity is increased after immune training, and oxidative phosphorylation is associated with immune tolerance. Innate immune memory is closely associated with neurodegenerative diseases, brain tumors, brain damage and psychosis. Further study on the mechanism of microglia-mediated innate immune memory is helpful to understand the occurrence and development of central nervous system diseases and provide new options for the treatment of central nervous system diseases.

Predicting mortality in acute ischaemic stroke treated with mechanical thrombectomy: analysis of a multicentre prospective registry.

OBJECTIVES: We aimed to determine predictors of mortality within 90 days and develop a simple score for patients with mechanical thrombectomy (MT). DESIGN: Analysis of a multicentre prospective registry. SETTING: In six participating centres, patients who had an acute ischaemic stroke (AIS) treated by MT between March 2017 and May 2018 were documented prospectively. PARTICIPANTS: 224 patients with AIS were treated by MT. RESULTS: Of 224 patients, 49 (21.9%) patients died, and 87 (38.8%) were independent. Variables associated with 90-day mortality were age, previous stroke, admission National Institutes of Health Stroke Scale (NIHSS), fasting blood glucose and occlusion site. Logistic regression identified four variables independently associated with 90-day mortality: age ≥80 years (OR 3.26, 95% CI 1.45 to 7.33), previous stroke (OR 2.33, 95% CI 1.04 to 5.21), admission NIHSS ≥18 (OR 2.37, 95% CI 1.13 to 4.99) and internal carotid artery or basilar artery occlusion (OR 2.92, 95% CI 1.34 to 6.40). Using these data, we developed predicting 90-day mortality of AIS with MT (PRACTICE) score ranging from 0 to 6 points. The receiver operator curve analysis found that PRACTICE score (area under the curve (AUC)=0.744, 95% CI 0.669 to 0.820) was numerically better than iScore (AUC=0.661, 95% CI 0.577 to 0.745) and Predicting Early Mortality of Ischemic Stroke score (AUC=0.638, 95% CI 0.551 to 0.725) for predicting 90-day mortality. CONCLUSIONS: We developed a simple score to estimate the 90-day mortality of patients who had an AIS treated with MT. But the score needs to be prospectively validated. TRIAL REGISTRATION NUMBER: Chinese Clinical Trial Registry (ChiCTR-OOC-17013052).

Morinda citrifolia (Noni) Juice Suppresses A549 Human Lung Cancer Cells via Inhibiting AKT/Nuclear Factor-κ B Signaling Pathway.

OBJECTIVE: To study the mechanism of the anti-tumor effect of Morinda citrifolia (noni). METHODS: The influences of noni juice on cell proliferation, apoptosis, invasion, migration and the activity of AKT/nuclear factor- κ B (NF- κ B) signaling pathway in A549 human lung cancer cells were detected by MTT, cell counting kit-8, colony formation, Annexin V/PI double labeling, transwell, scratch test and immunoblotting assay, respectively. A549 cells were inoculated into the right axilla of nude mice, followed by noni juice treatment. The body weight of the nude mice was weighed, and the tumor volume and weight were measured. Cell proliferation and expression of apoptosis-related proteins were measured by immunohistochemistry, and the activity of NF- κ B signaling pathway was measured by immunoblotting. RESULTS: The in vitro studies showed that noni juice inhibited the A549 cells proliferation, migration and invasion. Noni juice also promoted cells apoptosis in A549 cells. Immunoblotting assay showed that the phosphorylation level of AKT, p50, and STAT3 proteins was inhibited to different extents after noni juice treatment. The in vivo studies showed that noni juice effectively suppressed tumor formation of A549 cells in nude mice. Noni juice treatment inhibited the expression of Ki67, PCNA, and Bcl-2 protein in the tumor; while promoted the expression of caspase-3 protein. Additionally, we also found that noni juice treatment could restrain the activity of AKT/NF- κ B signaling pathway in the tumor tissue. CONCLUSION: Noni juice inhibited the proliferation of A549 lung cancer cells, induced apoptosis, and inhibited cell invasion and migration via regulating AKT/NF- κ B signaling pathway.

Programming changes in GLUT1 mediated the accumulation of AGEs and matrix degradation in the articular cartilage of female adult rats after prenatal caffeine exposure.

Osteoarthritis is associated with intrauterine growth retardation (IUGR) and abnormal glucose metabolism. Our laboratory previously reported that prenatal caffeine exposure (PCE) can induce intrauterine maternal glucocorticoid (GC) overexposure in IUGR offspring and increase susceptibility to osteoarthritis after birth. In the present study, we demonstrated the essential role of glucose transporter 1 (GLUT1) programming changes in the increased matrix degradation of articular cartilage and susceptibility to osteoarthritis in female PCE adult offspring. In vivo, we found that PCE decreased the matrix content but did not significantly change the expression of matrix degradation-related genes in the articular cartilage of female fetal rats. The decreased expression of IGF1 and GLUT1 and the content of advanced-glycation-end-products (AGEs) were also detected. At different postnatal stages (2, 6, and 12 weeks), the cartilage matrix content decreased while the degradation-related genes expression increased in the PCE group. Meanwhile, the expression of IGF1 and GLUT1 and AGEs content in the local cartilage increased. In vitro, the expression levels of IGF1 and GLUT1 were inhibited by corticosterone but remained unchanged under caffeine treatment. Exogenous IGF1 can reverse the corticosterone-induced decrease in GLUT1 expression and promote AGEs production, while mifepristone (a glucocorticoid receptor inhibitor) reversed the corticosterone-induced low expression of IGF1 and GLUT1. Exogenous AGEs can increase the expression of inflammatory factors (IL-6 and TNF-α) and degradation-related genes, and decrease the matrix synthesis-related genes expression in chondrocyte. In conclusion, the GC-IGF1-GLUT1 axis mediated intrauterine dysplasia of articular cartilage, increased accumulation of AGEs and matrix degradation after birth in PCE female offspring, thereby increasing their susceptibility to osteoarthritis in adulthood.

A hMTR4-PDIA3P1-miR-125/124-TRAF6 Regulatory Axis and Its Function in NF kappa B Signaling and Chemoresistance.

BACKGROUND AND AIMS: DNA damage-induced NF-κB activation is a major obstacle to effective antitumour chemotherapy. Long noncoding RNAs (lncRNAs) that regulate chemoresistance of cancer cells remain largely unknown. This study aimed to characterize the lncRNAs that may affect chemotherapy sensitivity. APPROACH AND RESULTS: We found that lncRNA PDIA3P1 (protein disulfide isomerase family A member 3 pseudogene 1) was up-regulated in multiple cancer types and following treatment with DNA-damaging chemotherapeutic agents, like doxorubicin (Dox). Higher PDIA3P1 level was associated with poorer recurrence-free survival of human hepatocellular carcinoma (HCC). Both gain-of-function and loss-of-function studies revealed that PDIA3P1 protected cancer cells from Dox-induced apoptosis and allowed tumor xenografts to grow faster and to be more resistant to Dox treatment. Mechanistically, miR-125a/b and miR-124 suppressed the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6), but PDIA3P1 bound to miR-125a/b/miR-124 and relieved their repression on TRAF6, leading to activation of the nuclear factor kappa B (NF-κB) pathway. Consistently, the effect of PDIA3P1 inhibition in promoting Dox-triggered apoptosis was antagonized by silencing the inhibitor of κBα (IκBα) or overexpressing TRAF6. Administration of BAY 11-7085, an NF-κB inhibitor attenuated PDIA3P1-induced resistance to Dox treatment in mouse xenografts. Moreover, up-regulation of PDIA3P1 was significantly correlated with elevation of TRAF6, phosphorylated p65, or NF-κB downstream anti-apoptosis genes in human HCC tissues. These data indicate that enhanced PDIA3P1 expression may confer chemoresistance by acting as a microRNA sponge to increase TRAF6 expression and augment NF-κB signaling. Subsequent investigations into the mechanisms of PDIA3P1 up-regulation revealed that human homologue of mRNA transport mutant 4 (hMTR4), which promotes RNA degradation, could bind to PDIA3P1, and this interaction was disrupted by Dox treatment. Overexpression of hMTR4 attenuated Dox-induced elevation of PDIA3P1, whereas silencing hMTR4 increased PDIA3P1 level, suggesting that Dox may up-regulate PDIA3P1 by abrogating the hMTR4-mediated PDIA3P1 degradation. CONCLUSION: There exists a hMTR4-PDIA3P1-miR-125/124-TRAF6 regulatory axis that regulates NF-κB signaling and chemoresistance, which may be exploited for anticancer therapy.

The serum level of CC chemokine ligand 18 correlates with the prognosis of non-small cell lung cancer.

BACKGROUND: CC chemokine ligand 18 (CCL18) is a chemotactic cytokine involved in the pathogenesis and progression of various cancers. Our previous research showed that the expression of CCL18 is obviously higher in non-small cell lung cancer (NSCLC) than in the adjacent normal tissues, suggesting its role in NSCLC. METHODS: We further examined the serum level of CCL18 in 80 NSCLC patients with enzyme-linked immunosorbent assay and simultaneously analyzed the survival curve of these patients by the Kaplan-Meier method, and then utilized a log-rank test to evaluate the correlation of CCL18 expression with the malignant progression of NSCLC. RESULTS: Our results showed that the median serum concentration of CCL18 was significantly elevated to 436.11 ng/mL in NSCLC patients compared to 41.97 ng/ml in healthy people (P<0.01), which was also positively related to the expression of lung cancer biomarkers carcinoma-embryonic antigen and cytokeratin fragment antigen 21-1. Moreover, correlation analysis showed that an increased level of serum CCL18 was associated with a worse survival time in NSCLC patients. CONCLUSION: Our findings suggest that the serum CCL18 level of NSCLC patients was negatively correlated with the prognosis, thus suggesting that CCL18 may serve as a potential circulating biomarker for NSCLC diagnosis.

Nitrogen-doped fluorescent carbon dots used for the imaging and tracing of different cancer cells.

Here, we report the synthesis of nitrogen-doped fluorescent carbon (C) dots using a one-pot hydrothermal method. Transmission electron microscopy results reveal that the particle size of the nitrogen-doped C-dots is very small, with an average diameter of 4.6 nm. After being kept in water for 10 days, the nitrogen-doped C-dots can still dissolve well in the water, showing good stability and compatibility in aqueous solution. The fluorescence spectra show that the nitrogen-doped C-dots exhibit emission-tunable color from blue to green upon excitation from 230 to 520 nm. Cell tests show that the C-dots are low in cytotoxicity and can be used for imaging, detecting and tracing between hepatoma and HeLa cells, because hepatoma and HeLa cells show different sensitivity to different fluorescent colors pumped at different excitation wavelengths.

Investigation of the molecular mechanisms underlying postoperative recurrence in prostate cancer by gene expression profiling.

The present study aimed to identify potential genes associated with prostate cancer (PCa) recurrence following radical prostatectomy (RP) in order to improve the prediction of the prognosis of patients with PCa. The GSE25136 microarray dataset, including 39 recurrent and 40 non-recurrent PCa samples, was downloaded from the Gene Expression Omnibus database. Differentially-expressed genes (DEGs) were identified using limma packages, and the pheatmap package was used to present the DEGs screened using a hierarchical cluster analysis. Furthermore, gene ontology functional enrichment analysis was used to predict the potential functions of the DEGs. Subsequently, Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed to analyze pathway enrichment of DEGs in the regulatory network. Lastly, a protein-protein interaction (PPI) network of the DEGs was constructed using Cytoscape software to understand the interactions between these DEGs. A total of 708 DEGs were identified in the recurrent and non-recurrent PCa samples. Functional annotation revealed that these DEGs were primarily involved in cell adhesion, negative regulation of growth, and the cyclic adenosine monophosphate and mitogen-activated protein kinase (MAPK) signaling pathways. Furthermore, five key genes, including cluster of differentiation 22, insulin-like growth factor-1, inhibin ß A subunit, MAPK kinase 5 and receptor tyrosine kinase like orphan receptor 1, were identified through PPI network analysis. The results of the present study have provided novel ideas for predicting the prognosis of patients with PCa following RP.

Loss of Sun2 promotes the progression of prostate cancer by regulating fatty acid oxidation.

The role of Sun2 has been described by previous studies in various types of cancers, including breast cancer and lung cancer. However, its role and potential molecular mechanism in the progression of prostate cancer have not been fully elucidated. In the present study, we found that Sun2 expression was reduced in prostate cancer tissues compared with paired normal tissues, and that low expression of Sun2 was significantly correlated with Higher Gleason scores, postoperative T stage (pT), Lymph nodal invasion and Clinical pathological stages. In addition, reduced Sun2 Expression predicts poor survival of prostate cancer patients and could serve as an independent predictor of prostate cancer patients overall survival (OS).Furthermore, Sun2 overexpression inhibits the prostate cancer cells growth, and Sun2 knockdown promotes the prostate cancer cells growth both in vitro and vivo. Mechanical silencing of , Sun2 promoted fatty acid oxidation (FAO) in prostate cancer, prostate cancer cells growth promoted by Sun2 silencing could be reversed by the FAO inhibitor Etomoxir. Additionally, we also showed that serum amyloid A1 (SAA1) play a vital role in FAO, ATP and cell growth promoted by Sun2 loss in prostate cancer. These results suggest that Loss of Sun2 promoted the prostate cancer progression by regulating FAO.

Bladder Acellular Matrix Grafts Seeded with Adipose-Derived Stem Cells and Incubated Intraperitoneally Promote the Regeneration of Bladder Smooth Muscle and Nerve in a Rat Model of Bladder Augmentation.

The objective of this study was to investigate the feasibility of bladder acellular matrix grafts (BAMGs) seeded with adipose-derived stem cells (ASCs) followed by intraperitoneal incubation for bladder reconstruction in a rat model of bladder augmentation, and to explore the underlying mechanism. Autologous CM-DiI-labeled ASC-seeded (experimental group) and unseeded (control group) BAMGs were incubated in the peritoneum of male rats for 2 weeks and then harvested for bladder augmentation. Histological analysis of the incubated BAMGs revealed numerous cells growing in homogeneous collagen bundles in both groups. In the control BAMGs, these cells were mesenchyme derived, while in the ASC-seeded BAMGs, myofibroblasts and mesothelial cells were found inside and on the surface of the scaffold, respectively. Immunofluorescence analysis demonstrated that some of the myofibroblasts were transdifferentiated from the ASCs after 2 weeks of intraperitoneal incubation. The greater bladder capacity was found in the experimental group than the control group both 4 and 14 weeks postoperatively. Histological analysis revealed that the entire urothelium regenerated well both in the experimental group and the control group without significant difference 4 weeks and 14 weeks postoperatively. From the quantitative data of immunohistochemical and immunofluorescence staining, the smooth muscle cells (SMCs) regenerated significantly better in the experimental group than the control group both 4 weeks and 14 weeks postoperatively. Also significantly more nerve cells were found in the experimental group 14 weeks postoperatively. At 4 weeks postoperatively, the immunofluorescence double staining revealed that some SMCs in the BAMG were transdifferentiated from the implanted ASCs, but no CM-DiI labeling of ASCs was detected 14 weeks postoperatively. Taken together, our results demonstrate that ASC-seeded and peritoneally incubated BAMGs promote not only the morphological regeneration of the bladder smooth muscle and nerve, but also the bladder capacity, which indicates their potential for bladder regeneration.

Relationship of long noncoding RNA and viruses.

Long noncoding (lnc)RNAs comprise a diverse group of transcripts including large intervening noncoding (linc)RNAs, natural antisense transcripts (NATs) and intronic lncRNAs. The functions and mechanisms of more than 200 lncRNAs have been studied in vitro and the results suggest that lncRNAs may be molecular markers of prognosis in cancer patients. Some lncRNAs can promote virus replication and allow escape from cytosolic surveillance to suppress antiviral immunity. For example, lncRNA can cause persistent infection by Theiler's virus, and microRNA (miR)-27a/b is important for efficient murine cytomegalovirus (MCMV) replication. The available evidence suggests that lncRNAs may be potential targets of novel antiviral drugs.

MicroRNA-1285 Regulates 17ß-Estradiol-Inhibited Immature Boar Sertoli Cell Proliferation via Adenosine Monophosphate-Activated Protein Kinase Activation.

This study investigated the capacity of 10 µM 17ß-estradiol to inhibit immature boar Sertoli cell (SC) proliferation and the involvement of microRNA (miR)-1285 in this process. SC viability and cell cycle progression were investigated using a cell counting kit-8 and flow cytometry, respectively. Expression of AMP-activated protein kinase (AMPK), S phase kinase-associated protein 2 (Skp2), and miR-1285 was analyzed by real-time RT-PCR and Western blotting. 17ß-Estradiol (10 µM) reduced SC viability and miR-1285 expression and promoted AMPK phosphorylation. A double-stranded synthetic miR-1285 mimic promoted SC viability, increased levels of ATP, and phosphorylated mammalian target of rapamycin (mTOR) and Skp2 mRNA and protein, whereas p53 and p27 expression decreased, and 17ß-estradiol-mediated effects on SCs were significantly attenuated. A single-stranded synthetic miR-1285 inhibitor produced the opposite effects on these measures. Activation of AMPK inhibited SC viability, reduced levels of ATP, phosphorylated mTOR and Skp2 mRNA and protein, and increased p53 and p27 expression. An AMPK inhibitor (compound C) attenuated the effects of 17ß-estradiol on SCs. This indicated that 17ß-estradiol (10 µM) reduced SC proliferation by inhibiting miR-1285 and thus activating AMPK. Phosphorylated AMPK is involved in the regulation of 17ß-estradiol-mediated inhibition of SC viability through increasing p53 and p27 expression and inhibiting mTOR and Skp2 expression. Our findings also implicated Skp2 as the downstream integration point of p53 and mTOR. These findings indicated that miR-1285 may represent a target for the manipulation of boar sperm production.

EGCG decreases the expression of HIF-1α and VEGF and cell growth in MCF-7 breast cancer cells.

PURPOSE: To investigate the effects of epigallocatechin-3-gallate (EGCG) on the expression of HIF-1α and vascular endothelial growth factor (VEGF) and cell growth in MCF-7 breast cancer cells. METHODS: MCF-7 human breast cancer cells were pretreated with different concentrations of EGCG (25, 50, 100 mg/L) for 48 h. The growth and proliferation of cells were analyzed by trypan blue staining in the pretreated MCF-7 cells. Furthermore, mRNA expression of HIF-1α and VEGF was detected by reverse transcriptase polymerase chain reaction (RT-PCR) analysis in the pretreated MCF-7 cells. Protein expression of HIF-1α was detected by Western blot, and the secreted protein level of VEGF in the supernatant of the culture medium was analyzed by enzyme linked immuno- sorbent assay (ELISA) in the MCF-7 cells pretreated with different concentrations of EGCG. RESULTS: Cell growth decreased dramatically in MCF-7 cells treated with different concentrations of EGCG, compared with untreated (control) cells. Moreover, protein expression of HIF-1α and VEGF declined in a dose-dependent manner in MCF-7 cells pretreated with increasing concentrations of EGCG. CONCLUSIONS: EGCG inhibits cell growth and proliferation of MCF-7 breast cancer cells, possibly by inhibiting the protein expression of HIF-1α and VEGF.

Construction and identification of the recombinant lentiviral expression vector targeting human BAX inhibitor-1 gene.

PURPOSE: The aim of this study was to construct a recombinant lentiviral expression vector targeting human BAX inhibitor- 1(BI-1) gene and observe its expression in NIH3T3 cells. METHODS: Human BI-1 gene was amplified by polymerase chain reaction (PCR), and then cloned into the vector pLCMV- IG using DNA recombinant technique. After the inserted sequences in the recombinant plasmids were identified by PCR, and double digesting and DNA sequencing analysis, the recombinant lentivirus was packaged and administered into NIH3T3 cells. The BI-1 mRNA and protein expression were examined by real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. RESULTS: PCR double digesting analysis and DNA sequencing confirmed that the BI-1 DNA sequences were successfully inserted into the lentiviral vectors. After transfection with the recombinant lentivirus, BI-1 expression in NIH3T3 cells was significantly increased at both mRNA and protein levels. CONCLUSION: The lentiviral vector expressing BI-1 has been successfully constructed, which allowed for the subsequent analysis of the role of BI-1 in cell growth and transduction.

Identification of H-2d restricted T cell epitope of foot-and-mouth disease virus structural protein VP1.

BACKGROUND: Foot-and-mouth disease (FMD) is a highly contagious and devastating disease affecting livestock that causes significant financial losses. Therefore, safer and more effective vaccines are required against Foot-and-mouth disease virus(FMDV). The purpose of this study is to screen and identify an H-2d restricted T cell epitope from the virus structural protein VP1, which is present with FMD. We therefore provide a method and basis for studying a specific FMDV T cell epitope. RESULTS: A codon-optimized expression method was adopted for effective expression of VP1 protein in colon bacillus. We used foot-and-mouth disease standard positive serum was used for Western blot detection of its immunogenicity. The VP1 protein was used for immunizing BALB/c mice, and spleen lymphocytes were isolated. Then, a common in vitro training stimulus was conducted for potential H-2Dd, H-2Kd and H-2Ld restricted T cell epitope on VP1 proteins that were predicted and synthesized by using a bioinformatics method. The H-2Kd restricted T cell epitope pK1 (AYHKGPFTRL) and the H-2Dd restricted T cell epitope pD7 (GFIMDRFVKI) were identified using lymphocyte proliferation assays and IFN-γ ELISPOT experiments. CONCLUSIONS: The results of this study lay foundation for studying the FMDV immune process, vaccine development, among other things. These results also showed that, to identify viral T cell epitopes, the combined application of bioinformatics and molecular biology methods is effective.

Screening and identification of B cell epitopes of structural proteins of foot-and-mouth disease virus serotype Asia1.

The objective of this study was to screen and identify the B cell epitopes of structural proteins of foot-and-mouth disease virus (FMDV) serotype Asia1. The complete amino acid sequence of all the four structural proteins (P1 region) was analyzed using the DNAStar Protean system. Seventeen peptides were predicted and selected as potential B cell epitopes. The potential B cell epitope genes were cloned into the pGEX-6P-1 plasmid, then expressed and purified. The resulting 17 glutathione S-transferase (GST) fusion peptides were detected by Western blot and ELISA for evaluation of their antigenicity. Six of the 17 fusion peptides were identified successfully by sera from rabbits immunized with the purified P1 polyprotein of FMDV type Asia1. The six fusion proteins were epi1-1 (VP1:(1)TTTTGESADPVT(12)), epi1-2 (VP1:(17)NYGGETQTARRLH(29)), epi1-6 (VP1:(194)TTQDRRKQEIIAPEKQTL(211)), epi2-2 (VP2:(40)EDAVSGPNTSG(50)), epi3-1 (VP3:(26)YGKVSNPPRTSFPG(39)), and epi4-2 (VP4:(30)YQNSMDTQLGDN(41)). The results of this study lay a foundation for further study of the structure and function of the structural proteins and may aid in the design of an epitope vaccine against foot-and-mouth disease (FMD) type Asia1. This study has also shown that the bioinformatics method, in combination with molecular biology methods can be used to map the B cell epitopes on viral proteins.

[Expression of inflammatory factors in lung tissue of acute paraquat poisoned rats].

OBJECTIVE: To investigate the expression of inflammatory factors in lung tissue of acute paraquat (PQ) poisoned rats. METHODS: Fifty male SD rats were randomized divided into two groups: the normal control group (NC group, n = 10) and the PQ group (n = 40). The 1 ml saline was administered once in normal control group. The PQ group was administered with 20 mg/kg 1% PQ by intraperitoneal injection to establish the model of PQ induced lung injury. At six hours, at the first, the third and the seventh day the PQ group were sacrificed, while at the first day the normal control group was sacrificed. The level of tumor necrosis factor alpha (TNF-alpha) mRNA, interleukin 10 (IL-10) mRNA, high mobility group box 1 (HMGB-1) mRNA in lung of rats were detected. Meanwhile, pathological changes of the lung were examined under optical microscope. RESULTS: Compared with that in normal control group, TNF-alpha mRNA expression in lung tissue of PQ group reached the peak at the six hour and decreased slowly at the first day [(0.740 +/- 0.100) and (0.584 +/- 0.049) respectively]. At the six hour and the first day in PQ group it was significantly higher than that in normal control group (P < 0.05 or P < 0.01). IL-10 mRNA expression in lung tissue of PQ group was elevated at the six hour, reached the peak at the first day, at the third day [(0.551 +/- 0.016) and (0.524 +/- 0.010) respectively] and the seventh day also higher than that in normal control group. At the first and the seventh day in the PQ group it was significantly higher than that in normal control group (P < 0.01). Meanwhile, HMGB-1 mRNA expression in lung tissue of PQ group was also elevated at the six hour, reached the peak at the first day, at the third [(0.695 +/- 0.060), (0.871 +/- 0.154) and (0.819 +/- 0.188) respectively] and the seventh day also higher than that in normal control group. At six hour, the first and the third day in the PQ group it was significantly higher than that in normal control group (P < 0.01). The histological changes such as alveolar edema, hemorrhage and inflammatory cell infiltration in the PQ group were more than those in the normal control group. CONCLUSION: In rats after PQ intoxication the levels of the inflammatory factors TNF-alpha, IL-10 and HMGB-1 are higher than normal rats, and inflammatory could play an important role in lung injury of poisoned rats.