Integration of metabolomics and transcriptomics reveals the regulation mechanism of the phenylpropanoid biosynthesis pathway in insect resistance traits in Solanum habrochaites.

Solanum habrochaites (SH), a wild species closely related to 'Ailsa Craig' (AC), is an important germplasm resource for modern tomato breeding. Trichomes, developed from epidermal cells, have a role in defense against insect attack, and their secretions are of non-negligible value. Here, we found that the glandular heads of type VI trichomes were clearly distinguishable between AC and SH under cryo-scanning electron microscopy, the difference indicating that SH could secrete more anti-insect metabolites than AC. Pest preference experiments showed that aphids and mites preferred to feed near AC compared with SH. Integration analysis of transcriptomics and metabolomics data revealed that the phenylpropanoid biosynthesis pathway was an important secondary metabolic pathway in plants, and SH secreted larger amounts of phenylpropanoids and flavonoids than AC by upregulating the expression of relevant genes in this pathway, and this may contribute to the greater resistance of SH to phytophagous insects. Notably, virus-induced silencing of Sl4CLL6 not only decreased the expression of genes downstream of the phenylpropanoid biosynthesis pathway (SlHCT, SlCAD, and SlCHI), but also reduced resistance to mites in tomato. These findings provided new genetic resources for the synthesis of phenylpropanoid compounds and anti-insect breeding in S. habrochaites and a new theoretical basis for the improvement of important traits in cultivated tomato.

Identification and characterization of circRNAs as competing endogenous RNAs for miRNA-mRNA in colorectal cancer.

BACKGROUND: Recent studies showed that circRNAs are involved in the biological process of some human cancers. However, little is known about their functions in colorectal cancer (CRC). METHODS: Here we first revealed the expression profiles of circRNAs in the CRC tissues and the adjacent non-tumorous tissues using high-throughput sequencing. The sequence feature, chromosome location, alternative splicing and other characteristics of the circRNAs were also explored. The miRNA and mRNA expression profiles were then obtained by analyzing relevant CRC data retrived from the TCGA database. We obtained and analyzed the competing endogenous RNA (ceRNA) network of the top three pairs of the largest up-regulated and down-regulated circRNAs. RESULTS: In this study, we obtained 50,410 circRNAs in the CRC tissue and the adjacent non-tumor tissues, of which 33.7% (16,975) were new, and revealed differential changes in circRNA expression during colorectal carcinogenesis. We have identified six potential key circRNAs (circPIEZO1-3, hsa_circ_0067163, hsa_circ_0140188, hsa_circ_0002632, hsa_circ_0001998 and hsa_circ_0023990) associated with CRC, which play important roles in carcinogenesis as ceRNA for regulation of miRNA-mRNA network. In the subsequent KEGG analysis, several CRC-related pathways were found. CONCLUSIONS: Our findings advance the understanding of the pathogenesis of CRC from the perspective of circRNAs and provide some circRNAs as candidate diagnostic biomarkers or potential therapeutic targets.

Newly synthesized podophyllotoxin derivative, LJ12, induces apoptosis and mitotic catastrophe in non-small cell lung cancer cells in vitro.

Deoxypodophyllotoxin (DPT), an active compound isolated from a number of herbs and used in traditional medicine, has been reported to exhibit promising anti­tumor activity. A newly synthesized derivative, N-(1-oxyl­4'-demethyl-4-deoxyp odophyllic)-L­methine-4'-piperazine carbamate (LJ12) may have improved antitumor activity and fewer side effects. The present study assessed the effect of LJ12 on cell viability, apoptosis, cell cycle distribution and mitotic catastrophe in A549 human lung cancer cells in vitro. The molecular mechanisms underlying the antitumor activity of LJ12 were also examined. The results demonstrated that LJ12 reduced A549 cell viability in a time­ and dose­dependent manner, with a lower half maximal inhibitory concentration of ~0.1 µM, compared with another known DPT derivative, etoposide (10 µM). Flow cytometric analysis showed that LJ12 induced tumor cell arrest at the G2/M phase of the cell cycle. The present study also observed an expected concomitant decrease in the numbers of cells cells in the G0/G1 and S phases. LJ12 was found to upregulate the protein expression levels of Cdc2 and Cyclin B1. Furthermore, LJ12 induced tumor cell apoptosis and the protein expression of B cell lymphoma­2­associated X protein, caspase­3 and p53. The present study also observed the formation of giant, multinucleated cells, indicating that LJ12 induced mitotic catastrophe in the tumor cells. These results indicated that LJ12 has anti­non­small cell lung cancer activity in vitro. Further investigations aim to develop LJ12 as a therapeutic agent for the treatment of lung cancer.

Antinociceptive effects of incarvillateine, a monoterpene alkaloid from Incarvillea sinensis, and possible involvement of the adenosine system.

Incarvillea sinensis is a Bignoniaceae plant used to treat rheumatism and relieve pain in traditional Chinese medicine. As a major component of I. sinensis, incarvillateine has shown analgesic activity in mice formalin tests. Using a series of animal models, this study further evaluated the effects of incarvillateine against acute, inflammatory, and neuropathic pain. Incarvillateine (10 or 20 mg/kg, i.p.) dose-dependently attenuated acetic acid-induced writhing, but did not affect thermal threshold in the hot plate test. In a Complete Freund's Adjuvant model, incarvillateine inhibited both thermal hyperalgesia and paw edema, and increased interleukin-1ß levels. Additionally, incarvillateine attenuated mechanical allodynia induced by spared nerve injury or paclitaxel, whereas normal mechanical sensation was not affected. Incarvillateine did not affect locomotor activity and time on the rotarod at analgesic doses, and no tolerance was observed after 7 consecutive daily doses. Moreover, incarvillateine-induced antinociception was attenuated by theophylline, 1,3-dipropyl-8-cyclopentylxanthine, and 3,7-dimethyl-1-propargylxanthine, but not naloxone, indicating that the effects of incarvillateine on chronic pain were related to the adenosine system, but not opioid system. These results indicate that incarvillateine is a novel analgesic compound that is effective against inflammatory and neuropathic pain, and that its effects are associated with activation of the adenosine system.

Kinetics and Immunodominance of Virus-Specific T Cell Responses During Hantaan Virus Infection.

Immunodominant T cell responses are important for protection against virus challenge. However, studies screening for the immunodominant T cell responses and following their kinetics in acute Hantaan virus (HTNV) infection are very limited. Herein, the HTNV nucleocapsid protein-specific T cell responses were longitudinally screened in 15 patients with acute HTNV infection, eight of whom had a particularly severe hemorrhagic fever with renal syndrome (HFRS). An extremely impaired IFN-γ-producing T cell response was observed in patients with severe HFRS at the early stage of infection, especially to the immunodominant epitopes detected in the mild to moderate group, namely peptides N127-141, N139-153, N241-255, and N355-369. The initially insufficient T cell response to the immunodominant epitopes may play a role in influencing the severity of HTNV infection. These findings provide information that may aid the design of future vaccines against hantaviruses.

PNAS-4, an Early DNA Damage Response Gene, Induces S Phase Arrest and Apoptosis by Activating Checkpoint Kinases in Lung Cancer Cells.

PNAS-4, a novel pro-apoptotic gene, was activated during the early response to DNA damage. Our previous study has shown that PNAS-4 induces S phase arrest and apoptosis when overexpressed in A549 lung cancer cells. However, the underlying action mechanism remains far from clear. In this work, we found that PNAS-4 expression in lung tumor tissues is significantly lower than that in adjacent lung tissues; its expression is significantly increased in A549 cells after exposure to cisplatin, methyl methane sulfonate, and mitomycin; and its overexpression induces S phase arrest and apoptosis in A549 (p53 WT), NCI-H460 (p53 WT), H526 (p53 mutation), and Calu-1 (p53(-/-)) lung cancer cells, leading to proliferation inhibition irrespective of their p53 status. The S phase arrest is associated with up-regulation of p21(Waf1/Cip1) and inhibition of the Cdc25A-CDK2-cyclin E/A pathway. Up-regulation of p21(Waf1/Cip1) is p53-independent and correlates with activation of ERK. We further showed that the intra-S phase checkpoint, which occurs via DNA-dependent protein kinase-mediated activation of Chk1 and Chk2, is involved in the S phase arrest and apoptosis. Gene silencing of Chk1/2 rescues, whereas that of ATM or ATR does not affect, S phase arrest and apoptosis. Furthermore, human PNAS-4 induces DNA breaks in comet assays and γ-H2AX staining. Intriguingly, caspase-dependent cleavage of Chk1 has an additional role in enhancing apoptosis. Taken together, our findings suggest a novel mechanism by which elevated PNAS-4 first causes DNA-dependent protein kinase-mediated Chk1/2 activation and then results in inhibition of the Cdc25A-CDK2-cyclin E/A pathway, ultimately causing S phase arrest and apoptosis in lung cancer cells.

Inhibitory effects of the attenuated Salmonella typhimurium containing the IL-2 gene on hepatic tumors in mice.

To observe the inhibitory effects of an attenuated S. typhimurium strain carrying IL-2 gene (TPI) on hepatoma cell line (HepG2) and transplanted tumors in mice. TPI, TPG (an attenuated S. typhimurium strain carrying green fluorescent protein gene), and TP (an attenuated S. typhimurium strain) strains were transfected into HepG2 cells. At 48 h after transfecting, the transfection rate was 82.58 ± 1.74%. The expression level of IL-2 was (99.5 ± 12.2) ng/1 × 10(6) cells. Compared with TPG, TP, and normal mouse groups, the proportion of CD4(+) T and CD8(+) T cells in the blood from the TPI group was higher, the levels of IgM and IgG(1) were significantly increased, and the proliferation activity of splenic lymphocyte was significantly stronger. The transplanted tumor weight in the TPI group was significantly smaller than that in the other two groups. The infiltration of lymphocytes increased in the tumor from TPI group mice. TPI was effectively transfected into cancer cells, which expressed the protein of interest. Oral administration of TPI prolonged survival of mice transplanted with hepatoma cell tumours.

Effect of oral hepatocyte growth factor gene mediated by attenuated salmonella on 2-, 4-, 6-trinitro-benzene-sulfonic-acid-induced ulcerative colitis in rat.

BACKGROUND AND AIM: In order to explore a new therapeutic method, we investigated the effects of exogenously expressed hepatocyte growth factor mediated by attenuated salmonella (TPH) on rats with ulcerative colitis (UC) induced by 2-, 4-, 6-trinitro-benzene-sulfonic acid. METHODS: The UC rats were treated with TPH, attenuated salmonella with a eukaryotic expression vector (TP) or sodium bicarbonate (model control [MC]) every other day. Cluster of differentiation (CD)4(+) and CD8(+) T cells and immunoglobulins in the blood were analyzed by flow cytometry. The HGF expression was determined by immunohistochemistry. A macroscopic-scale observation of the colon and a histological assessment were also carried out. RESULTS: The CD4(+) T counts and the CD4(+) /CD8(+) ratio in the TPH group were significantly lower than that in the MC group. The immunoglobulin M and immunoglobulin G(1) levels in the TPH group were significantly lower than that in the MC group and TP group. After treatment with TPH, the symptoms of the ulcerative rats were significantly alleviated. The colonic lesion grades in the TPH group were lower than that in the TP group and MC group. Significant improvement occurred after the TPH treatment, as evidenced by alleviated mucosal inflammation. At 7 days post-treatment, the HGF expression in the colonic tissues that were treated with TPH was stronger than that in the samples treated with TP. CONCLUSIONS: TPH inhibits the proliferation of T lymphocytes and the antibody production of B lymphocytes. Furthermore, it ameliorates mucosal inflammation and promotes the regeneration of mucosa and the healing of the colonic ulceration.

[Bortezomib combined with autologous peripheral blood hematopoietic stem cell transplantation for therapy of patients with multiple myeloma].

This study was aimed to evaluate the therapeutic efficacy of bortezomib combined with autologous peripheral blood hematopoietic stem cell transplantation (autoPBSCT) for patients with multiple myeloma (MM). 5 patients underwent autologous hematopoietic stem cell transplantation. Bortezomib treatment was supplied for patients before autoPBSCT and in the conditioning of transplantation, it was also used in maintaining treatment. Patients with transplantation adopted bortezomib plus melphalan conditioning regimen. The number of infused MNC and number of CD34(+) cells were 4.06×10(8) (4.09×10(8) - 4.37×10(8))/kg and 3.98×10(6) (2.49×10(6) - 8.2×10(6))/kg respectively. The results showed that hematopoiesis was reconstituted in 5 patients, with a neutrophil cell count more than 0.5×10(9)/L at day 14 (13 - 25 days) after transplantation and platelet count more than 50×10(9)/L at day 28 (21 - 41 days) after transplantation. Transplantation-associated death was not observed. 5 patients were disease-free survival. In conclusion, treatment of bortezomib combined with autologous peripheral hematopoietic stem cell transplantation is an effective method for patients with multiple myeloma. Use of bortezomib after transplantation might still be favourable to MM patients, for survival prolongation and life quality improvement.

Identification of three novel CTL epitopes within nucleocapsid protein of Hantaan virus.

Hantaan virus (HTNV) is a member of the Hantavirus genus that causes human hemorrhagic fever with renal syndrome (HFRS) in humans. The CTL response seems to play a key role in control of viral infection, but only a few HTNV epitopes recognized by the CTLs have been reported. Herein, we screened a panel of overlapping peptides covering the HTNV nucleocapsid protein by ELISPOT assays for those that can elicit IFN-γ production in vitro. Three novel CD8(+) CTL epitopes, N197-205 (RYRTAVCGL), N245-253 (KLLPDTAAV), and N258-266 (GPATNRDYL), were defined on the nucleocapsid protein and were found to be restricted by various HLA alleles including A11, A24, and B7. The epitopes were highly conserved among the reported HTNV strains and other hantanviruses, including Dobrava-Belgrade virus and Seoul virus, supporting their potential use in vaccine designs.

[Application of Epstein-Barr virus-transformed B lymphoblastic cells in identification of CTL epitopes specific for Hantaan virus].

AIM: To establish Epstein-Barr virus (EBV)-transformed B lymphoblastic cell lines (B-LCL) to present peptides as antigen-presenting cells (APC) and stimulate short-cultured T cells secreting IFN-gamma, by which the T cell epitopes are identified. METHODS: PBMCs from patients with hemorrhagic fever with renal syndrome (HFRS) were transformed using EBV from supernatant of B95-8 cells. ELISPOT assay was then employed to evaluate the IFN-gamma production of short-cultured G9L-specific CD8(+) T cells stimulated with peptide-pulsed autologous B-LCL cells. RESULTS: B-LCL pulsed with G9L or G9L-nested V15R can stimulate G9L-specific CD8(+) T cells producing IFN-gamma, but not B-LCL pulsed with non-homologous I15P. CONCLUSION: B-LCL can efficiently and specifically present peptides to peptide-specific T cells as non-professional APC.

Cellular immune response to Hantaan virus nucleocapsid protein in the acute phase of hemorrhagic fever with renal syndrome: correlation with disease severity.

BACKGROUND: The cellular immune response to Hantaan virus (HTNV) is incompletely understood, especially in humans. METHODS: To investigate the cellular immunity during acute HTNV infection, the magnitude of the CD4(+) and CD8(+) T cell responses to HTNV nucleocapsid protein was quantitated by direct ex vivo interferon-gamma (IFN-gamma) enzyme-linked immunosorbent spot analysis, using an array of overlapping peptides. RESULTS: We found that the combined frequencies of HTNV-specific T cells at the earliest available time point (5-8 days after fever onset) were significantly higher in patients who had mild or moderate hemorrhagic fever with renal syndrome (HFRS) than in those who had severe or critical HFRS (P= .006). Moreover, these frequencies were higher in patients with subsequent mild renal failure (maximum serum creatinine level, 707 micromol/L) (P= .006). Kinetic analysis showed that a decrease in the serum creatinine level during the acute phase of illness was often accompanied by an increase in the magnitude of IFN-gamma-producing T cells. CONCLUSION: Taken together with published data on the similar associations with neutralizing antibody, these data suggest that IFN-gamma-producing T cells may help reduce the risk of progression to acute renal failure caused by HTNV infection.

[Identification of HTNV-NP-specific T lymphocyte epitopes and analysis of the epitope-specific T cell response].

AIM: To identify the nucleocapsid protein of Hantaan virus (HTNV-NP)-specific T lymphocyte epitopes and analyze the epitope-specific T cell response during hemorrhagic fever with renal syndrome (HFRS). METHODS: T lymphocyte epitopes and frequencies of epitope-specific T cells were determined by ELISPOT using PBMCs from HFRS patients stimulated by individual or mixture of overlapping 15-mer peptides spanning the amino acid sequence of HTNV-NP. RESULTS: Out of 10 peptide mixtures, 8 elicited strong HTNV-NP-specific responses in 18 of 47 HFRS patients, and the T cell response was found at early stage of HFRS. Moreover, 17 HTNV-NP-specific T lymphocyte epitopes were identified in 11 patients, and most epitopes were clustered near the center of NP in linear structure. Among them, 14 T lymphocyte epitopes were described for the first time. CONCLUSION: HTNV-NP-specific T cell response can be elicited at early stage of HFRS and T lymphocyte epitopes mainly located in the center of NP, suggesting that it may play an important role in immune protection during HTNV infection.