Human ß-defensin-1 affects the mammalian target of rapamycin pathway and autophagy in colon cancer cells through long non-coding RNA TCONS_00014506.

BACKGROUND: Colorectal cancer has a low 5-year survival rate and high mortality. Human ß-defensin-1 (hBD-1) may play an integral function in the innate immune system, contributing to the recognition and destruction of cancer cells. Long non-coding RNAs (lncRNAs) are involved in the process of cell differentiation and growth. AIM: To investigate the effect of hBD-1 on the mammalian target of rapamycin (mTOR) pathway and autophagy in human colon cancer SW620 cells. METHODS: CCK8 assay was utilized for the detection of cell proliferation and determination of the optimal drug concentration. Colony formation assay was employed to assess the effect of hBD-1 on SW620 cell proliferation. Bioinformatics was used to screen potentially biologically significant lncRNAs related to the mTOR pathway. Additionally, p-mTOR (Ser2448), Beclin1, and LC3II/I expression levels in SW620 cells were assessed through Western blot analysis. RESULTS: hBD-1 inhibited the proliferative ability of SW620 cells, as evidenced by the reduction in the colony formation capacity of SW620 cells upon exposure to hBD-1. hBD-1 decreased the expression of p-mTOR (Ser2448) protein and increased the expression of Beclin1 and LC3II/I protein. Furthermore, bioinformatics analysis identified seven lncRNAs (2 upregulated and 5 downregulated) related to the mTOR pathway. The lncRNA TCONS_00014506 was ultimately selected. Following the inhibition of the lncRNA TCONS_00014506, exposure to hBD-1 inhibited p-mTOR (Ser2448) and promoted Beclin1 and LC3II/I protein expression. CONCLUSION: hBD-1 inhibits the mTOR pathway and promotes autophagy by upregulating the expression of the lncRNA TCONS_00014506 in SW620 cells.

Oxalic Acid in Root Exudates Enhances Accumulation of Perfluorooctanoic Acid in Lettuce.

Perfluorooctanoic acid (PFOA) is bioaccumulative in crops. PFOA bioaccumulation potential varies largely among crop varieties. Root exudates are found to be associated with such variations. Concentrations of low-molecular-weight organic acids (LMWOAs) in root exudates from a PFOA-high-accumulation lettuce variety are observed significantly higher than those from PFOA-low-accumulation lettuce variety (p < 0.05). Root exudates and their LMWOAs components exert great influences on the linear sorption-desorption isotherms of PFOA in soils, thus activating PFOA and enhancing its bioavailability. Among root exudate components, oxalic acid is identified to play a key role in activating PFOA uptake, with >80% attribution. Oxalic acid at rhizospheric concentrations (0.02-0.5 mM) can effectively inhibit PFOA sorption to soils by decreasing hydrophobic force, electrostatic attraction, ligand exchange, and cation-bridge effect. Oxalic acid enhances dissolution of metallic ions, iron/aluminum oxides, and organic matters from soils and forms oxalate-metal complexes, based on nuclear magnetic resonance spectra, ultraviolet spectra, and analyses of metal ions, iron/aluminum organometallic complexes, and dissolved organic carbon. The findings not only reveal the activation process of PFOA in soils by root exudates, particularly oxalic acid at rhizospheric concentrations, but also give an insight into the mechanism of enhancing PFOA accumulation in lettuce varieties.

[Electroacupuncture Intervention Improves Cartilage Degeneration and Subchondral Bone Osteoporosis of Knee-joint Possibly by Adjusting OPG/RANK/RANKL Signaling in Ovariectomized Rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on histopathological changes of cartilage and subchondral bone and osteoprotegerin (OPG)，receptor activator of nuclear factor-κB (RANK), and RANK ligand (RANKL) (OPG/RANK/RANKL) signaling and the expression of matrix metalloproteinase-13 (MMP-13) in ovariectomized(OVX)rats, so as to explore its mechanism underlying improvement of osteoporosis. METHODS: Three-month female SD rats were randomly divided into sham operation, model and EA groups (n＝8 in each group). The ovoariectomy model was established by resection of bilateral ovaries. Rats of the sham group were treated by simple removal of a piece of adipose tissue around the bilateral ovaries. EA (3 Hz/15 Hz, 1 mA) was applied to bilateral "Sanyinjiao" (SP 6), "Yanglingquan" (GB 34), "Yinlingquan" (SP 9) and "Zusanli" (ST 36) for 30 min, once daily (except the weekends) for 12 weeks. The histopathological changes of the subchondral bone of the right knee-joint were observed after Saffron O dyeing and evaluated by Mankin's score, and its anatomical structure including the bone volume fraction (bone volume/tissue volume, BV/TV), trabecular bone number (Tb. N) and trabecular thickness (Tb.Th), trabecular separation (Tb.Sp) was observed by using Micro CT imaging. The urine C-terminal cross-linking telopeptide of type Ⅰ collagen (CTX-Ⅰ), CTX-Ⅱ (two bone resorption markers) and serum estrogen (E 2) contents were assayed by ELISA, and the expression levels of OPG, RANKL and MMP-13 mRNAs in the cartilage tissue of the left knee-joint were detected by quantitative RT-PCR. RESULTS: Following modeling, the BV/TV, Tb. N and Tb.Th levels were significantly decreased (P<0.01) and Tb.Sp and Mankin's score obviously increased in the model group compared with the sham group (P<0.01), suggesting a formation of osteoporosis and degeneration of the cartilage tissue. The serum E 2 content and OPG mRNA level in the cartilagetissue were significantly down-regulated (P<0.01), and urine CTX-Ⅰ and CTX-Ⅱ contents and RANKL mRNA and MMP-13 mRNA expression levels cartilagetissue were considerably up-regulated in the model group relevant to the sham group (P<0.01). After EA intervention, modeling-induced decrease of BV/TV, Tb.N, Tb.Th, E 2 and OPG mRNA levels and OVX-induced increase of Tb.Sp, Mankin's score, CTX-Ⅰ， CTX-Ⅱ， RANKL mRNA and MMP-13 mRNA levels were all completely reversed in the EA group relevant to the model group (P<0.01，P<0.05)．. CONCLUSION: EA intervention can inhibit subchondral bone osteoporosis and articular cartilage degeneration of knee-joint in OVX rats, which is closely associated with its effects in inhibiting the down-regulation of serum E 2 and OPG mRNA expression and up-regulation of CTX-Ⅰ， CTX-Ⅱ， RANKL mRNA and MMP-13 mRNA levels, including adjusting OPG/RANK/RANKL signaling.

Cultivar-Dependent Accumulation and Translocation of Perfluorooctanesulfonate among Lettuce ( Lactuca sativa L.) Cultivars Grown on Perfluorooctanesulfonate-Contaminated Soil.

Perfluorooctanesulfonate (PFOS) is a toxic and persistent organic pollutant that can be widely detected in agricultural soils. In this study, two lettuce cultivars with low PFOS accumulation were screened out to reduce the exposure of PFOS to the human body via vegetable consumption. The screened low-PFOS cultivars may help to ensure food safety, despite planting in highly PFOS-polluted soils (1.0 mg/kg), due to their high tolerance to PFOS and 4.4-5.7 times lower shoot PFOS concentration than the high-PFOS cultivars. Protein content and protein-mediated transpiration played key roles in regulating PFOS accumulation in the lettuce cultivars tested. Lower protein content, lower stomatal conductance, and lower transpiration rate resulted in low PFOS accumulation. This study reveals the mechanism of forming low-PFOS accumulation of lettuce cultivars at physiological and biochemical levels and lays a foundation for developing a cost-effective and safe approach to grow vegetables in PFOS-polluted soils.

Systematic module approach identifies altered genes and pathways in four types of ovarian cancer.

The present study aimed to identify altered genes and pathways associated with four histotypes of ovarian cancer, according to the systematic tracking of dysregulated modules of reweighted protein­protein interaction (PPI) networks. Firstly, the PPI network and gene expression data were initially integrated to infer and reweight normal ovarian and four types of ovarian cancer (endometrioid, serous, mucinous and clear cell carcinoma) PPI networks based on Spearman's correlation coefficient. Secondly, modules in the PPI network were mined using a clique­merging algorithm and the differential modules were identified through maximum weight bipartite matching. Finally, the gene compositions in the altered modules were analyzed, and pathway functional enrichment analyses for disrupted module genes were performed. In five conditional­specific networks, universal alterations in gene correlations were revealed, which leads to the differential correlation density among disrupted module pairs. The analyses revealed 28, 133, 139 and 33 altered modules in endometrioid, serous, mucinous and clear cell carcinoma, respectively. Gene composition analyses of the disrupted modules revealed five common genes (mitogen­activated protein kinase 1, phosphoinositide 3­kinase­encoding catalytic 110­KDα, AKT serine/threonine kinase 1, cyclin D1 and tumor protein P53) across the four subtypes of ovarian cancer. In addition, pathway enrichment analysis confirmed one common pathway (pathways in cancer), in the four histotypes. This systematic module approach successfully identified altered genes and pathways in the four types of ovarian cancer. The extensive differences of gene correlations result in dysfunctional modules, and the coordinated disruption of these modules contributes to the development and progression of ovarian cancer.

[Effects of cadmium on protein tyrosine phosphorylation of mouse spermatozoa and the protective role of EGTA]. / é•‰å¯¹å°é¼ ç²¾å­è›‹ç™½é ªæ°¨é ¸ç£·é ¸åŒ–ä¿®é¥°çš„å½±å“åŠEGTA的保护作用.

In this study, we explored the effects of cadmium (Cd) on mouse sperm motility parame-ters, protein tyrosine phosphorylation and the location of tyrosine-phosphorylated targets using computer-assisted sperm analysis (CASA), western blot (WB) and immunofluorescence technique coupled to sperm in vitro culture method, respectively. The results showed sperm motility was inhibi-ted by Cd in a dose-dependent manner and when Cd increased to 1.0 µmol·L-1, sperm motility was inhibited significantly (P<0.05). Simultaneously, protein tyrosine phosphorylation was enhanced by Cd and in particular, the tyrosine phosphorylation of ～55 kDa proteins was greatly promoted when Cd concentrations were greater or equal to 1.0 µmol·L-1 (P<0.05). Importantly, these tyrosine-phosphorylated proteins were mainly localized in the middle piece of mouse sperm. However, when sperm was incubated with 30 µmol·L-1 ethylene glycol tetraacetic acid (EGTA) and 10 µmol·L-1 Cd concurrently, both the tyrosine phosphorylation of ～55 kDa proteins and sperm motility were not changed obviously (P>0.05). These results suggested that Cd may inhibit sperm motility by inducing the tyrosine phosphorylation of ～55 kDa proteins in the middle piece and EGTA could chelate Cd ions to relieve its toxicity. This study demonstrated that Cd induced the tyrosine phosphorylation of a specific subset of proteins and thus decreased sperm motility. Interes-tingly, EGTA acted as an inhibitor to block Cd from entering the sperm, which provided a novel research method for revealing the molecular mechanisms of reproductive toxicity caused by Cd.

[The induced IL-21 levels in CD4(+) T cells of peripheral blood from the different clinical types of patients infected with hepatitis B virus].

AIM: To investigate the levels of interleukin (IL)-21 in CD4(+);T cells of peripheral blood from the different types of patients infected with hepatitis B virus (HBV) and elucidate its role in the hepatitis B pathogenesis. METHODS: Peripheral blood mononuclear cells (PBMCs) from the patients infected with HBV and healthy individuals were stimulated with or without PMA coupled with ionomycin. The levels of IL-21 in CD4(+) T cells and Th17 cells were analyzed by flow cytometry. RESULTS: PMA and ionomycin induced the expression of IL-21, and IL-21 was mainly produced by CD4(+); T cells, but IL-17A(+) IL-21(+) CD4(+) T cells were not detected. The frequencies of IL-21(+) CD4(+) T cells in the patients of acute hepatitis B and chronic asymptomatic HBV carriers were higher than in healthy controls and severe chronic hepatitis B patients; there were no remarkable differences in the proportion of Th17 cells among the different groups of patients. Furthermore, the proportion of IL-21(+) CD4(+) T cells correlated with Th17 cells in all groups except for the acute hepatitis B patients. CONCLUSION: IL-21 levels, which correlated with Th17 cells, were different in CD4(+);T cells from the different types of patients infected with hepatitis B virus (HBV). The results showed that IL-21 may play a role in the hepatitis B pathogenesis.

Overexpression of Toll-like receptor 2/4 on monocytes modulates the activities of CD4(+)CD25(+) regulatory T cells in chronic hepatitis B virus infection.

The significance of TLR expression and Tregs in HBV infection has not been clearly described. In this report, flow cytometry was performed to assess TLR2/4 expression on monocytes and circulating CD4(+)CD25(+)CD127(low/-) Tregs frequency of 16 acute hepatitis B (AHB), 42 chronic hepatitis B (CHB), 22 asymptomatic HBV carriers (AsC), and 20 normal controls (NC). We found that TLR2 and TLR4 were overexpressed on CD14(+) monocytes in HBV-infected patients as compared with NCs. Upregulation of TLR2 in NCs and TLR4 in CHBs was observed following HBeAg incubation. However, TLR2 and TLR4 expression decreased after HBcAg stimulation. The difference in the proportion of Tregs between NCs and CHBs was significant. Both Pam3Csk4 (TLR2 agonist)- and lipopolysaccharide (TLR4 agonist)-activated CD4(+)CD25(+) Tregs showed enhanced suppression function in CHBs. These results suggest that overexpression of TLR2 and TLR4 may modulate the suppressive function of Tregs, which contribute to the immunotolerance of chronic HBV infection.

Down-regulation of CXCR4 expression by SDF-KDEL in CD34(+) hematopoietic stem cells: An anti-human immunodeficiency virus strategy.

CXCR4 plays an essential role as the first discovered coreceptor for the entry of T cell tropic isolates of HIV-1. Blocking the surface expression of this receptor may be a potential strategy to prevent HIV-1 infection. A lentiviral vector, pLenti6/V5-S-K, expressing a SDF-KDEL fusion protein was constructed and a replication-incompetent lentiviral stock was produced. The lentiviral stock was transduced into CD34(+) hHSC and the transient expression of the recombinant protein, SDF-1, was assayed using indirect immunofluorescence. The surface expression of CXCR4 in CD34(+) hHSC pretreated with different amounts of recombinant lentiviral vectors was detected by flow cytometric analysis. A marked down-regulation of CXCR4 expression in the cells transduced with recombinant lentiviral vectors pLenti6/V5-S-K was observed by flow cytometry with PE-conjugated anti-human CXCR4 monoclonal antibodies which showed the percentages of the inhibition effects of CXCR4-SDF-1 mediated syncytium formation are presented by concentration. P24 antigen levels of cell culture supernatants were detected on the 4th, 7th, and 10th day, with 10(3) TCID50 HIV-1 infected CD34(+) hHSC to evaluate the inhibitory effect of pLenti6/V5-S-K transduction on HIV-1 infection. The cells transfected with pLenti6/V5-S-K had a significant reduction of HIV-1 DP27 infection compared to controls (P<0.05).

Expression of L protein of Hantaan virus 84FLi strain and its application for recovery of minigenomes.

Hantaan virus (HTNV) is an Old World hantavirus associated with hemorrhagic fever with renal syndrome (HFRS). To visualize the localization of the L protein of HTNV strain 84FLi within cells, a fusion protein composed of enhanced green fluorescent protein and L protein, EGFP-L, was expressed in Vero cells. The 273 KDa expressed fusion protein of EGFP-L localized in the perinuclear region. We also described the development of a reverse genetics system for HTNV strain 84FLi. The RNA polymerase I (pol I)-mediated transcription system was used to generate artificial viral RNA genome segments (minigenomes), which contained the chloramphenicol acetyltransferase (CAT) reporter gene in antisense (virus RNA) or sense (virus-complementary RNA) orientation flanked by the noncoding regions of HTNV 84FLi L segment. CAT could be detected in cells after transfection, indicating the successful encapsidation, transcription and replication of the pol I-derived minigenomes. The passaged transfer of CAT demonstrates that recombinant virus containing packaged pol I-derived minigenomes has been produced. This system may be helpful in studying the gene function and pathogenesis of HTNV.

[Study on the mechanism of HLA-I-expressing regulation caused by the core region mutation of HBV adr subtype].

OBJECTIVE: To study the influence and mechanism of HBV core region mutation on HLA-I expression. METHODS: Eukaryotic expression vectors of HBV core region mutations L97, G87 and V60 were constructed and transfected into HepG2 cells. Then the expressions of HLA-I were detected by RT-PCR and Western blot. The mRNA of antigen-presentation-associated genes, including LMP2, TAP1 and tapasin, were measured using RT-PCR. RESULTS: Different levels of HBsAg in the supernatants of transfected cells were detected by ELISA. The HBsAg of the mutated groups was markedly higher than that of the wild ones. All the transfected cells expressed HLA-I molecules, especially the L97 group. It was also found that the mRNA of TAP1 gene was up-regulated, while the mRNA of LMP and tapasin genes had no changes. CONCLUSION: The core region mutation of HBV can lower the expression of HBsAg; mutated groups and wild ones both can increase the expression of HLA-I molecules. The up-regulation of TAP1 gene expression might be the cause of these changes.