Evolutionary law and regulatory technology of roof migration on gob-side entry retaining.

In order to study the evolutionary law of roof migration on Gob-Side Entry Retaining, this paper takes the gob-side entry retaining in the comprehensive mining face of the Ningtiaota coal mine as the engineering background, and analyzes the evolutionary law of the overlying rock layer on the roof at different locations during the roadway stay and the stress distribution around the roadway through numerical simulation software, which shows that there is a concentration of stress inside the Flexible formwork concrete wall, and therefore the maximum settlement of the roof on the side of Flexible formwork concrete wall is 35.35 mm, due to the existence of "arch-shaped" decompression area from the working face. Therefore, the maximum settlement of the roof slab on the side of flexible formwork concrete wall was 35.35 mm. Due to the existence of "arch-shaped" decompression area on the roof and floor of roadway, the settlement of the roof slab on both sides of the roadway gradually increased when it was from - 20 to 10 m away from the working face, and the central position had the following pattern of firstly decreasing and then gradually increasing, and then exceeding the top of the roadway. After decreasing and then gradually increasing, after 10 m ahead of the working face, the two sides of the roadway roof subsidence law and the central part of the roadway to maintain the same; the use of cutting the top of the flexible mold concrete wall support technology as a means of controlling the top of the roof along the empty roadway subsidence, the analysis shows that the roof after roof cutting of the amount of subsidence have been reduced, the maximum difference in the rate of change of the displacement is 0.011%, and the maximum difference in the amount of subsidence of 4.98 mm; through the field monitoring data analysis of the pressure of mining The peak value of the influence curve of the working face is located at 19 m of the working face, 9 m of the lagging working face and 19 m of the roadway outside the working face are less affected by the additional mining stress field, comparing the fracture brokenness of the roadway roof before and after the roof cutting, the fracture area in the uncut section is much larger than that in the section of the roof cutting.

Characteristics of surrounding rock damage and control technology of a facing-mining excavating roadway in north Shaanxi mining area.

In a coal mine in the northern region of Shaanxi Province, China, a facing-mining excavating roadway exists, which is intended to be retained for subsequent working face safety services. This paper investigates the deformation and damage characteristics of the surrounding rock in different stages using FLAC 3D numerical simulation, taking the facing-mining excavating roadway of this coal mine as the research context. At 20 m ahead of the working face, a discontinuous plastic zone appears in the surrounding rock of the roadway, a phenomenon attributed to the varying hardness of the lithologyand termed 'plastic zone jumping.' The numerical simulation results have been were verified using drill hole peeping. Real-time monitoring of the roadway's stability is conducted on-site, showing that the roadway is significantly affected by mining at the 50 m point ahead of the working face. Based on the numerical simulation and on-site monitoring results, the support strength was increased at 50 m from the working face along the roadway, and a new support scheme was adopted. In the lagging section of the roadway, where mining pressure is strongly evident, differentiated reinforcement using anchor rods, anchor ropes, and W steel belts has been employed, resulting in a satisfactory on-site effect.

Destrin Contributes to Lung Adenocarcinoma Progression by Activating Wnt/ß-Catenin Signaling Pathway.

Lung cancer, especially lung adenocarcinoma, is one of the most common neoplasms worldwide. However, the mechanisms underlying its initiation, development, and metastasis are still poorly understood. Destrin (DSTN) is a member of ADF/cofilin family. Its detailed biological function remains unknown, although it is reported that DSTN is involved in cytoskeleton remodeling and regulation of actin filament turnover. Recent evidence has shown that high expression of cofilin-1 is associated with invasion and poor prognosis of several types of human tumors, but the detailed mechanism is still entirely unclear, particularly in lung cancer tumorigenesis and malignancy. Here, we report that DSTN was highly expressed in a mouse lung cancer model induced by urethane and in clinical lung adenocarcinoma tissue samples. Its expression level was positively correlated with cancer development, as well as metastasis to the liver and lymph nodes. Consistently, it was directly associated with the poor prognosis of lung adenocarcinoma patients. Furthermore, we also found that DSTN promotes cell proliferation, invasion, and migration in vitro, and facilitates subcutaneous tumor formation and lung metastasis via intravenous injection in vivo. Mechanically, DSTN associates with and facilitates nuclear translocation of ß-catenin, which promotes epithelial-to-mesenchymal transition (EMT). Taken together, our results indicated that DSTN enhances lung cancer malignancy through facilitating ß-catenin nuclear translocation and inducing EMT. Combined with multivariate analyses, DSTN might potentially serve as a therapeutic target and an independent prognostic marker of lung adenocarcinoma. IMPLICATIONS: This finding indicates that DSTN facilitates ß-catenin nuclear translocation and promotes malignancy in lung adenocarcinoma.

Effect of PAK1 gene silencing on proliferation and apoptosis in hepatocellular carcinoma cell lines MHCC97-H and HepG2 and cells in xenograft tumor.

This study intends to explore the effect of the PAK1 gene silencing on apoptosis and proliferation of hepatocellular carcinoma (HCC) MHCC97-H and HepG2 cells and cells in xenograft tumor. MHCC97-H and HepG2 cells and mice with xenograft tumor in vivo were randomly divided into control, empty vector and PAK1 shRNA groups. Morphology and the expression of green fluorescent protein of MHCC97-H and HepG2 cells and cells in xenograft tumor were observed. MTT assay and flow cytometry were used to detect proliferation, cell cycle and apoptosis of MHCC97-H and HepG2 cells and cells in xenograft tumor. The expressions of PAK1, PCNA, Ki67, Cyclin E, CDK2, p21, p53, Bax and Bcl-2 were measured using the quantitative reverse transcription polymerase chain reaction and western blotting. Compared with the control and empty vector groups, number of adherent cells of MHCC97-H and HepG2 cells and cells in xenograft tumor was reduced, and green fluorescent cells became round and reduced in the PAK1 shRNA group. Cell proliferation, the cells at S phase, the mRNA and protein expressions of PAK1, PCNA, Ki67, Cyclin E, CDK2 and Bcl-2 of MHCC97-H and HepG2 cells and cells in xenograft tumor were decreased, while the cells at G1 phase, apoptosis rate, the mRNA and protein expressions of p21, p53 and Bax of MHCC97-H and HepG2 cells and cells in xenograft tumor were increased in the PAK1 shRNA group. PAK1 gene silencing decreases proliferation of MHCC97-H cells, HepG2 cells and cells in xenograft tumor through the p53/p21 pathway.

Development and evaluation of a MAb-based ELISA for detection of Chlamydophila pneumoniae infection with variable domain 2 and 3 of the major outer membrane protein.

OBJECTIVE: This paper aims to develop a monoclonal antibodies (MAbs)- based ELISA for detecting Chlamydophila pneumoniae (C. pneumoniae) antigens in humans with the variable domains (VD) 2 and 3 of the major outer membrane protein (MOMPVD2-VD3) and to assess its sensitivity and specificity by comparing with a widely used MAb that is able to recognize the elementary bodies of C. pneumoniae. METHODS: MOMPVD2-VD3 were overexpressed in Escherichia coli and purified by affinity chromatography. Mice were immunized with the recombinant antigen, and hybridomas secreting MAbs were screened. Three stable hybridomas clones were selected and named 5D6, 7G3, and 8C9. The MAbs-based ELISA was scrutinized for species-specific recognition with a number of human throat swab samples from Group I (156 patients with typical respiratory illness clinically confirmed before) and Group II (57 healthy donors). RESULTS: In Group I, 55 positive cases were detected by anti-EB MAb-based ELISA, 51 cases were positive by MAbs 5D6-based ELISA, and 33 and 38 cases were positive by MAb 8C9 and 7G3-based ELISA respectively. Of the 57 samples from Group II "healthy donors", 5 were positive and 52 were negative with both anti-EB and 5D6-based tests, while 2 and 3 positive cases were identified by the other two MAb-based ELISAs respectively. CONCLUSION: The novel MOMPVD2-VD3 MAb-based assay may have higher specificity than the anti-EB MAb, which may possibly be used as an alternative tool for the diagnosis of C. pneumoniae infection.

A novel agonistic anti-human death receptor 5 monoclonal antibody with tumoricidal activity induces caspase- and mitochondrial-dependent apoptosis in human leukemia Jurkat cells.

An agonistic antibody against TNF-related apoptosis-inducing ligand death receptor 5 (DR5) is a practicable candidate drug for antitumor therapy. In this study, a novel murine anti-human DR5 monoclonal antibody, mDRA-6(IgG1-κ), has been generated. This study aimed to explore the caspase-dependent and mitochondrial mechanisms of mDRA-6 in inducing apoptosis in human leukemia Jurkat cells. The apoptotic effects of mDRA-6 on Jurkat cells, which express DR5 on the cell surface, were detected by flow cytometry and western blot after exposure to different doses of mDRA-6 and at fixed doses of mDRA-6 at different times. It was demonstrated that mDRA-6 can induce Jurkat cell apoptosis via caspase- and mitochondrial-dependent pathways. These results indicate that the novel antibody mDRA-6 against DR5 has an antitumor function and may provide a new reagent for tumor therapy.

Caspase-dependent molecular mechanisms of anti-human DR5 monoclonal antibody mDRA-6 inducing apoptosis of human leukemia Jurkat cells.

BACKGROUND AND OBJECTIVE: Both tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and some monoclonal agonistic antibodies against TRAIL receptors have antitumor activity. We have previously prepared a novel monoclonal agonistic antibody against human death receptor 5 (DR5) and designated it as mDRA-6. This study was to explore the Caspase-dependent molecular mechanisms of mDRA-6 inducing apoptosis of human leukemia Jurkat cells. METHODS: After exposure to different doses of mDRA-6, DNA fragmentation of Jurkat cells was detected by agarose gel electrophoresis, cell proliferation was detected by MTT assay, and cell apoptosis was detected by flow cytometry after Annexin V-FITC/PI double staining. Jurkat cells were further treated with the inhibitors for Caspase-10, -9, -8 and -3. The active cleavage products of Caspase-10, -9, -8, -3 and poly ADP-ribose polymerase (PARP), BH3 interacting domain death agonist (Bid), truncated Bid (tBid) and cytochrome c (Cyto c), were analyzed by western blot. RESULTS: After mDRA-6 treatment, DNA fragmentation was detected in Jurkat cells. mDRA-6 inhibited cell proliferation in a dose-dependent manner. When treated with 2.0 microg/mL mDRA-6, the apoptosis rates of Jurkat cells were 16.2% at 0.25 h, 28.3% at 0.5 h, 69.2% at 1 h and 78.2% at 2 h. Interestingly, the mDRA-6-induced apoptosis was repressed by 77.9% by Caspase-8 inhibitor ZIF, 54.2% by Caspase-3 inhibitor ZDF, and 8.7% by Caspase-9 inhibitor ZLF, but was not repressed by Caspase-10 inhibitor ZAF. After mDRA-6 exposure, the proenzymes of Caspase-8, -9 and -3 were reduced and their active cleavage products were increased along with the increase of exposure time, the cleavage products of PARP were also increased, Bid was degraded to tBid, and an abundance of Cyto c was released from mitochondria, but the proenzyme of Caspase-10 showed no change and no cleavage products of Caspase-10 were detectable. CONCLUSION: mDRA-6 can induce apoptosis of Jurkat cells via the Caspase-dependent and mitochondrial pathways.

Detachment of esophageal carcinoma cells from extracellular matrix causes relocalization of death receptor 5 and apoptosis.

AIM: To investigate the effect of detachment of esophageal cancer cells from extracellular matrix on the localization of death receptor 5 (DR5) and apoptosis. METHODS: Anchorage-dependent EC9706 cells of esophageal squamous cell carcinoma were pretreated or not treated with brefeldin A. Detached cells were harvested by ethylenediaminetetraacetic acid digestion. Expression and localization of DR5 in these cells were determined by immunocytochemical and immunofluorescence assays, as well as flow cytometry analysis. Apoptosis of EC9706 cells was detected by flow cytometry after stained with fluorescein isothiocyanate-labeled annexin V/propidium iodide. Activation of caspase 8 was detected by Western blot analysis. RESULTS: Immunocytochemical assay indicated that DR5 was predominantly perinuclear in adherent cells but was mainly localized in cell membrane in detached cells. In addition, immunofluorescence assay also confirmed the above-mentioned results, and further demonstrated that DR5 was present in the form of coarse granules in detached cells, but in the form of fine granules in adherent cells. Cytometry analysis revealed higher levels of DR5 expression on the surfaces of brefeldin-A-untreated cells than on the surfaces of brefeldin-A-treated cells, but brefeldin A treatment did not affect the total DR5 expression levels. Moreover, nocodazole did not influence the extracelluar DR5 expression levels in EC9706 cells. Apoptosis assay revealed that detached cells were more sensitive to DR5 antibody-induced apoptosis than adherent cells. Western blotting showed that caspase 8 was activated in temporarily detached cells 4 h earlier than in adherent cells. CONCLUSION: Progress from adhesion to detachment of EC9706 cells causes DR5 relocalization, and promotes cytoplasmic translocation of DR5 to cell surfaces via a Golgi-dependent pathway. Moreover, it might also result in DR5 aggregation to render apoptosis of detached cells.

[Apoptosis induced by NNAMB, a novel polyamine conjugate, in human erythroleukemia K562 cells and its mechanism].

OBJECTIVE: To investigate the apoptosis-inducing effects of NNAMB, a novel polyamine conjugate, in erythroleukemia K562 cells and its molecular mechanism. METHODS: Cell viability was assessed by MTT assay and trypan blue dye exclusion method. The cell morphology was observed by fluorescence microscopy. The cell cycle distribution, apoptosis and mitochondrial membrane potential were measured by flow cytometry. The expression of caspase-3, -8, -9, cytochrome c in the K562 cells was detected by Western blot. RESULTS: NNAMB inhibited the proliferation of K562 cells. The cells treated with NNAMB showed a typical apoptotic morphology, Sub-G1 peak and loss of mitochondrial membrane potential. Western blot assay showed that NNAMB increased the expression of caspase-3, -9, cytochrome c but not caspase-8 in a dose-and time-dependent manner. CONCLUSION: NNAMB induces apoptosis via mitochondrial pathway in K562 cells.

Proteomic identification of malignant transformation-related proteins in esophageal squamous cell carcinoma.

Esophageal cancer (EC) persists to be a leading cancer-related death in northern China. Clinical outcome of EC is the most dismal among many types of digestive tumors because EC at early stage is asymptomatic. The current study used 2-DE-based proteomics to identify differentially expressed proteins between esophageal cancer cell lines and immortal cell line. Fifteen proteins were identified with differences of more than five folds, comprising the down-regulation of annexin A2, histone deacetylase 10 isoform beta and protein disulfide-isomerase ER-60 precursor, and the up-regulation of heat shock 70 kDa protein 9B precursor, solute carrier family 44 Member 3, heterogeneous nuclear ribonucleoprotein L (hnRNP L), eukaryotic translation initiation factor 4A isoform 2, triosephosphate isomerase1 (TPI), peroxiredoxin1 (PRX1), forminotransferase cyclodeaminase form (FTCD), fibrinogen gamma-A chain precursor, kinesin-like DNA binding protein, lamin A/C, cyclophilin A (CypA), and transcription factor MTSG1. Expression pattern of annexin A2 was verified by Western blotting, immunocytochemistry and immunohistochemistry analysis. The implication of these protein alterations correlated to the esophageal malignant transformation is discussed.

Synergistic antitumor effects of anthracenylmethyl homospermidine and alpha-difluoromethylornithine on promyelocytic leukemia HL60 cells.

The present study was designed to assess the synergistic antitumor effects of anthracenylmethyl homospermidine (ANTMHspd), a novel polyamine conjugate, with alpha-difluoromethylornithine (DFMO) and to elucidate the mechanism of these effects on human leukemia HL60 cells. Cell proliferation was assessed by the MTT assay. Cell cycle, apoptosis and mitochondria membrane potential (MMP) were evaluated by flow cytometry. Caspases and cytochrome c were detected by Western Blot analysis. The combination treatment strongly inhibited cell proliferation, induced cell apoptosis and caused an accumulation in the G1 phase with an accompaniment decrease in S phase. Moreover, reduction of MMP, release of cytochrome c and activation of caspase-3 and caspase-9 but not caspase-8 were observed during the combination-mediated apoptosis. All these findings demonstrated that the combination treatment with DFMO and ANTMHspd resulted in synergistic antitumor effects on HL60 cells.

A novel homospermidine conjugate inhibits growth and induces apoptosis in human hepatoma cells.

AIM: To elucidate the mechanism responsible for the antiproliferative effects of a novel homospermidine conjugate, anthracenylmethyl homospermidine (ANTMHspd), in the human hepatoma BEL-7402 cell line. METHODS: The viability of the cells was assessed by MTT assay and the trypan blue dye exclusion method. Morphological changes were observed by fluorescence microscopy with Hoechst 33258 staining. Cell cycle distribution, apoptosis, and mitochondrial membrane potential were measured by flow cytometry. Protein expression was detected by Western blot analysis. RESULTS: ANTMHspd strongly decreased BEL-7402 cell proliferation in a dose- and time-dependent manner. Hoechst 33258 staining and the flow cytometry assay showed that ANTMHspd induced cell apoptosis and cell cycle perturbation. Furthermore, ANTMHspd could induce mitochondrial membrane potential loss and cytochrome c release and enhance cleaved caspase-3, cleaved caspase-9, and Bax protein expression without caspase-8 activation. ANTMHspd could also decrease the expression of Bcl-2 and cytochrome c in mitochondria. In addition, the specific inhibitors of caspase-9 and caspase-3 almost abolished the ANTMHspd-induced caspase-9 and caspase-3 activation, respectively. CONCLUSION: ANTMHspd could induce BEL-7402 cell apoptosis via the mitochondrial/caspase-dependent pathway and the Bcl-2 family was involved in the control of apoptosis.

[Leukemic cell apoptosis induced by anti-human DR5 monoclonal antibody mDRA-6].

AIM: To investigate the apoptotic effect of anti-human DR5 (death receptor 5 of TRAIL) monoclonal antibody mDRA-6 on leukemic cells. METHODS: The morphological changes of leukemic cells were observed by fluorescence microscope. The cytotoxic and apoptotic effects of mDRA-6 on Jurkat, HL-60 and K562 cells were detected by MTT analysis and flow cytometry with Annexin V-FITC/PI staining. RESULTS: Chromatin condensation, budding and apoptotic bodies were observed in Jurkat and HL-60 cells treated by mDRA-6. Death and apoptosis of leukemic cells treated by mDRA-6 were increased, but the effect of mDRA-6 on K562 cells was not obvious. CONCLUSION: Apoptosis of leukemic cells can be induced by anti-human DR5 monoclonal antibody mDRA-6. Different leukemic cell lines are of different sensitivity to mDRA-6.

[Adriamycin enhances anti-human DR5 monoclonal antibody (mDRA-6) induced HL-60 cells apoptosis].

OBJECTIVE: To investigate synergistic killing effect of anti-human DR5 (death receptor 5 of TRAIL) monoclonal antibody (mDRA-6) and adriamycin(Adr) on HL-60 cells. METHODS: mDRA-6 was prepared by immunizing BALB/c mice with DR5 protein. DR5 expression on Adr-treated HL-60 cells was detected by flow cytometry. Morphologic changes of HL-60 cells were observed under fluorescence microscope. Cytotoxic and apoptotic effects of mDRA-6 and Adr on HL-60 cells were measured by MTT analysis. DNA fragmentation was detected by agarose gel electrophoresis. RESULTS: Adr induce DR5 expression on HL-60 cells. Cell budding, chromatin condensation and apoptotic body formation were observed in HL-60 cells treated by mDRA-6 and Adr. Death and apoptosis of these cells and DNA ladder were exhibited on agarose gel electrophoresis. CONCLUSION: mDRA-6 and Adr have synergistic killing effect on HL-60 cells.

[Cytotoxic mechanism of anti-human death receptor 5 monoclonal antibody mDRA-6].

AIM: To investigate the cytotoxic action and its mechanism of a novel anti-human DR5 monoclonal antibody (mAb mDRA-6). METHODS: The cytotoxic action of mAb mDRA-6 on Jurkat cells and the effects of inhibitors of caspase 8 and caspase 9 on apoptosis of Jurkat cells induced with mAb mDRA-6 were detected by flow cytometry. The effects of mAb mDRA-6 on the morpha of Jurkat cells was observed by fluorescence microscope. The apoptosis of Jurkat cells was detected by flow cytometry with Annexin V-FITC/PI staining. The DNA fragmentation in Jurkat cells was analysed by agrose gel electrophoresis. RESULTS: mAb mDRA-6 exerted cytotoxicity on Jurkat cells in dose-dependent and time-dependent manner. Jurkat cells treated with mDRA-6 exhibited typical apoptostic features in morphology, namely, membrane crenation, bubbling, chromatin condensation, and formation of apoptotic bodies. The flow cytometry analysis showed that phosphatidylserine (PS) was highly expressed in Jurkat cells treated with mDRA-6. Agrose gel electrophoresis indicated that DNA fragmentation occurred in Jurkat cells. Inhibitor of caspase 8 inhibited the apoptosis of Jurkat cells induced with mDRA-6 while Inhibitor of caspase 9 showed less effect. CONCLUSION: mDRA-6 may exert cytotoxicity by inducing Jurkat cell apoptosis through signal transduction pathway of death receptors, which may be a useful tool in treating tumors with DR5 as target molecule and exploring the functional domain of DR5.

[Effect of anti-human DR5 monoclonal antibody on the apoptosis of human hepatocyte HL7702 cell lines].

AIM: To evaluate the effect of a novel anti-human DR5 monoclonal antibody (mAb mDRA-6) on the apoptosis of human hepatocyte HL7702 cell lines. METHODS: DR5 expression on HL7702 cell surface was determined by flow cytometry(FCM). The effect of mAb mDRA-6 on the morphous of HL7702 cells was observed by fluorescence microscope. mDRA-6 cytotoxicity on HL7702 cells was detected by using MTT analysis. The rate of apoptosis was detected by FCM with Annexin V-FITC/PI staining. RESULTS: HL7702 cells treated with mDRA-6 exhibited some typical apoptotic features in morphology. MTT analysis showed the death rate of HL7702 cells was 39% in the presence of 40 mg/L mDRA-6 for 6 hours. FCM detection indicated the apoptosis rate of the cells was 25.5% in the presence of 3 mg/L mDRA-6 for 6 hours with Annexin V-FITC/PI staining. CONCLUSION: mDRA-6 can induce the apoptosis of HL7702 cells.

[Preparation and characterization of monoclonal antibody against DR5].

AIM: To prepare monoclonal antibodies(mAb) against DR5 and characterize their properties. METHODS: BALB/c mice were immunized with DR5, and then mAb was prepared by hybridoma technique. Ig subclass and specificity of mAbs was analyzed by ELISA and flow cytometry, respectively. The titres of mAbs in ascitic fluid, relative affinity and epitopes recognized by mAbs were determined by indirect ELISA. RESULTS: Four hybridoma cell lines secreting anti DR5 mAbs were obtained. Their Ig subclass belonged to IgG1. The titers of 4 mAbs in ascitic fluid were 1x10(-4) - 5x10(-6). Affinity constant of mAbs were 1x10(9). They recognized 2 different epitopes on DR5 molecule. CONCLUSION: Four mAbs against DR5 are prepared successfully, which provides useful reagent for clinical diagnosis and further research.