Insights into the reaction of anammox to exogenous pyridine: Long-term performance and micro mechanisms.

Some industrial wastewaters contain high amounts of toxic nitrogen-containing heterocyclic compounds, which may inhibit the efficiency of biological treatment. This work systematically investigated how exogenous pyridine affected the anaerobic ammonia oxidation (anammox) system and discussed the microscopic response mechanisms based on genes and enzymes. The anammox efficiency was not seriously inhibited by pyridine less than 50 mg/L. Bacteria secreted more extracellular polymeric substances to resist pyridine stress. After 6 days stress with 80 mg/L pyridine, the nitrogen removal rate of anammox system lost 47.7%. Long-term stress of pyridine reduced anammox bacteria by 7.26% and the expression of functional genes by 45%. Pyridine could actively bind to hydrazine synthase and ammonium transporter. This work fills a research gap in the ongoing threat of pyridines to anammox, and has guiding value for the application of anammox process in the treatment of ammonia-rich wastewater containing pyridine.

[Bone Marrow Adipocytes Promote the Survival of Multiple Myeloma Cells and Up-Regulate Their Chemoresistance].

OBJECTIVE: To investigate the effect of adipocytes in the bone marrow microenvironment of patients with multiple myeloma (MM) on the pathogenesis of MM. METHODS: Bone marrow adipocytes (BMA) in bone marrow smears of health donors (HD) and newly diagnosed MM (ND-MM) patients were evaluated with oil red O staining. The mesenchymal stem cells (MSC) from HD and ND-MM patients were isolated, and in vitro co-culture assay was used to explore the effects of MM cells on the adipogenic differentiation of MSC and the role of BMA in the survival and drug resistance of MM cells. The expression of adipogenic/osteogenic differentiation-related genes PPAR-γ, DLK1, DGAT1, FABP4, FASN and ALP both in MSC and MSC-derived adipocytes was determined with real-time quantitative PCR. The Western blot was employed to detect the expression levels of IL-6, IL-10, SDF-1α, TNF-α and IGF-1 in the supernatant with or without PPAR-γ inhibitor. RESULTS: The results of oil red O staining of bone marrow smears showed that BMA increased significantly in patients of ND-MM compared with the normal control group, and the BMA content was related to the disease status. The content of BMA decreased in the patients with effective chemotherapy. MM cells up-regulated the expression of MSC adipogenic differentiation-related genes PPAR-γ, DLK1, DGAT1, FABP4 and FASN, but the expression of osteogenic differentiation-related gene ALP was significantly down-regulated. This means that the direct consequence of the interaction between MM cells and MSC in the bone marrow microenvironment is to promote the differentiation of MSC into adipocytes at the expense of osteoblasts, and the cytokines detected in supernatant changed. PPAR-γ inhibitor G3335 could partially reverse the release of cytokines by BMA. Those results confirmed that BMA regulated the release of cytokines via PPAR-γ signal, and PPAR-γ inhibitor G3335 could distort PPAR-γ mediated BMA maturation and cytokines release. The increased BMA and related cytokines effectively promoted the proliferation, migration and drug resistance of MM cells. CONCLUSION: The BMA and its associated cytokines are the promoting factors in the survival, proliferation and migration of MM cells. BMA can protect MM cells from drug-induced apoptosis and plays an important role in MM treatment failure and disease progression.

Renal venous sampling assisted the diagnosis of juxtaglomerular cell tumor: a case report and literature review.

Juxtaglomerular cell tumor (JCT) is an endocrine tumor marked by elevated renin levels and high blood pressure. This case report presents the clinical findings of a 47-year-old woman with a history of recurrent hypokalemia, headaches, hypertension, and increased plasma renin activity (PRA). Dynamic enhanced magnetic resonance imaging (MRI) revealed a small nodule on the upper part of the right kidney. Selective renal venous sampling indicated a higher PRA only in the right upper pole renal vein. The patient underwent surgical removal of the right kidney mass, and the pathology results confirmed the diagnosis of JCT. This case underscores the importance of conducting selective renal venous sampling for accurate JCT diagnosis.

Responses of anammox to long-term p-nitrophenol stress: From apparent and microscopic phenomena to mechanism simulation.

p-Nitrophenol is usually present in ammonia-rich wastewaters produced by some chemical plants. In this work, the response of anammox process to long-term p-nitrophenol stress was investigated. The changes in the efficiency, sludge characteristics, and microorganisms of the anammox system under different levels of p-nitrophenol stress were examined, and the potential stress mechanisms of p-nitrophenol on anammox were further speculated. The results showed that 10-50 mg/L p-nitrophenol had no obvious impact on nitrogen removal efficiency, but stimulated the secretion of more extracellular polymeric substances. 60 mg/L p-nitrophenol caused the nitrogen removal efficiency to decrease by 64.5% in 5 days. Long-term exposure to p-nitrophenol led to 8.6% reduction in Candidatus_Kuenenia abundance and 18.4%-35.9% decrease in the expression level of anammox bacterial functional genes. Molecular simulation indicated that p-nitrophenol could bind to key enzymes of anammox. This study provides new insights into the treatment of wastewater containing p-nitrophenol or phenol by anammox.

How phenol stresses anammox for the treatment of ammonia-rich wastewater: Phenomena, microbial community evolution and molecular modeling.

Phenol is a biotoxic organic compound and found in large quantities in ammonia-rich wastewater discharged from coking and petrochemical industries. In this work, phenol was fed to the system of anaerobic ammonia oxidation (anammox), and the possible inhibitory mechanism was speculated using the characterization of granular sludge, analysis of microbial community and molecular docking simulations. The results showed that phenol (0-300 mg/L) did not significantly inhibit anammox. However, phenol did activate denitrification, which increased the nitrogen removal rate (NRR) by 0.94 kg N/(m3·d). Moreover, when phenol concentration reached t400 mg/L, the NRR was inhibited by 70%, while the extracellular polymeric substance (EPS) of granular sludge was reduced. Phenol resulted in the reduction of Candidatus_Kuenenia and promoted the proliferation of phenol-degrading denitrifying bacteria, Azoarcus and Thauera. Molecular docking indicated that phenol, 2-nitrophenol and 4-nitrophenol could bind the nitrite reductase (NirS), which prevented the first step of the anammox reaction.

Diffuse large B-cell lymphoma-derived exosomes push macrophage polarization toward M2 phenotype via GP130/STAT3 signaling pathway.

Growing evidence shows that cancer progression links with both heterogeneity of the tumor microenvironment and dysregulated activity of immune cells. Cancer-secreted exosomes are being recognized as indispensable mediators of the exchange cargo between cancer and immune cells. The M2-phenotype tumor-associated macrophages have the function of promoting tumor progression and drug resistance. Diffuse large B-cell lymphoma(DLBCL) is a highly heterogeneous and very common malignant non-Hodgkin's lymphoma. Here, we demonstrate that different subtype DLBCL cell-derived exosomes are internalized by macrophages, which can affect macrophages polarization. The mechanism of DLBCL-derived exosomes on macrophage polarization remains unclear currently. This study showed that DLBCL-secreted exosomes could induce the transformation of macrophages to a protumor M2-like phenotype, and block the drug-induced apoptosis of DLBCL cells in an indirect co-culture system. Different DLBCL-derived exosomes could change the phenotype of macrophages through the STAT3 signaling, which upregulated the expression of oncogenic genes and classical markers of M2-like phenotype macrophages, such as IL-10, CD206, and CD163. The addition of DLBCL-derived exosomes resulted in the activation of the STAT3 signaling pathway of M0/M2 macrophages in an indirect co-culture system. GP130 was highly enriched in DLBCL-derived exosomes, which triggered the activation of STAT3 of macrophages and subsequently induced the downstream targets such as BCL2, SURVIVIN, and BAX. The parallel changes of STAT3 and GP130 in macrophages confirmed that GP130 of DLBCL-derived exosomes promoted macrophage polarization by activating STAT3 signaling. Furthermore, all of these effects could be reversed by the GP130 inhibitor SC144. The data indicated that DLBCL-derived exosomes could trigger macrophages polarization into a pro-survival M2-like phenotype, which was at least partially through the GP130/STAT3 signaling pathway. Collectively, this study showed that DLBCL-derived exosomes could promote macrophages transformation to protumor M2-like phenotype in the tumor microenvironment.

[Effect of the Connexin 43 Coupling to the Biobehavior of Multiple Myeloma Cells].

OBJECTIVE: To investigate the effect of gap junction intercellular communication (GJIC) combined by connexin43 (Cx43) and its signal to the biobehavior of multiple myeloma (MM) cells, and its possible mechanism. METHODS: The mesenchymal stem cell (MSC) cells were isolated and cultured from patients with MM and normal donors. The expression of connexin43 (Cx43) in MSC cells from different sources was detected by RT-PCR and Western blot. The side population (SP) cells were sorted by flow cytometry (FCM). The effect of MSC cells from different sources to the cell cycle, Cx43 expression, colony formation in vitro, stem cell related genes expression, cytokines secretion and chemoresistance in MM SP cells as well as with or without Cx43 inhibitor 18α-glycyrrhetinic acid (18α-GA) was observed. RESULTS: There was no significantly difference between the MSC isolated from normal donor and MM patients. Western blot showed that Cx43 expression in SP cells was up-regulated when the cells were incubated with MSC, and medium containing 18α-GA could partially inhibit it, moreover, it was more significant in MSC cells of MM patients. The ability of colony formation of SP cells in vitro was higher than those of MM cells and MM-MSC could promote the colony formation in a co-culture manner. The effect of MM-MSC to SP cells was down-regulated after 18α-GA was added. RT-PCR showed that there was several important stem cell-related genes including c-myc, Oct-4 Klf-4, and Sox-2 were found in RPMI 8226 cells, but those cells were up-regulated in SP cells (P<0.001). Meanwhile, MM-MSC could up-regulate the expression of c-myc, Klf-4 and Sox-2 (P<0.001), but down-regulate Oct-4 gene in the SP cells. The expression of those genes decreased after 18α-GA was added, but showed no significant difference (P>0.05). Cytometry bead array assays showed that MM-MSCs could secrete high level of IL-6, but the levels of IL-6, IL-10 and TGF-ß increased significantly when the MM-MSCs were co-cultured with SP cells (P<0.05), especially the levels of IL-6 and IL-10 were significantly higher than cultured alone. There was no significant change in the levels of bFGF and IL-17 before and after co-cultured. The levels of IL-6, IL-10 and TGF-ß in supernatant decreased significantly after GJ inhibitor 18α-GA was added. PI/Annexin V assay showed that MM cells were sensitive to bortezomib (BTZ)-induced apoptosis, but the sensitivity for SP cells was weaker. The ratio of cell apoptosis was 75.2%±0.77% and 8.12%±0.86% (P<0.001), respectively. MM-MSC could down-regulate the cell apoptosis induced by BTZ, while the sensitivity of MM cells to BTZ could be partially recovered after GJ inhibitor was added. CONCLUSION: MSC derived from MM patients can enhance GJIC to maintain its "hematopoiesis" by up-regulating the expression of Cx43 in MM cells, and at the same time promote cell proliferation and drug recistance by secreting multiple cytokines, which finally contributes to the relapse of MM.

Exosomal miR-1910-3p promotes proliferation, metastasis, and autophagy of breast cancer cells by targeting MTMR3 and activating the NF-κB signaling pathway.

Exosomes are key mediators of intercellular communication and play a role in the pathogenesis and progression of cancer. Exosomes in circulating body fluids serve as molecular markers for cancer diagnosis. This study aimed to investigate the role of exosomal microRNA (miR)-1910-3p in breast cancer and determine its clinical diagnostic value. MiR-1910-3p promoted proliferation and migration of breast cancer cells in vitro and in vivo. In vitro, exosomes enriched in miR-1910-3p transferred miR-1910-3p to mammary epithelial cells and breast cancer cells, promoting proliferation and migration, inhibiting apoptosis, and inducing autophagy. In vivo, exosomes enriched in miR-1910-3p promoted the proliferation and migration of breast cancer cells. MiR-1910-3p downregulated myotubularin-related protein 3, activated the NF-κB and wnt/ß-catenin signaling pathway, and promoted breast cancer progression. Serum miR-1910-3p in exosomes was an effective diagnostic marker that improved the sensitivity of breast cancer diagnosis when used in combination with the traditional tumor marker CA153. In conclusion, breast cancer cell-derived exosomes promoted the growth, metastasis, and autophagy of breast cancer cells by transferring miR-1910-3p. MiR-1910-3p in serum exosomes may serve as a novel molecular marker for breast cancer diagnosis.

The role of follicular T helper cells in patients with malignant lymphoid disease.

OBJECTIVES: To investigate the dynamic change of follicular T helper cells (TFH) in patients with malignant lymphoid disease (MLD) and to explore its clinical significance. METHODS: The dynamic change of TFH cells, ICOS+- and PD-1+ TFH cells at pretreatment and different treatment periods was determined by flow cytometry in 85 MLD patients. Concentration of interleukin 21 (IL-21) was evaluated by ELISA, and the correlation between clinical prognosis and the ratio of TFH cells was analyzed. RESULTS: Significantly increased ICOS+- and PD-1+ TFH cells were found in MLD patients at pretreatment compared to healthy controls. Decreased or even close to normal levels of ICOS+- and PD-1+ TFH cells were found at the end of treatment. However, in the patients with progressive disease, high levels of ICOS+- and PD-1+ TFH cells were found. Moreover, a significantly increased plasma IL-21 level was found in MLD patients. Negative correlation was found between the level of ICOS+-, PD-1+ TFH cells, as well as IL-21 and the prognosis of MLD. CONCLUSIONS: Significantly increased TFH cell ratios were found in patients with MLD, and decreased TFH cells ratios could be expected in those treatment-effective patients, which could be used as the therapeutic efficacy index.

[Effects of Bone Marrow Mesenchymal Stem Cells on the Biological Characteristics of Migrating and Homing in Multiple Myeloma Cells].

OBJECTIVE: To construct a co-culture system for bone marrow mesenchymal stem cells (BMMSC) and multiple myeloma (MM) cells, and to investigate the effects of co-cultured BMMSC on the migrating and homing of multiple myeloma cells. METHODS: The BMMSC from the transgenic mice with green fluorescent protein (GFP) fetal bone were cultured by adherent screening. A co-culture system of BMMSC and MM cell line XG-7 cells was constracted, the proliferation and apoptosis of cells were determined by trypan blue exclusion and Annexin V/PI, respectively, MDC staining was employed to detect the autophagy. The moving direction distribution of molecule in BMMSC and XG-7 cells labeled with PE-CD138 in co-culture process were observed dinamically by confocal microscopy. RESULTS: After co-culture with GFP-BMMSC, the resistance of XG-7 cells to apoptosis and autophagy were enhanced; at the same time, their proliferation increased. Apoptosis rates of XG-7 cells directly and indirectly co-cultured with BMMSC were (6.23 ± 0.12)% and (6.97 ± 0.03)% respectively, which were lower than that of XG-7 cells cultured alone (17.90 ± 1.46)% (P < 0. 01). There was low level of autophagy in XG-7 cells co-cultured with BMMSC. XG-7 cells are highly polarized and contained a specialized membrane domain with specific protein and lipid components to contact with BMMSC under confocal microscope. After methyl-ß-cyclodextrin treatment, the molecules were normally enriched in the specialized domain. CONCLUSION: BMMSC can protect XG-7 cells from apoptosis and autophagy, and obviously promote the proliferation of XG-7 cells, and can influence the migrating and homing of multiple myeloma cells.

Impact of Global and Gene-Specific DNA Methylation in de Novo or Relapsed Acute Myeloid Leukemia Patients Treated with Decitabine.

In this investigation, global DNA methylation patterns and the specific methylation status of 5 genes were studied in DNA from peripheral blood (PB) and impact on progression free survival (PFS) and overall-survival (OS) in patients with de novo or relapsed acute myeloid leukemia (AML) treated with decitabine-based regimens waas assessed. DNA was isolated from PB samples at the time of -1, 1, and 7 days of chemotherapy. Global methylation was determined by ELISA, and the CpG island DNA methylation profile of 5 genes using a DNA methylation PCR system. Our data demonstrated that patients with a high level of 5-mC had a poor prognosis after demethylation therapy and those who have low levels of 5-mC in PB achieved higher CR and better SO, but there was no significant correlation found between the 5-mC levels and other clinical features before treatment except the disease status. Higher methylation status of Sox2 and Oct4 genes was associated with differential response to demethylation therapy. A relatively low methylation percentage in one or both of these two genes was also associated with longer OS after decitabine based chemotherapy. We also suggest that global DNA and Oct-4/Sox2 methylation might impact on the pathogenesis of leukemia and play an important role in the initiation and progression. Moreover, dynamic analysis of 5-mC and Oct-4/Sox2 in peripheral blood nucleated cells of leukemia patients may provide clues to important molecular diagnostic and prognostic targets.

[Effect of Curcumol on the Biological Behavior of Multiple Myeloma Cells].

Objective To observe the effect of curcumol on the biological behavior of multiple myeloma (MM) cells, thus studying its possible mechanisms for MM treatment. Methods Bone marrow mesenchymal stem cells (BMSCs) and multiple myeloma cell line 8226 (RPMI 8226) were taken as sub- jects, which were then divided into the RMPI 8226 group (cultured by RMPI 8226 alone) and the BMSCs + RMPI 8226 group (cultured by BMSCs and RMPI 8226). Curcumol in different concentrations (0. 1, 0.5, 1. 0 , 10.0 µg/mL) was added to cells in the two groups respectively. Cell proliferation, cell cycle, and ap' optosis induced by curcumol were examined by flow cytometry. The expressions of receptor activator of nuclear factor Kb ligand ( RANKL ) and osteoprotegerin ( OPG) were detected using reverse tran- scriptase-polymerase chain reaction (RT-PCR). Results Curcumol induced arrested cell cycle of RMPI 8226. The arrest of RMPI 8226 cell cycle was more obviously in the RMPI 8226 group than in the BMSCs + RMPI 8226 group. After curcumol treatment the cell proliferation of RPMI 8226 was significantly inhibited (P <0. 01) and its apoptosis was increased (P <0. 01). Co-cultured with BMSCs decreased curcumol in- duced apoptosis of RPMI 8226. Curcumol down-regulated the expression of osteogenic differentiation related gene RANKL, and up-regulated the expression of OPG. Conclusions Curcumol disturbed the cell cycle and induced apoptosis of RPMI 8226 cells. Curcumol up-regulated the expression of OPG as well as down-regulated the expression of RANKL. Co-culture with BMSCs could obviously inhibit curcumol in- duced apoptosis of MM cells, which might be associated with drug resistance of MM cells.

[Sensitivity to bortezomib of RPIM8226 cells after co-cultured with down-regulated Cav-1 expression HUVECs].

OBJECTIVE: To investigate the sensitivity to bortezomib of RPMI8226 cells after co-cultured with down-regulated Caveolin (Cav)-1 expression of HUVECs by transfection with Cav-1 shRNA (HUVECs(Cav-1 low)). METHODS: Exposure to bortezomib with or without 50 nmol/L dexamethasone at different concentration, the proliferation of RPMI8226 was analyzed by MTT assay when it was cultured alone or co-cultured with HUVECs(Cav-1 low). Cav-1 expression was detected by using of Western blot and cell cycle, apoptosis and the level of reactive oxygen species (ROS) were analyzed by flow cytometry. RESULTS: Cav-1 expression was notably down-regulated in HUVECs(Cav-1 low) (0.2199±0.0288 vs 1.3195±0.2393) (P<0.01). The IC(50) of bortezomib for RPMI8226 cultured alone, co-cultured with HUVECs orHUVECCav- 1 low were 20 nmol/L, 50 nmol/L and 65 nmol/L, respectively. The percentages of G0/G1 phase in RPMI8226 cultured alone, co-cultured with HUVECs and HUVECs(Cav-1 low) were 28.49%, 30.41%, and 36.15% respectively. The protection of RPMI 8226 against apoptosis by HUVECs was demonstrated that the apoptosis/death rates were 66.8%, 10.7% and 8.6% in RPMI8226 cultured alone, co-cultured with HUVECs and HUVECs(Cav-1 low) after exposure to 20 nmol/L bortezomib for 24 h. RPMI8226 could induce the oxidative stress of HUVECs before and after co-culture. The ROS level was raised from 15.0% to 35.2% in RPMI8226, from 80.4% to 91.0% in HUVECs, and from 84.6% to 96.8% in HUVECs(Cav-1 low). CONCLUSION: The down-regulated Cav-1 expression of HUVECs could promote proliferation and induce apoptosis of RMPI8226 cells, lead to G0/G1 phase arrest, and reduce the sensitivity to bortezomib.

[Alterations of connexin 43 expression and SDF-1α secretion of bone marrow mesenchymal stem cells co-cultured with myeloma cells].

OBJECTIVE: To construct a co- culture system of mesenchymal stem cells (MSC) and multiple myeloma (MM) cells and investigate the alterations of connexin 43 (CX43) expression and stromal derived growth factor (SDF)- 1α secretion of MSC. METHODS: CX43 expression and SDF- 1α secretion of MM cell lines (RPMI8226) and human primary MM cells were analyzed by western blot and immunofluorescence. Western blot, RT- PCR and immunofluorescence were employed to detect the alterations of CX43 expression and distribution in MSC directly and indirectly co-cultured with myeloma cells. Lucifer yellow dye spread was utilized to evaluate gap junctional intercellular communication (GJIC) between co- cultured MSC. Transwell was applied to study the transmigration of RPMI8266 induced by MSC under the condition of 18α- glycyrrhetinic acid (18α-GA). The level of SDF- 1α was detected by EILSA. RESULTS: RPMI8266, U266 and one-third primary MM cells expressed CX43 at low or moderate levels. CX43 wasn't expressed in XG- 4 and XG- 7 cells but highly expressed in MSC. The expressions of CX43 mRNA of MSC were up- regulated after directly and indirectly co- cultured with RPMI8226, 1.36 and 2.10 times that of MSC cultured alone respectively. Western blot analysis showed that CX43 protein expression of MSC was also up-regulated, mainly distributed in cytoplasm. Lucifer yellow dye spread showed that GJIC was up-regulated in MSC. SDF-1α concentration in supernatant of MSC directly and indirectly co-cultured with RPMI8226 were (373.02±10.11)pg/ml and (309.71±10.71)pg/ml respectively, which were higher than that of MSC cultured alone (237.84±9.23)pg/ml (P<0.01), and could be inhibited by 18α-GA [(237.84±9.23)pg/ml and (94.31±6.44)pg/ml] respectively (P<0.01). 18α-GA could inhibit the transmigration of RPMI8226 induced by MSC, decrease from (8.00±0.67)% to (4.82±0.19)%. CONCLUSION: CX43 expression of MSC was up-regulated after directly and indirectly co-cultured with MM cells, which could improve the level of SDF-1α secretion of MSC. GJ inhibitor could downregulate SDF-1α secretion of MSC and inhibit the transmigration of MM cells induced by MSC.

[Influence of rosiglitazone and all-trans-retinoic acid on angiogenesis and growth of myeloma xenograft in nude mice].

OBJECTIVE: To observe the effect of rosiglitazone (RGZ) and all-trans-retinoic acid (ATRA) on the growth of myeloma xenograft in nude mice and to explore the influence of RGZ and ATRA on VEGF expression and angiogenesis in the tumor. METHODS: VEGF gene expression in myeloma cell line U266 cells was analyzed by semi-quantitative RT-PCR after incubation with RGZ, ATRA, or RGZ + ATRA for 24 h. Myeloma xenograft was established by subcutaneous injection of 10(7) U266 cells in the scapula area of 4-week old nude mice. 7 days later, the nude mice were administered with RGZ, ATRA or RGZ + ATRA, respectively, by intraperitoneal injection once every day for 21 days. The control mice were given equal volume of normal saline instead of the drug. On the 21(st) day of treatment, the mice were sacrificed and the tumors were taken off, and the tumor volume and weight were measured. The tumors were examined by histopathology with HE staining, and microvessel density (MVD), CD34 and VEGF expression in the tumors were analyzed by immunohistochemical staining. RESULTS: VEGF mRNA was highly expressed in U266 cells and was decreased in a dose-dependent manner after incubation with RGZ. The VEGF mRNA level was further more decreased after RGZ + ATRA treatment. Xenografts of U266 cells were developed in all nude mice. The volume and weight of xenografts in the RGZ group were (785 ± 262) mm(3) and (1748 ± 365) mg, respectively, significantly lower than those of the control group (both P < 0.01). More significant inhibition was in the RGZ + ATRA group, (154 ± 89) mm(3) and (626 ± 102) mg, respectively, both were P < 0.05 vs. the RGZ group. RGZ inhibited the angiogenesis in U266 xenografts and immunohistochemical staining showed that the tumor MVD and VEGF expression were significantly decreased by RGZ treatment, and further more inhibited in the RGZ + ATRA group. VEGF protein was expressed in all xenografts in the nude mice. Its immunohistochemical staining intensity was 2.20 ± 0.40 in the control group, significantly higher than that of 1.48 ± 0.37 in the RGZ group (P < 0.01), and that of RGZ + ATRA group was 0.58 ± 0.26, further significantly lower than that of the RGZ group (P < 0.01). CD34 was expressed in all xenografts, most highly in the control group and lowest in the RGZ + ATRA group. The microvessel density (MVD) was highest in the control group (56.4 ± 15.2), significantly lower in the RGZ group (44.6 ± 11.2) (P < 0.05), and lowest in the RGZ + ATRA group (21.5 ± 8.6, P < 0.01). CONCLUSIONS: The growth of myeloma cells can also be inhibited by RGZ and ATRA in nude mice in vivo. In addition to differentiation and apoptosis induction, RGZ can inhibit the formation of myeloma xenograft probably also through the downregulation of VEGF expression and subsequent angiogenesis.

[Performance of microbial fuel cells with Fe/C catalyst carbon felt air-cathode for treating landfill leachate].

Ferric nitrate/activated carbon powder catalyst was obtained through impregnation and Fe/C catalyst was adsorbed on carbon felt as air cathode electrodes. Effects of activated carbon powder dosage and ferric nitrate concentration on electricity generation of MFC with landfill leachate as fuel were measured. Performances of cathodes obtained at different ferric nitrate concentrations were evaluated by cyclic voltammetry tests. The results showed that with the increase of activated carbon powder dosage or the iron nitrate concentration, MFC produce electrical properties showed a decreasing trend after the first rise. When the activated carbon powder dosage was 1 g and the iron nitrate concentration was 0.25 mol x L(-1), it was proved to be an optimum cell performance for 4199.8 mW x m(-3) output power and 465 omega apparent resistance. Under the optimal ratio rang between ferric nitrate and activated carbon powder, MFC apparent resistance decreased and the power density increased respectively with the increase of catalyst total dosage. The best produce electrical properties of MFC with Fe/C catalyst for 0.25 mol x L(-1) iron nitrate and 1 g activated carbon powder dosage was observed by cyclic voltammetry tests. The output power of MFC and the removal quantity increased with the concentration of inlet and the maximum values were respectively 5478.92 mW x m(-3) and 1505.2 mg x L(-1). the maximum removal rates of COD achieved at 89.1%.

[The influence of induced autophagy in vitro on proliferation of multiple myeloma cells].

OBJECTIVE: To investigate the influence of autophagy on the survival and proliferation of multiple myeloma (MM) cells. METHODS: Multiple myeloma (MM) cell line U266 cell autophagy was induced by serum-free culture condition, and adding rapamycin or 3-MA respectively. The cells proliferation was observed. U266 cells, lymphoma cell Jurket under normal culture condition, and serum-free cultured Jurket cell were used as control group. The proliferation and apoptosis of cells were determined by CCK8 and flow cytometry, respectively. MDC staining were employed to detect the autophagy. The mRNA expression of Mtor and Beclin1 gene of U266 cells were assayed by RT-PCR. Protein LC3I/LCII and LAMP1 was analyzed by western blot. RESULTS: There was low level of autophagy in U266 cells, sera starvation increased the level of autophagy. Rapamycin upregulated autophagy of the U266 cells and stimulated their proliferation. But the autophagy level of sera starvation and rapamycin group declined when culture for 96h.3-MA had the same effects on U266 cells, although it was on 24 h. But rapamycin and 3-MA could inhibit cell proliferation under normal culture condition. Compared with normal culture condition, apoptosis of U266 cells increased significantly after 24h incubation in medium without sera \[(1.33 ± 0.09)% and (17.90 ± 1.46)%, respectively\] (P < 0.01). Rapamycin and 3-MA could inhibit the serum-free induced apoptosis \[(6.23 ± 0.12)% and (6.97 ± 0.03)%, respectively\](P < 0.01), but cell apoptosis was at the same level after 72 hour incubation \[(30.37 ± 0.27)%, (30.13 ± 1.93)% and (28.57 ± 2.83)%, respectively\] (P > 0.05). However, apoptosis of U266 cells decreased to 18.7% and 12.6% after removal of rapamycin and 3-MA. CONCLUSION: There is basically level of autophagy in MM cells which is higher than those in the Jurkat cells. Both Rapamycin and 3-MA can inhibit the cells proliferation under normal culture condition. Up-regulated autophagy promotes survival and proliferation of MM cells under sera deletion. Rapamycin strengthens this effect with limited duration. 3-MA has dual effects on cell autophagy.

[Inhibition of hypoxia-inducible factor-1 alpha in RPMI8226 myeloma cells results in reduced tumor growth in nude mice].

OBJECTIVE: To explore the influence of inhibition of hypoxia-inducible factor-1 alpha (HIF-1 alpha) by RNA interference (RNAi) on tumorigenesis of human myeloma cell line (HMCL) RPMI8226 cells in nude mice. METHOD: RNAi vector of HIF-1 alpha was constructed with commercial shRNA expression vector pSilencer 2. 1-U6 hygro. RT-PCR and western blot were used to detect HIF-1 alpha mRNA and protein expression respectively. Vascular endothelial growth factor (VEGF) secretion and cell cycle changes were detected by ELISA and flow cytometry respectively. Expression of target gene of HIF-1 alpha, VEGF and Glut-1 were tested under hypoxia condition. Tumorigenesis was observed after transfected cells were injected subcutaneously in nude mice. RESULTS: After interference, expression of HIF-1 alpha decreased significantly at both mRNA and protein level. Under normoxia condition, VEGF concentrations in HIF-la inhibited cells (RPMI8226-il and RPMI8226-i2) and non-inhibited cells (RPMI8226-c and RPMI8226) showed no differences. While under hypoxia condition, VEGF concentration in the above four cells was (506.0 +/- 53.2), (494.7 +/- 63.1), (984.4 +/- 61.9) and (938.2 +/- 62.2) pg/ml, respectively, being significantly lower in RPMI8226-il and RPMI8226-i2 cells than in RPMI8226-c and RPMI8226 cells (P <0.05). HIF-1 alpha interference was found to suppress the cells shift from S-phase to G1 induced by hypoxia. VEGF and Glut-1 expressions were markedly attenuated (P <0.05). The growth rate of HIF-1 alpha inhibition tumors in subcutaneous xenograft model decreased drastically. CONCLUSIONS: RNAi inhibits HIF-1 alpha expression. Reduced tumor growth by HIF-1 alpha inhibition may partly through inhibiton of angiogenesis and glycolysis metabolism.

[Effects of RNA interference targeting hypoxia-inducible factor-1alpha (HIF-1alpha) on chemosensitivity of leukemia K562 cells towards homoharringtonine].

BACKGROUND & OBJECTIVE: Hypoxia-inducible factor-1alpha (HIF-1alpha) is a key transcription factor under anoxic circumstances. Little is known about changes in biological characters of hematological malignancies, especially leukemia. This study was to explore the influence of RNA interference (RNAi) targeting HIF-1alpha on sensitivity of human chronic myelogeneous leukemia (CML) K562 cells towards homoharringtonine (HHT). METHODS: HIF-1alpha short hairpin RNA (shRNA) was constructed using pSilencer 2.1-U6 hygro vector and transfected into K562 cells. Positive clones were screened using hygromycin. After inhibition of HIF-1alpha, expressions of its target genes such as vascular endothelial growth factor (VEGF), glucose transporter-1 (Glut-1), phosphoglycerate kinase (PGK), and P-glycoprotein (P-gp) were detected by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Sensitivity of K562 cells to HHT was detected by MTT assay. RESULTS: HIF-1alpha expression was inhibited at both mRNA and protein levels after transfection of RNAi HIF-1alpha, which subsequently caused a dramatic decrease in VEGF, Glut-1, PGK, and P-gp under hypoxic conditions. In addition, HIF-1alpha inhibition was found to increase drug sensitivity of K562 cells to HHT. CONCLUSION: HIF-1alpha inhibition may result in a decrease of genes related to angiogenesis and glycolysis metabolism and an increase of drug sensitivity to HHT in K562 cells.

[Effects of the osteoclast in pathogenesis of multiple myeloma].

OBJECTIVE: To investigate the effects of myeloma cells on the differentiation of osteoclast precursors (pOCs) into OCs in different culture systems in vitro and the interaction between OCs and myeloma cells. METHODS: Myeloma cell lines 8226, XG1 and XG7 and pOCs were cocultured in different culture system. OCs was examined by TRAP staining. RT-PCR was used to evaluate the expression of receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG) of myeloma cells and the effects of myeloma cells on RANKL/OPG expression in coculture. The role of OCs in myeloma cells cycle was measured by FCM with PI staining. The supportive effects of OCs on myeloma cells survival were determined by FCM with double staining for annexin V and PI. RESULTS: 8226 and XG1 cells could directly stimulate the differentiation of pOCs into TRAP+ multinuclear mature OCs. Myeloma cells, which expressed neither RANKL nor OPG, upregulated RANKL expression and decreased OPG expression in mouse primary bone marrow stromal cells (pBMSC). When OCs were co-cultured with myeloma cells, all OCs apparently remained alive after 7 days while devoid of sRANKL and M-CSF. OCs stimulated the proliferation of myeloma cells in co-culture systems,the cell number increased to (3.8 +/- 0.1) x 10(5)/well, (3.9 +/- 0.1) x 10(5)/well, (4.0 +/- 0.1) x 10(5)/well, and to (8.7 +/- 0.1) x 10(5)/well, (9.1 +/- 0.1) x 10(5)/well, (9.0 +/- 0.1 ) x 10(5)/well after co-culture for 3 days and 7 days for XG1 cells, XG7 cells and 8226 cells, respectively (P <0.01). However, OCs could counteract cytotoxic effects of dexamethasone. The proportion of Annexin V-/PI- cells were 57.71%, 82.18% and 90.92% for 8226 cells, XG1 and XG7 cells after co-culture with OCs (P <0.01). CONCLUSION: Myeloma cells stimulated the differentiation of pOCs into TRAP+ multinuclear mature OCs by directly and/or indirectly disrupting the balance of RANKL/OPG, OCs promoted MM cells growth and survival, thus maintaining a vicious circle between myeloma cells and osteoclasts.