[Experimental study on the therapeutic effect and mechanism of erlotinib on non-proliferative diabetic retinopathy].

Objective: To investigate the therapeutic effect and mechanism of erlotinib, an epidermal growth factor receptor (EGFR) inhibitor, on non-proliferative diabetic retinopathy (NPDR). Methods: An experimental research was conducted. Human retinal Müller cells (RMC) were MIO-M1 cells from Moorfields Ophthalmology Hospital and the Institute of Ophthalmology at London University College. MIO-M1 cells were divided into normal, hypertonic, high glucose, high glucose+dimethyl sulfoxide (DMSO), high glucose+erlotinib 0.5 mmol/L, high glucose+erlotinib 1 mmol/L, and high glucose+erlotinib 2 mmol/L groups using a random number table method. Detection of the effect of erlotinib on the proliferation of MIO-M1 cells under high glucose conditions was performed by 5-ethynyl-2'-deoxyuridine (EdU) method. Western blotting (WB) was used to detect the effect of erlotinib on the activation markers of glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS) protein levels in MIO-M1 cells under high glucose conditions. WB was used to detect the effect of erlotinib on the protein levels of nerve growth factor receptor (p75NTR), vimentin, and cell retinol binding protein (CRALBP) in RMC under high glucose conditions. MIO-M1 cells were divided into normal group, high glucose group, high glucose+DMSO group, and high glucose+erlotinib (1 mmol/L) group using random number table method. The effect of erlotinib on EGFR nuclear translocation under high glucose conditions was detected by cell immunofluorescence staining. Immunoprecipitation was used to detect the effect of erlotinib on the interaction between EGFR and transcription intermediate factor 2 (TIF2) in MIO-M1 cells under high glucose conditions. MIO-M1 cells were randomly divided into normal group, high glucose group, high glucose+DMSO group, high glucose+Myc-DDK empty body group, high glucose+erlotinib group, high glucose+erlotinib+human doublet protein group, high glucose+erlotinib+TIF2 plasmid group, and high glucose+erlotinib+human doublet protein+TIF2 plasmid group. Cell immunofluorescence staining was used to detect the effect of erlotinib on the binding of EGFR and TIF2 in MIO-M1 cells under high glucose conditions through the EGFR/TIF2 axis. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect the regulatory effect of EGFR and TIF2 binding on cyclin D1 transcription in MIO-M1 cells under high glucose conditions. The mouse model of diabetes retinopathy (DR) was constructed and divided into normal group, DR group, DR+DMSO group, DR+erlotinib 0.25 mg·kg-1·d-1 group, DR+erlotinib 0.5 mg·kg-1·d-1 group and DR+erlotinib 1 mg·kg-1·d-1 group. 25 mice in total, 5 in each group. Tissue immunofluorescence staining was used to detect the expression of RMC activation marker GFAP. The FITC-dextran injection experiment was used to detect the effect of erlotinib on retinal vascular leakage in a murine DR model. Results: Compared with the normal group (32.4%±3.0%), the proportion of EdU positive cells in RMC in the high glucose group (59.2%±3.8%) increased (P<0.001). Compared with the high glucose group (59.2%±3.8%), the proportion of EdU positive cells in the high glucose+1 mmol/L erlotinib group (37.6%±4.4%) decreased (P<0.001). Compared with the normal group, the expression of GFAP in RMC in the high glucose group increased (1 in the normal group, 2.27±0.11 in the high glucose group, P<0.001), while the expression of GS decreased (1 in the normal group, 0.32±0.03 in the high glucose group, P<0.001). 1 mmol/L erlotinib treatment reduced the expression of GFAP in RMC under high glucose conditions (1.32±0.13 and 2.27±0.11, respectively; P<0.001), and increased the expression of GS (0.71±0.06 and 0.32±0.03, respectively; P<0.001). The colocalization of EGFR and DAPI in RMC of the high glucose+1 mmol/L erlotinib group was lower than that of the high glucose group (52.2%±4.1% and 76.4%±5.7%, respectively; P<0.001). The expression of TIF2 or EGFR both increased while using EGF or TIF2 antibodies to precipitate TIF2 or EGFR under high glucose conditions compared to the normal group (1 in the normal group, 2.27±0.20 in the high glucose group, 2.17±0.21 in the EGFR, all P<0.05). And the expression of TIF2 (1.38±0.10) or EGFR (1.32±0.13) in the high glucose+erlotinib group was lower than that in the high glucose group (2.27±0.20) and the high glucose group (2.17±0.21) (all P<0.05). The colocalization of EGFR and TIF2 (17.2%±3.9%) and the mRNA level of Cyclin D1 (1.32±0.16) in the RMC of the high glucose+erlotinib group were lower than those in the high glucose group (54.6%±3.7% of EGFR and TIF2 colocalization ratio, 2.58±0.19 of Cyclin D1 mRNA level,all P<0.05). The high glucose+erlotinib+AREG (EGFR agonist) group, high glucose+erlotinib+Myc DDK-TIF2 plasmid group and high sugar+erlotinib+AREG+Myc-DDK-TIF2 plasmid group EGFR colocalization with TIF2 (colocalization ratios 24.1%±1.9%, 26.0%±2.3%, 35.3%±2.5%) and TIF2 mRNA levels (1.71±0.16, 1.72±0.18, 2.20±0.18). Compared with the high glucose+erlotinib group, The increases were statistically significant (all P<0.05). Compared to the normal group, the expression of GFAP in mouse retina tissue was increased in the DR group (1 in the normal group, 3.07±0.19 in the DR group, P<0.001), and 0.5 mg·kg-1·d-1 erlotinib (1.73±0.30) significantly reduced the expression of GFAP in the retina of DR group mice (P<0.05). Compared to the normal group (3.97±0.47), the DR group (23.13±2.15) showed an increase in fluorescein leakage, while the DR+erlotinib group (11.66±1.45) showed a significant decrease in leakage compared to the DR group (all P<0.05). Conclusions: Erlotinib inhibits the proliferation and activation of RMC induced by high glucose, inhibits the entry of EGFR into the nucleus, inhibits the binding of EGFR to TIF2 in RMC, and reduces the transcription of Cyclin D1 in RMC by inhibiting the interaction between EGFR and TIF2. At the same time, erlotinib inhibits the proliferation and activation of RMC in the mouse DR model, ameliorating retinal vascular leakage in mice. These results suggest that erlotinib inhibits the activation and proliferation of RMC by downregulating the EGFR/TIF2/Cyclin D1 pathway under high glucose conditions, thereby alleviating the progression of NPDR.

[The association between apparent temperature and hand, foot, and mouth disease and its spatial heterogeneity in Guangdong, Anhui and Jilin provinces].

Objective: To study the association between apparent temperature (AT) and the incidence of hand,foot, and mouth disease (HFMD) and its spatial heterogeneity in 46 cities in Guangdong, Anhui and Jilin provinces, and provide scientific evidence for the early warning of HFMD. Methods: The data of HFMD incidence and meteorological factors from 2009 to 2018 in Guangdong province, 2009 to 2015 in Anhui province, and 2013 to 2018 in Jilin province were collected. Distributed lag non-linear models were constructed to investigate the association between AT and the incidence of HFMD in 46 cities from three provinces in China. Meta-analysis was used to pool the city-specific estimates, and Meta-regression was applied to analyze the factors that may cause spatial heterogeneity. Results: The relationship between daily AT and the incidence of HFMD in 46 cities appeared nonlinear. The association in Guangdong was similar to that in Jilin, and the risk of HFMD increased with the increase of AT. While the risk of HFMD in Anhui first increased with the increase of AT, and peaked at 18.1 ℃ and then went down. AT on different levels showed different lag impacts and the higher AT showed greater and longer lag impact. The spatial heterogeneity of associations may have been caused by latitude, longitude, average temperature, and average sunshine hours. Conclusions: AT is a comprehensive index to evaluate the association between temperature, relative humidity and wind speed and the incidence of HFMD. Higher AT may increase the risk of HFMD. The AT and HFMD relationship across spatial heterogeneity varies depending on geographic location and meteorological conditions.

Elastosonography and two-dimensional ultrasonography in diagnosis of axillary lymph node metastasis in breast cancer.

AIM: To compare diagnostic value of two-dimensional (2D) ultrasonography and elastosonography for suspected axillary lymph node metastasis of breast cancer. MATERIALS AND METHODS: Elastosonography and 2D ultrasonography were performed on 78 axillary lymph nodes of 78 patients with suspected breast cancer. Scores of shape, long- to short-axis ratio, cortical thickness, and lymph node hilum were summed as the score of each lymph node at 2D ultrasonography, while a four-point scale was adopted for elasticity scoring. The combined score of each lymph node was obtained by summing the score at 2D ultrasonography and that at elasticity scoring. The strain ratio was calculated by comparison of the average strain of the lymph node with that of the subcutaneous tissue. Diagnostic efficacies of 2D ultrasonography, elasticity scoring, and the combined method were compared. RESULTS: There were 78 axillary lymph nodes, including 34 non-metastatic and 44 metastatic nodes. The elasticity scores of non-metastatic and metastatic axillary lymph nodes were 1.44±0.82 and 3.11±0.75, respectively (p<0.05). The difference in area under the operating characteristic curve (AUC) was statistically significant between 2D ultrasonography and the combined method (p<0.05). The sensitivity, specificity, and accuracy of 2D ultrasonography and elasticity scoring were 77.3% versus 86.4%, 76.5% versus 85.3%, and 76.9% versus 85.9%, respectively (all p>0.05), and those of the combined method were 93.2%, 73.5%, and 84.6%, respectively. There was a significant difference in sensitivity between 2D ultrasonography and the combined method (p<0.05). CONCLUSIONS: Combined application of 2D ultrasonography with elastosonography can improve the diagnostic capability for metastatic axillary lymph node characterisation in breast cancer.

The relationship between lung function and the clinical and histopathological features in Chinese patients with nasal polyps.

OBJECTIVE: To investigate lung function in Chinese patients suffering from chronic rhinosinusitis with nasal polyps and examine its association with histopathological features. METHODS: The lung function of 99 patients with nasal polyps was measured. Haematoxylin and eosin and immunohistochemistry staining were performed to evaluate any inflammatory cells and epithelial tissue remodelling. RESULTS: Predicted maximal expiratory flow rate at 25 per cent vital capacity was reduced (p < 0.05) in epithelial hyperplasia, and predicted maximal expiratory flow rate at 50 per cent vital capacity was reduced (p < 0.05) in goblet cell hyperplasia. Both peripheral blood eosinophilia and tissue eosinophilia nasal polyps manifested significantly reduced: forced expiratory volume in 1 second/forced vital capacity ratio, predicted maximal expiratory flow rate at 25, 50 and 75 per cent of vital capacity, and predicted maximal mid-expiratory flow. Peripheral blood eosinophils were negatively correlated with predicted maximal expiratory flow rate at 25 and 50 per cent of vital capacity, and predicted maximal mid-expiratory flow. Eosinophils in tissue were negatively correlated with all lung function parameters investigated except predicted forced vital capacity. CONCLUSION: Clinicians should be aware of lung function decline in nasal polyps patients, especially in those with tissue eosinophilia.

[Expression of signaling pathway of mammalian target of rapamycin in articular cartilage cell induced by interleukin 1ß].

Objective: To observe effect of interleukin(IL)-1ß on the expression of signaling pathway of mammalian target of rapamycin(mTOR) of articular cartilage. Methods: Articular cartilage of rats was isolated under sterile technique, cells were digested by type Ⅱ collagenase and trypsin and cultured in vitro, pre-culture the Ⅱ cells for three days, different concentrations of IL-1ß were added for 24 hours.The cells were stained with toluidine blue and HE, to observe morphological changes of cells.RT-PCR was used to detect the mRNA expression of typeⅡcollagen gene, aggrecan gene, mTOR gene and P70S6K gene, Western blotting was used to detect the expression of protein related to mTOR. Results: With increasing concentrations of IL-1ß, the phenotype of cells appeared polygon into a spindle, the mRNA expression of gene of type Ⅱ collagen (the control group: 0.821±0.014; 1 ng/ml: 0.614±0.014; 10 ng/ml: 0.549±0.009; 100 ng/ml: 0.520±0.008), aggrecan(0.867±0.005; 0.857±0.001; 0.554±0.008; 0.538±0.004) and mTOR(0.845±0.015; 0.785±0.009; 0.569±0.025; 0.518±0.014) reduced, but P70S6K(0.465±0.024; 0.566±0.022; 0.663±0.022; 0.896±0.015) increased by PCR .Expression of protein detected by Western blotting was similar to the trend of PCR. Conclusion: mTOR signaling pathway may play an important role on the degeneration of articular cartilage, regulating mTOR signaling pathway may provides a new idea of delaying the degeneration process of cells.

[The expression of long non-coding RNA LINC00261 in laryngeal carcinoma tissue and their clinical significance].

Objective:To investigate the expression of LINC00261 in laryngeal carcinoma and to discuss their relevance and the roles in carcinogenesis and development of laryngeal carcinoma. Method:The expressions of LINC00261 in laryngeal carcinoma tissue and paired adjacent normal tissue was determined by real-time PCR. The relationship between the expressions of LINC00261 and the clinic pathological characteristics including clinical stage, pathological type, histological grade and lymph node metastasis in LSCC was analysed according to the clinical data. Result:①The expression of LINC00261 was significantly decreased in the LSCC tissue compared with the normal laryngeal tissue(P<0.01).②In clinical stage grouping, there were no statistical differences of the quantity of LINC00261 expression among supraglottic, glottic and subglottic LSCC(P>0.05).In histological differentiation grouping, LINC00261 had no significant changes in poorly differentiated LSCC compared with the well and moderately differentiated LSCC(P>0.05). In histological grade grouping, the expression of LINC00261 in T1+T2 stages was significantly higher than T3+T4 stages(P<0.05). Moreover, the expression of LINC00261 in LSCC with lymph node metastasis was significantly lower than that without of lymph node metastasis(P<0.05). Conclusion:Down regulation of LINC00261 in laryngeal carcinoma may contribute to the carcinogenesis and development of laryngeal carcinoma. The decreased expression of LINC00261 maybe relative to T term degree and lymphamatic metastasis of laryngeal carcinoma.

Comparison of biological effects of modulated electro-hyperthermia and conventional heat treatment in human lymphoma U937 cells.

Loco-regional hyperthermia treatment has long history in oncology. Modulated electro-hyperthermia (mEHT, trade name: oncothermia) is an emerging curative treatment method in this field due to its highly selective actions. The impedance-matched, capacitive-coupled modulated radiofrequency (RF) current is selectively focused in the malignant cell membrane of the cancer cells. Our objective is studying the cell-death process and comparing the cellular effects of conventional water-bath hyperthermia treatment to mEHT. The U937 human histiocytic lymphoma cell line was used for the experiments. In the case of conventional hyperthermia treatment, cells were immersed in a thermoregulated water bath, whereas in the case of mEHT, the cells were treated using a special RF generator (LabEHY, Oncotherm) and an applicator. The heating dynamics, the maximum temperature reached (42 °C) and the treatment duration (30 min) were exactly the same in both cases. Cell samples were analysed using different flow cytometric methods as well as microarray gene expression assay and western blot analysis was also used to reveal the molecular basis of the induced effects. Definite difference was observed in the biological response to different heat treatments. At 42 °C, only mEHT induced significant apoptotic cell death. The GeneChip analysis revealed a whole cluster of genes, which are highly up-regulated in case of only RF heating, but not in conventional heating. The Fas, c-Jun N-terminal kinases (JNK) and ERK signalling pathway was the dominant factor to induce apoptotic cell death in mEHT, whereas the cell-protective mechanisms dominated in case of conventional heating. This study has clearly shown that conventional hyperthermia and RF mEHT can result in different biological responses at the same temperature. The reason for the difference is the distinct, non-homogenous energy distribution on the cell membrane, which activates cell death-related signalling pathways in mEHT treatment but not in conventional heat treatment.

[Expression of Has-miR-93-5p in laryngeal squamous cell carcinoma and its clinical significance].

Objective:To investigate the expression of has-miR-93-5p on human laryngeal squamous cell carcinoma and the influence on malignant phenotype of Hep-2 cell.Method:The expression of has-miR-93-5p of paraffin samples in LSCC was determined by looped-primer Real-time PCR, and the relationship between the expression and the clinical pathological parameters was analysed. The has-miR-93-5p Inhibitor sequence was transfected into Hep-2 cells as the Inhibitor group. Using the MTS assay, Edu and colony formation assay to investigate the change of cell viability,proliferation and clone formation ability after transfection. Transwell invasion assay was used to detect the changes of cell migration and invasion ability. Flow cytometry was used to detect the changes of cell cycle and apoptosis.Result:The relative expression of has-miR-93-5p in LSCC was 11.148±1.141,which was higher than in normal tissues of adjacent to carcinoma1(985±4.547)（P <0.01）.The constituent ratio of has-miR-93-5p high expression in the group of low differentiation, T3+T4 and lymphatic metastasis was 69.8%、76.5%and 89.5%,which was higher than the group of high differentiation,T1+T2 and nonlymphatic metastasis respectively（P <0.05）, Inhibition the expression of has-miR-93-5p in vitro in Hep-2 cells could obviously inhibit the cell vitality, proliferation, clone, migration, and invasion ability, also could retardant the cells in G2/M phase, and promote its apoptosis.Conclusion: has-miR-93-5p might be an important molecule in pathogenesis of laryngeal squamous cell carcinoma. It could inhibit malignant phenotype of laryngeal squamous cancer cells when has-miR-93-5p expressionwas suppressed in vitro.

Positive association between PPARD rs2016520 polymorphism and coronary heart disease in a Han Chinese population.

PPARD encodes peroxisome proliferator-activated re-ceptor delta, which has been shown to play an important role in control-ling lipid metabolism and atherosclerosis. In this case-control study, we explored the relationship between PPARD rs2016520 polymorphism and coronary heart disease (CHD) in a Han Chinese population. A to-tal of 657 CHD cases and 640 controls were included in the associa-tion study. rs2016520 polymorphism genotyping was performed using the melting temperature-shift polymerase chain reaction method. The PPARD rs2016520-G allele reduced CHD risk by 17.9% (χ(2) = 5.061, P = 0.025, OR = 0.821, 95%CI = 0.692-0.975). Furthermore, a signifi-cant difference in CHD risk was observed for the PPARD rs2016520 polymorphism in the dominant model (AG + GG vs AA: χ(2) = 4.751, degrees of freedom (df) = 1, P = 0.029, OR = 0.784, 95%CI = 0.631- 0.976). Analysis by age suggested that the G-allele decreased CHD risk by 14.8% in ages greater than 65 years (χ(2) = 4.446, P = 0.035, OR = 0.852, 95%CI = 0.684-1.060). In contrast, meta-analysis of PPARD rs2016520 among 3732 cases and 5042 controls revealed no associa-tion between PPARD rs2016520 and CHD (P = 0.19). We found that the PPARD rs2016520-GG genotype decreased CHD risk in a Han Chinese population. Moreover, we found an association between serum high-density lipoprotein cholesterol level and PPARD rs2016520 in senior individuals aged ≥ 65 years. The meta-analysis revealed no association between PPARD rs2016520 and CHD, suggesting ethnic differences in the association between the PPARD locus and CHD.

Changes in quantity and spectroscopic properties of water-extractable organic matter during soil aquifer treatment.

The aim of this study was to identify qualitative and quantitative changes in the character of water-extractable organic matter (WEOM) in soils as a consequence of soil aquifer treatment (SAT). Soil samples were obtained from a soil-column system with a 2-year operation, and divided into seven layers from top to bottom: CS1 (0-12.5 cm), CS2 (12.5-25 cm), CS3 (25-50 cm), CS4 (50-75 cm), CS5 (75-100 cm), CS6 (100-125 cm) and CS7 (125-150 cm). A sample of the original soil used to pack the columns was also analysed to determine the effects of SAT. Following 2 years of SAT operation, both soil organic carbon and water-extractable organic carbon were shown to accumulate in the top soil layer (0-12.5 cm), and to decrease in soil layers deeper than 12.5 cm. The WEOM in the top soil layer was characterized by low aromaticity index (AI), low emission humification index (HIX) and low fluorescence efficiency index (F(eff)). On the other hand, the WEOM in soil layers deeper than 12.5 cm had increased values of HIX and F(eff), as well as decreased AI values relative to the original soil before SAT. In all soil layers, the percentage of hydrophobic and transphilic fractions decreased, while that of the hydrophilic fraction increased, as a result of SAT. The production of the amide-2 functional groups was observed in the top soil layer. SAT operation also led to the enrichment of hydrocarbon and amide-1 functional groups, as well as the depletion of oxygen-containing functional groups in soil layers deeper than 12.5 cm.

An artificially constructed radiation-responsive promoter is activated by doxorubicin.

We previously developed an artificially constructed promoter that was activated in response to X-ray irradiation in LNCap, a prostate cancer cell line. Anticancer drugs were examined to see whether some of them could stimulate the activity of the promoter. It was found that doxorubicin (Dox) treatment to LNCap transfected with a gene cassette of the luciferase gene under control of the promoter-enhanced luciferase activity in a dose-dependent manner, indicating that the promoter could be controlled by Dox. When the luciferase gene was replaced with the fcy::fur gene whose product facilitates conversion of 5-fluorocytosine into 5-fluorouracil that is highly toxic, Dox stimulated the expression of the gene product, resulting in facilitation of cell killing effect in the presence of 5-fluorocytosine. These results suggest that therapeutic gene expression controlled with an anticancer drug may lead to a more effective cancer therapy with less hazardous side effects.

Regulation of gene expression in prostate cancer cells with an artificially constructed promoter responsive to radiation.

A promoter library was developed that is composed of DNA fragments constructed by randomly elongating the cis-acting elements of transcription factors presumably activated in prostate cancer by radiation, and linking to the TATA-box sequence. One promoter with the strongest reactivity to X-ray in the LNCap cells of the library was chosen and improved by the introduction of random mutations. The resultant promoter was designated clone 880-8, showing the highest dose-dependent activity enhancement with X-ray irradiation (X-irradiation). A recombinant retrovirus expressing the luciferase gene under the control of clone 880-8 was infected into LNCap cells that showed 9.12±0.36-fold enhancement of luciferase activity 12 h after X-irradiation at 10 Gy. When the infected cells were inoculated onto nude mice, enhancement of luciferase expression was 4.27±1.36-fold 12 h after X-irradiation at 10 Gy. When LNCap was infected with another recombinant carrying the fcy::fur gene downstream from clone 880-8, fcy::fur expression was enhanced by X-irradiation. It was also shown to increase the dose-dependent cell killing ratio with 5-FC as compared with a counterpart without X-irradiation. These results suggest that the method used in this study is effective to construct a promoter responsive to stimulation. Such promoters can be used for stimulation-controlled gene therapies.

Effect of ultrasonic and alkaline pretreatment on sludge degradation and electricity generation by microbial fuel cell.

Both ultrasonic and alkaline pretreatment of excess sewage sludge were investigated to enhance organic degradation and electricity generation from sludge by the subsequent microbial fuel cell (MFC). The ultrasonic pretreatment showed that the degree of sludge disintegration was directly related to the energy input, ultrasonic density and duration. Alkaline pretreatment demonstrated that more soluble organic matters were released from the sludge with more NaOH dose and longer reaction time, and the degree of sludge disintegration within 30 min accounted for 45-76% of that for 24 h. When ultrasonic and alkaline pretreatment were combined, the released chemical oxygen demand (COD) was higher than those with ultrasonic or alkaline pretreatment alone. Ultrasonic and alkaline (pH=11) pretreatment could enhance electricity generation from sludge by the subsequent MFC, resulting in more degradation of total COD (TCOD) and volatile solids (VS). Slight change in power output from the MFC was observed due to the higher soluble chemical oxygen demand (SCOD) in the pretreated sludge. By using the combined ultrasonic and alkaline pretreatment of sludge, the removal efficiencies of TCOD and VS were increased from 27.1% to 61.0% and 35.2% to 62.9% in comparison with raw sludge, respectively, and the power output in MFC was slightly increased from 10.3 W/m(3) to 12.5 W/m(3).

Transformation of nitrogen and distribution of nitrogen-related bacteria in a polluted urban stream.

Most researchers focused on either nitrogen species or microbial community for polluted urban stream while ignoring the interaction between them and its effect on nitrogen transformation, which restricted the rational selection of an effective and feasible remediation technology. Taking Buji stream in Shenzhen (China) as target stream, the distribution of nitrogen-related bacteria was investigated by most probable number (MPN) besides analysis of nitrogen species etc. The nitrogen-related bacteria in sediment were 10(2) times richer than those in water. Owing to their faster growth, the MPN of ammonifying bacteria and denitrifying bacteria were 10(5) and 10(2) times higher than those of nitrifying bacteria, respectively. The ammonifying bacteria numbers were significantly related to BOD5 in water, while nitrifying bacteria in sediment correlated well with nitrate in water. Thus, nitrification occurred mainly in sediment surface and was limited by low proportion of nitrifying bacteria. The denitrifying bacteria in sediment had good relationship with BOD5 and nitrite and nitrate in water. Low DO and rich organic compounds were beneficial to denitrification but unfavourable to nitrification. Denitrification was restricted by low nitrite and nitrate concentration. These results could be served as a reference for implementing the remediation scheme of nitrogen polluted urban stream.

Continuous electricity production from leachate in a novel upflow air-cathode membrane-free microbial fuel cell.

In this study, a novel microbial fuel cell, i.e. upflow air-cathode membrane-free microbial fuel cell (UAMMFC) was reported and its performance in electricity generation from original leachate was examined. The experimental results demonstrated that the UAMMFC could continuously generate electricity from leachate (0.3V; REX=150 Omega) for an operational period of time (50 h). The maximum volumetric power reached 12.8 W/m3 at current density of 41 A/m3 (93 Omega). NH4+-N elimination from the leachate was shown to be a consequence of electrochemistry-independent oxidation occurred in the MFC. Increasing organic loading rate from 0.65 to 5.2 kgCOD/m3 d resulted in a decrease of overall Coulombic efficiency (CE) from 14.4% to 1.2%. The low CE obtained here should be attributed to severe oxygen diffusion from the open-to-air cathode.

Enhancement of macrosphelide-induced apoptosis by mild hyperthermia.

Hyperthermia is a useful adjunct in cancer therapy as it can increase the effectiveness and decrease the toxicity of currently available cancer treatments such as chemotherapy and radiation. In the present study, we investigated whether 41 degrees C hyperthermia (mild HT) for 20 min can enhance macrosphelide (MS5)-induced apoptosis in human lymphoma U937 cells. Our results revealed that, compared with MS5 (5 microM) and mild HT alone, the combined treatment exhibited significant enhancement in apoptosis at 6 h, which was evaluated by observing morphological changes and DNA fragmentation. Marked increase in the reactive oxygen species (ROS) generation was observed immediately after the combined treatment. Significant increase in Fas externalization, caspase-8 and caspase-3 activation, and loss of mitochondrial membrane potential (MMP) was found after the combined treatment compared with MS5 and mild HT alone. Moreover, this combination can also alter the expression of apoptosis-related proteins as evident by the cleavage of Bid and down-regulation of Bcl-2 while no change in the expression of Bax was observed. Furthermore, an immediate rise in the intracellular calcium ion ([Ca(2+)]i) concentration was observed after the combined treatment, which continuously increased in a time-dependent manner. In addition, mild HT treatment alone also increases [Ca(2+)]i concentration without inducing apoptosis. Our data indicate that early increase in ROS generation is mainly responsible for the enhancement of apoptosis after the combined treatment.

Murine dendritic cells modified with CXCL10 gene and tumour cell lysate mediate potent antitumour immune responses in mice.

The aim of our present study was to estimate the effect of a therapeutic vaccine against tumour based on dendritic cells (DC) vaccine modified with tumour cell lysate and chemokine CXCL10 gene. In this study, mouse bone marrow DC were pulsed with tumour cell (RM-1) lysate and then transfected with a plasmid vector expressing CXCL10 cDNA by DOTAP liposome. The protective and therapeutic effects of the DC vaccine in RM-1 tumour model were assessed (divided into CXCL10/Lysate-DC, CXCL10/DC, pcDNA/Lysate-DC, Lysate-DC, pcDNA-DC, DC and PBS). The DC transfected with CXCL10 gene were capable of synthesizing and secreting CXCL10 chemokine. The highest CTL activity against RM-1 cells was induced in mice immunized with DC vaccine that was modified with RM-1 lysate and CXCL10 gene (CXCL10/Lysate-DC) when compared with its counterpart in mice. The CXCL10/Lysate-DC immunized mice also exhibited resistance to tumour challenge most effectively. In the RM-1 tumour model, immunization of CXCL10/Lysate-DC inhibited the tumour growth most significantly when compared with other groups and the survival time of the mice treated with CXCL10/Lysate-DC was greatly extended. These findings provide a potential strategy to improve the efficacy of DC-based tumour vaccine.

A hydrogen peroxide-generating agent, 6-formylpterin, enhances heat-induced apoptosis.

The enhancement of heat-induced apoptosis by 6-formylpterin, an intra-cellular generator of hydrogen peroxide (H2O2), was examined in human myelomonocytic lymphoma U937 cells. The cells were treated with either 6-formylpterin alone at a nontoxic concentration of 300 microM (37 degrees C), heat shock (44 degrees C per 20 min) alone or a combination of the two, then incubated at 37 degrees C for 6 h. Assessments of apoptosis, mitochondrial membrane potential and caspase-3 activation were performed by flow cytometry. Moreover, caspase-8 activation and changes in the intra-cellular Ca2+ concentration ([Ca2+]i) were examined. Bax, Bcl-2, Bcl-XL, Bid, cytochrome c and PKCd were detected by Western blotting. The induction of heat-induced apoptosis evaluated by morphological observation and DNA fragmentation were promoted by the addition of 6-formylpterin. Mitochondrial membrane potential was decreased and the activation of caspase-3 and -8 was enhanced in the cells treated with the combination. A decreased-expression of Bid was noted, although no significant changes in Bax, Bcl-2 and Bcl-XL expression were observed after the combined treatment. Furthermore, both the release of cytochrome c from mitochondria to cytosol and the translocation of PKCd from cytosol to mitochondria, which were induced by heat shock, were enhanced by the addition of 6-formylpterin. The number of cells with a higher [Ca2+]i was also increased by the addition of 6-formylpterin. These findings suggest that the increase in [Ca2+]i, the activation of the mitochondria-caspase dependent pathway and the translocation of PKCd to mitochondria play principal roles in the enhancement of heat-induced apoptosis by 6-FP.

Oral immunization with a live recombinant attenuated Salmonella typhimurium protects mice against Toxoplasma gondii.

The natural site of infection for T. gondii is the mucosal surface of the intestine, so the protective immunity obtained after natural infection with T. gondii points to the importance of developing a vaccine that stimulates mucosal defences. In this study, an aroA- and aroD- attenuated strain of Salmonella typhimurium (BRD509) has been used to deliver the recombinant eukaryotic plasmid pSAG(1-2)/CTA2/B expressing a multi-antigenic gene encoding SAG1 and SAG2 of T. gondii linked to A2/B subunits of cholera toxin as a candidate oral T. gondii vaccine. Immunoblot analysis showed compound gene expression in HeLa cells in vitro and intragastric immunization of mice with the recombinant salmonella resulted in the induction of humoral and Th1 type cellular immune responses and afforded protection against RH strain T. gondii challenge. Anti-T. gondii IgG values increased markedly in the BRD509/pSAG(1-2)-CTA2/B immunized group; these values were significantly higher than those in the negative controls (P = 0.008). With CTA2/B genetic adjuvant, the T. gondii-specific response was predominantly Th1, indicating that the CTA(2)/B genetic adjuvant was able to overcome the strong Th2-bias of the antigen (IgG2a >> IgG1). Antigen-specific T cell proliferative responses and CTL activity were significantly enhanced when cholera toxin CTA2/B genetic adjuvant was used (P = 0.009; P = 0.006). Culture supernatants from antigen-stimulated splenocytes from mice in these groups were also examined by ELISA for Th1- and Th2-type cytokines; mean IFN-gamma levels produced after oral immunization with BRD509/pSAG(1-2)-CTA2/B were about nine-fold higher than after immunization with BRD509/pSAG(1-2) (P = 0.007). On the other hand, the levels of IL-4 were low for all groups and no increase was seen in the presence of CTA2/B genetic adjuvant. When the immunized mice were intraperitoneally challenged with 10(3) tachyzoites of the highly virulent RH strain, the survival time of the mice immunized with BRD509/pSAG(1-2)-CTA2/B was markedly longer than other groups (P = 0.003) and a 40% survival rate was achieved. This is the first report that demonstrates that an oral attenuated salmonella DNA vaccine can induce protective immunity against the acute phase of T. gondii infection.

Analysis of gene expression in apoptosis of human lymphoma U937 cells induced by heat shock and the effects of alpha-phenyl N-tert-butylnitrone (PBN) and its derivatives.

Hyperthermia, a modality of cancer therapy, has been known as a stress to induce apoptosis. However, the molecular mechanism of heat shock-induced apoptosis, especially on roles of intracellular oxidative stress, is not fully understood. First, when human lymphoma U937 cells were treated with heat shock (44 degrees C, 30 min), the fraction of apoptosis, revealed by phosphatidylserine externalization, increased gradually and peaked at 6 hr after the treatment. In contrast, intracellular superoxide formation increased early during the heat shock treatment and peaked at 30 min after the treatment. When the cells were treated with heat shock in the presence of alpha -phenyl-N-tert-butylnitrone (PBN) and its derivatives, which are potent antioxidants, the DNA fragmentation was inhibited in an order according to the agents' hydrophobicity. PBN showing the highest inhibitory effects suppressed not only intracellular superoxide formation but also various apoptosis indicators. cDNA microarray was employed to analyze gene expression associated with heat shock-induced apoptosis, and the time-course microarray analysis revealed 5 groups showing changes in their pattern of gene expression. Among these genes, c-jun mRNA expression showed more than 40 fold increase 2 hr after heat treatment. The expression level of c-jun mRNA verified by quantitative real-time PCR was about 20 fold increase, and c-jun expression was similarly suppressed by PBN and its derivatives. These results suggest that the change of c-jun expression is an excellent molecular marker for apoptosis mediated by intracellular oxidative stress induced by heat shock.