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Knoevenagel condensation of acetylacetone and aromatic aldehyde catalyzed by L-proline in the choline chloride-based deep eutectic solvents was achieved. High yields are up to 96.6%. Mild reaction conditions, enhanced rates, improved yields, recyclability of deep eutectic solvents containing L-proline, and reagents’ reactivity which is different from that in standard organic solvents are the remarkable features observed in deep eutectic solvents. The deep eutectic solvents containing catalyst was recycled in four subsequent runs with no decrease in activity.

The Knoevenagel condensation of acetylacetone and aromatic aldehyde catalyzed by lipase from porcine pancreas(PPL) and α-amylase from hog pancreas in ionic liquids and deep eutectic solvents was achieved for the first time. The highest yields is up to 96.1%. Mild reaction conditions, enhanced rates, improved yields and reagents’ reactivity which is different from that in standard organic solvents are the remarkable features observed in ionic liquids and deep eutectic solvents.

Oxidative stress and apoptosis play important roles in the pathogenesis of various degenerative diseases. Previous studies have shown that naringin can exert therapeutic effects in multiple degenerative diseases by resisting oxidative stress and inhibiting apoptosis. Although naringin is effective in treating degenerative disc disease, the underlying mechanism remains unclear. This study is aimed at investigating the effects of naringin on oxidative stress, apoptosis, and intervertebral disc degeneration (IVDD) induced by cyclic stretch and the underlying mechanisms in vitro and in vivo. Abnormal cyclic stretch was applied to rat annulus fibrosus cells, which were then treated with naringin, to observe the effects of naringin on apoptosis, oxidative stress, mitochondrial function, and the nuclear factor- (NF-) κB signaling pathway. Subsequently, a rat model of IVDD induced by dynamic and static imbalance was established to evaluate the effects of naringin on the degree of degeneration (using imaging and histology), apoptosis, and oxidative stress in the serum and the intervertebral disc. Naringin inhibited the cyclic stretch-induced apoptosis of annulus fibrosus cells, reduced oxidative stress, improved mitochondrial function, enhanced the antioxidant capacity, and suppressed the activation of the NF-κB signaling pathway. Additionally, it reduced the degree of IVDD (evaluated using magnetic resonance imaging) and the level of oxidative stress and inhibited apoptosis and p-P65 expression in the intervertebral discs of rats. Thus, naringin can inhibit cyclic stretch-induced apoptosis and delay IVDD, and the underlying mechanism may be related to the inhibition of oxidative stress and activation of the NF-κB signaling pathway. Naringin may be an effective drug for treating degenerative disc disease.

In the present work, the influence of temperature effect on polaron in a quantum well (QW) with asymmetric Gaussian potential (AGP). By employing strongly coupling method of Pekar variational (SCMPV), the ground state (GS), the first-excited state (FES) energies and transition frequency (TF) of the polaron are derived. The state-energies and the TF as relationship of the temperature T and the electron phonon coupling strength (EPCS) are obtained by using quantum statistics theory (QST). We have found that the state-energies and the TF increase when the temperature is increasing. The states-energies and the TF are decreased with raising the EPCS.

A stretchable, skin-mountable and wearable strain sensor is important because of its possible applications in monitoring our daily life and work, such as robotic intelligent equipment, health-monitoring, human motion detection and remote precise diagnosis. A linen fabric-derived carbon (LDC) has been rationally integrated with 2D graphene and 1D silver nanowires by impregnation of the linen fabric with a graphene oxide suspension followed by carbonization and final impregnation with a suspension of silver nanowires. The Ag nanowires/graphene/linen-derived carbon (Ag-GLDC) composite sensor exhibits excellent stretchability (>60%), good cycling stability, and high sensitivity with gauge factors of 11.2, 36.8 and 74.5for strains of 0–20%, 20%–40%, 40%–60%, respectively. The composite sensor has been successfully used to monitor the vigorousmotion of human body joints (wrist, knee and elbow), suggesting its potential use in human motion detection. This work provides a new method for the preparation of flexible and wearable composite strain sensors with high performance.

The catalytic promiscuity of MML, lipase from Mucor miehei, was reported to catalyze direct aldol reactions between aromatic aldehydes and cyclic ketones in water, ionic liquids, and deep eutectic solvents at 50 °C. Water can promote the catalytic activity of MML. A wide range of aromatic aldehydes reacted with cyclic ketones to provide the corresponding aldol products with high yields (up to 87.2%) and moderate to good diastereoselectivity (up to 3:97 dr).

Bovine pancreatic lipase (BPL) is reported to catalyze direct asymmetric aldol reactions between aromatic aldehydes and cyclic ketones in water at 50 °C, and water can greatly promote the catalytic activity and selectivity of BPL. A wide range of aromatic aldehydes reacted with cyclic ketones to provide the corresponding aldol products with high yields (up to 93.7%) and moderate to good stereoselectivity (up to 99% ee and 1:99 dr).

Pepsin was found to catalyze asymmetric cross aldol reactions of aromatic and polycyclicaromatic aldehydes with cyclic ketones in ionic liquids and deep eutectic solvents for the first time. Pepsin exhibited high catalytic activity and excellent stereoselectivity in ionic liquids and deep eutectic solvents in the presence of moderate water. High yields of up to 93.1%, excellent enantioselectivities of up to 96% ee, and good diastereoselectivities of up to 5:95 dr were achieved.

We academically explore the properties of the coherence time (CT) of an electron powerful coupled to the phonons in a RbCl quantum pseudodot (QPD) qubit with an exterior electric field alongside the ρx direction. By employing the Pekar type variational method (PTVM) and the Fermi golden rule, the CT changes with the electric field, the chemical potential of the two-dimensional electron gas (CPTDEG), the zero point of the pseudoharmonic potential (ZPPHP) and the polaron radius is studied. The examined results indicate ① that the CT of RbCl QPD qubit decays with aggrandizing electric field, ② that the CT is a decreasing function of the CPTDEG, the ZPPHP and the polaron radius, ③ that here we can find that we can adjust the CT by changing the external electric field, the CPTDEG, the ZPPHP and the polaron radius.

Background The identification of the important elements that control hepatic stellate cell (HSC) activation will expand our understanding of the mechanism of liver fibrosis induced by hypoxia and affect the outcome of clinical treatment. A previous research demonstrated that N-Myc downstream-regulated gene 2 (NDRG2) is a potential regulator of fibrosis and a downstream target gene of hypoxia-inducible factor 1 (HIF-1). In this research, we studied the expression and function of NDRG2 in liver fibrosis induced by hypoxia. Methods LX-2 cells/NF-κB-silenced LX-2 cells were exposed to hypoxic conditions (1% O2) to activate HSCs in vitro. The protein and mRNA expression levels of NDRG2, α-SMA and transforming growth factor beta 1 (TGF-β1) were evaluated by western blotting and real-time polymerase chain reaction (RT-PCR), respectively. Functional studies were performed using adenovirus-mediated gene upregulation. Results The NDRG2 mRNA and protein levels were reduced under hypoxic conditions in LX-2 cells and overexpression of NDRG2 resulted in a decrease in the expression of TGF-β1 and α-SMA. Interestingly, no relationship was observed between NDRG2 and TGF-β1 when the NF-κB pathway was blocked, which indicates that NDRG2 can regulate the expression of TGF-β1 in LX-2 cells via the NF-κB pathway under hypoxic conditions. Conclusions NDRG2 may regulate the expression of TGF-β1 via the NF-κB pathway and may be a novel therapeutic target for liver fibrosis induced by hypoxia.

By employing the variational method of Pekar type, we study the effects of electric field on RbCl quantum pseudodot (QPD) qubits. Our results confirm that (1) the electron oscillates in the RbCl QPD with a certain period; (2) the electron’s probability density is a raising function of electric field; (3) the oscillating frequency is an increasing one of the electric field and the two-dimensional electron gas chemical potential. Two ways are found for prolonging the lifetime of the qubit and suppressing the decoherence in the quantum information field.

The degradation of PAEs (DMP, DEP and DBP) at low concentration (μg·L-1 range) by the O3 oxidation and H2O2/O3 oxidation process were investigated in this study. The experiment results showed that the H2O2/O3 ratio was 2.0, the pH value was 10, the PAEs removal rates were 90%~95% in model wastewater, and were over 70% in the tail water from municipal sewage plant with the H2O2/O3 oxidation process.

A method for the detection of PAEs (16 kinds ) in the childrens products using ultrasonic extraction and gas chromatography were investigated in this study. The method was linear between 0.500 and 10.000 μg ml-1, R2 between 0.995 and 0.999, LOQ between 0.704 and 1.585 μg ml-1. The method repeatability was between 1.2% and 13% RSD. The experiment results showed that the children's products samples containing different kinds of PAEs. DIBP and DCHP were frequency found in all sample. The PAEs concentration of the childrens products sample was between 0.019 and 6.301 μg ml-1.

The removal of high concentration ammonia in wastewater was investigated by an indirect electrochemical oxidation method using titanium electrodes coated with ruthenium and iridium (RuO2-IrO2-TiO2/Ti). The effect of different initial pH on ammonia removal by electrochemical oxidation was studied. The concentrations variation of ammonia, total nitrogen, nitrate nitrite, and free cholorines, chloramines was analyzed under the conditions with and without pH controlling. The results indicate that ammonia removal efficiency was higher under the moderate alkaline condition than that under neutral one. During the electrolysis process, nitrate and nitrite concentrations were very low, even below their detecting limits. The concentrations of free chlorines and chloramines were significantly affected by pH, as trichloramine and free chlorines were mainly produced in the reaction without pH controlling, while monochloramine was mainly produced in a stable alkalescene.

A pilot study was carried out to evaluate the incorporation of air sparging for enhancing ultrafiltration membrane filterability in treating soybean whey. The results showed that the decline of membrane permeate flux was slowed down by air sparging. The filtration could continue for 1200 minutes with acceptable permeate flux under the optimal parameters as temperature of 45°C, pH of 7.0, transmembrane pressure (TMP) of 0.05MPa and air sparging intensity of 5.0m3/h. The experiment also revealed that air sparging was advantageous to hydrodynamic flushing and chemical cleaning to reclaim membrane permeate flux. Moreover, the effect of air sparging on the retaining ratios of protein and sugar was investigated.

When exterior wall adopts solar chimney thermal channel, natural ventilation comes into being in the thermal channel, which plays the role of reducing the heat indoor. In this article, we use FLUENT to explore the heat transfer and flow quality of the solar chimney thermal channel in Changsha area in the summer. The research mainly studies how different thermal channel widths, inlet and outlet vent heighst and solar radiation intensities can impact on ventilation effect and further concludes the rules of thermal channel flow field and ventilation rate changing of the solar chimney.

A pilot study was carried out to evaluate the application of aerobic submerged MBR in treating polyester wastewater and a full scale system in which MBR was adopted as the key aspect was put forward to treat the polyester wastewater of Zhuhai Yuhua Polyester co., Ltd., China. The pilot study revealed that HRT could be affected by the influent COD, and a HRT larger than 36h was proposed for treating polyester wastewater by MBR. Volumetric loading rate and sludge loading rate ranged in 0.7-2.0 kgCOD/m3•d and 0.09~0.24 kgCOD/ kgMLSS•d respectively were of small influence on the COD removal. DO in the range of 2.5-4.0 mg/L seemed to be optimal for avoiding limitations due to oxygen concentration and creating an effective turbulence. The modified full scale system which includes anaerobic digesting tank and MBR followed by a stabilization pond was verified to be effective to treat the polyester wastewater in the latest two years. The results illustrated that the effluent water quality could meet the discharge limits of water pollutants (DB 44/26-2001) which was established in Guangdong, China. Observed sludge yield of the system fluctuated between 0.10-0.18 gMLSS/gCOD and averaged at 0.137 gMLSS/gCOD. The membrane permeate at around 10 L/m2h, TMP of less than 0.7bar, and total mixed liquor suspended solids (MLSS) between 6-7g/L were suitable to operate the MBR.

Gr-1(+)CD11b(+)F4/80(+) cells play important roles in tumor development and have a negative effect on tumor immunotherapy. So far, the mechanisms underlying the regulation of their immunosuppressive phenotype by classical and alternative macrophage activation stimuli are not well elucidated. In this study, we found that molecules from necrotic tumor cells (NTC-Ms) stimulated Gr-1(+)CD11b(+)F4/80(+) cells to induce apoptosis of activated T cells but not nonstimulated T cells. The apoptosis-inducing capacity was determined by higher expression levels of arginase I and IL-10 relative to those of NO synthase 2 and IL-12 in Gr-1(+)CD11b(+)F4/80(+) cells, which were induced by NTC-Ms through TLR4 signaling. The apoptosis-inducing capacity of NTC-Ms-stimulated Gr-1(+)CD11b(+)F4/80(+) cells could be enhanced by IL-10. IFN-gamma may reduce the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells only if their response to IFN-gamma was not attenuated. However, the potential of Gr-1(+)CD11b(+)F4/80(+) cells to express IL-12 in response to IFN-gamma could be attenuated by tumor, partially due to the existence of active STAT3 in Gr-1(+)CD11b(+)F4/80(+) cells and NTC-Ms from tumor. In this situation, IFN-gamma could not effectively reduce the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells. Tumor immunotherapy with 4-1BBL/soluble programmed death-1 may significantly reduce, but not abolish the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells in local microenvironment. Blockade of TLR4 signaling could further reduce the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells and enhance the suppressive effect of 4-1BBL/soluble form of programmed death-1 on tumor growth. These findings indicate the relationship of distinct signaling pathways with apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells and emphasize the importance of blocking TLR4 signaling to prevent the induction of T cell apoptosis by Gr-1(+)CD11b(+)F4/80(+) cells.

To evaluate the therapeutic effect of heated (60 degrees C) lipiodol via hepatic artery administration in a rabbit model of VX2 liver cancer.Thirty male New Zealand white rabbits were randomly divided into three groups with 10 rabbits assigned to each group. VX2 carcinoma cells were surgically implanted into the left hepatic lobe. The tumors were allowed to grow for 2 weeks, and studies were performed until the diameter of the tumors detected by ultrasonograph reached 2-3cm. Under anesthesia, trans-catheter hepatic arterial embolization was performed and doxorubicin-lipiodol (37 degrees C) (1mL), lipiodol (60 degrees C) (1mL) or control (physiological saline (37 degrees C) (1mL)) solution was injected into the hepatic arteries of animals in the three groups. One week later, the volume of the tumor was measured by ultrasonograph again. The serum of all rabbits was collected before injection and at 4 and 7 days after injection, and the level of aspartate aminotransferase (AST) was checked. The survival period of the three groups of rabbits after treatment was also recorded. During the last course of their disease, the rabbits were given analgesics to relieve suffering.The tumor growth rate in the lipiodol (60 degrees C) group (0.92+/-0.21, tumor volume from 1811+/-435 to 1670+/-564mm(3)) was significantly lower than that in the control group (3.48+/-1.17, tumor volume from 1808+/-756 to 5747+/-1341mm(3)) (P0.05) and in the doxorubicin-lipiodol (37 degrees C) group (1.69+/-0.26, tumor volume from 1881+/-641 to 2428+/-752mm(3)) (P0.05). Consequently, the survival period of the animals in the lipiodol (60 degrees C) group (41.0+/-3.0 days) was significantly greater than that in the doxorubicin-lipiodol (37 degrees C) group (38.0+/-2.5 days) (P0.05). On the other hand, there was no statistically significant difference in serum AST levels between the lipiodol (60 degrees C) group (148.2+/-11.3UL(-1)) and the doxorubicin-lipiodol (37 degrees C) group (139.7+/-12.3UL(-1)) (P0.05). However, the serum AST level in the lipiodol (60 degrees C) group was significantly higher at 4 days after injection (P0.05) than in the control group (68.6+/-6.6UL(-1)).Treatment with lipiodol (60 degrees C) resulted in an effect on serum AST levels similar to that caused by treatment with doxorubicin-lipiodol (37 degrees C). Thus, lipiodol (60 degrees C) treatment could greatly prolong the survival period of rabbits with VX2 cancer by inhibiting tumor growth.

Cytotoxic CD8(+) T cells are key effectors in the immunotherapy of malignant and viral diseases. However, autologous T cell responses to tumor antigens presented by self-MHC are usually weak and ineffective. Allo-restricted T cells represent a potent source of tumor-specific T cells for adoptive immunotherapy. This study reports in vivo anti-melanoma efficacy of the pTRP2-specific allo-restricted CTLs expanded from the BALB/c splenocytes by multiple stimulations with aAPCs made by coating H-2K(b)-Ig/pTRP2 dimeric complexes, anti-CD28 antibody, 4-1BBL molecules and CD83 molecules to cell-sized latex beads. The induced allo-restricted CTLs exhibited specific lysis against RMA-S cells pulsed with the peptide pTRP2 and H-2K(b+) melanoma cells expressing TRP2, while a murine Lewis lung carcinoma cell line 3LL could not be recognized by the CTLs. The peptide-specific activity was inhibited by anti-H-2K(b) monoclonal antibody Y3. Adoptive transfer of the allo-restricted CTLs specific for malignant melanoma expanded by the aAPCs can mediate effective anti-melanoma response in vivo. These results suggested that the specific allo-restricted CTLs expanded by aAPCs coated with an MHC-Ig/peptide complex, anti-CD28 antibody, 4-1BBL and CD83 could be a potential option of specific immunotherapy for patients with malignant melanoma.

Transvenous embolization has become the treatment of choice for such lesions We evaluated Onxy for patients with cavernous dural arteriovenous fistulae (CDAVFs) who underwent transvenous embolization via different transvenous approaches. Case records of six patients with symptomatic CDAVFs, treated between October 2006 and November 2007 were reviewed. A total of seven transvenous procedures were performed in the six patients with CDAVFs. All the patients with CDAVFs of the cavernous sinus were symptom free following embolization. The approach via the internal jugular vein and the inferior petrosal sinus was possible in four of the six patients, with complete occlusion of the fistula. In the remaining two patients, the approach was via the facial vein. Transient bradyarrythmia without morbidity was the only complication in two patients.

Cytotoxic CD8 + T cells are key effectors in the immunotherapy of malignant and viral diseases. However, the lack of efficient methods for their in vitro priming and expansion has become a bottleneck to the development of vaccines and adoptive transfer strategies. Synthetic artificial antigen-presenting cells (aAPCs) are now emerging as an attractive tool for eliciting and expanding CTL responses. This study reported a novel approach for targeting malignant melanoma with pTRP2-specific cytotoxic T lymphocytes (CTLs) expanded from the C57BL/6 splenocytes by multiple stimulations with aAPCs made by coating H-2K b -Ig/pTRP2 dimeric complexes, anti-CD28 antibody, 4-1BBL molecules and CD83 molecules to cell-sized latex beads. The induced CTLs exhibited specific lysis against RMA-S cells pulsed with the peptide pTRP2 and H-2K b+ melanoma cells expressing TRP2, while a murine Lewis lung carcinoma cell line 3LL could not be recognized by the CTLs. The peptide-specific activity was inhibited by anti-H-2K b monoclonal antibody Y3. Adoptive Transfer of CTLs specific for malignant melanoma expanding by the aAPCs can mediate effective anti-melanoma response. These results suggested the bead-based aAPCs coated with an MHC-Ig/peptide complex, anti-CD28 antibody, 4-1BBL and CD83 could provide a useful tool for the reproducible expansion of specific CTLs for adoptive immunotherapy.

E10A, a recombinant adenovirus type 5 vector carrying the human endostatin gene, may be a promising gene therapy drug in the treatment of solid tumors by antiangiogenesis, but a preclinical safety evaluation of E10A has not yet been performed. With high and low doses equivalent to 30 and 7.5 times the human curative dose, respectively, intramuscular injections of E10A were given once daily, 6 days/week, for 3 months, followed by a 1-month recovery period. As of 4 months, all experimental animals appeared generally healthy: normal behavior and eating habits, no nausea, vomiting, or salivation, no abnormal changes in urination or defecation, and increased body weight with the time of experiment. Urinalysis, hemogram, blood biochemistry, electrocardiogram, macroscopic and microscopic studies of organs and tissues were done before treatment, at month 3 of treatment, and 1 month posttreatment. At all time points, no significant abnormal toxic effects were noted. Preliminary investigation of E10A immunotoxicity in dogs indicated that anti-adenoviral antibodies were generated, in a dose- and time-independent manner, after E10A injection. Our data demonstrated that, long term, high-dose intramuscular administration of recombinant human endostatin-carrying adenovirus (E10A) was not notably toxic and might be safe for clinical therapeutic use, although additional long-term toxicity studies by other administration routes are still necessary.

T-bet (T box expressed in T cells), a Th1-specific T box transcription factor, controls many kinds of immune cells, such as Th1, NK, CD8+, dendritic cells, and B cells. This study was to explore potential effects of T-bet gene on biological functions of mouse macrophage Raw264.7 cells in vitro.The eukaryotic expression vector carrying mouse T-bet (pcDNA3.0-mT-bet) was constructed and identified by consequence analysis, double restrictive endonucleases digestion and polymerase chain reaction (PCR). The gene expression in Raw264.7 cells was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. PBS, pcDNA3.0, and pcDNA3.0-mT-bet were transiently transfected into Raw264.7 cells respectively; cell cycle, MHC I/II expression levels, phagocytic activity of FITC-dextran, nitric oxide (NO) secretion level, and the cytotoxicity of Raw264.7 cells to mouse leukemia cell line L1210 were evaluated 48 hours after transfection.Eukaryotic expression vector which could express T-bet protein in Raw264.7 cells was successfully constructed. There was no difference in cell cycle between pcDNA3.0 group and pcDNA3.0-mT-bet group. There was significant difference in MHC I expression level between pcDNA3.0 group (20.8+/-0.7) and pcDNA3.0-mT-bet group (24.8+/-0.6, P0.05), but not in MHC II expression level; there was also difference in mean fluorescence intensity of phagocytized dextran between pcDNA3.0 group (28.2+/-0.4) and pcDNA3.0-mT-bet group (32.8+/-0.8, P0.05); there was also significant difference in NO secretion level between pcDNA3 group (0 pmol) and pcDNA3.0-mT-bet group [(1.7+/-0.6) pmol, P0.05] without lipopolysaccharide (LPS) stimulation; meanwhile, significant difference was also observed between pcDNA3.0 group [(10.5 +/-1.3) pmol] and pcDNA3.0-mT-bet group [(15.6+/-1.6) pmol, P0.05] under the stimulation of LPS (10 microg/ml) for 20 h; there was also difference in cytotoxicity of Raw264.7 cells to L1210 cells in vitro between pcDNA3 group [(35.6+/-2.1)%] and pcDNA3.0-mT-bet group [(51.9+/-3.5)%, P0.05].T-bet up-regulates MHC I expression and NO secretion level in Raw264.7 cells, increases their cytotoxicity to L1210 cells, but has no influences on the cell cycle and MHC II expression.

To study the effect of HLA-G1 molecule expressed by an endothelial cell line (ECV304) on the cytotoxic activity of allogeneic NK cells.ECV304 cells were transfected with recombinant plasmid pcDNA3-HLA-G1 by the liposome transfection, and the expressed HLA-G1 on the cell surface was detected by indirect immunofluorescent assay and flow cytometry. The cytotoxic activity of allogeneic NK cells against ECV304 cells was analyzed by the MTT method.HLA-G1 was expressed on the surface of the transfected ECV304 cells. The specific lysis of NK cells against plasmid pcDNA3 transfected ECV304 was (50.6+/-18.1)%, while the specific lysis against pcDNA3-HLA-G1 transfected ECV304 was (29.7+/-11.4)%, which was significantly lower than the former (P0.001).HLA-G1 expressed by the ECV304 cells can inhibit cytotoxicity of allogeneic NK cells.

To construct the expression vector of biotin-protein ligase (BirA enzyme) gene and express the BirA enzyme with bioactivity in E.coli BL-21 (DE3).The BirA gene was amplified from E.coli genome by PCR and cloned into pGEX-4T-2 to construct the recombinant plasmid pGEX-BirA. After being verified by DNA sequencing, the fusion protein was expressed under IPTG induction in the E.coli BL-21 (DE3). The expressed product was purified through Glutathione-agarose chromatography column. The enzyme activity of the expressed product was identified by ELISA and Western blot.The recombinant prokaryotic expression vector pGEX-BirA was constructed and the fusion protein GST-BirA was expressed successfully. The results of ELISA and Western blot showed that the purified BirA enzyme biotinylated HLA-A2 peptide complex.BirA enzyme with bioactivity is prepared successfully, which can be used for studying the interaction between protein and protein.

To form soluble HLA-G1-peptide complex by refolding in vitro, and to study its immune function.The heavy chain and beta(2m) of sHLA-G1 were expressed as insoluble aggregates in E. coli, and then the two subunits were refolded to form HLA-G1-peptide complex by dilution method in the presence of specific peptide. The refolded product was purified through Sephadex G-75 gel filtration. The purified product was identified by Western blot with mAb W6/32. The function of soluble HLA-G1 was explored from following three aspects, namely, the influences on cytotoxicity of NK cells, on proliferation of T cells in mixed lymphocyte culture and apoptosis of activated T cells.The refolded complex was recognized by mAb W6/32. It effectively inhibited cytotoxicity of NK cells and proliferation of T cells, and induced apoptosis of activated T cells.The refolding of soluble HLA-G1-peptide complex has been successfully realized in vitro. The complex can inhibit the functions of NK cells and T cells.

To improve the refolding efficiency of soluble HLA-A2-peptide complex in vitro.The heavy chain (HC) of MHC class I was extracted from bacteria under denaturing and non-reducing conditions. Anion-exchange and (NH4)2SO4 precipitation were applied to purify the HC. Then the purified HC, beta2m and an antigenic peptide (N-YMDGTMSQV-COOH of Try(369-377)) were refolded to form an HLA-A2-peptide complex by dilution method in the buffer of pH 6.6. The refolded products were detected by Western blot and ELISA with W6/32 and anti-human beta2m antibody.The refolded products consisted of HLA-A2-peptide complex, beta2m, and a little amount of HC polymer. The refolding efficiency was 2.5 fold higher than that of the conventional method.This study confirmed that the refolding efficiency of the method reported in this paper is higher as compared with the conventional method, which is of importance to the preparation of HLA-peptide tetramers and artificial antigen presenting cells.

Inhibition of tumor growth by endostatin has been shown to be an effective strategy in cancer therapy in mice. However, its widespread application has been hampered by difficulties in a large-scale production of the recombinant endostatin protein, rapid loss bioactivity of the protein, and the cumbersome daily administration. These limitations could be resolved by in vivo delivery and expression of the endostatin gene. In this study, we observed the effect and advantage of endostatin gene therapy mediated by a recombinant adenoviral vector (Ad/hEndo) on the growth of hepatocellular carcinoma BEL-7402 xenografted tumors, comparison with recombinant endostatin protein.Hepatocellular carcinoma BEL-7402 cells were inoculated subcutaneously in the flank of Balb/c nude mice. Nine days after tumor cell inoculation, animals were given a cycle of four courses of intra-tumoral injections of Ad/hEndo of 5 x 10(8) pfu (low-dose group) and 1 x 10(9) pfu (high-dose group) at intervals of six days, respectively. Recombinant human endostatin protein (rhEndo) was administrated daily subcutaneously at a dose of 10 mg.kg(-1).d(-1) at a site nearby the tumor for ten days. The expression of endostatin mRNA in tumor tissue was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) after Ad/hEndo injection. Dynamic changes of concentration of endostatin protein in tumor tissue were quantitated by enzyme-linked immunosorbent assay (ELISA).After 4 courses of treatment, the tumor growth rates of high-dose treated group with 1 x 10(9) pfu of Ad/hEndo were inhibited by 42.26% compared with the Ad/LacZ control group (P = 0.001) and by 46.26% compared with the NIH buffer control group (P = 0.003), respectively. However, in this study, Ad/hEndo at low dose of 5 x 10(8) pfu failed to demonstrate significant inhibition of tumor growth, compared with control groups. After daily administration of recombinant human endostatin protein (rhEndo) for 9 days, the ratio of T/C (rhEndo group versus PBS group) was less than 47%. However, two days after rhEndo treatment ceased, the ratio of T/C was more than 50%. The peak of expression of endostatin mRNA in tumor tissue was at 2 or 3 days after administration intratumorally with Ad/hEndo of 1 x 10(9) pfu and gradually dropped undetectable by day 7. Dynamic analysis of endostatin concentration in tumor tissue showed that the highest level of mRNA is up at the third day after injection, and dropped to basal level three weeks later.Endostatin gene therapy mediated by a recombinant adenoviral vector had significantly inhibited the growth of hepatocellular carcinoma BEL-7402 xenografted tumors at a high dose of 1 x 10(9) pfu compared with other groups. The analysis of dynamic expression of endostatin in vivo indicated that Ad/hEndo had acquired a high-level, relatively long-term expression in vivo and bioactivity capability.

Endostatin, a 20-kDa carboxyl-terminal fragment of collagen XVIII, is a potent inhibitor of endothelial cell proliferation and tumor angiogenesis. We have constructed replication-deficient recombinant adenovirus (Ad-rhE), which encoded secreted human endostatin, and our previous studies showed that Ad-rhE had a potent suppression of tumor growth in vivo. In the present study, we investigated the dynamic distribution and expression of human endostatin gene in vivo using fluorogenic real-time quantitative PCR and enzyme-linked immunosorbent assay(ELISA), respectively, with an injection of 2.0 x10(9)pfu of Ad-rhE. After injection, the Ad-rhE DNAs decreased sharply, but lasted a relative long-term at low concentration (10,000--20,000 copies/mg tissues). Whereas the expressed endostatin rose up rapidly, and reached to the top on day 5 after injection of Ad-rhE, and then decreased sharply, but endostatin in tumors sustained to over 9 days at a certain level. Both Ad-rhE DNAs and endostatin mainly enriched in tumors in vivo, and then in livers. These results suggest that endostatin gene delivered by adenoviral vector can generate a high expression in vivo, and both the metabolism pathways of Ad-rhE DNAs and endostatin in vivo are through the systems of livers.

To refold and biotinylate HLA-A2-peptide complex in-vitro.The BirA substrate peptide (BSP) containing H chain of HLA-A2 and beta(2m) were expressed highly as insoluble aggregates in E.coli, and then the two subunits were refolded to form an HLA-A2-peptide complex by dilution method in the presence of an antigenic peptide (NH(2)-CLGGLLTMV-COOH of EB virus latent membrane protein 2A LMP2A). Then the BirA enzyme was used to biotinylate the refolded complex. The refolded and biotinylated products were detected by ELISA and Western blot with mAb W6/32 and rabbit anti-human beta(2m) antibody and streptavidin.The refolded complex was composed of H chain aggregate, HLA-A2-peptide complex and beta(2m). Both HLA-A2-peptide complex and the H chain aggregate could be biotinylated.The refolding and biotinylation of HLA-A2-peptide complex were successfully performed and the products were confirmed by our practical immunological method. This study laid the foundation for the preparation of HLA-peptide tetramer and artificial antigen presenting cells.

A heuristic search algorithm for image registration is proposed, in which no threshold sequence is needed. The results show that the algorithm is reasonable and efficient, and has higher speed than sequential similarity detection algorithm in some cases. >

AIM: To study the induction of T cellular immune responses in BALB/c mice immunized with uric acid and dendritic cells (DCs) pulsed with hepatitis B virus surface antigen (HBsAg). METHODS: DCs were generated from bone-marrow cells of BABL/c mice, and then pulsed or unpulsed with HBsAg protein (HBsAg-pulsed-DCs or unpulsed-DCs) in vitro. BABL/c mice were immunized with HBsAg-pulsed-DCs (1 × 106) and uric acid, injected through the tail vein of each mouse. The mice in control groups were immunized with HBsAg-pulsed-DCs alone, unpulsed-DCs alone or 200 μg uric acid alone or PBS alone. The immunization was repeated 7 d later. Cytotoxic T lymphocytes (CTLs) in vivo were determined by the CFSE labeled spleen lysis assay. Spleen cells or spleen T cells were isolated, and re-stimulated in vitro with HBsAg for 120 h or 72 h. Production of IFN-γ and IL-4 secreted by spleen cells were determined by ELISA method; proliferation of spleen T cells were detected by flow cytometry. RESULTS: The cytotoxicities of HBsAg-specific-CTLs, generated after immunization of HBsAg-pulsed-DCs and uric acid, were 68.63% ± 11.32% and significantly stronger than that in the control groups (P < 0.01). Compared with control groups, in mice treated with uric acid and HBsAg-pulsed-DCs, the spleen T cell proliferation to HBsAg re-stimulation was stronger (1.34 ± 0.093 vs 1.081 ± 0.028, P < 0.01), the level of IFN-γ secreted by splenocytes was higher (266.575 ± 51.323 vs 135.223 ± 32.563, P < 0.01) , and IL-4 level was lower (22.385 ± 2.252 vs 40.598 ± 4.218, P < 0.01). CONCLUSION: Uric acid can strongly enhance T cell immune responses induced by HBsAg-pulsed-DCs vaccine. Uric acid may serve as an effective adjuvant of DC vaccine against HBV infection.