Tuning Topological Orders by a Conical Magnetic Field in the Kitaev Model.

We show that a conical magnetic field H=(1,1,1)H can be used to tune the topological order and hence, anyon excitations of the Z_{2} quantum spin liquid in the isotropic antiferromagnetic Kitaev model. A novel topological order, featured with Chern number C=4 and Abelian anyon excitations, is induced in a narrow range of intermediate fields H_{c1}≤H≤H_{c2}. On the other hand, the C=1 Ising-topological order with non-Abelian anyon excitations, as previously known to be present at small fields, is found here to survive up to H_{c1}. The results are obtained by developing and applying a Z_{2} mean field theory that works at finite fields and is asymptotically exact in the zero field limit and the associated variational quantum Monte Carlo.

Implantation of bFGF-treated islet progenitor cells ameliorates streptozotocin-induced diabetes in rats.

AIM: To examine whether implantation of islet preparation-derived proliferating islet cells (PIC) could ameliorate diabetes in rats. METHODS: PIC were expanded from rat islet preparation by supplementation of basic fibroblast growth factor (bFGF) and implanted into rats with streptozotocin (STZ)-induced diabetes through the portal vein. Body weight and blood glucose levels were measured. Serum insulin levels were measured by radioimmunoassay. The presence of insulin-positive cells was determined by hematoxylin and immunohistochemical staining. RESULTS: Cultured islet cells (CIC) were demonstrated to dedifferentiate in vitro, and the apoptosis ratios reached more than 50% by the 15th day post-isolation. PIC cells treated with bFGF (20 ng/mL) continued growing within 30 days after isolation, and no apoptotic cells were detected. Implantation of PIC into diabetic rats was capable of ameliorating diabetes, in terms of the restoration of euglycemia, weight gain, improved glucose response and elevated serum insulin levels for up to 130 days. Livers derived from PIC-implanted rats were examined for insulin expression and single insulin-positive cells. In addition, most islets of PIC-implanted STZ-induced diabetic rats were intact at 130 days post-transplantation and comparable to those of normal rats. CONCLUSION: Implantation of bFGF-treated proliferating islet cells is a promising cellular therapeutic approach for diabetes.

A gutless adenoviral vector expressing full-length anti-Her2 antibody.

1. Therapeutic monoclonal antibodies are increasingly being used in clinical cancer treatment, but their complex technology and high cost limit their use. Helper-dependent (HD) adenoviruses are among the most efficient and safe gene therapy vectors capable of mediating long-term expression. 2. Using Gateway (Invitrogen, San Diego, CA, USA) cloning technology, we constructed an HD­trastuzumab (TAb) plasmid carrying the full-length anti-HER2 antibody gene. Using an efficient recombinase, namely in vitro-evolved Flippase-expressing recombinase, to excise the helper virus packaging signal in producer cells, we developed a scalable HD vector production method. Antibody expression of HD-TAb in vitro was detected by ELISA and western blot. 3. The full-length antibody gene delivery system allowed for continuous production of a full-length antibody at a high concentration. Bioactive antibody macromolecules were generated via gene transfer in vitro. 4. In conclusion, HD adenoviral vectors can stably express a full-length antibody for prolonged periods without the difficulties associated with sophisticated antibody manufacture techniques and at a much lower cost. As a promising tool for gene therapy, this novel system can shorten the duration and reduce the expense of antibody development.

RNA interference targeting ORC1 gene suppresses the proliferation of vascular smooth muscle cells in rats.

BACKGROUND: The proliferation of vascular smooth muscle cells (VSMCs) plays an important role in the pathogenesis of vascular diseases such as atherosclerosis and postangioplasty restenosis. The largest subunit of the origin recognition complex (ORC), ORC1, plays a critical role during the initiation of DNA replication in eukaryotes. However, the involvement of ORC1 in the initiation of DNA replication in VSMCs has not been studied yet. OBJECTIVE: The aim of this study was to silence ORC1 gene selectively by using RNA interference and analyze the effects of ORC1 gene on the proliferation and apoptosis of rat VSMCs. METHODS: Freshly isolated rat VSMCs were transfected with siRNA targeting ORC1 gene capsulated in liposome. ORC1 protein expression was determined by Western blotting and ORC1 mRNA level by RT-PCR. DNA synthesis was analyzed by (3)H thymidine ((3)H-TdR) incorporation and cell proliferative activity and cell cycle distribution by flow cytometry. Two apoptosis-related proteins, Bax and Bcl-2, were examined immunohistochemically. RESULTS: Down-regulation of ORC1 mRNA and protein expression was observed in rat VSMCs at 24 h after transfection with the three pairs of siRNA targeting ORC1 gene and this reduction persisted at least 7 days post-transfection. Down-regulation of ORC1 mRNA (60%) and protein (80%) expression was observed at 72 h post-transfection in the cells transfected with B-ORC1 siRNA. A significant decrease in (3)H thymidine incorporation was observed in rat VSMCs with ORC1 gene silencing after serum challenge, but not in the non-silenced control. A significant increase in the proliferation index and a significant decrease in the percentage of cells at G(0)/G(1) phase after serum challenge were observed in the non-silenced control, but not in ORC1 gene silenced cells. A significant increase in the ratio of Bcl-2/Bax was observed after serum challenge in the non-silenced control, but only a slight increase was found in the ORC1 gene silenced cells. ORC1 gene silencing disappeared 7 days after transfection. Continuous serum challenge stimulated VSMCs to synchronously reenter the cell cycle as evidenced by increases in [(3)H] thymidine incorporation, the proliferation index, and the ratio of Bcl-2/Bax, as non-silenced cells were induced to resume cell cycle progression by the addition of 15% fetal bovine serum to the culture medium. CONCLUSION: ORC1 gene silencing causes rat VSMCs to enter a reversible G(0) quiescent, growth arrested state; thus, ORC1 gene may be an important new target for suppressing VSMCs proliferation.

[Expression of origin recognition complex 1 gene in DNA replication of vascularsmooth muscle cells].

OBJECTIVE: To explore the expression of origin recognition complex 1 (ORC1) during the DNA replication of vascular muscle cells (VSMC). METHODS: VSMC of thoracic aorta in rats were obtained by the adherence method of tissue culture. The cell synchrony was obtained by the method of double-thymidine block, colchicine treatment and serum starvation. The expression of ORC1 mRNA at different cell cycles of VSMC was determined by RT-PCR and the protein expression of ORC1 was analyzed by Western blot. RESULTS: Cultured VSMC were identified by light microscope and immunocytochemistry. Significant expression of ORC1 mRNA and protein in a quiescent stage of VSMC were not observed. Upon synchronization, the expression of ORC1 mRNA was significantly higher at G(1)/S phase of VSMC than that at S and G(2)/M phases. The expression of ORC1 protein followed same changes as the ORC1 mRNA expression at different stages of cell cycles. CONCLUSION: ORC1 may be an important regulatory factor at the initiation of proliferative process of VSMC.

[Protective effects of short-term and long-term exercise preconditioning on myocardial injury in rats].

OBJECTIVE: To study the role and mechanism of myocardial apoptosis after short-term and long-term exercise preconditioning. METHODS: Forty-eight male SD rats were randomly divided into control group (C), exhaust group (E), short-exercise preconditioning (S-EP) and long-term exercise preconditioning group (L-EP). Short-term and long-term exercise preconditioning were conducted for 3 days and 3 weeks of repeated intermittent swimming training program. The changes of myocardial cells were observed under light microscope. The serum levels of ischemia-modified albumin(IMA) and creatine kinase-isoenzyme(CK-MB) were detected by ELISA. Real time fluorescence quantitative PCR and Western blot were used to detect the expressions of tumor necrosis factor-α(TNF-α),Caspase-8, Caspase-3 genes and proteins in myocardial tissue. The apoptosis of cardiomyocytes was observed by TUNEL method. RESULTS: Compared with group C, group E had serious myocardial injury. The levels of serum IMA, CK-MB and the expressions of TNF-α, Caspase-8 and Caspase-3 in myocardium were increased (P<0.05). Compared with group E, serum CK-MB and TNF-α and Caspase-8 mRNA in S-EP group were significantly lower than those in group E (P<0.05), but there was no significant difference in serum IMA and Caspase-3 mRNA and protein (P>0.05). The levels of serum IMA, CK-MB and TNF-α, Caspase-8 and Caspase-3 mRNA in L-EP group were significantly lower than those in control group (P<0.05). The apoptosis of cardiomyocytes in group E was obvious. Short-term and long-term exercise preconditioning could inhibit apoptosis. Compared with S-EP group, the apoptosis of L-EP group was significantly decreased. CONCLUSIONS: Short-term and long-term exercise preconditioning can reduce myocardial injury after exhaustive exercise, but short-term exercise preconditioning does not alter the expression of Caspase protease. Long-term exercise preconditioning significantly inhibits Caspase-8, 3 mRNA expression and reduces protein synthesis. The inhibitive effects of long-term exercise preconditioning on myocardial cell apoptosis were stronger than those of short-term exercise preconditioning.

[Gene therapy for ovarian cancers by adenovirus-mediated complete antibody gene].

OBJECTIVE: To study the feasibility of adenoviral transduction of Herceptin complete antibody gene and its effect on Her2 over-expressing cancer. METHODS: The genes of VH and VL from the monoclonal antibody Herceptin were cloned into the genome of replication-defective adenovirus by viral recombination technology to produce the recombinant adenovirus Ad-SG-Her. Normal human liver cells of the line L-02 were transfected with Ad-SG-Her and ELISA was used to detect the expression of Herceptin antibody 3 and 7 days after. Forty BALB/c nude mice were inoculated with Her2 high-expressing oophoroma cells of SK-OV-3 line and were randomized into 4 equal groups: group A injected with Ad-SG-Her at the dosage of: 2 x 10(9) plaque forming unit (pfu) through the caudal vein, group B injected with Ad-SG-Her at the dosage of 1 x 10(9) pfu, group C injected with Ad-SG-Her at the dosage of 5 x 10(8) pfu, and control group. On the days 3, 7, 10, 14, 21, 28, and 35 after the injection of virus, the antibody expression in the serum was measured by ELISA and the size of tumor was measured vernier caliper. Western blot and IFA was used to detect the specificity for Her2-overexpressing ovarian cancer cell lines SK-OV-3 and the integrity of complete antibody. Anti-tumor effects were also observed in nude mice bearing SK-OV-3 tumors. RESULTS: The constructed recombinant adenovirus Ad-SG-Her could express Herceptin efficiently both in vitro and in vivo. The biological activity of the expressed antibody was similar to that of the commercial Herceptin as shown by Western blotting, IFA, and ELISA. Herceptin expression of Ad-SG-Her was detected since day 3 after treatment in the groups A, B, and C and reached the peak on days 7 - 10. The expression lasted for four weeks or so. The expression level was significantly different between group A and the groups B and C (all P < 0.05), however, without a significant difference between the group B and group C. The antibody expression of group A might increase to 103.5 micro g/ml, high enough to inhibit tumor growth and induce tumor cell apoptosis. The antibody expression of the group B was below 40 micro g/ml, and that of the group C was below 30 micro g/ml. Furthermore the expressed antibody doses were statistically significantly different at different time points. Almost no tumor growth was seen in the group A during the observation period in comparison with the groups B and C and the control group (all P < 0.05). The tumor growth was almost not influenced in the group B and C and the control group. CONCLUSION: Ad-SG-Her efficiently expresses humanized complete Herceptin with effective bioactivity and induces long-term stable expression both in vitro and in vivo. The system may serve as a new antitumoral gene therapy strategy in antibody field.

[Effect of miR-146a on IL-18 expression in mouse macrophage].

AIM: To investigate the effect of miR-146a on the Th1/Th2 cytokine expression in mouse RAW264.7 cell line and primary peritoneal macrophage. METHODS: miR-146a mimics, mimics negative control (NC mimics), inhibitor miR-146a and inhibitor negative control (NC inhibitor) were transfected into RAW264.7 cells and freshly isolated peritoneal macrophage. IL-18, IL-5 and IL-10 expressions in the cells were measured by real time PCR. RESULTS: MiR-146a mimics suppressed IL-18 expression (P<0.05), and miR-146a specific inhibitor increased IL-18 expression significantly (P<0.05). However, IL-5 and IL-10 expressions were not affected by both miR-146a mimics and miR-146a inhibitor transfections. CONCLUSION: These data demonstrate at first time that miR-146a can regulate Th1 cytokine IL-18 expression, but not affect Th2 cytokine IL-5 and IL-10 expressions.

Reversal of hyperglycemia by protein transduction of NeuroD in vivo.

AIM: To test whether the neurogenic differentiation (NeuroD) protein could alleviate symptoms of diabetes mellitus by its transduction activity in vivo. METHODS: Type 1 diabetes mellitus in mice was induced by ip (intraperitoneal) injection of streptozotocin (150 mg/kg). One group of diabetic mice were intravenously injected with the NeuroD-EGFP (Enhanced Green Fluorescent Protein) (5 mg/kg, n=6) and the other group with EGFP (5 mg/kg, n=5). After the transduction of NeuroD-EGFP, the distribution of the protein was examined by means of frozen section under fluorescent microscope observation. We conducted RT-PCR and Real-time quantitative PCR to measure the transcription levels of insulin mRNA. Immunohistochemistry was utilized to detect the insulin protein. Radioimmunoassay was conducted to determine the serum insulin levels. Blood glucose levels and body weights were regularly recorded after the protein administration. RESULTS: The NeuroD protein can be transduced into cells in vivo with a high efficiency of nearly 100%. Insulin mRNA was highly expressed in NeuroD-treated diabetic mice, 38-fold higher than that of control group (P<0.05). Immunohistochemistry revealed enteric insulin expression in the NeuroD-treated diabetic mice. The fasting serum insulin level of the NeuroD-EGFP group (n=6) was 337+/-39 pg/mL, significantly higher than that of the control diabetic mice (n=5) which was 84+/-23 pg/mL (P<0.01, t-test). Records of blood glucose level also displayed alleviation of hyperglycemia after NeuroD administration (P<0.01, t-test, n=6). CONCLUSION: In vivo-transduced NeuroD in the small intestine remained functionally active and could ameliorate the non-fasting glucose levels of streptozotocin-induced, diabetic mice by inducing enteric insulin expression.