Anti-NSCLC activity in vitro of Hsp90N inhibitor KW-2478 and complex crystal structure determination of Hsp90N-KW-2478.

KW-2478 is a promising anti-cancer lead compound targeting to the molecular chaperone heat shock protein 90 N (Hsp90N). Absence of complex crystal structure of Hsp90N-KW-2478, however, hampered further structure optimization of KW-2478 and understanding on the molecular interaction mechanism. Herein, a high-resolution complex crystal structure of Hsp90N-KW-2478 was determined by X-ray diffraction (XRD, resolution limit: 1.59 Å; PDB ID: 6LT8) and their molecular interaction was analyzed in detail, which suggested that KW-2478 perfectly bound in the N-terminal ATP-binding pocket of Hsp90 to disable its molecular chaperone function, therefore suppressed or killed cancer cells. The results from thermal shift assay (TSA, ΔTm, 18.82 ± 0.51 °C) and isothermal titration calorimetry (ITC, Kd, 7.30 ± 2.20 nM) suggested that there is an intense binding force and favorable thermodynamic changes during the process of KW-2478 binding with Hsp90N. Additionally, KW-2478 exhibited favorable anti-NSCLC activity in vitro, as it inhibited cell proliferation (IC50, 8.16 µM for A549; 14.29 µM for H1975) and migration, induced cell cycle arrest and promoted apoptosis. Thirty-six novel KW-2478 derivatives were designed, based on the complex crystal structure and molecular interaction analysis of Hsp90N-KW-2478 complex. Among them, twenty-two derivatives exhibited increased binding force with Hsp90N evaluated by molecular docking assay. The results would provide new guidance for anti-NSCLC new drug development based on the lead compound KW-2478.

LncRNA UCA1 Promotes Mitochondrial Function of Bladder Cancer via the MiR-195/ARL2 Signaling Pathway.

BACKGROUND/AIMS: This study aims to identify whether Urothelial Cancer Associated 1 (UCA1) regulates mitochondrial metabolic reprogramming in bladder cancer, and to explore how UCA1 participates in mitochondrial metabolism by the UCA1/miR-195/ARL2 signaling pathway; these findings may be aid in the development of tumor diagnostic and therapeutic strategies. METHODS: Bladder tissues were obtained from patients. Stable cell lines were constructed, with ectopic expression of UCA1 in UMUC2 cells and knockdown of UCA1 in 5637 cells. The expression levels of UCA1, miR-195, and ARL2 were detected by real-time PCR, western blotting, and immunohistochemistry Cell viability was detected by Cell Counting Kit-8 (CCK8) assay; mitochondrial DNA copy numbers were tested by realtime PCR; ATP level was evaluated by ATP assay kit; mitochondrial membrane potential was analyzed by 5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolylcarbocyanine iodide (JC-1) fluorescent probe. miRNAs between UCA1 and ARL2 were predicted by TargetScan and RNAHybrid, and then determined by real-time PCR. Dual-luciferase activity assay and RNA immunoprecipitation (RIP) assay were used to verify the relationship between UCA1 and miR-195. The expression level of ARL2 was silenced by small interfering RNA(siRNA). For in vivo experiments, UCA1-silencing 5637 cells were subcutaneously injected into BALB/C nude mice to evaluate the effects of UCA1 on tumor progression by the regulation of miR-195 and ARL2. RESULTS: We demonstrate here that UCA1 enhances mitochondrial function in bladder cancer cells. UCA1 contributes to ARL2-induced mitochondrial activity, which plays an important role in mitochondrial function. UCA1, as a competing endogenous RNA (ceRNA), regulates mitochondrial function through upregulating ARL2. In this way, it inhibited the miR-195 signaling pathway to enhance mitochondrial function in bladder cancer. Additionally, ARL2 is a direct target of miR-195 and can be repressed by either miR-195 overexpression or UCA1 inhibition. Knockdown of ARL2 was analogous to the inhibition of UCA1 and the upregulation of miR-195. Animal experiments further indicated that UCA1 promoted bladder tumor growth by regulating miR-195 /ARL2. CONCLUSION: These data suggest that UCA1 enhanced mitochondrial function and cell viability through the UCA1/miR-195/ARL2 axis in vitro and in vivo. The elucidation of this signaling network provides a more adequate theoretical basis for understanding the molecular pathology of bladder cancer, and also UCA1 as a potential diagnosis and treatment target for bladder cancer.

Long non-coding RNA UCA1 promotes glutamine metabolism by targeting miR-16 in human bladder cancer.

OBJECTIVE: Long non-coding ribonucleic acid urothelial carcinoma-associated 1 has been found to be a participant in cancer development and glucose metabolism in bladder cancer. However, the role of urothelial carcinoma-associated 1 in metabolic reprogramming in cancer remains to be clarified. In this study, we aim to elucidate the molecular mechanism underlying the regulation of glutamine metabolism by urothelial carcinoma-associated 1 in bladder cancer. METHODS: The RNA levels of urothelial carcinoma-associated 1, GLS2 and miR-16 in bladder tissues and cell lines were examined by real-time reverse transcriptase-polymerase chain reaction. The protein levels of GLS2 were detected by western blot analysis. Reactive oxygen species generation was examined by the fluorescein isothiocyanate mean value and fluorescence microscope. Glutamine consumption was analyzed using the glutamine assay kit. Additionally, we performed luciferase reporter assays to validate urothelial carcinoma-associated 1 sequence whether contains miR-16 binding site and the interaction between the 3'UTR sequence of GLS2 and mature miR-16. RESULTS: Real-time reverse transcriptase-polymerase chain reaction demonstrated that the RNA level of urothelial carcinoma-associated 1 and GLS2 was positively correlated in bladder cancer tissues and cell lines. The expression of GLS2 mRNA and protein increased in cells which overexpression of urothelial carcinoma-associated 1 and decreased in cells which knocked-down of urothelial carcinoma-associated 1 cell lines. urothelial carcinoma-associated 1 reduced ROS production, and promoted mitochondrial glutaminolysis in human bladder cancer cells. Furthermore, luciferase reporter assays indicated that there was a miR-16 binding site in urothelial carcinoma-associated 1, and it showed appreciable levels of sponge effects on miR-16 as readouts in a dose-dependent manner. Moreover, the 'seed region' of miR-16 directly bound to the 3'UTR of GLS2 mRNA and regulated GLS2 expression level. CONCLUSIONS: Together, our results revealed that urothelial carcinoma-associated 1 regulated the expression of GLS2 through interfering with miR-16, and repressed ROS formation in bladder cancer cells.

Complex Crystal Structure Determination of Hsp90N-NVP-AUY922 and In Vitro Anti-NSCLC Activity of NVP-AUY922.

New targeted chemotherapy agents greatly improved five-year survival in NSCLC patients, but which were susceptible to drug resistance. NVP-AUY922, terminated in phase II clinical trials, exhibited promising anti-NSCLC (non-small-cell lung cancer) activity targeting to Hsp90N (heat shock protein), which demonstrated advantages in overcoming drug resistance as a broad-spectrum anti-cancer target. It was expected to develop novel anti-NSCLC drugs to overcome drug resistance by the structural optimization of NVP-AUY922. However, the absence of high-resolution complex crystal structure of Hsp90N-NVP-AUY922 blocked the way. Herein, 1.59 Å-resolution complex crystal structure of Hsp90N-NVP-AUY922 (PDB ID 6LTI) was successfully determined by X-ray diffraction. Meanwhile, there was a strong binding capability between NVP-AUY922 and its target Hsp90N verified by TSA (ΔTm, -15.56 ± 1.78°C) and ITC (K d, 5.10 ± 2.10 nM). Results by the complex crystal structure, TSA and ITC verified that NVP-AUY922 well accommodated in the ATP-binding pocket of Hsp90N to disable the molecular chaperone activity of Hsp90. Therefore, NVP-AUY922 exhibited approving inhibitory activity on NSCLC cell line H1299 (IC50, 2.85 ± 0.06 µM) by inhibiting cell proliferation, inducing cell cycle arrest and promoting cell apoptosis. At the basis of the complex crystal structure and molecular interaction analysis, thirty-two new NVP-AUY922 derivatives were further designed, and among which twenty-eight new ones display enhanced binding force with Hsp90N by molecular docking evaluation. The results would promote anti-NSCLC new drug development to overcome drug resistance based on the lead compound NVP-AUY922.

New paeonol derivative C302 reduces hypertension in spontaneously hypertensive rats through endothelium-dependent and endothelium-independent vasodilation.

Hypertension is a major risk factor for cardiovascular disease and Chinese herb monomers could provide new structural skeletons for anti-hypertension new drug development. Paeonol is a Chinese herbal monomer extracted from Cortex moutan, exhibited some anti-hypertensive activity. The study focused on the structural optimization of paeonol to provide promising lead compounds for anti-hypertension new drug development. Herein, twelve new paeonol derivatives (PD) were designed and synthesized and their vasodilation activity was evaluated by in vitro vasodilation drug screening platform based on Myograph. Its anti-hypertension activity, PD-C302 (2-hydroxy-4-methoxyvalerophenone) as a representative with the optimal vasodilation activity, was determined by its response to blood pressure in spontaneously hypertensive rats (SHR) in vivo. Moreover, its molecular mechanism was probed by the vasodilation activity of rat superior mesenteric artery rings with or without endothelium pre-contracted by potassium chloride (KCl) or phenylephrine hydrochloride (PE). It was indicated that PD-C302 significantly reduced the blood pressure in SHR, which would involve in PD-C302-induced vasodilation. Furthermore, endothelium-dependent pathways and endothelium-independent pathways both contributed importantly to PD-C302-induced vasodilation at low concentration of PD-C302. Endothelium-independent pathways (vascular smooth muscle cell-mediated vasodilation), were mainly responsible for the PD-C302-induced vasodilation at high concentration of PD-C302, which involved in opening multiple K+ channels to restrain Ca2+ channels, and then triggered vasodilation to reduce blood pressure. PD-C302 has a simple structure and favorable anti-hypertensive activity in vivo, which could be a promising lead compound for anti-hypertension new drug development.

Role of Long Non-coding RNAs on Bladder Cancer.

Bladder cancer (BC) is the most common malignant tumor in the urinary system, and its early diagnosis is conducive to improving clinical prognosis and prolonging overall survival time. However, few biomarkers with high sensitivity and specificity are used as diagnostic markers for BC. Multiple long non-coding RNAs (lncRNAs) are abnormally expressed in BC, and play key roles in tumorigenesis, progression and prognosis of BC. In this review, we summarize the expression, function, molecular mechanisms and the clinical significance of lncRNAs on bladder cancer. There are more than 100 dysregulated lncRNAs in BC, which are involved in the regulation of proliferation, cell cycle, apoptosis, migration, invasion, metabolism and drug resistance of BC. Meanwhile, the molecular mechanisms of lncRNAs in BC was explored, including lncRNAs interacting with DNA, RNA and proteins. Additionally, the abnormal expression of thirty-six lncRNAs is closely associated with multiple clinical characteristics of BC, including tumor size, metastasis, invasion, and drug sensitivity or resistance of BC. Furthermore, we summarize some potential diagnostic and prognostic biomarkers of lncRNA for BC. This review provides promising novel biomarkers in early diagnosis, prognosis and monitoring of BC based on lncRNAs.

Atheroprotective Effects and Molecular Mechanism of Berberine.

Cardiovascular diseases remain the leading cause of morbidity and mortality worldwide. Atherosclerosis is the main pathological basis of cardiovascular diseases and it is closely associated with hyperlipidemia, endothelial injury, macrophage-derived foam cells formation, proliferation and migration of vascular smooth muscle cells (VSMCs), platelet aggregation, and altered gut microbiota. Various symptomatic treatments, that are currently used to inhibit atherosclerosis, need to be administered in long term and their adverse effects cannot be ignored. Berberine (BBR) has beneficial effects on atherosclerosis through regulating multiple aspects of its progression. This review highlights the recent advances in understanding the anti-atherosclerosis mechanism of BBR. BBR alleviated atherosclerosis by attenuation of dyslipidemia, correction of endothelial dysfunction, inhibition of macrophage inflammation and foam cell formation, activation of macrophage autophagy, regulation of the proliferation and migration of VSMCs, attenuation of platelet aggregation, and modulation of gut microbiota. This review would provide a modern scientific perspective to further understanding the molecular mechanism of BBR attenuating atherosclerosis and supply new ideas for atherosclerosis management.

Rational Design and Synthesis of 3-Morpholine Linked Aromatic-Imino-1H-Indoles as Novel Kv1.5 Channel Inhibitors Sharing Vasodilation Effects.

Atrial fibrillation (AF) is the most common clinical sustained arrhythmia; clinical therapeutic drugs have low atrial selectivity and might cause more severe ventricle arrhythmias while stopping AF. As an anti-AF drug target with high selectivity on the atrial muscle cells, the undetermined crystal structure of Kv1.5 potassium channel impeded further new drug development. Herein, with the simulated 3D structure of Kv1.5 as the drug target, a series of 3-morpholine linked aromatic amino substituted 1H-indoles as novel Kv1.5 channel inhibitors were designed and synthesized based on target-ligand interaction analysis. The synthesis route was practical, starting from commercially available material, and the chemical structures of target compounds were characterized. It was indicated that compounds T16 and T5 (100 µM) exhibited favorable inhibitory activity against the Kv1.5 channel with an inhibition rate of 70.8 and 57.5% using a patch clamp technique. All compounds did not exhibit off-target effects against other drug targets, which denoted some selectivity on the Kv1.5 channel. Interestingly, twelve compounds exhibited favorable vasodilation activity on pre-contracted arterial rings in vitro using KCl or phenylephrine (PE) by a Myograph. The vasodilation rates of compounds T16 and T4 (100 µM) even reached over 90%, which would provide potential lead compounds for both anti-AF and anti-hypertension new drug development.

Multiple functions of autophagy in vascular calcification.

BACKGROUND: Vascular calcification is a closely linked to cardiovascular diseases, such as atherosclerosis, chronic kidney disease, diabetes, hypertension and aging. The extent of vascular calcification is closely correlate with adverse clinical events and cardiovascular all-cause mortality. The role of autophagy in vascular calcification is complex with many mechanistic unknowns. METHODS: In this review, we analyze the current known mechanisms of autophagy in vascular calcification and discuss the theoretical advantages of targeting autophagy as an intervention against vascular calcification. RESULTS: Here we summarize the functional link between vascular calcification and autophagy in both animal models of and human cardiovascular disease. Firstly, autophagy can reduce calcification by inhibiting the osteogenic differentiation of VSMCs related to ANCR, ERα, ß-catenin, HIF-1a/PDK4, p62, miR-30b, BECN1, mTOR, SOX9, GHSR/ERK, and AMPK signaling. Conversely, autophagy can induce osteoblast differentiation and calcification as mediated by CREB, degradation of elastin, and lncRNA H19 and DUSP5 mediated ERK signaling. Secondly, autophagy also links apoptosis and vascular calcification through AMPK/mTOR/ULK1, Wnt/ß-catenin and GAS6/AXL synthesis, as apoptotic cells become the nidus for calcium-phosphate crystal deposition. The failure of mitophagy can activate Drp1, BNIP3, and NR4A1/DNA­PKcs/p53 mediated intrinsic apoptotic pathways, which have been closely linked to the formation of vascular calcification. Additionally, autophagy also plays a role in osteogenesis by regulating vascular calcification, which in turn regulates expression of proteins related to bone development, such as osteocalcin, osteonectin, etc. and regulated by mTOR, EphrinB2 and RhoA. Furthermore, autophagy also promotes vitamin K2-induced MC3T3 E1 osteoblast differentiation and FGFR4/FGF18- and JNK/complex VPS34-beclin-1-related bone mineralization via vascular calcification. CONCLUSION: The interaction between autophagy and vascular calcification are complicated, with their interaction affected by the disease process, anatomical location, and the surrounding microenvironment. Autophagy activation in existent cellular damage is considered protective, while defective autophagy in normal cells result in apoptotic activation. Identifying and maintaining cells at the delicate line between these two states may hold the key to reducing vascular calcification, in which autophagy associated clinical strategy could be developed.

Complex crystal structure determination and anti-non-small-cell lung cancer activity of the Hsp90N inhibitor Debio0932.

Debio0932 is a promising lead compound in phase I clinical trials targeting the N-terminal ATP-binding pocket of the molecular chaperone heat-shock protein 90 (Hsp90N). The absence of a crystal structure of the Hsp90N-Debio0932 complex, however, has impeded further structural optimization of Debio0932 and understanding of the molecular-interaction mechanism. Here, a high-resolution crystal structure of the Hsp90N-Debio0932 complex was successfully determined (resolution limit 2.20 Å; PDB entry 6lr9) by X-ray diffraction and the molecular-interaction mechanism was analysed in detail, which suggested that Debio0932 suppresses cancer cells by accommodating itself in the ATP-binding pocket of Hsp90N, disabling its molecular-chaperone capability. The results of a thermal shift assay (ΔTm = 8.83 ± 0.90°C) and isothermal titration calorimetry (Kd = 15.50 ± 1.30 nM) indicated strong binding and favourable thermodynamic changes in the binding of Hsp90N and Debio0932. Based on the crystal structure of the complex and on molecular-interaction analysis, 30 new Debio0932 derivatives were designed and nine new derivatives exhibited increased binding to Hsp90N, as determined by molecular-docking evaluation. Additionally, Debio0932 suppressed cell proliferation (IC50 values of 3.26 ± 2.82 µM for A549, 20.33 ± 5.39 µM for H1299 and 3.16 ± 1.04 µM for H1975), induced cell-cycle arrest and promoted apoptosis in three non-small-cell lung cancer (NSCLC) cell lines. These results provide novel perspectives and guidance for the development of new anti-NSCLC drugs based on the lead compound Debio0932.

Complex Crystal Structure Determination and in vitro Anti-non-small Cell Lung Cancer Activity of Hsp90 N Inhibitor SNX-2112.

SNX-2112, as a promising anticancer lead compound targeting heat shock protein 90 (Hsp90), absence of complex crystal structure of Hsp90 N -SNX-2112 hindered further structural optimization and understanding on molecular interaction mechanism. Herein, a high-resolution complex crystal structure of Hsp90 N -SNX-2112 was successfully determined by X-ray diffraction, resolution limit, 2.14 Å, PDB ID 6LTK, and their molecular interaction was analyzed in detail, which suggested that SNX-2112 was well accommodated in the ATP-binding pocket to disable molecular chaperone activity of Hsp90, therefore exhibiting favorable inhibiting activity on three non-small cell lung cancer (NSCLC) cell lines (IC50, 0.50 ± 0.01 µM for A549, 1.14 ± 1.11 µM for H1299, 2.36 ± 0.82 µM for H1975) by inhibited proliferation, induced cell cycle arrest, and aggravated cell apoptosis. SNX-2112 exhibited high affinity and beneficial thermodynamic changes during the binding process with its target Hsp90 N confirmed by thermal shift assay (TSA, ΔTm, and -9.51 ± 1.00°C) and isothermal titration calorimetry (K d , 14.10 ± 1.60 nM). Based on the complex crystal structure and molecular interaction analysis, 32 novel SNX-2112 derivatives were designed, and 25 new ones displayed increased binding force with the target Hsp90 N verified by molecular docking evaluation. The results would provide new references and guides for anti-NSCLC new drug development based on the lead compound SNX-2112.

Nϵ-Carboxymethyl-Lysine Deteriorates Vascular Calcification in Diabetic Atherosclerosis Induced by Vascular Smooth Muscle Cell-Derived Foam Cells.

Nϵ-carboxymethyl-lysine (CML), an advanced glycation end product, is involved in vascular calcification (VC) in diabetic atherosclerosis. This study aimed to investigate the effects of CML on VC in diabetic atherosclerosis induced by vascular smooth muscle cell (VSMC)-derived foam cells. Human studies, animal studies and cell studies were performed. The human study results from 100 patients revealed a poor blood glucose and lipid status and more severe coronary lesions and stenosis in patients with coronary artery disease and diabetes mellitus. Intraperitoneal injection of streptozotocin combined with a high-fat diet was used to build a diabetic atherosclerosis model in ApoE-/- mice. The animal study results indicated that CML accelerated VC progression in diabetic atherosclerosis by accelerating the accumulation of VSMC-derived foam cells in ApoE-/- mice. The cell study results illustrated that CML induced VSMC-derived foam cells apoptosis and aggravated foam cells calcification. Consistent with this finding, calcium content and the expression levels of alkaline phosphatase, bone morphogenetic protein 2 and runt-related transcription factor 2 were significantly elevated in A7r5 cells treated with oxidation-low-density lipoprotein and CML. Thus, we concluded that CML promoted VSMC-derived foam cells calcification to aggravate VC in diabetic atherosclerosis, providing evidence for the contribution of foam cells to diabetic VC.

Research Progress on PARP14 as a Drug Target.

Poly-adenosine diphosphate-ribose polymerase (PARP) implements posttranslational mono- or poly-ADP-ribosylation modification of target proteins. Among the known 18 members in the enormous family of PARP enzymes, several investigations about PARP1, PARP2, and PARP5a/5b have been launched in the past few decades; more specifically, PARP14 is gradually emerging as a promising drug target. An intact PARP14 (also named ARTD8 or BAL2) is constructed by macro1, macro2, macro3, WWE, and the catalytic domain. PARP14 takes advantage of nicotinamide adenine dinucleotide (NAD+) as a metabolic substrate to conduct mono-ADP-ribosylation modification on target proteins, taking part in cellular responses and signaling pathways in the immune system. Therefore, PARP14 has been considered a fascinating target for treatment of tumors and allergic inflammation. More importantly, PARP14 could be a potential target for a chemosensitizer based on the theory of synthetic lethality and its unique role in homologous recombination DNA repair. This review first gives a brief introduction on several representative PARP members. Subsequently, current literatures are presented to reveal the molecular mechanisms of PARP14 as a novel drug target for cancers (e.g., diffuse large B-cell lymphoma, multiple myeloma, prostate cancer, and hepatocellular carcinoma) and allergic inflammatory. Finally, potential PARP inhibitor-associated adverse effects are discussed. The review could be a meaningful reference for innovative drug or chemosensitizer discovery targeting to PARP14.

Localization Analysis of Heterophilic Antigen Epitopes of H1N1 Influenza Virus Hemagglutinin.

Previous studies have indicated that two monoclonal antibodies (mAbs; A1-10 and H1-84) of the hemagglutinin (HA) antigen on the H1N1 influenza virus cross-react with human brain tissue. It has been proposed that there are heterophilic epitopes between the HA protein and human brain tissue (Guo et al. in Immunobiology 220:941-946, 2015). However, characterisation of the two mAbs recognising the heterophilic epitope on HA has not yet been performed. In the present study, the common antigens of influenza virus HA were confirmed using indirect enzyme-linked immunosorbent assays and analysed with DNAMAN software. The epitopes were localized to nine peptides in the influenza virus HA sequence and the distribution of the peptides in the three-dimensional structure of HA was determined using PyMOL software. Key amino acids and variable sequences of the antibodies were identified using abYsis software. The results demonstrated that there were a number of common antigens among the five influenza viruses studied that were recognised by the mAbs. One of the peptides, P2 (LVLWGIHHP191-199), bound both of the mAbs and was located in the head region of HA. The key amino acids of this epitope and the variable regions in the heavy and light chain sequences of the mAbs that recognised the epitope are described. A heterophilic epitope on H1N1 influenza virus HA was also introduced. The existence of this epitope provides a novel perspective for the occurrence of nervous system diseases that could be caused by influenza virus infection, which might aid in influenza prevention and control.

[Case-control study on the iliac bone flap transplantation with deep circumflex iliac artery and quadratus femoris bone flap transplantation for the treatment of Garden III/IV femoral neck fracture of young and middle-aged patients].

OBJECTIVE: To compare the clinical effects between hip anterior S-P approach combined with iliac bone flap transplantation with deep circumflex iliac artery and posterior K-L approach combined with quadratus femoris bone flap transplantation for the treatment of femoral neck fracture of Garden III-IV in young and middle-aged patients. METHODS: From January 2004 to January 2011,46 patients with femoral neck fractures were treated by two kinds of operation. Among them, 20 cases were treated with anterior S-P approach combined with iliac bone flap transplantation with deep circumflex iliac artery, included 12 males and 8 females with an average age of (32.1 ± 7.3) years old, involved 12 cases of Garden III and 8 cases of Garden IV. The other 26 cases were treated with posterior K-L approach combined with quadratus femoris bone flap transplantation, included 20 males and 6 females with an average age of (37.8 ± 6.9) years old, involved 16 cases of Garden III and 10 cases of Garden IV. The index of hospitalization (hospitalization time, total cost, operative time, intraoperative blood loss, postoperative complications), the quality index of operation (fracture reduction, position of internal fixation, fracture healing time, nonunion and femoral head necrosis) of two groups were observed and compared. Hip joint function were evaluated by Harris score. RESULTS: All patients were followed up from 28 to 41 months with an average of 36 months. The intraoperative blood loss of group S-P (92.3 ± 10.4) ml was less than that of group K-L (132.4 ± 11.2) ml, there was significant difference between two groups (P < 0.05). The operation time of group S-P (81.4 ± 9.2) min was more than that of group K-L (67.1 ± 4.5) min, the difference was statistically significant (P < 0.05). One case in group S-P and 9 cases in group K-L appeared postoperative complications, there was significant difference between two groups (P < 0.05). The fracture healing time of S-P group (83.5 ± 7.3) d was shorter than that of group K-L (103.2 ± 12.6) d, there was significant difference between two groups (P < 0.05). At 30 months after operation, there were significant difference in Harris scoring between two groups (P < 0.05). CONCLUSION: Anterior S-P approach combined with iliac bone flap transplantation with deep circumflex iliac artery for treatment of femoral neck fracture of Garden III-IV of young and middle-aged patients, it has characteristics in clear anatomic and easy to operate. As compared with K-L approach, S-P approach can better reserve residual blood supply of femoral neck. While combining with the iliac bone flap transplantation with deep circumflex iliac artery, it could better reconstruct the blood supply of femoral neck to promote fracture healing.

[Preparation and partial characterization of monoclonal antibodies against HA protein of H1 subtype influenza virus].

AIM: Preparation of H1 subtype of influenza virus HA protein monoclonal antibody (mAbs), and to analyse their reactivity. METHODS: H1N1 influenza virus vaccine (2009) and seasonal A1 influenza virus vaccine as antigen, conventional immunization, fusion, cloning, access to the antigen-specific mAbs. To study the reactivity and specificity of mAbs by ELISA, HI test and Western blot. RESULTS: We have obtained 97 hybridoma of stable secreting anti-H1 subtype of influenza virus HA protein. According to their different reactivity, these mAbs can be divided into four categories: 39 strains for strain-specific, of which 29 have HI activity; 7 strains for subtype-specific, of which 5 have HI activity; 16 strains for the 2009 pandemic strain and the seasonal strains to common antigens, of which 9 have HI activity; 35 strains common for influenza virus antigens, of which 22 have HI activity. CONCLUSION: Both vaccines have better immunogenicity and immune protection activity. Preparation of four types antibody against HA protein of H1 subtype of influenza virus, can provide experimental data for preparation subtype-specific diagnostic kits and influenza A and B virus diagnostic kit, and lay a foundation for further studying of H1N1 influenza virus HA epitope.

[Comparison study of small splint fixation and plaster slab fixation for the treatment of distal radius fractures].

OBJECTIVE: To study the advantage of fir bark splint for the treatmet of distal radius fracture. METHODS: From January 2006 to June 2008, 80 cases were randomly divided into two groups and treated by fir bark splint fixation or plaster fixation. There were 40 cases in the fir bark splint group, including 18 males and 22 females with an average age of 45.2 years ranging from 20 to 66 years. There were 40 cases in the plaster group, including 19 males and 21 females with an average age of 44.1 years ranging from 23 to 65 years. Four weeks after manipulative reduction and external fixation, the fixtion effect, function recovery, bone mineral density of secondly metacarpus were obsearved by X-ray film, and the clinical effect were evaluated according to healing time ahead of schedule. RESULTS: As compared between the two groups, the fixation effect of the fir bark splint group was better than that of the plaster group (Z = -2.844, P = 0.004 < 0.05); the function recovery of the fir bark splint group was better than that of the plaster group (Z = -3.196, P = 0.001 < 0.05); the bone mineral density of secondly metacarpus of the fir bark splint group were better than that of plaster group (t = 4.56, P < 0.001); the curative effect of fir bark splint group was better than that of the plaster group (Z = -2.601, P = 0.009 < 0.05). CONCLUSION: Fir bark splint could effectively keep fixation stability and promote fracture healing as well as function recovery.

[Construction and expression of recombinant adenovirus vector carrying human endostatin, K5 and endostatin-K5 gene].

AIM: To construct the recombinant adenoviral vectors expressing human endostatin, K5 and endostatin-K5 gene respectively, and study their bioactivity in vitro. METHODS: Human endostatin, K5 and endostatin-K5 gene were amplified by PCR, which were then subcloned into shuttle vector pAd5-CMV-H1H2-MCS-6His by enzyme and ligation respectively. The positive recombinant plasmids linearized by Pac I were cotransfected into HEK 293 cells with the Pac I linearized adenoviral backbone plasmid using calcium phosphate precipitation method. The recombinant viruses were purified by CsCl density gradient centrifugation. The protein expression at different time points (24 h, 48 h and 72 h) was determined by Western blot. The inhibitory effect of the protein on ECV-304 growth was detected by MTT. RESULTS: The recombinant adenoviral vectors expressing human endostatin, K5 and endostatin-K5 gene were successfully constructed in HEK293 cells. Protein expression was detected by Western blot. The level of protein expression was increased with the prolonged incubation of the infected HeLa cells. Three kinds of the protein expressed by the recombinant adenoviral vectors showed obvious inhibitory effect on ECV-304 cell growth. CONCLUSION: The protein expressed by adenoviral vectors carrying endostatin, K5 and endostatin-K5 gene has an obvious inhibitory effect on ECV-304 cell growth.

[Construction of an expression vector for high expression of siRNA].

AIM: To construct the vector for efficient expression of siRNA using pre-mir30 backbone. METHODS: By chemical synthesis method, pre-mir30 backbone introduced an appropriate restriction enzyme site for foreign shRNA inserting was cloned into an expressing vector containing U6 promoter. The silencing efficiency of a new siRNA expressing vector was detected by transfection and Western blot. RESULTS: The new vector containing pre-mir30 backbone expressing siRNA against GFP could markedly inhibit the expression of GFP compared with the vector expressing control siRNA. CONCLUSION: siRNA expressing vector constructed by pre-mir30 backbone could highly express foreign siRNA.